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Background: Transplacentally transferred antibodies induced by maternal pertussis vaccination interfere with infant immune responses to pertussis primary vaccination. We evaluated whether this interference remains in toddlers after booster vaccination. Methods: In a prior phase IV, observer-blind, placebo-controlled, randomized study (NCT02377349), pregnant women in Australia, Canada and Europe received intramuscular tetanus-reduced-antigen-content diphtheria-three-component acellular pertussis vaccine (Tdap group) or placebo (control group) at 270/7–366/7 weeks’ gestation, with crossover immunization postpartum. Their infants were primed (study NCT02422264) and boosted (at 11–18 months; current study NCT02853929) with diphtheria-tetanus-three-component acellular pertussis-hepatitis B virus-inactivated poliovirus/Haemophilus influenzae type b vaccine (DTaP-HepB-IPV/Hib) and 13-valent pneumococcal conjugate vaccine. Immunogenicity before and after booster vaccination, and reactogenicity and safety of the booster were evaluated descriptively. Results: 263 (Tdap group) and 277 (control group) toddlers received a DTaP-HepB-IPV/Hib booster. Pre-booster vaccination, observed geometric mean concentrations (GMCs) for the three pertussis antigens and diphtheria were 1.4–1.5-fold higher in controls than in the Tdap group. No differences were observed for the other DTaP-HepB-IPV/Hib antigens. One month post-booster vaccination, booster response rates for pertussis antigens were ≥ 92.1% and seroprotection rates for the other DTaP-HepB-IPV/Hib antigens were ≥ 99.2% in both groups (primary objective). Higher post-booster GMCs were observed in controls versus the Tdap group for anti-filamentous hemagglutinin (1.2-fold), anti-pertussis toxoid (1.5-fold) and anti-diphtheria (1.4-fold). GMCs for the other DTaP-HepB-IPV/Hib antigens were similar between groups. Serious adverse events were reported for three toddlers (controls, not vaccination-related). One death occurred pre-booster (Tdap group, not vaccination-related). Conclusions: As a consequence of interference of maternal pertussis antibodies with infant immune responses to pertussis primary vaccination, pertussis antibody concentrations were still lower in toddlers from Tdap-vaccinated mothers before DTaP-HepB-IPV/Hib booster vaccination. After the booster, antibody concentrations were lower for filamentous hemagglutinin and pertussis toxoid but not for pertactin. The clinical significance of this interference requires further evaluation. Clinical Trial Registration. ClinicalTrials.gov: NCT02853929. publishedVersion

This study explores the structural and functional changes associated with a low-temperature thermal transition of 2 engineered bacterial uricase mutants. Uricase has a noncovalent homotetrameric structure, with 4 active sites located at the interface of subunits. Using differential scanning calorimetry, a low-temperature transition was identified at 42°C for mutant A and at 33°C for mutant B. This transition was stabilized by the uricase inhibitor, oxonic acid, suggesting a strong structural relationship to the active site. For mutant B, there was a reversible loss of enzymatic activity above the low-temperature transition. Spectroscopic measurements demonstrated that there was also a reversible loss of secondary and tertiary structures and an increase in surface hydrophobicity. However, the hydrophobic core environment and the tetrameric structure were not altered over the low-temperature transition suggesting that the changes occurred primarily at the surface of the enzyme. The protein became aggregation-prone at temperatures approaching the cluster of higher-temperature melting transitions at 84°C, indicating these transitions represent a global unfolding of the protein. Our findings shed light on the structural changes that affect the uricase mechanism of action and provide new insights into how enzyme therapeutic development may be approached.

The overexpression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations, retroviral insertion during gene therapy, or in transgenic mice models, leads to the onset of T-cell leukemias. LMO2 comprises two protein-binding LIM domains that allow LMO2 to interact with multiple protein partners, including LIM domain-binding protein 1 (Ldb1, also known as CLIM2 and NLI), an essential cofactor for LMO proteins. Sequestration of Ldb1 by LMO2 in T-cells may prevent it binding other key partners, such as LMO4. Here, we show using protein engineering and enzyme-linked immunosorbent assay (ELISA) methodologies that LMO2 binds Ldb1 with a twofold lower affinity than does LMO4. Thus, excess LMO2 rather than an intrinsically higher binding affinity would lead to sequestration of Ldb1. Both LIM domains of LMO2 are required for high-affinity binding to Ldb1 (K(D) = 2.0 x 10(-8) M). However, the first LIM domain of LMO2 is primarily responsible for binding to Ldb1 (K(D) = 2.3 x 10(-7) M), whereas the second LIM domain increases binding by an order of magnitude. We used mutagenesis in combination with yeast two-hybrid analysis, and phage display selection to identify LMO2-binding "hot spots" within Ldb1 that locate to the LIM1-binding region. The delineation of this region reveals some specific differences when compared to the equivalent LMO4:Ldb1 interaction that hold promise for the development of reagents to specifically bind LMO2 in the treatment of leukemia.

SOCS3 is essential for regulating the extent, duration, and specificity of cellular responses to cytokines such as G-CSF and IL-6. Here we describe the solution structure of SOCS3, the first structure determined for any SOCS protein, in complex with a phosphotyrosine-containing peptide from the IL-6 receptor signaling subunit gp130. The structure of the complex shows that seven peptide residues form a predominantly hydrophobic binding motif. Regions outside the SOCS3 SH2 domain are important for ligand binding, in particular, a single 15 residue alpha helix immediately N-terminal to the SH2 domain makes direct contacts with the phosphotyrosine binding loop and, in part, determines its geometry. The SH2 domain itself is remarkable in that it contains a 35 residue unstructured PEST motif insertion that is not required for STAT inhibition. The PEST motif increases SOCS3 turnover and affects its degradation pathway, implying that it has an important regulatory role inside the cell.

Results are reported concerning the synergetic effect observed in the oxidation of propane to acrylic acid over the orthorhombic M1 and hexagonal M2 phases present in the most active and selective MoVTe(Sb)NbO catalysts. The pure phases and phase mixtures containing either tellurium or antimony have been prepared and individually tested as catalysts. The results obtained confirm that the phase responsible for the catalytic properties of the efficient catalysts is phase M1, and that M2 is poorly active. Mechanical mixtures of the pure phases have also been prepared and tested. All of the catalysts have been characterized before and after the catalytic reaction by X-ray diffraction, X-ray photoelectron spectroscopy, and high-resolution electron microscopy with EDS analyses. Although the synergetic effect previously described (bifunctional catalysis with the oxidative dehydrogenation of propane molecules on the orthorhombic M1 phase and the subsequent oxidation of propene on the hexagonal M2 phase) was observed, another cause related to migration of tellurium from the M2 phase to the surface of the M1 phase was indicated. This migration should balance a loss of tellurium in the active phase occurring under the conditions of a catalytic test, but may also create new dehydrogenation sites for propene and/or anneal total oxidation sites. The hexagonal M2 phase would thus play a role of tellurium reservoir for the active M1 phase. This effect was not reversible and concerned only the tellurium. Antimony, which is less volatile, should not be lost by the active phase. Furthermore, it was shown not to diffuse at the surface of the phases.

The adsorption of pyridine on the surface of the M1 active phase of the MoVTe(Sb)O catalysts has been studied for the determination of the Bronsted and Lewis acid sites. The results obtained showed medium Lewis and Bronsted acidity for antimony containing phases and low Lewis and Bronsted acidity for tellurium ones. The incorporation of niobium in the M1 phases resulted in a decrease of both acidities. The samples characterized have been tested as catalysts in the oxidation of propane to acrylic acid. The results obtained allowed to propose correlations between the acidic properties and the catalytic selectivity. 2005 Elsevier B.V. All rights reserved.

Different spectroscopic techniques like XANES, ESR, XPS and Mossbauer spectroscopy have been used to determine the oxidation state of the various cations in the M1 and M2 phases of the MoVTe(Sb)NbO catalysts used for the oxidation or ammoxidation of propane. It was observed that the tellurium or antimony M 1o rM2 phases contained mainly Te IV , and Nb V. Molybdenum, vanadium and antimony were shown to be present as Mo VI and Mo V ,V V and V IV and Sb V and Sb III . The M1 phase which is responsible for the high efficiency of catalysts, corresponds to the total stoichiometry (AO)2� x(A2O)xM20O56 with A = Sb or Te, M = Mo, V and Nb and 0 � x � 1. It has a structure with hexagonal channels occupied by the A cations and oxides. This channel may contain an excess of oxygen which constitutes a reservoir for the catalysts and which likely plays a role in the reoxidation of the catalytic sites. The balance of the charges introduced by these oxides anions was achieved by the partial oxidation of Sb III to Sb V in M1(Sb) and by the oxidation of Mo V to Mo VI in M1(Te), Te remaining always Te IV as shown from Mossbauer spectroscopy data. The characterization of the solids after catalytic test showed very few changes in the solids structures and compositions. The study allowed concluding that the lone pair elements does not have only a role as constituents of the surface catalytic sites but also as bulk components to store oxygen in the hexagonal channels and contributes to its rapid diffusion to the surface. # 2004 Elsevier B.V. All rights reserved. reported as (Te2O)2M20O56 and (SbO)2M20O56 with M = Mo, V, Nb. The V/Mo and Nb/Mo ratios can vary, but are most frequently close to 0.3 and 0.1, respectively (4,5). The phase containing Te has an oxide over- stoichiometry while the one containing Sb, an oxide sub- stoichiometry, they may better be reported with the general composition (AO)2� x(A2O)xM20O56 with A = Te or Sb and 0 � x � 1. The first structure model for the M1 phases has been proposed in 2002 based on an isomorphism with the phase Cs0.7(Nb,W)5O14 (4). The structure of the M1 phase has been described as a corner sharing MO6 (M = Mo, V, Nb) octahedra network with tellurium or antimony cations and oxygen anions occupying sites in hexagonal channels formed by the octahedra. Besides hexagonal channels, pentagonal and heptagonal channels are present and oriented in the same direction. The heptagonal channels have been

Leukemia inhibitory factor (LIF)-induced cell signaling occurs following sequential binding to the LIF receptor alpha-chain (LIFR), then to the gp130 co-receptor used by all members of the interleukin-6 family of cytokines. By monovalently displaying human LIF on the surface of M13 phage and randomizing clusters of residues in regions predicted to be important for human LIFR binding, we have identified mutations, which lead to significant increases in affinity for binding to LIFR. Six libraries were constructed in which regions of 4-6 amino acids were randomized then panned against LIFR. Mutations identified in three distinct clusters, residues 53-57, 102-103, and 150-155, gave rise to proteins with significantly increased affinity for binding to both human and mouse LIFR. Combining the mutations for each of these regions further increased the affinity, such that the best mutants bound to human LIFR with1000-fold higher affinity than wild-type human LIF. NMR analysis indicated that the mutations did not alter the overall structure of the molecule relative to the native protein, although some local changes occurred in the vicinity of the substituted residues. Despite increases in LIFR binding affinity, these mutants did not show any increase in activity as agonists of LIF-induced proliferation of Ba/F3 cells expressing human LIFR and gp130 compared with wild-type LIF. Incorporation of two additional mutations (Q29A and G124R), which were found to abrogate cell signaling, led to the generation of highly potent antagonists of both human and murine LIF-induced bioactivity.

Suppressor of cytokine signaling (SOCS)-2 is a member of a family of intracellular proteins implicated in the negative regulation of cytokine signaling. The generation of SOCS-2-deficient mice, which grow to one and a half times the size of their wild-type littermates, suggests that SOCS-2 may attenuate growth hormone (GH) signaling. In vitro studies indicate that, while SOCS-2 can inhibit GH action at low concentrations, at higher concentrations it may potentiate signaling. To determine whether a similar enhancement of signaling is observed in vivo or alternatively whether increased SOCS-2 levels repress growth in vivo, we generated and analyzed transgenic mice that overexpress SOCS-2 from a human ubiquitin C promoter. These mice are not growth-deficient and are, in fact, significantly larger than wild-type mice. The overexpressed SOCS-2 was found to bind to endogenous GH receptors in a number of mouse organs, while phosphopeptide binding studies with recombinant SOCS-2 defined phosphorylated tyrosine 595 on the GH receptor as the site of interaction. Together, the data implicate SOCS-2 as having dual effects on GH signaling in vivo.

Total chemical synthesis by convergent chemical ligation was used to prepare a ‘backbone engineered’ 202-residue covalent dimer asymmetric form of the HIV-1 protease molecule. The Gly49-Ile50 peptide bond backbone –N(H)– atom, critically involved in H-bonding to substrates, was specifically replaced by an –O– atom in one flap only . The resulting enzyme analogue retained full intrinsic activity , demonstrating that enzyme–substrate hydrogen bonding at the Gly49-Ile50 peptide bond in only a single flap is sufficient for normal catalytic function.

Members of the suppressor of cytokine signalling (SOCS) family of proteins have been shown to inhibit cytokine signalling via direct interactions with JAK kinases or activated cytokine receptors. In addition to their novel amino-terminal regions and SH2 domains that mediate these interactions, the SOCS proteins also contain carboxy-terminal regions of homology called the SOCS box. The SOCS box serves to couple SOCS proteins and their binding partners with the elongin B and C complex, possibly targeting them for degradation. Several other families of proteins also contain SOCS boxes but differ from the SOCS proteins in the type of domain or motif they contain upstream of the SOCS box. We report here the cloning, characterization, mapping and expression analysis of four members of the ankyrin repeat and SOCS box-containing (Asb) protein family.

Historically, total chemical synthesis had been used to prepare native proteinase from the human immunodeficiency virus (HIV-1 PR) for structural studies by X-ray crystallography. More recently, several functionally-relevant analogues of HIV-1 PR have also been obtained by total chemical synthesis. The results of structural and biochemical studies of the backbone engineered analogues put in question the established belief of the importance of an internal, tetrahedrally coordinated water molecule (water 301) in mediating catalytically important flap–substrate interaction. An enzyme analogue in which the peptide bond between residues Gly 51 and Gly 52 was replaced by a thioester moiety displayed normal enzymatic activity, while the crystal structure of its complex with the inhibitor MVT101 (solved at 2.5 A resolution as mirror image, D-enantiomer) did not show the presence of water 301. The enzyme analogue in which the ability to donate hydrogen bonds to substrate was deleted (by substitution of Ile 50 –N(H)– by a sulfur atom) in both flaps was 2500-fold less active. By contrast, the covalent dimer form of the enzyme with the Gly 49 –Ile 50 peptide bond –N(H)– atom specifically replaced by an –O– atom in one flap only retained normal enzymatic activity. The combined data from these studies strongly indicate that flap–substrate hydrogen bonds from only one flap are sufficient for full enzymatic activity of the HIV-1 PR, and raise the possibility that the retroviral enzyme may make use of only one flap in catalysis. This result may have profound implications for drug design targeted at HIV-1 PR.

The products of oxidation of the alpha-tocopherol model compound, 2,2,5,7,8-pentamethyl-6-chromanol (PH) by t-butyl hydroperoxide in chloroform varied with the amount of water present. In the presence of a trace of water, the main products were the spirodimer (PSD) and spirotrimer (PST). As the content of water increased, the main product became 2-(3-hydroxy-3-methylbutyl)-3,5,6-trimethyl-1,4-benzoquinone (PQ). Oxidation of PH in aqueous liposome suspension also produced PQ as the major product. These results suggested that, in aqueous solutions, the major oxidation product of PH would be PQ and of alpha-tocopherol (TH) would be alpha-tocopheryl quinone (TQ). The ease of reduction of PQ and TQ was studied in chemical and biological systems. PQ, TQ, and ubiquinone-10 (UQ) were rapidly reduced to their respective hydroquinones (PQH2, TQH2, and UQH2) at pH 7.3 by NADH plus FAD. Whole blood reduced PQ rapidly at 37 degrees C to PQH2 but did not reduce TQ to TQH2. Human peripheral blood mononuclear cells took up TQ from a bovine serum albumin complex and reduced it to TQH2. Ingestion of TQ (350 mg) by one of us (PSK) resulted in the formation of TQH2 during a 5 h period. These results demonstrate that several biological systems are able to reduce TQ to TQH2 and that it is a reaction that may occur normally in vivo.