Botulinum neurotoxins (BoNTs) and tetanus toxin (TeTX) are the deadliest biological substances that cause botulism and tetanus, respectively. Their astonishing potency and capacity to enter neurons and interfere with neurotransmitter release at presynaptic terminals have attracted much interest in experimental neurobiology and clinical research. Fused with reporter proteins or labelled with fluorophores, BoNTs and TeTX and their non-toxic fragments also offer remarkable opportunities to visualize cellular processes and functions in neurons and synaptic connections. This study presents the state-of-the-art optical probes derived from BoNTs and TeTX and discusses their applications in molecular and synaptic biology and neurodevelopmental research. It reviews the principles of the design and production of probes, revisits their applications with advantages and limitations and considers prospects for future improvements. The versatile characteristics of discussed probes and reporters make them an integral part of the expanding toolkit for molecular neuroimaging, promoting the discovery process in neurobiology and translational neurosciences.
- Klíčová slova
- Advanced biomaterials, Fluorescent probes, Fusion proteins, Molecular trafficking, Optical imaging, Retrograde transport, SNARE proteins,
- MeSH
- botulotoxiny chemie MeSH
- fluorescenční barviva chemie MeSH
- lidé MeSH
- molekulární sondy chemie MeSH
- neurony * metabolismus MeSH
- neurotoxiny * MeSH
- neurozobrazování * metody MeSH
- synapse * metabolismus MeSH
- tetanový toxin * chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- botulotoxiny MeSH
- fluorescenční barviva MeSH
- molekulární sondy MeSH
- neurotoxiny * MeSH
- tetanový toxin * MeSH
Activin receptor-like kinases 1-7 (ALK1-7) regulate a complex network of SMAD-independent as well as SMAD-dependent signaling pathways. One of the widely used inhibitors for functional investigations of these processes, in particular for bone morphogenetic protein (BMP) signaling, is LDN-193189. However, LDN-193189 has insufficient kinome-wide selectivity complicating its use in cellular target validation assays. Herein, we report the identification and comprehensive characterization of two chemically distinct highly selective inhibitors of ALK1 and ALK2, M4K2234 and MU1700, along with their negative controls. We show that both MU1700 and M4K2234 efficiently block the BMP pathway via selective in cellulo inhibition of ALK1/2 kinases and exhibit favorable in vivo profiles in mice. MU1700 is highly brain penetrant and shows remarkably high accumulation in the brain. These high-quality orthogonal chemical probes offer the selectivity required to become widely used tools for in vitro and in vivo investigation of BMP signaling.
- MeSH
- aktivinové receptory typu I antagonisté a inhibitory metabolismus MeSH
- aktivinové receptory typu II * metabolismus antagonisté a inhibitory MeSH
- inhibitory proteinkinas farmakologie chemie MeSH
- kostní morfogenetické proteiny metabolismus MeSH
- lidé MeSH
- molekulární sondy chemie MeSH
- myši MeSH
- objevování léků MeSH
- pyrazoly chemie farmakologie chemická syntéza MeSH
- signální transdukce účinky léků MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aktivinové receptory typu I MeSH
- aktivinové receptory typu II * MeSH
- inhibitory proteinkinas MeSH
- kostní morfogenetické proteiny MeSH
- molekulární sondy MeSH
- pyrazoly MeSH
Surgery is an efficient way to treat localized prostate cancer (PCa), however, it is challenging to demarcate rapidly and accurately the tumor boundary intraoperatively, as existing tumor detection methods are seldom performed in real-time. To overcome those limitations, we develop a fluorescent molecular rotor that specifically targets the prostate-specific membrane antigen (PSMA), an established marker for PCa. The probes have picomolar affinity (IC50 = 63-118 pM) for PSMA and generate virtually instantaneous onset of robust fluorescent signal proportional to the concentration of the PSMA-probe complex. In vitro and ex vivo experiments using PCa cell lines and clinical samples, respectively, indicate the utility of the probe for biomedical applications, including real-time monitoring of endocytosis and tumor staging. Experiments performed in a PCa xenograft model reveal suitability of the probe for imaging applications in vivo.
- MeSH
- antigeny povrchové chemie metabolismus MeSH
- buňky PC-3 MeSH
- endocytóza MeSH
- fluorescenční spektrometrie metody MeSH
- glutamátkarboxypeptidasa II chemie metabolismus MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sondy chemie metabolismus MeSH
- myši inbrední BALB C MeSH
- myši nahé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory prostaty diagnóza metabolismus MeSH
- optické zobrazování metody MeSH
- proteinové domény MeSH
- transplantace heterologní MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- antigeny povrchové MeSH
- FOLH1 protein, human MeSH Prohlížeč
- glutamátkarboxypeptidasa II MeSH
- molekulární sondy MeSH
The influence of experimental conditions on chromatographic behaviour of promising oligodeoxynucleotide double-labelled molecular probes containing an azaphthalocyanine macrocycle as a perspective dark quencher was studied. A recently introduced new stationary phase based on styrene-divinylbenzene copolymer was tested. The planar and hydrophobic structure of the azaphthalocyanine is considerably different from those of currently used fluorophores and quenchers. Thus, the most challenging issue was the separation of the double-labelled probe from its main impurity represented by a mono-labelled probe, containing only the azaphthalocyanine macrocycle. The absorbance measurement cannot simply determine this impurity, and its presence fundamentally compromises the biological assay. The commonly used gradient elution was not suitable and isocratic conditions seemed to be more appropriate. The azaphthalocyanine moiety influences the properties of the modified oligodeoxynucleotides substantially, and thus their chromatographic behaviour was determined predominantly by this quencher. Acetonitrile was the preferred organic solvent for the analysis of probes containing the azaphthalocyanine quencher and the effect of ion-pairing reagents was dependent on the probe structure. The temperature seemed to be an effective parameter for fine-tuning of the separation and mass transfer improvement. Generally, our findings could be helpful in method development for purity evaluation of double-labelled oligodeoxynucleotide probes and semipreparative methods.
- Klíčová slova
- azaphthalocyanines, chromatography, ion-pairing, molecular probes, oligodeoxynucleotides,
- MeSH
- acetonitrily chemie MeSH
- aza sloučeniny * analýza chemie MeSH
- fluorescenční barviva * analýza chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- molekulární sondy * analýza chemie MeSH
- oligodeoxyribonukleotidy * analýza chemie MeSH
- rozpouštědla MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetonitrile MeSH Prohlížeč
- acetonitrily MeSH
- aza sloučeniny * MeSH
- fluorescenční barviva * MeSH
- molekulární sondy * MeSH
- oligodeoxyribonukleotidy * MeSH
- rozpouštědla MeSH
The function of enzymatic proteins is given by their ability to bind specific small molecules into their active sites. These sites can often be found in pockets on a hypothetical boundary between the protein and its environment. Detection, analysis, and visualization of pockets find its use in protein engineering and drug discovery. Many definitions of pockets and algorithms for their computation have been proposed. Kawabata and Go defined them as the regions of empty space into which a small spherical probe can enter but a large probe cannot and developed programs that can compute their approximate shape. In this article, this definition was slightly modified in order to capture the existence of large internal holes, and a Voronoi-based method for the computation of the exact shape of these modified regions is introduced. The method first puts a finite number of large probes on the protein exterior surface and then, considering both large probes and atomic balls as obstacles for the small probe, the method computes the exact shape of the regions for the small probe. This is all achieved with Voronoi diagrams, which help with the safe navigation of spherical probes among spherical obstacles. Detected regions are internally represented as graphs of vertices and edges describing possible movements of the center of the small probe on Voronoi edges. The surface bounding each region is obtained from this representation and used for visualization, volume estimation, and comparison with other approaches. © 2019 Wiley Periodicals, Inc.
- Klíčová slova
- Voronoi diagram, binding site, cavity, large probe, pocket, small probe,
- MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- molekulární sondy analýza chemie MeSH
- povrchové vlastnosti MeSH
- proteiny analýza MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- molekulární sondy MeSH
- proteiny MeSH
The strength of an excitatory synapse depends on its ability to release glutamate and on the density of postsynaptic receptors. Genetically encoded glutamate indicators (GEGIs) allow eavesdropping on synaptic transmission at the level of cleft glutamate to investigate properties of the release machinery in detail. Based on the sensor iGluSnFR, we recently developed accelerated versions of GEGIs that allow investigation of synaptic release during 100-Hz trains. Here, we describe the detailed procedures for design and characterization of fast iGluSnFR variants in vitro, transfection of pyramidal cells in organotypic hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser-scanning microscopy. As the released glutamate spreads from a point source-the fusing vesicle-it is possible to localize the vesicle fusion site with a precision exceeding the optical resolution of the microscope. By using a spiral scan path, the temporal resolution can be increased to 1 kHz to capture the peak amplitude of fast iGluSnFR transients. The typical time frame for these experiments is 30 min per synapse.
- MeSH
- biosenzitivní techniky metody MeSH
- hipokampální oblast CA3 cytologie MeSH
- konfokální mikroskopie MeSH
- kultivované buňky MeSH
- kyselina glutamová analýza chemie metabolismus MeSH
- lidé MeSH
- molekulární sondy analýza chemie genetika metabolismus MeSH
- nervový přenos genetika fyziologie MeSH
- optické zobrazování MeSH
- transfekce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kyselina glutamová MeSH
- molekulární sondy MeSH
Lanthanide ions are widely used as luminescent probes for structural studies of various biomolecules, including DNA. Latest developments of circularly polarized luminescence (CPL) methodology further boosted interest to luminescence techniques. However, an effect of the lanthanide probes themselves on the DNA structure and conformation was investigated only partially and not for all lanthanides. In the present work, we performed a detailed spectroscopic study of Eu3+ complexes with native double-stranded DNA and compared them to the relevant complexes with single-stranded DNA. We employed infrared (IR), vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopic methods to investigate Eu3+ effect on DNA structure and conformational transitions. It was shown that Eu3+ ions can induce significant alteration of the native DNA structure at the concentrations often used in luminescence studies. While no DNA denaturation was observed at these metal ion concentrations, significant unstacking of the base pairs and disordering of the sugar-phosphate backbone, partial appearance of the A-form backbone geometry, and DNA transition into condensed ψ-type form took place. Eu3+ binding to single-stranded DNA was more pronounced than the binding to double-stranded DNA. We detected the main Eu3+ binding sites and determined the metal ion concentration range in which DNA geometry remains largely unaltered. The results obtained in the current study could be used for tuning the luminescence and CPL structural studies of DNA utilizing Eu3+ ions as probes.
- Klíčová slova
- DNA condensation, DNA structure, Infrared (IR), Lanthanide ions, Luminescence probe, Vibrational circular dichroism (VCD),
- MeSH
- cirkulární dichroismus MeSH
- DNA chemie MeSH
- europium chemie MeSH
- konformace nukleové kyseliny * MeSH
- luminiscence * MeSH
- molekulární sondy chemie MeSH
- ryby MeSH
- spektrofotometrie infračervená MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA MeSH
- europium MeSH
- molekulární sondy MeSH
Over the last few years, the development and relevance of 19F magnetic resonance imaging (MRI) for use in clinical practice has emerged. MRI using fluorinated probes enables the achievement of a specific signal with high contrast in MRI images. However, to ensure sufficient sensitivity of 19F MRI, fluorine probes with a high content of chemically equivalent fluorine atoms are required. The majority of 19F MRI agents are perfluorocarbon emulsions, which have a broad range of applications in molecular imaging, although the content of fluorine atoms in these molecules is limited. In this review, we focus mainly on polymer probes that allow higher fluorine content and represent versatile platforms with properties tailorable to a plethora of biomedical in vivo applications. We discuss the chemical development, up to the first imaging applications, of these promising fluorine probes, including injectable polymers that form depots that are intended for possible use in cancer therapy.
- Klíčová slova
- 19F MRI probe, Fluorine, Magnetic resonance imaging (MRI), Molecular imaging, Polymer,
- MeSH
- fluor chemie MeSH
- fluorokarbony chemie MeSH
- koncentrace vodíkových iontů MeSH
- kontrastní látky chemie MeSH
- lidé MeSH
- molekulární sondy chemie MeSH
- molekulární zobrazování přístrojové vybavení metody MeSH
- myši MeSH
- polymery chemie MeSH
- radiační rozptyl MeSH
- reaktivní formy kyslíku metabolismus MeSH
- světlo MeSH
- teplota MeSH
- zobrazování fluorovou magnetickou rezonancí metody trendy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- fluor MeSH
- fluorokarbony MeSH
- kontrastní látky MeSH
- molekulární sondy MeSH
- polymery MeSH
- reaktivní formy kyslíku MeSH
In the last few years, magnetically labeled cells have been intensively explored, and non-invasive cell tracking and magnetic manipulation methods have been tested in preclinical studies focused on cell transplantation. For clinical applications, it is desirable to know the intracellular pathway of nanoparticles, which can predict their biocompatibility with cells and the long-term imaging properties of labeled cells. Here, we quantified labeling efficiency, localization, and fluorescence properties of Rhodamine derivatized superparamagnetic maghemite nanoparticles (SAMN-R) in mesenchymal stromal cells (MSC). We investigated the stability of SAMN-R in the intracellular space during a long culture (20 days). Analyses were based on advanced confocal microscopy accompanied by atomic absorption spectroscopy (AAS) and magnetic resonance imaging. SAMN-R displayed excellent cellular uptake (24 h of labeling), and no toxicity of SAMN-R labeling was found. 83% of SAMN-R nanoparticles were localized in lysosomes, only 4.8% were found in mitochondria, and no particles were localized in the nucleus. On the basis of the MSC fluorescence measurement every 6 days, we also quantified the continual decrease of SAMN-R fluorescence in the average single MSC during 18 days. An additional set of analyses showed that the intracellular SAMN-R signal decrease was minimally caused by fluorophore degradation or nanoparticles extraction from the cells, main reason is a cell division. The fluorescence of SAMN-R nanoparticles within the cells was detectable minimally for 20 days. These observations indicate that SAMN-R nanoparticles have a potential for application in transplantation medicine.
- Klíčová slova
- Confocal microscopy, Dual contrast agents, Intracellular fluorescent labels, Iron oxide nanoparticles, Mesenchymal stromal cells, Rhodamine, Stem cell tracking,
- MeSH
- dextrany metabolismus MeSH
- fluorescenční spektrometrie MeSH
- intracelulární prostor metabolismus MeSH
- lidé MeSH
- magnetické nanočástice chemie MeSH
- mezenchymální kmenové buňky cytologie metabolismus MeSH
- molekulární sondy chemie metabolismus MeSH
- molekulární zobrazování metody MeSH
- rhodaminy chemie MeSH
- tuková tkáň cytologie MeSH
- viabilita buněk MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- dextrany MeSH
- ferumoxides MeSH Prohlížeč
- magnetické nanočástice MeSH
- molekulární sondy MeSH
- rhodaminy MeSH
