Tauopathies are characterised by intracellular deposits of fibrillar tau tangles. However, the interneuronal spread of pathological tau species precedes the development of major tau burdens. Two amyloid motifs, VQIINK in repeat 2 and VQIVYK in repeat 3, of tau repeat domain, assemble into β-sheet-rich fibrils on their own but alone do not form seed-competent fibrils. In contrast, the entire R3 region self-aggregates and forms seed-competent fibrils. Our study aimed to identify the minimal regions in the tau repeat domain that define seeding and its impact on intracellular tau phosphorylation and aggregation. Using peptides of individual repeats, we show that R2, like R3, forms seed-competent fibrils when assembled in the presence of heparin. However, R3, but not R2, forms seed-competent fibrils when assembled without heparin, even though both R2 and R3 have identical N-terminal hexapeptide and cysteine residue sequences. Moreover, cysteine to alanine substitution in R3 abrogates its self-aggregation and seeding potency. Tau RD P301S biosensor cells and Tau P301L (0N4R)-expressing HEK293 cells seeded with R2 and R3 fibrils show the induction of pathological phosphorylation of tau at Ser262/Ser396/Ser404 positions and oligomerisation of native tau. Protein fractions of biosensor cells seeded with R2 and R3 fibrils reseed endogenous tau aggregation when introduced into a fresh set of biosensor cells. Our findings suggest that R3 may be the minimal region for pathological seed generation under physiological conditions, whereas R2 might need polyanionic cofactors to generate pathogenic seeds. Lastly, R2 and R3 fibrils induce template-induced misfolding and pathological hyperphosphorylation of intracellular tau, making intracellular tau seed-competent.
- MeSH
- Alzheimer Disease * metabolism MeSH
- Cysteine MeSH
- HEK293 Cells MeSH
- Heparin MeSH
- Humans MeSH
- tau Proteins genetics metabolism MeSH
- Tauopathies * metabolism pathology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Long-term delivery of growth factors and immunomodulatory agents is highly required to support the integrity of tissue in engineering constructs, e.g., formation of vasculature, and to minimize immune response in a recipient. However, for proteins with a net positive charge at the physiological pH, controlled delivery from negatively charged alginate (Alg) platforms is challenging due to electrostatic interactions that can hamper the protein release. In order to regulate such interactions between proteins and the Alg matrix, we propose to complex proteins of interest in this study - CXCL12, FGF-2, VEGF - with polyanionic heparin prior to their encapsulation into Alg microbeads of high content of α-L-guluronic acid units (high-G). This strategy effectively reduced protein interactions with Alg (as shown by model ITC and SPR experiments) and, depending on the protein type, afforded control over the protein release for at least one month. The released proteins retained their in vitro bioactivity: CXCL12 stimulated the migration of Jurkat cells, and FGF-2 and VEGF induced proliferation and maturation of HUVECs. The presence of heparin also intensified protein biological efficiency. The proposed approach for encapsulation of proteins with a positive net charge into high-G Alg hydrogels is promising for controlled long-term protein delivery under in vivo conditions.
- MeSH
- Alginates chemistry MeSH
- Chemokine CXCL12 chemistry MeSH
- Human Umbilical Vein Endothelial Cells MeSH
- Fibroblast Growth Factor 2 chemistry MeSH
- Heparin chemistry MeSH
- Humans MeSH
- Microspheres MeSH
- Cell Line, Tumor MeSH
- Tissue Engineering MeSH
- Vascular Endothelial Growth Factor A chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
Krátce po celosvětovém zahájení očkování proti SARS-CoV-2 se objevily obavy z možné souvislosti mezi vakcinací, závažnou trombocytopenií a vznikem trombózy v atypických lokalizacích. Obavy se primárně týkaly vakcíny AstraZeneca (ChAdOx1 nCov-19), později vakcíny Johnson&Johnson (Ad26.COV2.S), ale také po aplikaci mRNA vakcín Pfizer-BioNTech a Moderna byla popsána akutní imunitní trombocytopenická purpura (ITP) provázená krvácením bez trombózy a žilní trombózy v atypických lokalizacích. Zkoumáním okolností vzniku těchto komplikací bylo zjištěno, že se jedná o obdobný mechanismus jako u heparinem indukované trombocytopenie 2. typu (HIT 2. typu), vzácného a závažného hyperkoagulačního stavu s žilními a tepennými trombózami. Příčinou HIT jsou IgG protilátky aktivující trombocyty, namířené proti antigenu, kterým je makromolekulární komplex tvořený destičkovým faktorem 4 (PF4) a heparinem. Pro odlišení obou stavů se začal razit nový termín vakcínou indukovaná (imunitní) trombotická trombocytopenie (VITT). Postižení pacienti měli vysoké hladiny protilátek proti imunitnímu komplexu tvořenému PF 4 a polyaniontovou složkou vakcíny (dvojvláknovou DNA). U pacientů, kteří mají po očkování trombózu v jakékoliv cévní lokalizaci, provázenou absolutní nebo relativní trombocytopenií a systémovými projevy, lze využít ELISA test k průkazu protilátek proti PF4 a funkční testy aktivace destiček k rychlé a spolehlivé diagnostice VITT a k jeho odlišení od žilní tromboembolické nemoci. Podání imunoglobulinu zvyšuje počet trombocytů, snižuje krevní srážlivost a inhibuje aktivaci trombocytů IgG protilátkami, podobně jako u HIT. Pro léčbu trombózy jsou vhodné přímé orální inhibitory F. Xa, přímé inhibitory trombinu a nepřímé inhibitory F.Xa.
Shortly after the worldwide initiation of vaccination against SARS-CoV-2, concerns emerged about a possible link between vaccination, severe thrombocytopenia, and the development of atypical venous thrombosis. Concerns were primarily about AstraZeneca (ChAdOx1 nCov-19), later Johnson & Johnson (Ad26.COV2.S), but cases of acute immune thrombocytopenic purpura (ITP) and bleeding without thrombosis and also atypical venous thrombosis after exposure to the messenger RNA-based vaccines produced by Pfizer-BioNTech and Moderna have been reported. Examination of the circumstances of these complications revealed that this is a similar mechanism to heparin-induced thrombocytopenia (HIT), a prothrombotic thrombocytopenic hypercoagulable disorder with venous and arterial thrombosis. HIT is caused by platelet-activating IgG antibodies directed against an antigen that is a macromolecular complex consisting of platelet factor 4 (PF4) and heparin. Naming this new entity vaccine-induced immune thrombotic thrombocytopenia (VITT) was suggested to avoid confusion with HIT. Patients had high levels of antibodies to the immune complex formed by PF 4 and the polyanionic component of the vaccine (double-stranded DNA). In patients with thrombosis at any vascular site after vaccination, accompanied by absolute or relative thrombocytopenia and systemic manifestations, HIT Ig ELISA assay to detect antibodies against PF4 and platelet-activating functional tests may be used for VITT recognition and differentiation from venous thromboembolic disease. Immune globulin impedes antibody-mediated platelet clearance and down-regulate platelet activation by immune complexes, as in HIT. It is prudent to choose from among the nonheparin antithrombotic agents - direct oral F.Xa inhibitors, direct thrombin inhibitors and indirect F.Xa inhibitors for the treatment of thrombosis.
- MeSH
- Platelet Activation drug effects MeSH
- Anticoagulants therapeutic use MeSH
- Immunoglobulins, Intravenous therapeutic use MeSH
- Humans MeSH
- Thrombocytopenia * chemically induced physiopathology therapy MeSH
- Platelet Factor 4 physiology adverse effects MeSH
- Thrombophilia chemically induced physiopathology prevention & control MeSH
- Thrombosis chemically induced physiopathology MeSH
- Vaccination MeSH
- COVID-19 Vaccines * blood adverse effects MeSH
- Check Tag
- Humans MeSH
A novel polyelectrolyte nanocarrier was synthesized via layer-by-layer self-assembly of polycationic and polyanionic chains. The nanocarrier is composed of polyglutamate grafted chitosan core, dextran sulfate as a complexing agent, and polyethyleneimine shell decorated with folic acid. This polyelectrolyte complex has unique physicochemical properties so that the core is considered as an efficient carrier for LTX-315 and melittin peptides, and the shell is suitable for delivery of miR-34a. The spherical nanocarriers with an average size of 123 ± 5 nm and a zeta potential of -36 ± 1 mV demonstrated controlled-release of gene and peptides ensured a synergistic effect in establishing multiple cell death pathways on chemoresistance human breast adenocarcinoma cell line, MDA-MB-231. In vitro cell viability assays also revealed no cytotoxicity for the nanocarriers, and an IC50 of 15 μg/mL and 150 μg/mL for melittin and LTX-315, respectively, after 48 h, whereas co-delivery of melittin with miR-34a increased smart death induction by 54%.
- MeSH
- Cell Death MeSH
- Chitosan * MeSH
- Humans MeSH
- Melitten pharmacology MeSH
- MicroRNAs administration & dosage genetics MeSH
- Cell Line, Tumor MeSH
- Breast Neoplasms * drug therapy MeSH
- Nanoparticles * MeSH
- Oligopeptides MeSH
- Polyelectrolytes MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.
- MeSH
- Alpharetrovirus physiology MeSH
- Betaretrovirus physiology MeSH
- Cell Membrane chemistry metabolism MeSH
- Gammaretrovirus physiology MeSH
- Gene Products, gag chemistry metabolism MeSH
- Host-Pathogen Interactions * MeSH
- Cells, Cultured MeSH
- Polyelectrolytes chemistry metabolism MeSH
- Retroviridae physiology ultrastructure MeSH
- Virus Assembly * MeSH
- Virion MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Proper assembly and disassembly of both immature and mature HIV-1 hexameric lattices are critical for successful viral replication. These processes are facilitated by several host-cell factors, one of which is myo-inositol hexaphosphate (IP6). IP6 participates in the proper assembly of Gag into immature hexameric lattices and is incorporated into HIV-1 particles. Following maturation, IP6 is also likely to participate in stabilizing capsid protein-mediated mature hexameric lattices. Although a structural-functional analysis of the importance of IP6 in the HIV-1 life cycle has been reported, the effect of IP6 has not yet been quantified. Using two in vitro methods, we quantified the effect of IP6 on the assembly of immature-like HIV-1 particles, as well as its stabilizing effect during disassembly of mature-like particles connected with uncoating. We analyzed a broad range of molar ratios of protein hexamers to IP6 molecules during assembly and disassembly. The specificity of the IP6-facilitated effect on HIV-1 particle assembly and stability was verified by K290A, K359A, and R18A mutants. In addition to IP6, we also tested other polyanions as potential assembly cofactors or stabilizers of viral particles.IMPORTANCE Various host cell factors facilitate critical steps in the HIV-1 replication cycle. One of these factors is myo-inositol hexaphosphate (IP6), which contributes to assembly of HIV-1 immature particles and helps maintain the well-balanced metastability of the core in the mature infectious virus. Using a combination of two in vitro methods to monitor assembly of immature HIV-1 particles and disassembly of the mature core-like structure, we quantified the contribution of IP6 and other small polyanion molecules to these essential steps in the viral life cycle. Our data showed that IP6 contributes substantially to increasing the assembly of HIV-1 immature particles. Additionally, our analysis confirmed the important role of two HIV-1 capsid lysine residues involved in interactions with IP6. We found that myo-inositol hexasulphate also stabilized the HIV-1 mature particles in a concentration-dependent manner, indicating that targeting this group of small molecules may have therapeutic potential.
- MeSH
- gag Gene Products, Human Immunodeficiency Virus chemistry genetics metabolism MeSH
- HIV-1 chemistry genetics MeSH
- Mutation, Missense MeSH
- Polymers chemistry MeSH
- Virus Assembly * MeSH
- Amino Acid Substitution MeSH
- Structure-Activity Relationship MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Polysaccharides based nanocomplexes have been developed for encapsulation, controlled delivery and to enhance the phototoxicity of the photosensitizer 5-aminolevulinic acid for application in photodynamic therapy. The nanocomplexes were prepared by coacervation in a solvent free environment using chitosan as polycation while alginic and polygalacturonic acid as polyanions. The complexes showed average dimension in the range 90-120nm, good stability in simulated physiological media and high drug encapsulation efficiency, up to 800μg per mg of carrier. Release studies demonstrate the possibility to tune the overall release rate and the intensity of the initial burst by changing the external pH. Cytotoxicity and photocytotoxicity tests confirmed the not toxicity of the used polysaccharides. Cell viability results confirmed the improvement of 5-aminolevulinic acid phototoxicity when loaded into the carrier compared to the free form. No effect of the irradiation on the nanocomplexes structure and on the release kinetics of the drug was observed. The results demonstrate that the prepared formulations have suitable properties for future application in photodynamic therapy and to ameliorate the therapeutic efficacy and overcome the side-effects related to the use of the photosensitizer 5-aminolevulinic acid.
- MeSH
- Chitosan chemistry MeSH
- Photosensitizing Agents chemistry toxicity MeSH
- HeLa Cells MeSH
- Hydrogen-Ion Concentration MeSH
- Aminolevulinic Acid chemistry toxicity MeSH
- Humans MeSH
- Nanoparticles chemistry MeSH
- Drug Carriers chemistry MeSH
- Light MeSH
- Temperature MeSH
- Drug Liberation MeSH
- Particle Size MeSH
- Cell Survival drug effects radiation effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Hyaluronan is a polyanionic, megadalton-scale polysaccharide, which initiates cell signaling by interacting with several receptor proteins including CD44 involved in cell-cell interactions and cell adhesion. Previous studies of the CD44 hyaluronan binding domain have identified multiple widespread residues to be responsible for its recognition capacity. In contrast, the X-ray structural characterization of CD44 has revealed a single binding mode associated with interactions that involve just a fraction of these residues. In this study, we show through atomistic molecular dynamics simulations that hyaluronan can bind CD44 with three topographically different binding modes that in unison define an interaction fingerprint, thus providing a plausible explanation for the disagreement between the earlier studies. Our results confirm that the known crystallographic mode is the strongest of the three binding modes. The other two modes represent metastable configurations that are readily available in the initial stages of the binding, and they are also the most frequently observed modes in our unbiased simulations. We further discuss how CD44, fostered by the weaker binding modes, diffuses along HA when attached. This 1D diffusion combined with the constrained relative orientation of the diffusing proteins is likely to influence the aggregation kinetics of CD44. Importantly, CD44 aggregation has been suggested to be a possible mechanism in CD44-mediated signaling.
Topical microbicides to stop sexually transmitted diseases, such as herpes simplex virus type 2 (HSV-2), are urgently needed. The emerging field of nanotechnology offers novel suitable tools for addressing this challenge. Our objective was to study, in vitro and in vivo, antiherpetic effect and antiviral mechanisms of several polyanionic carbosilane dendrimers with anti-HIV-1 activity to establish new potential microbicide candidates against sexually transmitted diseases. Plaque reduction assay on Vero cells proved that G2-S16, G1-S4, and G3-S16 are the dendrimers with the highest inhibitory response against HSV-2 infection. We also demonstrated that our dendrimers inhibit viral infection at the first steps of HSV-2 lifecycle: binding/entry-mediated events. G1-S4 and G3-S16 bind directly on the HSV-2, inactivating it, whereas G2-S16 adheres to host cell-surface proteins. Molecular modeling showed that G1-S4 binds better at binding sites on gB surface than G2-S16. Significantly better binding properties of G1-S4 than G2-S16 were found in an important position for affecting transition of gB trimer from G1-S4 prefusion to final postfusion state and in several positions where G1-S4 could interfere with gB/gH-gL interaction. We demonstrated that these polyanionic carbosilan dendrimers have a synergistic activity with acyclovir and tenofovir against HSV-2, in vitro. Topical vaginal or rectal administration of G1-S4 or G2-S16 prevents HSV-2 transmission in BALB/c mice in values close to 100%. This research represents the first demonstration that transmission of HSV-2 can be blocked by vaginal/rectal application of G1-S4 or G2-S16, providing a step forward to prevent HSV-2 transmission in humans.
- MeSH
- Acyclovir pharmacology MeSH
- Anti-Infective Agents pharmacology MeSH
- Antiviral Agents pharmacology MeSH
- Administration, Rectal MeSH
- Chlorocebus aethiops MeSH
- Dendrimers chemistry MeSH
- Epithelial Cells drug effects virology MeSH
- Herpes Simplex MeSH
- Hydrogen-Ion Concentration MeSH
- Humans MeSH
- Herpesvirus 2, Human drug effects MeSH
- Models, Molecular MeSH
- Mice, Inbred BALB C MeSH
- Polymers chemistry MeSH
- Rectum drug effects virology MeSH
- Silanes chemistry MeSH
- Tenofovir pharmacology MeSH
- Vagina drug effects virology MeSH
- Vero Cells MeSH
- Viral Proteins metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Nanotechnology-derived platforms, such as dendrimers, are very attractive in several biological applications. In the case of human immunodeficiency virus (HIV) infection, polyanionic carbosilane dendrimers have shown great potential as antiviral agents in the development of novel microbicides to prevent the sexual transmission of HIV-1. In this work, we studied the mechanism of two sulfated and naphthylsulfonated functionalized carbosilane dendrimers, G3-S16 and G2-NF16. They are able to inhibit viral infection at fusion and thus at the entry step. Both compounds impede the binding of viral particles to target cell surface and membrane fusion through the blockage of gp120-CD4 interaction. In addition, and for the first time, we demonstrate that dendrimers can inhibit cell-to-cell HIV transmission and difficult infectious synapse formation. Thus, carbosilane dendrimers' mode of action is a multifactorial process targeting several proteins from viral envelope and from host cells that could block HIV infection at different stages during the first step of infection.
- MeSH
- Antiviral Agents chemistry pharmacology MeSH
- Dendrimers chemistry MeSH
- Membrane Fusion drug effects MeSH
- HIV Infections prevention & control virology MeSH
- HIV Envelope Protein gp120 antagonists & inhibitors chemistry metabolism MeSH
- HIV-1 drug effects MeSH
- Humans MeSH
- Models, Molecular MeSH
- Polymers chemistry MeSH
- Virus Replication drug effects MeSH
- Silanes chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
