A novel peanut allergy immunotherapy: Plant-based enveloped Ara h 2 Bioparticles activate dendritic cells and polarize T cell responses to Th1.

Introduction: As the only market-authorized allergen immunotherapy (AIT) for peanut allergy is accompanied by a high risk of side effects and mainly induces robust desensitization without sustained efficacy, novel treatment options are required. Peanut-specific plant-derived eBioparticles (eBPs) surface expressing Ara h 2 at high density have been shown to be very hypoallergenic. Here, we assessed the dendritic cell (DC)-activating and T cell polarization capacity of these peanut-specific eBPs. Methods: Route and kinetics of eBP uptake were studied by (imaging) flow cytometry using monocyte-derived DCs incubated with fluorescently-labelled Ara h 2 eBPs or natural Ara h 2 (nAra h 2) in the presence or absence of inhibitors that block pathways involved in macropinocytosis, phagocytosis, and/or receptor-mediated uptake. DC activation was monitored by flow cytometry (maturation marker expression) and ELISA (cytokine production). T cell polarization was assessed by co-culturing DCs exposed to Ara h 2 eBPs or nAra h 2 with naïve CD4+ T cells, followed by flow cytometry assessment of intracellular IFNγ+ (Th1) and IL-13+ (Th2), and CD25+CD127-Foxp3+ regulatory T cells (Tregs). The suppressive activity of Tregs was tested using a suppressor assay. Results: Ara h 2 eBPs were taken up by DCs through actin-dependent pathways. They activated DCs demonstrated by an induced expression of CD83 and CD86, and production of TNFα, IL-6, and IL-10. eBP-treated DCs polarized naïve CD4+ T cells towards Th1 cells, while reducing Th2 cell development. Furthermore, eBP-treated DCs induced reduced the frequency of Foxp3+ Tregs but did not significantly affect T cell IL-10 production or T cells with suppressive capacity. In contrast, DC activation and Th1 cell polarization were not observed for nAra h 2. Conclusion: Ara h 2 eBPs activate DCs that subsequently promote Th1 cell polarization and reduce Th2 cell polarization. These characteristics mark Ara h 2 eBPs as a promising novel candidate for peanut AIT.

Study of NIT domain-containing chemoreceptors from two global phytopathogens and identification of NIT domains in eukaryotes.

Bacterial signal transduction systems are typically activated by the binding of signal molecules to receptor ligand binding domains (LBDs), such as the NIT LBD. We report here the identification of the NIT domain in more than 15,000 receptors that were present in 30 bacterial phyla, but also in 19 eukaryotic phyla, expanding its known phylogenetic distribution. The NIT domain formed part of seven receptor families that either control transcription, mediate chemotaxis or regulate second messenger levels. We have produced the NIT domains from chemoreceptors of the bacterial phytopathogens Pectobacterium atrosepticum (PacN) and Pseudomonas savastanoi (PscN) as individual purified proteins. High-throughput ligand screening using compound libraries revealed a specificity for nitrate and nitrite binding. Isothermal titration calorimetry experiments showed that PacN-LBD bound preferentially nitrate ( K D = 1.9 µM), whereas the affinity of PscN-LBD for nitrite ( K D = 2.1 µM) was 22 times higher than that for nitrate. Analytical ultracentrifugation experiments indicated that PscN-LBD is monomeric in the presence and absence of ligands. The R182A mutant of PscN did not bind nitrate or nitrite. This residue is not conserved in the NIT domain of the Pseudomonas aeruginosa chemoreceptor PA4520, which may be related to its failure to bind nitrate/nitrite. The magnitude of P. atrosepticum chemotaxis towards nitrate was significantly greater than that of nitrite and pacN deletion almost abolished responses to both compounds. This study highlights the important role of nitrate and nitrite as signal molecules in life and advances our knowledge on the NIT domain as universal nitrate/nitrite sensor module.

Synthesis of Nanocrystalline PuO2 by Hydrothermal and Thermal Decomposition of Pu(IV) Oxalate: A Comparative Study.

In recent years, the hydrothermal conversion of actinide (IV) oxalates into nanometric actinide dioxides (AnO2) has begun to be investigated as an alternative to the widely implemented thermal decomposition method. We present here a comparison between the hydrothermal and the conventional thermal decomposition of Pu(IV) oxalate in terms of particle size, morphology and residual carbon content. A parametric study was carried out in order to define the temperature and time applied in the hydrothermal conversion of tetravalent Pu-oxalate into PuO2 and to optimize the reaction conditions.

Preparation and Investigation of Crucial Oligomers in the Early Stages of Aß40 and Aß42 Aggregation.

Amyloid aggregation is a hallmark in many neuropathologies and other diseases of tremendous impact. It is increasingly evident that neuronal death associated with Alzheimer's disease (AD) is mainly produced by oligomers of the amyloid-ß (Aß) peptide. Yet little is known about the detailed structural and biophysical mechanisms of their formation. This lack of complete understanding comes from the labile nature and handling complexity of the oligomers. Consequently, providing reproducible and robust protocols for oligomer preparation is of particular importance.In this study, we describe detailed methods for the preparation and isolation of micellar oligomers of Aß that evolve towards larger and more stable oligomers enriched in beta-sheet structure and able to acquire a higher capacity to fibrillate. We also describe briefly some biophysical experiments allowing oligomer characterization.

A fast and easy one-step purification strategy for plant-made antibodies using Protein A magnetic beads.

A major difficulty to reach commercial- scale production for plant-made antibodies is the complexity and cost of their purification from plant extracts. Here, using Protein A magnetic beads, two monoclonal antibodies are purified in a one-step procedure directly from non-clarified crude plant extracts. This technique provides significant savings in terms of resources, operation time, and equipment.

Conformational flexibility of the conserved hydrophobic pocket of HIV-1 gp41. Implications for the discovery of small-molecule fusion inhibitors.

During HIV-1 infection, the envelope glycoprotein subunit gp41 folds into a six-helix bundle structure (6HB) formed by the interaction between its N-terminal (NHR) and C-terminal (CHR) heptad-repeats, promoting viral and cell membranes fusion. A highly preserved, hydrophobic pocket (HP) on the NHR surface is crucial in 6HB formation and, therefore, HP-binding compounds constitute promising therapeutics against HIV-1. Here, we investigated the conformational and dynamic properties of the HP using a rationally designed single-chain protein (named covNHR) that mimics the gp41 NHR structure. We found that the fluorescent dye 8-anilino-naphtalene-1-sulfonic acid (ANS) binds specifically to the HP, suggesting that ANS derivatives may constitute lead compounds to inhibit 6HB formation. ANS shows different binding modes to the HP, depending on the occupancy of other NHR pockets. Moreover, in presence of a CHR peptide bound to the N-terminal pockets in gp41, two ANS molecules can occupy the HP showing cooperative behavior. This binding mode was assessed using molecular docking and molecular dynamics simulations. The results show that the HP is conformationally flexible and connected allosterically to other NHR regions, which strongly influence the binding of potential ligands. These findings could guide the development of small-molecule HIV-1 inhibitors targeting the HP.

Rapid Conversion of Amyloid-Beta 1-40 Oligomers to Mature Fibrils through a Self-Catalytic Bimolecular Process.

The formation of fibrillar aggregates of the amyloid beta peptide (Aß) in the brain is one of the hallmarks of Alzheimer's disease (AD). A clear understanding of the different aggregation steps leading to fibrils formation is a keystone in therapeutics discovery. In a recent study, we showed that Aß40 and Aß42 form dynamic micellar aggregates above certain critical concentrations, which mediate a fast formation of more stable oligomers, which in the case of Aß40 are able to evolve towards amyloid fibrils. Here, using different biophysical techniques we investigated the role of different fractions of the Aß aggregation mixture in the nucleation and fibrillation steps. We show that both processes occur through bimolecular interplay between low molecular weight species (monomer and/or dimer) and larger oligomers. Moreover, we report here a novel self-catalytic mechanism of fibrillation of Aß40, in which early oligomers generate and deliver low molecular weight amyloid nuclei, which then catalyze the rapid conversion of the oligomers to mature amyloid fibrils. This fibrillation catalytic activity is not present in freshly disaggregated low-molecular weight Aß40 and is, therefore, a property acquired during the aggregation process. In contrast to Aß40, we did not observe the same self-catalytic fibrillation in Aß42 spheroidal oligomers, which could neither be induced to fibrillate by the Aß40 nuclei. Our results reveal clearly that amyloid fibrillation is a multi-component process, in which dynamic collisions between different interacting species favor the kinetics of amyloid nucleation and growth.

[Amoxicillin induced crystal nephropathy : Monitoring of residual plasma levels]. / Cristalluries sous amoxicilline : intérêt du monitoring des concentrations plasmatiques résiduelles.

Since 2010, a lot of cases of amoxicillin induced crystal nephropathy have been reported to the French pharmacovigilance centers partly due to the high doses recommended by infectious disease guidelines. Typical clinical presentation and exclusion of others toxics or immuno-allergic causes are mandatory to assess the diagnostic. Amoxicillin crystals are rarely found or searched and renal biopsy is not frequently performed due to technical reasons and prompt renal recovery after antibiotics withdrawal. Monitoring of residual plasma concentration is rarely used in clinical practice for diagnostic or prognostic interest. We present 9 consecutive cases of acute kidney injury suspected to be due to amoxicillin crystals with residuals plasma levels to disclose a predictive threshold of tubulopathy. All patients had a high residual rate at diagnosis but we cannot find a threshold that would allow to adapt the antibiotic dose, enhance hydratation and alkalinizide urine to increase the medication solubility and limit renal toxicity.

Extremely Thermostabilizing Core Mutations in Coiled-Coil Mimetic Proteins of HIV-1 gp41 Produce Diverse Effects on Target Binding but Do Not Affect Their Inhibitory Activity.

A promising strategy to neutralize HIV-1 is to target the gp41 spike subunit to block membrane fusion with the cell. We previously designed a series of single-chain proteins (named covNHR) that mimic the trimeric coiled-coil structure of the gp41 N-terminal heptad repeat (NHR) region and potently inhibit HIV-1 cell infection by avidly binding the complementary C-terminal heptad repeat (CHR) region. These proteins constitute excellent tools to understand the structural and thermodynamic features of this therapeutically important interaction. Gp41, as with many coiled-coil proteins, contains in core positions of the NHR trimer several highly conserved, buried polar residues, the role of which in gp41 structure and function is unclear. Here we produced three covNHR mutants by substituting each triad of polar residues for the canonical isoleucine. The mutants preserve their helical structure and show an extremely increased thermal stability. However, increased hydrophobicity enhances their self-association. Calorimetric analyses show a marked influence of mutations on the binding thermodynamics of CHR-derived peptides. The mutations do not affect however the in vitro HIV-1 inhibitory activity of the proteins. The results support a role of buried core polar residues in maintaining structural uniqueness and promoting an energetic coupling between conformational stability and NHR-CHR binding.

Probing Vulnerability of the gp41 C-Terminal Heptad Repeat as Target for Miniprotein HIV Inhibitors.

One of the therapeutic strategies in HIV neutralization is blocking membrane fusion. In this process, tight interaction between the N-terminal and C-terminal heptad-repeat (NHR and CHR) regions of gp41 is essential to promote membranes apposition and merging. We have previously developed single-chain proteins (named covNHR) that accurately mimic the complete gp41 NHR region in its trimeric conformation. They tightly bind CHR-derived peptides and show a potent and broad HIV inhibitory activity in vitro. However, the extremely high binding affinity (sub-picomolar) is not in consonance with their inhibitory activity (nanomolar), likely due to partial or temporal accessibility of their target in the virus. Here, we have designed and characterized two single-chain covNHR miniproteins each encompassing one of the two halves of the NHR region and containing two of the four sub-pockets of the NHR crevice. The two miniproteins fold as trimeric helical bundles as expected but while the C-terminal covNHR (covNHR-C) miniprotein is highly stable, the N-terminal counterpart (covNHR-N) shows only marginal stability that could be improved by engineering an internal disulfide bond. Both miniproteins bind their respective complementary CHR peptides with moderate (micromolar) affinity. Moreover, the covNHR-N miniproteins can access their target in the context of trimeric native envelope proteins and show significant inhibitory activity for several HIV pseudoviruses. In contrast, covNHR-C cannot bind its target sequence and neither inhibits HIV, indicating a higher vulnerability of C-terminal part of CHR. These results may guide the development of novel HIV inhibitors targeting the gp41 CHR region.

Thermodynamic dissection of the interface between HIV-1 gp41 heptad repeats reveals cooperative interactions and allosteric effects.

HIV-1 glycoprotein 41 (gp41) mediates fusion between virus and target cells by folding into a fusion active state, in which the C-terminal heptad repeat (CHR) regions associate externally to the N-terminal heptad repeat (NHR) trimer and form a very stable six-helix bundle coiled-coil structure. Therefore, interfering with the NHR-CHR interaction of gp41 is a promising therapeutic approach against HIV-1. However, a full understanding of the molecular and mechanistic details of this interaction is still incomplete. Here, we use single-chain, chimeric proteins (named covNHR) that reproduce accurately the CHR-NHR interactions to analyze the binding thermodynamics of several peptides with different length from the CHR region. The results indicate that cooperative binding involving two or more pockets of the NHR groove is necessary to obtain relevant affinities and that the binding energy is broadly distributed along the interface, underlining a crucial role of a middle pocket to achieve tight binding. In contrast, targeting only the deep hydrophobic pocket is insufficient to achieve significant affinity. Moreover, calorimetry experiments in combination with limited proteolysis performed using a mutant with occluded binding in the N-terminal pocket reveal the existence of an allosteric communication between the different regions. This study is the first detailed thermodynamic dissection of the NHR-CHR interaction in gp41 and contributes therefore to a better understanding of HIV fusion. These results are relevant for the development of potential fusion inhibitors.

Direct conversion of uranium dioxide UO2 to uranium tetrafluoride UF4 using the fluorinated ionic liquid [Bmim][PF6].

The industrial fluorination of UO2 to UF4 is based on a complex process involving the manipulation of a large amount of HF, a very toxic and corrosive gas. We present here a safer way to accomplish this reaction utilizing ionic liquid [Bmim][PF6] as a unique reaction medium and fluoride source.

Early mechanisms of amyloid fibril nucleation in model and disease-related proteins.

Protein amyloid aggregation is a hallmark in neuropathologies and other diseases of tremendous impact such as Alzheimer's or Parkinson's diseases. During the last decade, it has become increasingly evident that neuronal death is mainly induced by proteinaceous oligomers rather than the mature amyloid fibrils. Therefore, the earliest molecular events occurring during the amyloid aggregation cascade represent a growing interest of study. Important breakthroughs have been achieved using experimental data from different proteins, used as models, as well as systems related to diseases. Here, we summarize the structural properties of amyloid oligomeric and fibrillar aggregates and review the recent advances on how biophysical techniques can be combined with quantitative kinetic analysis and theoretical models to study the detailed mechanism of oligomer formation and nucleation of fibrils. These insights into the mechanism of early oligomerization and amyloid nucleation are of relevant interest in drug discovery and in the design of preventive strategies against neurodegenerative diseases.

Structural and Thermodynamic Analysis of HIV-1 Fusion Inhibition Using Small gp41 Mimetic Proteins.

Development of effective inhibitors of the fusion between HIV-1 and the host cell membrane mediated by gp41 continues to be a grand challenge due to an incomplete understanding of the molecular and mechanistic details of the fusion process. We previously developed single-chain, chimeric proteins (named covNHR) that accurately mimic the N-heptad repeat (NHR) region of gp41 in a highly stable coiled-coil conformation. These molecules bind strongly to peptides derived from the gp41 C-heptad repeat (CHR) and are potent and broad HIV-1 inhibitors. Here, we investigated two covNHR variants differing in two mutations, V10E and Q123R (equivalent to V38E and Q40R in gp41 sequence) that reproduce the effect of HIV-1 mutations associated with resistance to fusion inhibitors, such as T20 (enfuvirtide). A detailed calorimetric analysis of the binding between the covNHR proteins and CHR peptides (C34 and T20) reveals drastic changes in affinity due to the mutations as a result of local changes in interactions at the site of T20 resistance. The crystallographic structure of the covNHR:C34 complex shows a virtually identical CHR-NHR binding interface to that of the post-fusion structure of gp41 and underlines an important role of buried interfacial water molecules in binding affinity and in development of resistance against CHR peptides. Despite the great difference in affinity, both covNHR variants demonstrate strong inhibitory activity for a wide variety of HIV-1 strains. These properties support the high potential of these covNHR proteins as new potent HIV-1 inhibitors. Our results may guide future inhibition approaches.

An auxin controls bacterial antibiotics production.

The majority of clinically used antibiotics originate from bacteria. As the need for new antibiotics grows, large-scale genome sequencing and mining approaches are being used to identify novel antibiotics. However, this task is hampered by the fact that many antibiotic biosynthetic clusters are not expressed under laboratory conditions. One strategy to overcome this limitation is the identification of signals that activate the expression of silent biosynthetic pathways. Here, we report the use of high-throughput screening to identify signals that control the biosynthesis of the acetyl-CoA carboxylase inhibitor antibiotic andrimid in the broad-range antibiotic-producing rhizobacterium Serratia plymuthica A153. We reveal that the pathway-specific transcriptional activator AdmX recognizes the auxin indole-3-acetic acid (IAA). IAA binding causes conformational changes in AdmX that result in the inhibition of the expression of the andrimid cluster and the suppression of antibiotic production. We also show that IAA synthesis by pathogenic and beneficial plant-associated bacteria inhibits andrimid production in A153. Because IAA is a signalling molecule that is present across all domains of life, this study highlights the importance of intra- and inter-kingdom signalling in the regulation of antibiotic synthesis. Our discovery unravels, for the first time, an IAA-dependent molecular mechanism for the regulation of antibiotic synthesis.

Dynamic micellar oligomers of amyloid beta peptides play a crucial role in their aggregation mechanisms.

A deep understanding of the early molecular mechanism of amyloid beta peptides (Aß) is crucial to develop therapeutic and preventive approaches for Alzheimer's disease (AD). Using a variety of biophysical techniques, we have found that micelle-like dynamic oligomers are rapidly formed by Aß40 and Aß42 above specific critical concentrations. Analysis of the initial aggregation rates at 37 °C measured by thioflavin T and Bis-ANS fluorescence using a mass-action micellization model revealed a concentration-dependent switch in the nucleation mechanism. Bimolecular nucleation appears to occur at low peptide concentration while above the critical micellar concentration, the nucleation takes place more efficiently in the micelles. Upon incubation, these micelles mediate a rapid formation of larger, more stable oligomers enriched in beta-sheet structure. These oligomers formed from Aß40, enriched in amyloid nuclei, acquire a higher capacity to fibrillate than their micellar precursors. Aß42 can also form similar oligomers but they have lower beta-sheet structure content and lower capacity to fibrillate. On the other hand, a considerable fraction of the Aß42 peptide forms morphologically distinct oligomers that are unable to fibrillate and show significant effect on SH-SY5Y cell viability. Overall, our results highlight the importance of micellar structures as mediators of amyloid nucleation and contribute to the understanding of the differences between the aggregation pathways of Aß40 and Aß42.

Combining Structural Aggregation Propensity and Stability Predictions To Redesign Protein Solubility.

The aggregation propensity of each particular protein seems to be shaped by evolution according to its natural abundance in the cell. The production and downstream processing of recombinant polypeptides implies attaining concentrations that are orders of magnitude above their natural levels, often resulting in their aggregation; a phenomenon that precludes the marketing of many globular proteins for biomedical or biotechnological applications. Therefore, there is a huge interest in methods aimed to increase the proteins solubility above their natural limits. Here, we demonstrate that an updated version of our AGGRESCAN 3D structural aggregation predictor, that now takes into account protein stability, allows for designing mutations at specific positions in the structure that improve the solubility of proteins without compromising their conformation. Using this approach, we have designed a highly soluble variant of the green fluorescent protein and a human single-domain VH antibody displaying significantly reduced aggregation propensity. Overall, our data indicate that the solubility of unrelated proteins can be easily tuned by in silico-designed nondestabilizing amino acid changes at their surfaces.

Global Protein Stabilization Does Not Suffice to Prevent Amyloid Fibril Formation.

Mutations or cellular conditions that destabilize the native protein conformation promote the population of partially unfolded conformations, which in many cases assemble into insoluble amyloid fibrils, a process associated with multiple human pathologies. Therefore, stabilization of protein structures is seen as an efficient way to prevent misfolding and subsequent aggregation. This has been suggested to be the underlying reason why proteins living in harsh environments, such as the extracellular space, have evolved disulfide bonds. The effect of protein disulfides on the thermodynamics and kinetics of folding has been extensively studied, but much less is known on its effect on aggregation reactions. Here, we designed a single point mutation that introduces a disulfide bond in the all-α FF domain, a protein that, despite being devoid of preformed ß-sheets, forms ß-sheet-rich amyloid fibrils. The novel and unique covalent bond in the FF domain dramatically increases its thermodynamic stability and folding speed. Nevertheless, these optimized properties cannot counteract the inherent aggregation propensity of the protein, thus indicating that a high global protein stabilization does not suffice to prevent amyloid formation unless it contributes to hide from exposure the specific regions that nucleate the aggregation reaction.

Structural Basis for Polyamine Binding at the dCACHE Domain of the McpU Chemoreceptor from Pseudomonas putida.

Many bacteria can move chemotactically to a variety of compounds and the recognition of chemoeffectors by the chemoreceptor ligand binding domain (LBD) defines the specificity of response. Many chemoreceptors were found to recognize different amino and organic acids, but the McpU chemoreceptor from Pseudomonas putida was identified as the first chemoreceptor that bound specifically polyamines. We report here the three-dimensional structure of McpU-LBD in complex with putrescine at a resolution of 2.4 Å, which fitted well a solution structure generated by small-angle X-ray scattering. Putrescine bound to a negatively charged pocket in the membrane distal module of McpU-LBD. Similarities exist in the binding of putrescine to McpU-LBD and taurine to the LBD of the Mlp37 chemoreceptor of Vibrio cholerae. In both structures, the primary amino group of the respective ligand is recognized by hydrogen bonds established by two aspartate and a tyrosine side chain. This feature may be used to predict the ligands of chemoreceptors with unknown function. Analytical ultracentrifugation revealed that McpU-LBD is monomeric in solution and that ligand binding does not alter this oligomeric state. This sensing mode thus differs from that of the well-characterised four-helix bundle domains where ligands bind to two sites at the LBD dimer interface. Although there appear to be different sensing modes, results are discussed in the context of data, indicating that chemoreceptors employ the same mechanism of transmembrane signaling. This work enhances our understanding of CACHE domains, which are the most abundant sensor domains in bacterial chemoreceptors and sensor kinases.