Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic viruslike particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody

Perception de la désirabilité et de l’utilité sociales de l’homosexualité et de l’hétérosexualité au collège

The purpose of the current resarch was to compare adolescents’ stereotypes of homosexuality and heterosexuality from Beauvois’s theory of social value (1995 ; Dubois, 2005). We asked forty-three secondary school students to describe homosexuals and heterosexuals, from a list of personality traits, by giving their own judgments and by indicating how they thought that society perceived them. We expected them to think that the homosexuals were less desirable but at the same time just as socially useful as heterosexuals. We also expected the participants to feel that, in society, homosexuals were considered as less desirable and also less socially useful than heterosexuals. Those assumptions were only partially verified, especially among female participants. The results are discussed in terms of gender differences in the perception of homosexuality and in the consequences of these stereotypes

Perception de la désirabilité et de l’utilité sociales de l’homosexualité et de l’hétérosexualité au collège

The purpose of the current resarch was to compare adolescents’ stereotypes of homosexuality and heterosexuality from Beauvois’s theory of social value (1995 ; Dubois, 2005). We asked forty-three secondary school students to describe homosexuals and heterosexuals, from a list of personality traits, by giving their own judgments and by indicating how they thought that society perceived them. We expected them to think that the homosexuals were less desirable but at the same time just as socially useful as heterosexuals. We also expected the participants to feel that, in society, homosexuals were considered as less desirable and also less socially useful than heterosexuals. Those assumptions were only partially verified, especially among female participants. The results are discussed in terms of gender differences in the perception of homosexuality and in the consequences of these stereotypes

Tandem cores form VLPs when expressed in <i>N</i>. <i>benthamiana</i>.

<p>a) Western blot showing expression in <i>N</i>. <i>benthamiana</i> of monomeric (HBcAg), hetero-tandem (CoHe) and homo-tandem (CoHo) constructs. Lane C—empty vector control. b) Electron micrographs of monomeric (HBcΔ176), homo-tandem (CoHo) and hetero-tandem (CoHe) core particles produced in <i>N</i>. <i>benthamiana</i> and purified by sucrose gradient. Scale bar 100 nm. Arrows indicate smaller (T = 3) particles.</p

Plant-produced τGFP particles bind GFP.

<p>a) Ultracentrifuge tubes containing sucrose cushions photographed under UV light after ultracentrifugation. GFP-associated fluorescence remains in the supernatant when GFP-containing plant lysate is centrifuged alone or mixed with tEL-containing plant lysate; but migrates through the cushion when GFP-containing and τGFP-containing plant lysates are mixed. b) Detection of GFP by sandwich ELISA, after coating wells with τGFP (green), τglyc (orange) or an anti-GFP polyclonal IgG (blue) and adding GFP to the wells at four different concentrations after blocking. Detection is horseradish peroxidase—mediated ECL, and signal is net of background. Error bars are standard error. c) Electron micrograph of plant-produced τGFP particles in the presence of GFP, purified by sucrose cushion and size exclusion chromatography. Scale bar 100 nm.</p

Tandem core technology.

<p>a) The structure of a monomeric HBc VLP with one HBcAg dimer shown in a surface representation coloured yellow and green. b) Two HBcAg sequences fused together via a flexible linker makes a tandem core construct, with either full-length (hetero-tandem) or truncated (homo-tandem) C-terminus, and two modifiable major insertion regions (MIRs). c) Structure of a tandem core protein: N-terminal core 1 (in green) is fused via a flexible linker (red) to C-terminal core 2 (yellow). The two views are related by a 90° rotation.</p

EULAR points to consider for therapeutic drug monitoring of biopharmaceuticals in inflammatory rheumatic and musculoskeletal diseases

Objective To develop EULAR points-to-consider for therapeutic drug monitoring (TDM) of biopharmaceuticals in inflammatory rheumatic and musculoskeletal diseases (RMDs). Methods The points-to-consider were developed in accordance with EULAR standardised operation procedures by a multidisciplinary task force from eight European countries, based on a systematic literature review and expert consensus. Level of evidence and strength of the points-to-consider were determined, and mean levels of agreement among the task force were calculated using a 10-point rating scale. Results Six overarching principles and 13 points-to-consider were formulated. The level of agreement among the task force for the overarching principles and points-to-consider ranged from 8.4 to 9.9.The overarching principles define TDM and its subtypes, and reinforce the underlying pharmacokinetic/pharmacodynamic principles, which are relevant to all biopharmaceutical classes. The points-to-consider highlight the clinical utility of the measurement and interpretation of biopharmaceutical blood concentrations and antidrug antibodies in specific clinical scenarios, including factors that influence these parameters. In general, proactive use of TDM is not recommended but reactive TDM could be considered in certain clinical situations. An important factor limiting wider adoption of TDM is the lack of both high quality trials addressing effectiveness and safety of TDM and robust economic evaluation in patients with RMDs. Future research should focus on providing this evidence, as well as on further understanding of pharmacokinetic and pharmacodynamic characteristics of biopharmaceuticals. Conclusion These points-to-consider are evidence-based and consensus-based statements for the use of TDM of biopharmaceuticals in inflammatory RMDs, addressing the clinical utility of TDM

EULAR points to consider for therapeutic drug monitoring of biopharmaceuticals in inflammatory rheumatic and musculoskeletal diseases

ObjectiveTo develop EULAR points-to-consider for therapeutic drug monitoring (TDM) of biopharmaceuticals in inflammatory rheumatic and musculoskeletal diseases (RMDs).MethodsThe points-to-consider were developed in accordance with EULAR standardised operation procedures by a multidisciplinary task force from eight European countries, based on a systematic literature review and expert consensus. Level of evidence and strength of the points-to-consider were determined, and mean levels of agreement among the task force were calculated using a 10-point rating scale.ResultsSix overarching principles and 13 points-to-consider were formulated. The level of agreement among the task force for the overarching principles and points-to-consider ranged from 8.4 to 9.9.The overarching principles define TDM and its subtypes, and reinforce the underlying pharmacokinetic/pharmacodynamic principles, which are relevant to all biopharmaceutical classes. The points-to-consider highlight the clinical utility of the measurement and interpretation of biopharmaceutical blood concentrations and antidrug antibodies in specific clinical scenarios, including factors that influence these parameters. In general, proactive use of TDM is not recommended but reactive TDM could be considered in certain clinical situations. An important factor limiting wider adoption of TDM is the lack of both high quality trials addressing effectiveness and safety of TDM and robust economic evaluation in patients with RMDs. Future research should focus on providing this evidence, as well as on further understanding of pharmacokinetic and pharmacodynamic characteristics of biopharmaceuticals.ConclusionThese points-to-consider are evidence-based and consensus-based statements for the use of TDM of biopharmaceuticals in inflammatory RMDs, addressing the clinical utility of TDM.</jats:sec

Cryo-EM of plant-produced τGFP bound with GFP.

<p>a) Class averages computed using Relion of the τGFP particles. b) A 3D reconstruction (resolution estimate 25Å using the “gold standard” cross-FSC at cutoff 0.143) coloured by distance from the centre of the particle (red to blue). The map is shown viewed down a 5-fold axis with the He reconstruction on which the construct was based fitted within (grey surface). The projecting spikes represent density arising from the bound nanobody and GFP but do not occupy every position expected, instead appearing as an average of the density present with the highest intensity at the 2-fold (pseudo- 6-fold) axes and also at the 5-fold axis. These spikes are to some extent artefacts of the icosahedral symmetry imposed on the maps, but are reflected in the spikes also shown in the unaveraged class averages shown in a).</p