Imaging the Morphological Structure of Silk Fibroin Constructs through Fluorescence Energy Transfer and Confocal Microscopy

Silk fibroin is a well-known biopolymer that is used in several applications in which interactions with biological tissue are required. Fibroin is extremely versatile and can be shaped to form several constructs that are useful in tissue engineering applications. Confocal imaging is usually performed to test cell behavior on a construct, and, in this context, the fibroin intrinsic fluorescence is regarded as a problem. In addition, the intrinsic fluorescence is not intense enough to provide useful morphological images. In fact, to study the construct’s morphology, other techniques are used (i.e., SEM and Micro-CT). In this work, we propose a method based on fluorescence energy transfer (FRET) to suppress the fibroin intrinsic fluorescence and move it to a higher wavelength that is accessible to confocal microscopy for direct imaging. This was done by creating two FRET couples by dispersing two fluorophores (2,5-diphenyloxazole (PPO) and Lumogen F Violet 570 (LV)) into the fibroin matrix and optimizing their percentages to suppress the fibroin intrinsic fluorescence. With the optimized composition, we produced an electrospun mat, and the dimensions of the fibers were accurately determined by confocal microscopy.

Imaging the Morphological Structure of Silk Fibroin Constructs Through Fluorescence Energy Transfer and Confocal Imaging

Silk fibroin is a well-known biopolymer used in several applications in which the interaction with biological tissue is required. In fact, fibroin is extremely versatile and can be shaped to form several constructs useful in tissue engineering applications. Confocal imaging is usually per-formed to test the cells behaviour on the construct and in this context the fibroin autofluorescence is regarded as a problem. In addition, the autofluorescence is not intense enough to provide useful morphological images. In fact, to control study the constructs morphology other techniques are used (i.e. SEM, Micro-CT). In this work we propose a method based on the fluorescence energy transfer (FRET) to suppress the fibroin autofluorescence moving it to higher wavelength accessible to the confocal microscopy for a direct imaging.

Establishment of Novel Protein Interaction Assays between Sin3 and REST Using Surface Plasmon Resonance and Time-Resolved Fluorescence Energy Transfer

Repressor element-1 (RE-1) or neural restrictive silencer element (NRSE) bound with a zinc finger transcription repressor, RE-1 silencing transcription factor (REST, also known as neural restrictive silencer factor, NRSF) has been identified as a fundamental repressor element in many genes, including neuronal genes. Genes regulated by REST/NRSF regulate multifaceted neuronal phenotypes, and their defects in the machinery cause neuropathies, disorders of neuron activity), autism and so on. In REST repressions, the N-terminal repressor domain recruits Sin3B via its paired amphipathic helix 1 (PAH1) domain, which plays an important role as a scaffold for histone deacetylase 1 and 2. This machinery has a critical role in maintaining neuronal robustness. In this study, in order to establish protein–protein interaction assays mimicking a binding surface between Sin3B and REST, we selected important amino acids from structural information of the PAH1/REST complex and then tried to reconstitute it using recombinant short peptides derived from PAH1/REST. Initially, we validated whether biotinylated REST interacts with glutathione S-transferase (GST)-tagged PAH1 and whether another PAH1 peptide (PAH1-FLAG) competitively binds with biotinylated REST using surface plasmon resonance (SPR). We observed a direct interaction and competitive binding of two PAH1 peptides. Secondly, in order to establish a high-throughput and high-dynamic-range assay, we utilized an easily performed novel time-resolved fluorescence energy transfer (TR-FRET) assay, and closely monitored this interaction. Finally, we succeeded in establishing a novel high-quality TR-FRET assay and a novel interaction assay based on SPR.

Erratum: Liu, Y.-Z.; Jiang, X.-K.; Cao, W.-F.; Sun, J.-Y.; Gao, F. Detection of Thrombin Based on Fluorescence Energy Transfer between Semiconducting Polymer Dots and BHQ-Labelled Aptamers. Sensors 2018, 18, 589

The authors wish to make the following corrections to their paper [...]