The structure of the RbBP5 β-propeller domain reveals a surface with potential nucleic acid binding sites

The multi-protein complex WRAD, formed by WDR5, RbBP5, Ash2L and Dpy30, binds to the MLL SET domain to stabilize the catalytically active conformation required for histone H3K4 methylation. In addition, the WRAD complex contributes to the targeting of the activated complex to specific sites on chromatin. RbBP5 is central to MLL catalytic activation, by making critical contacts with the other members of the complex. Interestingly its only major structural domain, a canonical WD40 repeat -propeller, is not implicated in this function. Here, we present the structure of the RbBP5 -propeller domain revealing a distinct, feature rich surface, dominated by clusters of Arginine residues. Our nuclear magnetic resonance binding data supports the hypothesis that in addition to the role of RbBP5 in catalytic activation, its -propeller domain is a platform for the recruitment of the MLL complexes to chromatin targets through its direct interaction with nucleic acids

Favourable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

Background: Differences in humoral immunity to coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), between children and adults remain unexplained and the impact of underlying immune dysfunction or suppression unknown. Here, we sought to examine the antibody immune competence of children and adolescents with prevalent inflammatory rheumatic diseases, juvenile idiopathic arthritis (JIA), juvenile dermatomyositis (JDM) and juvenile systemic lupus erythematosus (JSLE), against the seasonal human coronavirus (HCoV)-OC43 that frequently infects this age group. // Methods: Sera were collected from JIA (n=118), JDM (n=49) and JSLE (n=30) patients, and from healthy control (n=54) children and adolescents, prior to the coronavirus disease-19 (COVID-19) pandemic. We employed sensitive flow cytometry-based assays to determine titres of antibodies that reacted with the spike and nucleoprotein of HCoV-OC43 and cross-reacted with the spike and nucleoprotein of SARS-CoV-2, and compared with respective titres in sera from patients with multisystem inflammatory syndrome in children and adolescents (MIS-C). // Findings: Despite immune dysfunction and immunosuppressive treatment, JIA, JDM and JSLE patients maintained comparable or stronger humoral responses than healthier peers, dominated by IgG antibodies to HCoV-OC43 spike, and harboured IgG antibodies that cross-reacted with SARS-CoV-2 spike. In contrast, responses to HCoV-OC43 and SARS-CoV-2 nucleoproteins exhibited delayed age-dependent class-switching and were not elevated in JIA, JDM and JSLE patients, arguing against increased exposure. // Conclusions: Consequently, autoimmune rheumatic diseases and their treatment were associated with a favourable ratio of spike to nucleoprotein antibodies

Reduced antibody cross-reactivity following infection with B.1.1.7 than with parental SARS-CoV-2 strains

Background: The degree of heterotypic immunity induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains is a major determinant of the spread of emerging variants and the success of vaccination campaigns, but remains incompletely understood. Methods: We examined the immunogenicity of SARS-CoV-2 variant B.1.1.7 (Alpha) that arose in the United Kingdom and spread globally. We determined titres of spike glycoprotein-binding antibodies and authentic virus neutralising antibodies induced by B.1.1.7 infection to infer homotypic and heterotypic immunity. Results: Antibodies elicited by B.1.1.7 infection exhibited significantly reduced recognition and neutralisation of parental strains or of the South Africa variant B.1.351 (Beta) than of the infecting variant. The drop in cross-reactivity was significantly more pronounced following B.1.1.7 than parental strain infection. Conclusions: The results indicate that heterotypic immunity induced by SARS-CoV-2 variants is asymmetric

Bat lung epithelial cells show greater host species-specific innate resistance than MDCK cells to human and avian influenza viruses

Background With the recent discovery of novel H17N10 and H18N11 influenza viral RNA in bats and report on high frequency of avian H9 seroconversion in a species of free ranging bats, an important issue to address is the extent bats are susceptible to conventional avian and human influenza A viruses. Method To this end, three bat species (Eidolon helvum, Carollia perspicillata and Tadarida brasiliensis) of lung epithelial cells were separately infected with two avian and two human influenza viruses to determine their relative host innate immune resistance to infection. Results All three species of bat cells were more resistant than positive control Madin-Darby canine kidney (MDCK) cells to all four influenza viruses. TB1-Lu cells lacked sialic acid α2,6-Gal receptors and were most resistant among the three bat species. Interestingly, avian viruses were relatively more replication permissive in all three bat species of cells than with the use of human viruses which suggest that bats could potentially play a role in the ecology of avian influenza viruses. Chemical inhibition of the JAK-STAT pathway in bat cells had no effect on virus production suggesting that type I interferon signalling is not a major factor in resisting influenza virus infection. Conclusion Although all three species of bat cells are relatively more resistant to influenza virus infection than control MDCK cells, they are more permissive to avian than human viruses which suggest that bats could have a contributory role in the ecology of avian influenza viruses

Prediction of Biological Functions on Glycosylation Site Migrations in Human Influenza H1N1 Viruses

Protein glycosylation alteration is typically employed by various viruses for escaping immune pressures from their hosts. Our previous work had shown that not only the increase of glycosylation sites (glycosites) numbers, but also glycosite migration might be involved in the evolution of human seasonal influenza H1N1 viruses. More importantly, glycosite migration was likely a more effectively alteration way for the host adaption of human influenza H1N1 viruses. In this study, we provided more bioinformatics and statistic evidences for further predicting the significant biological functions of glycosite migration in the host adaptation of human influenza H1N1 viruses, by employing homology modeling and in silico protein glycosylation of representative HA and NA proteins as well as amino acid variability analysis at antigenic sites of HA and NA. The results showed that glycosite migrations in human influenza viruses have at least five possible functions: to more effectively mask the antigenic sites, to more effectively protect the enzymatic cleavage sites of neuraminidase (NA), to stabilize the polymeric structures, to regulate the receptor binding and catalytic activities and to balance the binding activity of hemagglutinin (HA) with the release activity of NA. The information here can provide some constructive suggestions for the function research related to protein glycosylation of influenza viruses, although these predictions still need to be supported by experimental data

The Inviscid Limit and Boundary Layers for Navier-Stokes Flows

The validity of the vanishing viscosity limit, that is, whether solutions of the Navier-Stokes equations modeling viscous incompressible flows converge to solutions of the Euler equations modeling inviscid incompressible flows as viscosity approaches zero, is one of the most fundamental issues in mathematical fluid mechanics. The problem is classified into two categories: the case when the physical boundary is absent, and the case when the physical boundary is present and the effect of the boundary layer becomes significant. The aim of this article is to review recent progress on the mathematical analysis of this problem in each category.Comment: To appear in "Handbook of Mathematical Analysis in Mechanics of Viscous Fluids", Y. Giga and A. Novotn\'y Ed., Springer. The final publication is available at http://www.springerlink.co

Inferring stabilizing mutations from protein phylogenies : application to influenza hemagglutinin

One selection pressure shaping sequence evolution is the requirement that a protein fold with sufficient stability to perform its biological functions. We present a conceptual framework that explains how this requirement causes the probability that a particular amino acid mutation is fixed during evolution to depend on its effect on protein stability. We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny. This approach is able to predict published experimentally measured mutational stability effects (ΔΔG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach. As a further test, we use our phylogenetic inference approach to predict stabilizing mutations to influenza hemagglutinin. We introduce these mutations into a temperature-sensitive influenza virus with a defect in its hemagglutinin gene and experimentally demonstrate that some of the mutations allow the virus to grow at higher temperatures. Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin. This approach also makes a mathematical link between phylogenetics and experimentally measurable protein properties, potentially paving the way for more accurate analyses of molecular evolution

Dried blood spot UHPLC-MS/MS analysis of oseltamivir and oseltamivircarboxylate—a validated assay for the clinic

The neuraminidase inhibitor oseltamivir (Tamiflu®) is currently the first-line therapy for patients with influenza virus infection. Common analysis of the prodrug and its active metabolite oseltamivircarboxylate is determined via extraction from plasma. Compared with these assays, dried blood spot (DBS) analysis provides several advantages, including a minimum sample volume required for the measurement of drugs in whole blood. Samples can easily be obtained via a simple, non-invasive finger or heel prick. Mainly, these characteristics make DBS an ideal tool for pediatrics and to measure multiple time points such as those needed in therapeutic drug monitoring or pharmacokinetic studies. Additionally, DBS sample preparation, stability, and storage are usually most convenient. In the present work, we developed and fully validated a DBS assay for the simultaneous determination of oseltamivir and oseltamivircarboxylate concentrations in human whole blood. We demonstrate the simplicity of DBS sample preparation, and a fast, accurate and reproducible analysis using ultra high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer. A thorough validation on the basis of the most recent FDA guidelines for bioanalytical method validation showed that the method is selective, precise, and accurate (≤15% RSD), and sensitive over the relevant clinical range of 5–1,500 ng/mL for oseltamivir and 20–1,500 ng/mL for the oseltamivircarboxylate metabolite. As a proof of concept, oseltamivir and oseltamivircarboxylate levels were determined in DBS obtained from healthy volunteers who received a single oral dose of Tamiflu®

Efficacious Recombinant Influenza Vaccines Produced by High Yield Bacterial Expression: A Solution to Global Pandemic and Seasonal Needs

It is known that physical linkage of TLR ligands and vaccine antigens significantly enhances the immunopotency of the linked antigens. We have used this approach to generate novel influenza vaccines that fuse the globular head domain of the protective hemagglutinin (HA) antigen with the potent TLR5 ligand, flagellin. These fusion proteins are efficiently expressed in standard E. coli fermentation systems and the HA moiety can be faithfully refolded to take on the native conformation of the globular head. In mouse models of influenza infection, the vaccines elicit robust antibody responses that mitigate disease and protect mice from lethal challenge. These immunologically potent vaccines can be efficiently manufactured to support pandemic response, pre-pandemic and seasonal vaccines

Large-Scale Sequence Analysis of Hemagglutinin of Influenza A Virus Identifies Conserved Regions Suitable for Targeting an Anti-Viral Response

BACKGROUND: Influenza A viral surface protein, hemagglutinin, is the major target of neutralizing antibody response and hence a main constituent of all vaccine formulations. But due to its marked evolutionary variability, vaccines have to be reformulated so as to include the hemagglutinin protein from the emerging new viral strain. With the constant fear of a pandemic, there is critical need for the development of anti-viral strategies that can provide wider protection against any Influenza A pathogen. An anti-viral approach that is directed against the conserved regions of the hemaggutinin protein has a potential to protect against any current and new Influenza A virus and provide a solution to this ever-present threat to public health. METHODOLOGY/PRINCIPAL FINDINGS: Influenza A human hemagglutinin protein sequences available in the NCBI database, corresponding to H1, H2, H3 and H5 subtypes, were used to identify highly invariable regions of the protein. Nine such regions were identified and analyzed for structural properties like surface exposure, hydrophilicity and residue type to evaluate their suitability for targeting an anti-peptide antibody/anti-viral response. CONCLUSION/SIGNIFICANCE: This study has identified nine conserved regions in the hemagglutinin protein, five of which have the structural characteristics suitable for an anti-viral/anti-peptide response. This is a critical step in the design of efficient anti-peptide antibodies as novel anti-viral agents against any Influenza A pathogen. In addition, these anti-peptide antibodies will provide broadly cross-reactive immunological reagents and aid the rapid development of vaccines against new and emerging Influenza A strains