Reverse Engineering of a Pathogenic Antibody Reveals the Molecular Mechanism of Vaccine-Induced Immune Thrombotic Thrombocytopenia.

The massive COVID-19 vaccine roll-out campaign illuminated a range of rare side effects, the most dangerous of which─vaccine-induced immune thrombotic thrombocytopenia (VITT)─is caused by adenoviral (Ad)-vectored vaccines. VITT occurrence had been linked to the production of pathogenic antibodies that recognize an endogenous chemokine, platelet factor 4 (PF4). Mass spectrometry (MS)-based evaluation of the ensemble of anti-PF4 antibodies obtained from a VITT patient's blood indicates that the major component is a monoclonal antibody. Structural characterization of this antibody reveals several unusual characteristics, such as the presence of an N-glycan in the Fab segment and high density of acidic amino acid residues in the complementarity-determining regions. A recombinant version of this antibody (RVT1) was generated by transient expression in mammalian cells based on the newly determined sequence. It captures the key properties of VITT antibodies such as their ability to activate platelets in a PF4 concentration-dependent fashion. Homology modeling of the Fab segment reveals a well-defined polyanionic paratope, and the docking studies indicate that the polycationic segment of PF4 readily accommodates two Fab segments, cross-linking the antibodies to yield polymerized immune complexes. Their existence was verified with native MS by detecting assemblies as large as (RVT1)3(PF4)2, pointing out at FcγRIIa-mediated platelet activation as the molecular mechanism underlying VITT clinical manifestations. In addition to the high PF4 affinity, RVT1 readily binds other polycationic targets, indicating a polyreactive nature of this antibody. This surprising promiscuity not only sheds light on VITT etiology but also opens up a range of opportunities to manage this pathology.

Solution- and gas-phase behavior of decavanadate: implications for mass spectrometric analysis of redox-active polyoxidometalates.

Decavanadate (V10O28 6- or V10) is a paradigmatic member of the polyoxidometalate (POM) family, which has been attracting much attention within both materials/inorganic and biomedical communities due to its unique structural and electrochemical properties. In this work we explored the utility of high-resolution electrospray ionization (ESI) mass spectrometry (MS) and ion exclusion chromatography LC/MS for structural analysis of V10 species in aqueous solutions. While ESI generates abundant molecular ions representing the intact V10 species, their isotopic distributions show significant deviations from the theoretical ones. A combination of high-resolution MS measurements and hydrogen/deuterium exchange allows these deviations to be investigated and interpreted as a result of partial reduction of V10. While the redox processes are known to occur in the ESI interface and influence the oxidation state of redox-active analytes, the LC/MS measurements using ion exclusion chromatography provide unequivocal evidence that the mixed-valence V10 species exist in solution, as extracted ion chromatograms representing V10 molecular ions at different oxidation states exhibit distinct elution profiles. The spontaneous reduction of V10 in solution is seen even in the presence of hydrogen peroxide and has not been previously observed. The susceptibility to reduction of V10 is likely to be shared by other redox active POMs. In addition to the molecular V10 ions, a high-abundance ionic signal for a V10O26 2- anion was displayed in the negative-ion ESI mass spectra. None of the V10O26 cations were detected in ESI MS, and only a low-abundance signal was observed for V10O26 anions with a single negative charge, indicating that the presence of abundant V10O26 2- anions in ESI MS reflects gas-phase instability of V10O28 anions carrying two charges. The gas-phase origin of the V10O26 2- anion was confirmed in tandem MS measurements, where mild collisional activation was applied to V10 molecular ions with an even number of hydrogen atoms (H4V10O28 2-), resulting in a facile loss of H2O molecules and giving rise to V10O26 2- as the lowest-mass fragment ion. Water loss was also observed for V10O28 anions carrying an odd number of hydrogen atoms (e.g., H5V10O28 -), followed by a less efficient and incomplete removal of an OH• radical, giving rise to both HV10O26 - and V10O25 - fragment ions. Importantly, at least one hydrogen atom was required for ion fragmentation in the gas phase, as no further dissociation was observed for any hydrogen-free V10 ionic species. The presented workflow allows a distinction to be readily made between the spectral features revealing the presence of non-canonical POM species in the bulk solution from those that arise due to physical and chemical processes occurring in the ESI interface and/or the gas phase.

Decavanadate interactions with the elements of the SARS-CoV-2 spike protein highlight the potential role of electrostatics in disrupting the infectivity cycle.

Polyoxidometalates (POMs) exhibit a range of biological properties that can be exploited for a variety of therapeutic applications. However, their potential utility as antivirals has been largely overlooked in the ongoing efforts to identify safe, effective and robust therapeutic agents to combat COVID-19. We focus on decavanadate (V10), a paradigmatic member of the POM family, to highlight the utility of electrostatic forces as a means of disrupting molecular processes underlying the SARS-CoV-2 entry into the host cell. While the departure from the traditional lock-and-key approach to the rational drug design relies on less-specific and longer-range interactions, it may enhance the robustness of therapeutic agents by making them less sensitive to the viral mutations. Native mass spectrometry (MS) not only demonstrates the ability of V10 to associate with the receptor-binding domain of the SARS-CoV-2 spike protein, but also provides evidence that this association disrupts the protein binding to its host cell-surface receptor. Furthermore, V10 is also shown to be capable of binding to the polybasic furin cleavage site within the spike protein, which is likely to decrease the effectiveness of the proteolytic processing of the latter (a pre-requisite for the viral fusion with the host cell membrane). Although in vitro studies carried out with SARS-CoV-2 infected cells identify V10 cytotoxicity as a major factor limiting its utility as an antiviral agent, the collected data provide a compelling stimulus for continuing the search for effective, robust and safe therapeutics targeting the novel coronavirus among members of the POM family.

Ocsyn and mitochondrial-canalicular complexes in vestibular hair cells.

Ocsyn, a syntaxin-interacting protein characterized by Safieddine et al. [Safieddine, S., Ly, C.D., Wang, Y.-X., Kachar, B., Petralia, R.S., Wenthold, R.J., 2002. Ocsyn, a novel syntaxin-interacting protein enriched in the subapical region of inner hair cells. Mol. Cell. Neurosci., 20, 343-353] in the guinea pig organ of Corti was primarily identified in organelles located at the subapical region of inner hair cells. They proposed that in cochlear inner hair cells, ocsyn was involved in protein trafficking associated to recycling endosomes. Ocsyn happens to be highly homologous to syntabulin with an almost identical syntaxin-binding domain. Syntabulin is believed to attach syntaxin-containing vesicles to kinesin for their axonal transport along microtubules. The present study shows the distribution of ocsyn in guinea pig and rat vestibular hair cells using immunocytochemistry and confocal microscopy. Ocsyn was characterized by intense immunolabeled spots distributed exclusively in type I and II vestibular hair cells. The subcuticular region under the cuticular plate exhibited particularly densely packed spots. In the neck region of the sensory cells, where microtubules are abundant, there was no colocalization of ocsyn and alpha-tubulin. Ocsyn labeled spots were also present in the medial and basal hair cell regions, particularly in the supranuclear and infranuclear regions. Mitochondria are particularly numerous in these three regions (subcuticular, supranuclear and infranuclear). Double labeling of ocsyn and cytochrome c showed that ocsyn was present in the same zones that mitochondria. This, together with the great similarity of ocsyn and syntabulin, suggest that, akin to syntabulin, ocsyn is involved in addressing organelles. We propose that ocsyn is involved in the formation of the canalicular-mitochondrial complexes depicted by Spicer et al. [Spicer, S.S., Thomopoulos, G.N., Schulte, B.A., 1999. Novel membranous structures in apical and basal compartments of inner hear cells. J. Comp. Neurol., 409, 424-437].

Potential cellular receptors involved in hepatitis C virus entry into cells.

Hepatitis C virus (HCV) infects hepatocytes and leads to permanent, severe liver damage. Since the genomic sequence of HCV was determined, progress has been made towards understanding the functions of the HCV-encoded proteins and identifying the cellular receptor(s) responsible for adsorption and penetration of the virus particle into the target cells. Several cellular receptors for HCV have been proposed, all of which are associated with lipid and lipoprotein metabolism. This article reviews the cellular receptors for HCV and suggests a general model for HCV entry into cells, in which lipoproteins play a crucial role.

In search of a health education model: teachers' conceptions in four Mediterranean countries.

School programs are defined to promote the health of the pupils and to develop their competencies so that they can adopt behaviors favorable to their health. With the European project FP6 Biohead-Citizen (2004-2007), we analyzed the conceptions of teachers as regards health education, in France, Lebanon, Morocco and Tunisia, in reference to the biomedical model and the social health model. These four countries were selected because their school curricula represented different models of health education. Lebanon and Tunisia addressed health education with the biomedical model. In Morocco, the curriculum was also primarily based on the biomedical model and enclosed a few instructions issued from the social health model. In France, the health education curriculum declared an approach based on the health promotion model. Our study was based on multivariate statistical analyses of questionnaires filled out by 2537 in-service and pre-service teachers. Our analysis showed that the conceptions of the teachers concerning health education were not structured and related to a specific model. We also found that the dominating factors of influence on the choices expressed with regards to health education were, among different sociocultural variables, the religion, the home country, and, to a lesser extent, the level of training. Thus, the conceptions of the teachers were not integrated into comprehensive approaches but related to individual characteristics. Consequently health education implementation would require thorough training for pre-service and in-service teachers and should also explicitly take into account their conceptions and values.

Reverse transcriptase activity of hepatitis B virus polymerase in eukaryotic cell extracts in vitro.

In hepadnaviruses, reverse transcription is primed by the viral reverse transcriptase (RT) and requires the specific interaction between the RT and the viral RNA encapsidation signal termed e. To study the activity of the RT in vitro, the current procedure uses in vitro translated duck hepatitis B virus polymerase, but not the hepatitis B virus polymerase itself, in the rabbit reticulocyte lysate expression system. Here, the hepatitis B virus (HBV) polymerase has been successfully expressed in a translational extract that was obtained from monolayer human hepatocyte cells HuH-7. The translated polypeptide retained the RNA-directed polymerase (reverse transcriptase) activity on the viral RNA template containing the e signal. We suggest that the reverse transcription event of the viral RNA coding for the polymerase and containing an e structure is concomitant to the translation of the viral polymerase to the messenger RNA. In contrast to the duck polymerase, only a fraction of the reverse transcribed complementary DNA (cDNA) was covalently bound to the HBV polymerase in this system. When the e signal was missing on the mRNA, the translated full-length HBV polymerase could not reverse transcribe the viral RNA template. A truncated HBV polymerase that was lacking the YMDD catalytic active site for the initiation of reverse transcription was unable to reverse transcribe the viral mRNA template containing the e signal. The reverse transcription activity could also be partially inhibited by employing nucleoside analogues, such as 2'-3'-dideoxy-3'-thiacytidine (3TC; lamivudine) in the expression system. The procedure described here provides a method for the in vitro screening of new anti-HBV compounds directed against wild- type and mutants of this crucial viral protein, the HBV polymerase, without the use of animals (ducks) or animal extracts (rabbit reticulocyte lysate).

Reactivation of creatine kinase by dithiothreitol prior to use in an in vitro translation extract.

BACKGROUND: In vitro protein synthesis on exogenous messenger ribonucleic acids can be performed in various systems including cytoplasmic extract from eukaryotic cells, rabbit reticulocyte lysate and wheat germ extract. For optimal translation, an energy regeneration system based on creatine phosphate and creatine kinase is commonly employed for the regeneration of the endogenous adenosine triphosphate pools. Creatine kinase purchased from various commercial suppliers can be partially oxidised. Oxidised creatine kinase is not biologically active and might not allow the efficient initiation of translation of exogenous mRNAs in eukaryotic cell extracts in vitro. RESULTS: We successfully used dithiothreitol to reduce and therefore reactivate commercially available creatine kinase. When employed in cytoplasmic extracts obtained from eukaryotic cells grown in monolayers, the reactivated creatine kinase restored translation of the exogenous mRNAs. CONCLUSION: Lyophilised creatine kinase obtained from commercial suppliers can be purchased as an oxidised monomer. The reactivation of creatine kinase using a reducing agent such as dithiothreitol restores the biological activity of this enzyme. This procedure might therefore be extended to various other in vitro conditions and biological systems in which the maintenance of an efficient ATP-regenerating system is critical.

Generation of a Translational Extract from Eukaryotic Cells that have Grown as Monolayers.

The present article relates to the rapid and efficient generation of an in vitro translational extract that is obtained from the cytoplasmic fraction of eukaryotic cells that have grown as monolayers. The procedure is totally devoid of the use of animals or animal fluids. The cytoplasmic extract that is obtained allows efficient protein synthesis of exogenously added mRNAs. The latter mRNAs can be purified from prokaryotic or eukaryotic cells, or can be transcribed in vitro by employing any convenient RNA polymerase (for example, the bacteriophage SP6, T3 or T7 RNA polymerase). The described cytoplasmic preparation appears to be applicable to a large number of different eukaryotic cell lines. The cytoplasmic extract can be prepared freshly on a daily basis, or can be frozen and subsequently thawed for further use, without loss of activity. The preparation of the translation extract does not require living animals.

Latent hepatitis B virus (HBV) infection and HBV DNA integration is associated with further transformation of hepatoma cells in vitro.

Hepatitis B virus (HBV) is the major cause of chronic liver disease and hepatocellular carcinoma in the world, with more than 400 million people infected worldwide. To date, there is no reliable model for the study of the many aspects of HBV infection, despite the use of the chimpanzee. Although several alternative methods have been previously developed for the in vitro study of HBV infection, there is still an urgent need for new in vitro infection models, including for the ability of HBV to integrate into the host cell genome. Here we describe a process to improve infection of the human hepatoma cell lines HepG2 and HuH-7 in vitro with HBV originating from human blood. As shown previously for infection of hepatocytes with hepatitis C virus (HCV), the removal of the cell-bound lipoproteins prior to the addition of the viral inoculum to the cells could also be critical for the uptake of HBV via lipoprotein (LDL)-related receptors. Induction by insulin and dexamethasone led to an increase of HBsAg expression at the cell surface in association with the integration of the viral DNA into the host genome and HBx RNA detection. This integration process was also shown to be associated with cytopathic changes and further phenotypic transformations of the cells.