The pathologic role of autoantibodies in autoimmune disease is widely accepted. Recently, we reported that anti-myelin basic protein (MBP) serum Abs from multiple sclerosis (MS) patients exhibit ...proteolytic activity toward the autoantigen. The aim of this study is to determine MBP epitopes specific for the autoantibodies in MS and compare these data with those from other neuronal disorders (OND), leading to the generation of new diagnostic and prognostic criteria. We constructed a MBP-derived recombinant "epitope library" covering the entire molecule. We used ELISA and PAGE/surface-enhanced laser desorption/ionization mass spectroscopy assays to define the epitope binding/cleaving activities of autoantibodies isolated from the sera of 26 MS patients, 22 OND patients, and 11 healthy individuals. The levels of autoantibodies to MBP fragments 48-70 and 85-170 as well as to whole MBP and myelin oligodendrocyte glycoprotein molecules were significantly higher in the sera of MS patients than in those of healthy donors. In contrast, selective reactivity to the two MBP fragments 43-68 and 146-170 distinguished the OND and MS patients. Patients with MS (77% of progressive and 85% of relapsing-remitting) but only 9% of patients with OND and no healthy donors were positive for catalysis, showing pronounced epitope specificity to the encephalitogenic MBP peptide 81-103. This peptide retained its substrate properties when flanked with two fluorescent proteins, providing a novel fluorescent resonance energy transfer approach for MS studies. Thus, anti-MBP autoantibody-mediated, epitope-specific binding and cleavage may be regarded as a specific characteristic of MS compared with OND and healthy donors and may serve as an additional biomarker of disease progression.
The creation of effective bioscavengers as a pretreatment for exposure to nerve agents is a challenging medical objective. We report a recombinant method using chemical polysialylation to generate ...bioscavengers stable in the bloodstream. Development of a CHO-based expression system using genes encoding human butyrylcholinesterase and a proline-rich peptide under elongation factor promoter control resulted in self-assembling, active enzyme multimers. Polysialylation gives bioscavengers with enhanced pharmacokinetics which protect mice against 4.2 LD₅₀ of S-(2-(diethylamino) ethyl) O-isobutyl methanephosphonothioate without perturbation of long-term behavior.
Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to ...identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (VL) and variable heavy (VH) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes.
Search for the population of the low-energy continuum of tetraneutron system was performed for reactions of the \(^{8}\)He beam on deuterium target. These studies are performed for the data I.A. ...Muzalevskii et al., Phys. Rev. C 103, 044313 (2021), previously used for the studies of \(^{7}\)H and \(^{6}\)H in the \(^2\text{H}(^8\text{He},{^3\text{He}})^{7}\)H and \(^2\text{H}(^8\text{He},{^4\text{He}})^{6}\)H reactions. Evidence for a hump in the \(^4n\) continuum at \(3.5 \pm 0.7\) and \(3.2 \pm 0.8\) MeV, was observed in the \(^2\)H(\(^8\)He,\(^6\)Li)\(^4\)n and \(^2\)H(\(^8\)He,\(^3\)He)\(^7\)H\(\rightarrow ^3\)H+\(^4\)n reactions, respectively. The observed statistics is quite low (6 and up to 40 events) corresponding to very low cross sections of few microbarns or tens of microbarns. The background conditions for the \(^2\)H(\(^8\)He,\(^6\)Li)\(^4n\) reaction are shown to be good, favoring the physical nature of the observed events. The \(^2\)H(\(^8\)He,\(^3\)He)\(^7\)H\(\rightarrow ^3\)H+\(^4n\) process transforms to the \(^2\)H(\(^8\)He,\(^6\)Li\(^{\ast})^4n\) reaction in the limit of the highest \(^7\)H decay energies. The population of the low-energy region in the \(^{4}\)n spectrum is found to be perfectly correlated with the population of the lowest \(^{6}\)Li states in the \(^{3}\)He+\(^{3}\)H continuum. Theoretical calculations of \(^{8}\)He in a five-body \(\alpha\)+\(4n\) and of \(^{4}\)n in a four-body hyperspherical models are presented. The \(^{8}\)He wave function is shown to contain strong specific correlations, which may give rise to very low-energy structures in tetraneutron continuum in extreme-peripheral reaction scenarios.
Switchable targeting of solid tumors by BsCAR T cells Stepanov, Alexey V; Kalinin, Roman S; Shipunova, Victoria O ...
Proceedings of the National Academy of Sciences - PNAS,
11/2022, Letnik:
119, Številka:
46
Journal Article
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The development of chimeric antigen receptor (CAR) T cell therapy has become a critical milestone in modern oncotherapy. Despite the remarkable in vitro effectiveness, the problem of safety and ...efficacy of CAR T cell therapy against solid tumors is challenged by the lack of tumor-specific antigens required to avoid on-target off-tumor effects. Spatially separating the cytotoxic function of CAR T cells from tumor antigen recognition provided by protein mediators allows for the precise control of CAR T cell cytotoxicity. Here, the high affinity and capability of the bacterial toxin-antitoxin barnase-barstar system were adopted to guide CAR T cells to solid tumors. The complementary modules based on (1) ankyrin repeat (DARPin)-barnase proteins and (2) barstar-based CAR (BsCAR) were designed to provide switchable targeting to tumor cells. The alteration of the DARPin-barnase switches enabled the targeting of different tumor antigens with a single BsCAR. A gradual increase in cytokine release and tunable BsCAR T cell cytotoxicity was achieved by varying DARPin-barnase loads. Switchable BsCAR T cell therapy was able to eradicate the HER2
ductal carcinoma in vivo. Guiding BsCAR T cells by DARPin-barnase switches provides a universal approach for a controlled multitargeted adoptive immunotherapy.
Characteristics of a new separator of radioactive ion beams (RIBs) are described. It was recently commissioned at the Flerov Laboratory of Nuclear Reactions (FLNR JINR) and is in operation at the ...primary beam line of the U-400M heavy ion cyclotron providing RIBs in energy range of 10–50 MeV/nucleon. Preliminary results obtained in the first experiments performed with this separator in 2018 are presented. A medium-term program of scientific research is discussed.
Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we ...developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE–FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.
The proton and deuteron pickup reactions 2H(10Be,3He)9Li and 2H(10Be,4He)8Li radioactive beam produced by the new fragment separator ACCULINNA-2 at FLNR, JINR\@. These measurements were initially ...motivated as test reactions intended for the elucidation of results obtained in the study of the extremely neutron-rich 7H and 6H systems created in the 2H(10Be,3He)9Li and 2H(10Be,4He)8Li reactions using the same setup. In the 2H(10Be,3He)9Li reaction the 9Li ground-state (\(3/2^-\)) and its first excited state (2.69MeV, \(1/2^-\)) were identified in the low-energy region of its excitation spectrum. The differential cross sections for the 9Li g.~s.) population were extracted at forward center-of-mass angles (\(3^\circ-13^\circ\)) and compared with the FRESCO calculations. Spectroscopic factor of \(\sim 1.7\), derived by a model for the 10Be\( = p +\)9Li(g.s.) clustering was found in accord with the experimental data. The energy spectrum of 8Li populated in the 2H(10Be,4He)8Li reaction shows the strong peak which corresponds to excitation of the second excited state of 8Li (2.25 MeV, \(3^+\)). The fact that the ground and the first excited states of 8Li were not observed is fully consistent with Shell-Model calculations carried out for the 10Be g.\,s. and 8Li level structure applying momentum selection rules.
Conventional “bulk” PCR often yields inefficient and nonuniform amplification of complex templates in DNA libraries, introducing unwanted biases. Amplification of single DNA molecules encapsulated in ...a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.
Infection caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2) in many cases is accompanied by the release of a large amount of proinflammatory cytokines in an event known as ...“cytokine storm”, which is associated with severe coronavirus disease 2019 (COVID-19) cases and high mortality. The excessive production of proinflammatory cytokines is linked, inter alia, to the enhanced activity of receptors capable of recognizing the conservative regions of pathogens and cell debris, namely TLRs, TREM-1 and TNFR1. Here we report that peptides derived from innate immunity protein Tag7 inhibit activation of TREM-1 and TNFR1 receptors during acute inflammation. Peptides from the N-terminal fragment of Tag7 bind only to TREM-1, while peptides from the C-terminal fragment interact solely with TNFR1. Selected peptides are capable of inhibiting the production of proinflammatory cytokines both in peripheral blood mononuclear cells (PBMCs) from healthy donors and in vivo in the mouse model of acute lung injury (ALI) by diffuse alveolar damage (DAD). Treatment with peptides significantly decreases the infiltration of mononuclear cells to lungs in animals with DAD. Our findings suggest that Tag7-derived peptides might be beneficial in terms of the therapy or prevention of acute lung injury, e.g., for treating COVID-19 patients with severe pulmonary lesions.
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