Glioblastoma multiforme je mozkový nádor s obecně špatnou prognózou a incidencí 2-3/100000 obyvatel. V této studii chceme využít nový přístup k léčbě nádorů prostřednictvím fototermální terapie za použití zlatých nanotyčinek s potenciálem neinvazivního odstranění nádoru včetně radio- a chemo-rezistentních buněk. Lokalizovaná povrchová plazmonová rezonance nanotyčinek umožňuje přeměnit světelnou energii na teplo, jehož vhodná intenzita usmrtí nádorové buňky apoptózou a dodatečně stimuluje protinádorovou imunitní odpověď. Zlaté nanotyčinky mohou být současně naladěny na požadovanou vlnovou délku zdroje záření vhodného pro klinické použití. V rámci projektu vyvineme techniku pro cílení glioblastomu nanotyčkami za použití mesenchymálních a neurálních kmenových buněk jako jejich nosičů. Lečebná strategie bude otestována in vivo na myších pomocí techniky kranialního okna, kdy bude možné sledovat růst nádoru, distribuci nanotyčinek a efekt terapie v reálném čase.; Glioblastoma multiforme is a brain tumor with incidence of 2-3/100000 persons and generally poor prognosis. In this study we want to employ a new approach of photothermal cancer therapy using gold nanorods (GNRs) with potential to eradicate also the radio- and chemo-resistant cells. The localized surface plasmon resonance of GNRs allows effective transformation of light energy to heat that can be optimized to kill the cancer cells by apoptosis with additional stimulation of anti-tumor immune response. GNRs can be optically tuned to desired wavelength of the irradiation source suitable for clinical use. Mesenchymal or neural stem cells will be used as a vehicle for targeting of GNRs to brain tumors in mouse. Cranial window technique will be adapted for life imaging of tumor growth, GNRs distribution and detection of thermal effects of GNRs in one step.

• Keywords
• kmenové buňky, stem cells, glioblastoma, glioblastom, zlaté nanotyčky, fototermální terapie, kraniální okno, gold nanorods, photothermal therapy, cranial window,

• NML Publication type
• závěrečné zprávy o řešení grantu AZV MZ ČR

MXenes and their related nanocomposites with superior physicochemical properties such as high surface area, ease of synthesis and functionalization, high drug loading capacity, collective therapy potentials, pH-triggered drug release behavior, high photothermal conversion, and excellent photodynamic efficiency have been explored as alluring materials in photomedicine; the application of photons in medicine is facilitated for imaging and various disease treatment methods such as photothermal cancer/tumor ablation. Non-invasive theranostic strategies with synergistic activities have been developed using photothermal, photodynamic, and magnetic therapies together with remotely controlled drug/gene delivery for the diagnosis and treatment of various malignant diseases. Photothermal/photodynamic therapy and photoacoustic imaging using MXene-based structures have shown great promise in cancer phototherapy. However, hybridization and surface functionalization should be further explored to obtain biocompatible MXene-based composites/platforms with unique properties, high stability, and improved functionality in photomedicine. Toxicological and long-term biosafety assessments as well as clinical translation evaluations ought to be given high priority in research. Although some limited studies have revealed the excellent potentials of MXenes and their derivatives in photomedicine, further steps should be taken towards extensive research and detailed analysis in the field of optimizing the properties and improving the performance of these materials with a clinical and industrial outlook. Optical biosensing platforms have been developed along with electrochemical sensors and wearable sensors constructed from MXenes and their derivatives; future studies warrant the comprehensive analysis of optical transduction aspects such as colorimetry, electrochemiluminescence, photoluminescence, surface-enhanced Raman scattering, and surface plasmon resonance. Herein, the potentials of MXenes in photomedicine are deliberated encompassing important challenges and future research directions.

• MeSH
• Photochemotherapy * MeSH
• Phototherapy methods   MeSH
• Hyperthermia, Induced * methods   MeSH
• Humans MeSH
• Neoplasms * diagnostic imaging   drug therapy   MeSH
• Nanocomposites * chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

The respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica employ a type III secretion system (T3SS) to inject a 69-kDa BteA effector protein into host cells. This effector is known to contain two functional domains, including an N-terminal lipid raft targeting (LRT) domain and a cytotoxic C-terminal domain that induces nonapoptotic and caspase-1-independent host cell death. However, the exact molecular mechanisms underlying the interaction of BteA with plasma membrane (PM) as well as its cytotoxic activity in the course of Bordetella infections remain poorly understood. Using a protein-lipid overlay assay and surface plasmon resonance, we show here that the recombinant LRT domain binds negatively charged membrane phospholipids. Specifically, we determined that the dissociation constants of the LRT domain-binding liposomes containing phosphatidylinositol 4,5-bisphosphate, phosphatidic acid, and phosphatidylserine were ∼450 nM, ∼490 nM, and ∼1.2 μM, respectively. Both phosphatidylserine and phosphatidylinositol 4,5-bisphosphate were required to target the LRT domain and/or full-length BteA to the PM of yeast cells. The membrane association further involved electrostatic and hydrophobic interactions of LRT and depended on a leucine residue in the L1 loop between the first two helices of the four-helix bundle. Importantly, charge-reversal substitutions within the L1 region disrupted PM localization of the BteA effector without hampering its cytotoxic activity during B. bronchiseptica infection of HeLa cells. The LRT-mediated targeting of BteA to the cytosolic leaflet of the PM of host cells is, therefore, dispensable for effector cytotoxicity.

• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Bordetella bronchiseptica genetics   growth & development   metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Phagocytosis MeSH
• Phospholipids metabolism   MeSH
• HeLa Cells MeSH
• Humans MeSH
• Lipid Bilayers metabolism   MeSH
• Membrane Microdomains metabolism   MeSH
• Protein Domains MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: The Hsp70 proteins maintain proteome integrity through the capacity of their nucleotide- and substrate-binding domains (NBD and SBD) to allosterically regulate substrate affinity in a nucleotide-dependent manner. Crystallographic studies showed that Hsp70 allostery relies on formation of contacts between ATP-bound NBD and an interdomain linker, accompanied by SBD subdomains docking onto distinct sites of the NBD leading to substrate release. However, the mechanics of ATP-induced SBD subdomains detachment is largely unknown. METHODS: Here, we investigated the structural and allosteric properties of human HSPA1A using hydrogen/deuterium exchange mass spectrometry, ATPase assays, surface plasmon resonance and fluorescence polarization-based substrate binding assays. RESULTS: Analysis of HSPA1A proteins bearing mutations at the interface of SBD subdomains close to the interdomain linker (amino acids L399, L510, I515, and D529) revealed that this region forms a folding unit stabilizing the structure of both SBD subdomains in the nucleotide-free state. The introduced mutations modulate HSPA1A allostery as they localize to the NBD-SBD interfaces in the ATP-bound protein. CONCLUSIONS: These findings show that residues forming the hydrophobic structural unit stabilizing the SBD structure are relocated during ATP-activated detachment of the SBD subdomains to different NBD-SBD docking interfaces enabling HSPA1A allostery. GENERAL SIGNIFICANCE: Mutation-induced perturbations tuned HSPA1A sensitivity to peptide/protein substrates and to Hsp40 in a way that is common for other Hsp70 proteins. Our results provide an insight into structural rearrangements in the SBD of Hsp70 proteins and highlight HSPA1A-specific allostery features, which is a prerequisite for selective targeting in Hsp-related pathologies.

• MeSH
• Adenosine Triphosphate chemistry   genetics   MeSH
• Allosteric Regulation genetics   MeSH
• Protein Conformation * MeSH
• Humans MeSH
• Mutation genetics   MeSH
• Protein Domains genetics   MeSH
• HSP70 Heat-Shock Proteins chemistry   genetics   MeSH
• Protein Binding genetics   MeSH
• Binding Sites genetics   MeSH
• Deuterium Exchange Measurement MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The study of optical affinity biosensors based on plasmonic nanostructures has received significant attention in recent years. The sensing surfaces of these biosensors have complex architectures, often composed of localized regions of high sensitivity (electromagnetic hot spots) dispersed along a dielectric substrate having little to no sensitivity. Under conditions such that the sensitive regions are selectively functionalized and the remaining regions passivated, the rate of analyte capture (and thus the sensing performance) will have a strong dependence on the nanoplasmonic architecture. Outside of a few recent studies, there has been little discussion on how changes to a nanoplasmonic architecture will affect the rate of analyte transport. We recently proposed an analytical model to predict transport to such complex architectures; however, those results were based on numerical simulation and to date, have only been partially verified. In this study we measure the characteristics of analyte transport across a wide range of plasmonic structures, varying both in the composition of their base plasmonic element (microwires, nanodisks, and nanorods) and the packing density of such elements. We functionalized each structure with nucleic acid-based bioreceptors, where for each structure we used analyte/receptor sequences as to maintain a Damköhler number close to unity. This method allows to extract both kinetic (in the form of association and dissociation constants) and analyte transport parameters (in the form of a mass transfer coefficient) from sensorgrams taken from each substrate. We show that, despite having large differences in optical characteristics, measured rates of analyte transport for all plasmonic structures match very well to predictions using our previously proposed model. These results highlight that, along with optical characteristics, analyte transport plays a large role in the overall sensing performance of a nanoplasmonic biosensor.

• MeSH
• Nanotubes * MeSH
• Surface Plasmon Resonance * MeSH
• Models, Theoretical * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Allergy and Immunology MeSH
• Immune System MeSH
• Publication type
• Textbook MeSH

• Conspectus
• Patologie. Klinická medicína
• Učební osnovy. Vyučovací předměty. Učebnice
• NML Fields
• alergologie a imunologie

BACKGROUND: Galectin-3 (Gal-3) is a promising target in cancer therapy with a high therapeutic potential due to its abundant localization within the tumor tissue and its involvement in tumor development and proliferation. Potential clinical application of Gal-3-targeted inhibitors is often complicated by their insufficient selectivity or low biocompatibility. Nanomaterials based on N-(2-hydroxypropyl)methacrylamide (HPMA) nanocarrier are attractive for in vivo application due to their good water solubility and lack of toxicity and immunogenicity. Their conjugation with tailored carbohydrate ligands can yield specific glyconanomaterials applicable for targeting biomedicinally relevant lectins like Gal-3. RESULTS: In the present study we describe the synthesis and the structure-affinity relationship study of novel Gal-3-targeted glyconanomaterials, based on hydrophilic HPMA nanocarriers. HPMA nanocarriers decorated with varying amounts of Gal-3 specific epitope GalNAcβ1,4GlcNAc (LacdiNAc) were analyzed in a competitive ELISA-type assay and their binding kinetics was described by surface plasmon resonance. We showed the impact of various linker types and epitope distribution on the binding affinity to Gal-3. The synthesis of specific functionalized LacdiNAc epitopes was accomplished under the catalysis by mutant β-N-acetylhexosaminidases. The glycans were conjugated to statistic HPMA copolymer precursors through diverse linkers in a defined pattern and density using Cu(I)-catalyzed azide-alkyne cycloaddition. The resulting water-soluble and structurally flexible synthetic glyconanomaterials exhibited affinity to Gal-3 in low μM range. CONCLUSIONS: The results of this study reveal the relation between the linker structure, glycan distribution and the affinity of the glycopolymer nanomaterial to Gal-3. They pave the way to specific biomedicinal glyconanomaterials that target Gal-3 as a therapeutic goal in cancerogenesis and other disorders.

• MeSH
• Acrylamides chemistry   metabolism   MeSH
• Galectin 3 metabolism   MeSH
• Glycoconjugates chemistry   metabolism   MeSH
• Drug Delivery Systems * MeSH
• Humans MeSH
• Nanostructures chemistry   MeSH
• Drug Carriers chemistry   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Infections with shiga toxin-producing bacteria, like enterohemorrhagic Escherichia coli and Shigella dysenteriae, represent a serious medical problem. No specific and effective treatment is available for patients with these infections, creating a need for the development of new therapies. Recombinant lactic acid bacterium Lactococcus lactis was engineered to bind Shiga toxin by displaying novel designed albumin binding domains (ABD) against Shiga toxin 1 B subunit (Stx1B) on their surface. Functional recombinant Stx1B was produced in Escherichia coli and used as a target for selection of 17 different ABD variants (named S1B) from the ABD scaffold-derived high-complex combinatorial library in combination with a five-round ribosome display. Two most promising S1Bs (S1B22 and S1B26) were characterized into more details by ELISA, surface plasmon resonance and microscale thermophoresis. Addition of S1Bs changed the subcellular distribution of Stx1B, completely eliminating it from Golgi apparatus most likely by interfering with its retrograde transport. All ABD variants were successfully displayed on the surface of L. lactis by fusing to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA. Binding of Stx1B by engineered lactococcal cells was confirmed using flow cytometry and whole cell ELISA. Lactic acid bacteria prepared in this study are potentially useful for the removal of Shiga toxin from human intestine.

• MeSH
• Albumins metabolism   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• HeLa Cells MeSH
• Immobilized Proteins metabolism   MeSH
• Lactococcus lactis metabolism   MeSH
• Humans MeSH
• Cell Surface Display Techniques MeSH
• Protein Subunits metabolism   MeSH
• Surface Plasmon Resonance MeSH
• Protein Domains MeSH
• Flow Cytometry MeSH
• Recombination, Genetic genetics   MeSH
• Recombinant Proteins metabolism   MeSH
• Ribosomes metabolism   MeSH
• Sequence Homology, Amino Acid MeSH
• Shiga Toxin 1 chemistry   metabolism   MeSH
• Protein Transport MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1-TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1-TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex.

• MeSH
• Sodium Chloride pharmacology   MeSH
• DNA chemistry   genetics   metabolism   MeSH
• Fluorescence Polarization MeSH
• Spectrometry, Fluorescence MeSH
• Kinetics MeSH
• Binding, Competitive drug effects   MeSH
• Humans MeSH
• Surface Plasmon Resonance MeSH
• Telomeric Repeat Binding Protein 2 chemistry   genetics   metabolism   MeSH
• Telomere-Binding Proteins chemistry   genetics   metabolism   MeSH
• Static Electricity MeSH
• Telomere genetics   metabolism   MeSH
• Protein Binding drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

TRPV1 is a nonselective cation channel that integrates wide range of painful stimuli. It has been shown that its activity could be modulated by intracellular ligands PIP2 or calmodulin (CaM). The detailed localization and description of PIP2 interaction sites remain unclear. Here, we used synthesized peptides and purified fusion proteins of intracellular regions of TRPV1 expressed in E.coli in combination with fluorescence anisotropy and surface plasmon resonance measurements to characterize the PIP2 binding to TRPV1. We characterized one PIP2 binding site in TRPV1 N-terminal region, residues F189-V221, and two independent PIP2 binding sites in C-terminus: residues K688-K718 and L777-S820. Moreover we show that two regions, namely F189-V221 and L777-S820, overlap with previously localized CaM binding sites. For all the interactions the equilibrium dissociation constants were estimated. As the structural data regarding C-terminus of TRPV1 are lacking, restraint-based molecular modeling combined with ligand docking was performed providing us with structural insight to the TRPV1/PIP2 binding. Our experimental results are in excellent agreement with our in silico predictions.

• MeSH
• Ankyrins chemistry   MeSH
• Phosphatidylinositol Phosphates metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Calmodulin chemistry   metabolism   MeSH
• TRPV Cation Channels chemistry   genetics   metabolism   MeSH
• Protein Conformation MeSH
• Rats MeSH
• Ligands MeSH
• Liposomes metabolism   MeSH
• Mutation MeSH
• Recombinant Fusion Proteins chemistry   genetics   metabolism   MeSH
• Molecular Docking Simulation MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH