Biology subjects

Meszaros, T.

Publications and source records attributed to Meszaros, T..

9 recordsLinked to original sources

bioRxiv2026

Combining AlphaFold 3 with microscale thermophoresis for aptamer optimisation

Although use of AI-based tools has gained ground in molecular biology research, their reliability remains a matter of debate. Here, we combined AlphaFold 3 modelling with microscale thermophoresis to investigate the interactions between glucan-binding protein C and the biofilm-inhibiting aptamer and its five derivatives. Structural modelling identified a binding motif on the aptamer and suggested a mechanism of action; however, the predictions were only partially supported by the experimental results. The AlphaFold generated confidence metrics did not correlate directly with the measured binding affinities, indicating that confidence scores can help identify plausible interaction interfaces, but they are not reliable predictors of binding strength. These results highlight the importance of integrating structural prediction with biophysical validation to dissect aptamer-protein recognition.

molecular biology↗

bioRxiv2026

Hemoperfusion of pigs with a carbon-cellulose cartridge: a pilot study revealing a new animal model of extended anaphylactic shock

To investigate the immune mechanisms underlying extracorporeal circulation-associated anaphylactoid reactions, we inserted externally perfused cartridges into the venous circulation of pigs, including a cellulose-coated activated-charcoal adsorbent (Adsorba(R) 300C), a polysulfone hollow-fiber membrane hemofilter, and polypropylene hollow-fiber-based heart-lung machine gas-exchange oxygenators. Blood was circulated using a roller pump, and the animals were monitored for systemic and pulmonary arterial pressures (SAP, PAP) changes, blood levels of complement C3a, thromboxane B2, and hemoglobin, blood cell counts and hematocrit. None of the cartridges caused major changes in these endpoints except the Adsorba(R) 300C, which displayed a fulminant anaphylactoid reaction characterized by profound hypotension, maximal pulmonary hypertension, hemoconcentration, thrombocytopenia and a surge of C3a and thromboxane B2, i.e., hallmarks of complement activation-related pseudoallergy. Within 15 min, the reaction advanced to profound hemodynamic collapse, which was managed with norepinephrine and cardiopulmonary resuscitation. After brief rebound hypertension, shock recurred despite repeated rounds of resuscitation until death. Considering that major adverse reactions with overlapping symptoms have been reported in humans subjected to hemoperfusion with the same cartridge, this porcine model provides clinically relevant insights into the mechanisms of extracorporeal circulation-induced anaphylactoid responses. Furthermore, the Adsorba(R) 300C-induced physiological changes along the immune-cardiopulmonary axis represents a new animal model for irreversible cardiovascular collapse escalating into shock.

immunology↗

bioRxiv2025

Acute anaphylactic and multiorgan inflammatory effects of Comirnaty in pigs: evidence of spike protein mRNA transfection and paralleling inflammatory cytokine upregulation

Background and PurposeRare but serious adverse events (AEs) associated with mRNA-lipid nanoparticle (LNP) COVID-19 vaccines, such as Comirnaty and Spikevax, include anaphylaxis and acute multiorgan inflammatory syndrome. The mechanisms of these acute innate immune responses remain poorly understood. This study aimed to investigate these effects using an amplified porcine model. Experimental ApproachNaive and anti-PEG antibody-sensitized pigs were intravenously injected with Comirnaty. Acute anaphylactic responses were assessed through hemodynamic monitoring, hematological changes, and plasma inflammatory markers. Multiorgan inflammatory effects were evaluated by RT-qPCR detection of spike protein (SP) mRNA uptake and inflammatory cytokine gene expression in seven organs over 6 hours. Histopathology and immunohistochemistry were also performed. Key ResultsSevere cardiopulmonary distress developed within minutes of repeated intravenous injections of Comirnaty. RT-qPCR revealed predominant SP mRNA accumulation in liver and PBMCs, with spleen, kidney, lymph nodes, heart, and brain also affected in 40-90% of animals. Repetitive PBMC transfection showed reversible mRNA peaks at 15 minutes, followed by rapid decay. Expression of proinflammatory cytokines (IL-1RA, CXCL10, TNF-, CCL2) paralleled mRNA uptake, suggesting a causal relationship. Cytokine profiles varied by organ (e.g. IL-1RA in kidney, CCL2 in PBMCs). Histologic abnormalities and SP immunopositivity were noted in kidney, heart, and brain. Booster injections replicated PBMC transfection kinetics observed at first dose. Conclusions and ImplicationsThis porcine model reveals systemic anaphylactic reactivity and multiorgan SP mRNA transfection following intravenous injection of mRNA-LNPs, resulting in organ-specific inflammatory responses. These findings provide mechanistic insights into two rare vaccine-related acute AEs and may support future efforts to improve the safety of the mRNA-LNP platform technology.

immunology↗

bioRxiv2024

LIFERING, an E3 ligase, targets the acyl-CoA dehydrogenase-like protein IBR3, for ubiquitination and coordinates cell proliferation and cell death upon DNA damage

Plants rapidly respond to environmental changes to ensure an optimal balance between growth and survival with intact genome. Here, we show that a DNA damage response gene, which we named LIFERING, is under the direct and independent regulation of ATM/ATR-SOG1 and RBR pathways. We demonstrate that LIFERING upon DNA damage plays an essential role in maintaining the balance between cell proliferation and cell death. Downstream of RBR, it is required to maintain proliferation, and in response to DNA damaging agents it initiates a cell death accompanied by rapid elongation and differentiation of the transit amplifying cells in the root meristem. LIFERING is a RING-between-RING E3 ligase; its ligase activity is dependent on the Cys392 residue located in the BRcat domain, responsible for the coordination of zinc ion binding. Using proximity labelling, we identified the acyl-CoA dehydrogenase-like protein IBR3, which is involved in the conversion of indole-3-butyric acid (IBA) to indole-3-acetic acid (IAA), as one of its targets. We provide evidence that IBR3 is ubiquitinated at several Lys residues in the presence of LIFERING. Our findings show that LIFERING rapidly responds to DNA damage and via ubiquitination of IBR3, is likely to be involved in the regulation of free-auxin level, indicating a link between DNA damage response and auxin regulation in the root.

plant biology↗

bioRxiv2024

Selection of streptococcal glucan-binding protein C specific DNA aptamers to inhibit biofilm formation

Streptococcus mutans is a commensal bacterium in the oral cavity, but its overgrowth leads to biofilm formation and dental caries. S. mutans glucan-binding protein C (GbpC) is a cornerstone of the biofilm scaffold and thus a rational target for biofilm inhibition. Here we present a selection of DNA aptamers for GbpC to aid in the development of biofilm inhibiting drugs. During SELEX, we used the extracellular domain of GbpC as the target of selection and the structurally homologous antigen I/II protein and a GbpC-deficient strain of S. mutans as counter-targets. The aptamer candidates obtained were panned using a method based on primer-blocked asymmetric PCR and AlphaScreen. The interaction between the most promising candidates of panning and GbpC was analysed by biolayer interferometry and microscale thermophoresis. Finally, we tested the biofilm inhibitory effect of the aptamers on a wild-type and a GbpC-deficient strain of S. mutans. The measurements demonstrated both the efficacy and selectivity of the aptamers. Two of the aptamers studied reduced biofilm formation by 30% in the case of wild-type strains and no reduction was detected in the GbpC-deficient strain, confirming the suitability of the selection strategy presented and providing lead molecules for the development of biofilm inhibitors.

molecular biology↗

bioRxiv2024

mRNA-LNP COVID-19 vaccine lipids induce low level complement activation and production of proinflammatory cytokines: Mechanisms, effects of complement inhibitors, and relevance to adverse reactions

Messenger RNA-containing lipid nanoparticles (mRNA-LNPs) enabled widespread COVID-19 vaccination with a small fraction of vaccine recipients displaying acute or sub-acute inflammatory symptoms. The molecular mechanism of these adverse events (AEs) remains undetermined. Here we report that the mRNA-LNP vaccine, Comirnaty, triggers low-level complement (C) activation and production of inflammatory cytokines, which may be key underlying processes of inflammatory AEs. In serum, Comirnaty and the control PEGylated liposome (Doxebo) caused different rises of C split products, C5a, sC5b-9, Bb and C4d, indicating stimulation of the classical pathway of C activation mainly by the liposomes, while a stronger stimulation of the alternative pathway was equal with the vaccine and the liposomes. Spikevax had similar C activation as Comirnaty, but viral or synthetic mRNAs had no such effect. In autologous serum-supplemented peripheral blood mononuclear cell (PBMC) cultures, Comirnaty caused increases in the levels of sC5b-9 and proinflammatory cytokines in the following order: IL-1 < IFN-{gamma} < IL-1{beta} < TNF- < IL-6 < IL-8, whereas heatinactivation of serum prevented the rises of IL-1, IL-1{beta}, and TNF-. Clinical C inhibitors, Soliris and Berinert, suppressed vaccine-induced C activation in serum but did not affect cytokine production when applied individually. These findings suggest that the PEGylated lipid coating of mRNA-LNP nanoparticles can trigger C activation mainly via the alternative pathway, which may be causally related to the induction of some, but not all inflammatory cytokines. While innate immune stimulation is essential for the vaccines efficacy, concurrent production of C- and PBMC-derived inflammatory mediators may contribute to some of the AEs. Pharmacological attenuation of harmful cytokine production using C inhibitors likely requires blocking the C cascade at multiple points.

immunology↗

bioRxiv2023

The COVID-19 mRNA vaccine Comirnaty induces anaphylactic shock in an anti-PEG hyperimmune large animal model: Role of complement in cardiovascular, hematological, and inflammatory mediator changes

BackgroundComirnaty, Pfizer-BioNTechs polyethylene-glycol (PEG)-containing Covid-19 vaccine, can cause hypersensitivity reactions (HSRs) in a small fraction of immunized people which can, very rarely, culminate in life-threatening anaphylaxis. A role of anti-PEG antibodies (Abs) has been proposed, but causality has not yet been proven in an animal model. This study aimed to provide such evidence using anti-PEG hyperimmune pigs (i.e., pigs displaying very high levels of anti-PEG Abs). We also sought to find evidence for the role of complement (C) activation and thromboxane A2 (TXA2) release in blood as contributing effects to anaphylaxis. MethodsPigs (n=6) were immunized with 0.1 mg/kg PEGylated liposome (Doxebo) i.v. the rise of anti-PEG IgG and IgM was measured in serial blood samples with ELISA. After 2-3 weeks, during the height of seroconversion, the animals were injected i.v. with 1/3 human vaccine dose (HVD) of Comirnaty, and the hemodynamic (PAP, SAP), cardiopulmonary (HR, EtCO2,), hematological parameters (WBC, granulocyte, lymphocyte, and platelet counts) and blood immune mediators (anti-PEG IgM and IgG Abs, C3a and TXA2) were measured as endpoints of HSRs. ResultsA week after immunization of 6 pigs with Doxebo, the level of anti-PEG IgM and IgG rose 5-10-thousands-fold in all animals, and they all developed anaphylactic shock to i.v. injection of 1/3 HVD of Comirnaty. The reaction, starting within 1 min, led to the abrupt decline of SAP along with maximal pulmonary hypertension, decreased pulse pressure amplitude, tachycardia, granulo- and thrombocytopenia, and paralleling rises of plasma C3a and TXB2 levels. These vaccine effects were not observed in non-immunized pigs. ConclusionsConsistent with previous studies with PEGylated nano-liposomes, these data show a causal role of anti-PEG Abs in the anaphylaxis to Comirnaty. The reaction involves C activation, and, hence, it represents C activation-related pseudo-allergy (CARPA). The setup provides the first large-animal model for mRNA-vaccine-induced anaphylaxis in humans.

pharmacology and toxicology↗

bioRxiv2022

Tryptophan-like side chain holding aptamers inhibit respiratory syncytial virus infection of lung epithelial cells

Respiratory syncytial virus (RSV) is a leading cause of serious and even fatal acute lower respiratory tract infections in infants and in the elderly. Potent RSV neutralization has been achieved by antibodies that selectively bind the prefusion form of the viral fusion (F) protein. We hypothesised that similar potent neutralization could be achieved using F protein targeting aptamers. Aptamers have yet to reach their translational potential for therapeutics or diagnostics due to their short half-life and limited range of target-aptamer interactions; these shortcomings can, however, be ameliorated by application of amino acid-like side chain holding nucleotides. In this study, a stabilized version of the prefusion RSV F protein was targeted by aptamer selection using an oligonucleotide library holding a tryptophan-like side chain. This process resulted in aptamers that bound the F protein with high affinity and differentiated between pre- and postfusion conformation. Identified aptamers inhibited viral infection of lung epithelial cells. Moreover, introduction of modified nucleotides extended aptamer half-lives. Our results suggest that targeting aptamers to the surface of viruses could yield effective drug candidates, which could keep up with the pace of the continuously evolving pathogens.

molecular biology↗

bioRxiv2022

Zymosan-induced leukocyte and cytokine changes in pigs: a new model for streamlined drug testing against severe COVID-19

Injection of 0.1 mg/kg zymosan in pigs i.v. elicited transient hemodynamic disturbance within minutes, without major blood cell changes. In contrast, infusion of 1 mg/kg zymosan triggered maximal pulmonary hypertension with tachycardia, lasting for 30 min. This change was followed by a transient granulopenia with a trough at 1 h, and then, up to about 6 h, a major granulocytosis, resulting in a 3-4-fold increase of neutrophil-to-lymphocyte ratio (NLR). In parallel with the changes in WBC differential, qRT-PCR and ELISA analyses showed increased transcription and/or release of inflammatory cytokines and chemokines into blood, including IL-6, TNF-, CCL-2, CXCL-10, and IL-1RA. The expression of IL-6 peaked at already 1.5-2.5 h, and we observed significant correlation between lymphopenia and IL-6 gene expression. While these changes are consistent with zymosans known stimulatory effect on both the humoral and cellular arms of the innate immune system, what gives novel clinical relevance to the co-manifestation of above hemodynamic, hematological, and immune changes is that they represent independent bad prognostic indicators in terminal COVID-19 and other diseases involving cytokine storm. Thus, within a 6 h experiment, the model enables consecutive reproduction of a symptom triad that is characteristic of late-stage COVID-19. Given the limitations of modeling cytokine storm in animals and effectively treating severe COVID-19, the presented relatively simple large animal model may advance the R&D of drugs against these conditions. One of these disease markers (NLR), obtained from a routine laboratory endpoint (WBC differential), may also enable streamlining the model for high throughput drug screening against innate immune overstimulation.

immunology↗