scholarly journals FluCell-SELEX Aptamers as Specific Binding Molecules for Diagnostics of the Health Relevant Gut Bacterium Akkermansia muciniphila

2021 ◽  
Vol 22 (19) ◽  
pp. 10425
Author(s):  
Heinz Fabian Raber ◽  
Dennis Horst Kubiczek ◽  
Nicholas Bodenberger ◽  
Ann-Kathrin Kissmann ◽  
Deena D’souza ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Biochemical Characterization ◽  
Specific Binding ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Akkermansia Muciniphila ◽  
Excellent Starting Point ◽  
Starting Point ◽  
Specific Binding Molecules

Based on their unique properties, oligonucleotide aptamers have been named a gift of biological chemistry to life science. We report the development of DNA aptamers as the first high-affinity binding molecules available for fast and rapid labeling of the human gut bacterium Akkermansia muciniphila with a certain impact on Alzheimer´s disease. Fast and reliable analyses of the composition of microbiomes is an emerging field in microbiology. We describe the molecular evolution and biochemical characterization of a specific aptamer library by a FluCell-SELEX and the characterization of specific molecules from the library by bioinformatics. The aptamer AKK13.1 exerted universal applicability in different analysis techniques in modern microbiology, including fluorimetry, confocal laser scanning microscopy and flow cytometry. It was also functional as a specific binding entity hybridized to anchor primers chemically coupled via acrydite-modification to the surface of a polyacrylamide-hydrogel, which can be prototypically used for the construction of affinity surfaces in sensor chips. Together, the performance and methodological flexibility of the aptamers presented here may open new routes not only to develop novel Akkermansia-specific assays for clinical microbiology and the analyses of human stool samples but may also be an excellent starting point for the construction of novel electronic biosensors.

scholarly journals Characterization of activated sludge flocs by confocal laser scanning microscopy and image analysis

2021 ◽  
Author(s):  
Eva Nowak
Keyword(s):  
Image Analysis ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Reactor Performance ◽  
Sludge Flocs ◽  
P Limitation ◽  
Floc Structure ◽  
Compositional Changes

The purpose of this study was to characterize microbial floc structure and properties under phosphorus (P) limiting and non-limiting regimes. The P-limitation applied to the biomass did not significantly impact on reactor performance in terms of COD removal and MLSS. The composition of EPS was affected by the P-limitation with significantly increased accumulation of carbohydrates, uronic acids and proteins. CLSM and glycoconjugate mapping revealed that the relative abundance of α and β- N -acetylgalactosaminyl/galactopyranosyl and N -acetylglucosaminyl residues was affected by P-limitation, suggesting changes in microbial populations within the floc structure, which in turn could cause the compositional changes of EPS. The image analysis performed on CLSM images indicated that under non-limiting conditions the cell clumps within the floc were significantly smaller as compared to P-limiting conditions. The fractal dimension and porosity under limiting conditions were either significantly higher or lower than under P-rich conditions.

scholarly journals Development of Silica-Based Biodegradable Submicrometric Carriers and Investigating Their Characteristics as in Vitro Delivery Vehicles

2020 ◽  
Vol 21 (20) ◽  
pp. 7563
Author(s):  
Mikhail V. Zyuzin ◽  
Dingcheng Zhu ◽  
Wolfgang J. Parak ◽  
Neus Feliu ◽  
Alberto Escudero
Keyword(s):  
Cell Lines ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Bioactive Molecules ◽  
Confocal Laser ◽  
Electron Microscopies ◽  
Delivery Vehicles ◽  
Nanostructured Silica

Nanostructured silica (SiO2)-based materials are attractive carriers for the delivery of bioactive compounds into cells. In this study, we developed hollow submicrometric particles composed of SiO2 capsules that were separately loaded with various bioactive molecules such as dextran, proteins, and nucleic acids. The structural characterization of the reported carriers was conducted using transmission and scanning electron microscopies (TEM/SEM), confocal laser scanning microscopy (CLSM), and dynamic light scattering (DLS). Moreover, the interaction of the developed carriers with cell lines was studied using standard viability, proliferation, and uptake assays. The submicrometric SiO2-based capsules loaded with DNA plasmid encoding green fluorescence proteins (GFP) were used to transfect cell lines. The obtained results were compared with studies made with similar capsules composed of polymers and show that SiO2-based capsules provide better transfection rates on the costs of higher toxicity.

scholarly journals Environment-Sensitive Fluorescent Labelling of Peptides by Luciferin Analogues

2021 ◽  
Vol 22 (24) ◽  
pp. 13312
Author(s):  
Marialuisa Siepi ◽  
Rosario Oliva ◽  
Antonio Masino ◽  
Rosa Gaglione ◽  
Angela Arciello ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Emission Spectra ◽  
Water Concentration ◽  
Solvent Polarity ◽  
Firefly Luciferase ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Water Abundance ◽  
Proteins And Peptides

Environment-sensitive fluorophores are very valuable tools in the study of molecular and cellular processes. When used to label proteins and peptides, they allow for the monitoring of even small variations in the local microenvironment, thus acting as reporters of conformational variations and binding events. Luciferin and aminoluciferin, well known substrates of firefly luciferase, are environment-sensitive fluorophores with unusual and still-unexploited properties. Both fluorophores show strong solvatochromism. Moreover, luciferin fluorescence is influenced by pH and water abundance. These features allow to detect local variations of pH, solvent polarity and local water concentration, even when they occur simultaneously, by analyzing excitation and emission spectra. Here, we describe the characterization of (amino)luciferin-labeled derivatives of four bioactive peptides: the antimicrobial peptides GKY20 and ApoBL, the antitumor peptide p53pAnt and the integrin-binding peptide RGD. The two probes allowed for the study of the interaction of the peptides with model membranes, SDS micelles, lipopolysaccharide micelles and Escherichia coli cells. Kd values and binding stoichiometries for lipopolysaccharide were also determined. Aminoluciferin also proved to be very well-suited to confocal laser scanning microscopy. Overall, the characterization of the labeled peptides demonstrates that luciferin and aminoluciferin are previously neglected environment-sensitive labels with widespread potential applications in the study of proteins and peptides.

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