Luciferin-Bioinspired Click Ligation Enables Hydrogel Platforms with Fine-Tunable Properties for 3D Cell Culture.

There is an increasing interest in coupling reactions for cross-linking of cell-encapsulating hydrogels under biocompatible, chemoselective, and tunable conditions. Inspired by the biosynthesis of luciferins in fireflies, here we exploit the cyanobenzothiazole-cysteine (CBT-Cys) click ligation to develop polyethylene glycol hydrogels as tunable scaffolds for cell encapsulation. Taking advantage of the chemoselectivity and versatility of CBT-Cys ligation, a highly flexible gel platform is reported here. We demonstrate luciferin-inspired hydrogels with important advantages for cell encapsulation applications: (i) gel precursors derived from inexpensive reagents and with good stability in aqueous solution (>4 weeks), (ii) adjustable gel mechanics within physiological ranges (E = 180-6240 Pa), (iii) easy tunability of the gelation rate (seconds to minutes) by external means, (iv) high microscale homogeneity, (v) good cytocompatibility, and (iv) regulable biological properties. These flexible and robust CBT-Cys hydrogels are proved as supportive matrices for 3D culture of different cell types, namely, fibroblasts and human mesenchymal stem cells. Our findings expand the toolkit of click chemistries for the fabrication of tunable biomaterials.

Environment-Sensitive Fluorescent Labelling of Peptides by Luciferin Analogues.

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.

Near-infrared bioluminescence imaging of two cell populations in living mice.

Multicolor bioluminescence imaging using near-infrared emitting luciferases is an attractive application to detect two cell populations within one animal model. Herein, we describe how to distinguish dual-color bioluminescent signals co-localized in the same compartment. We tested CBG2 click beetle (λ = 660 nm) and CBR2 click beetle (λ = 730 nm) luciferases paired with NH2-NpLH2 luciferin. Following a spectral unmixing algorithm, single spectral contributions can be resolved and quantified, enabling the visualization of multiple cell types in deep tissue by injection of a single substrate. For complete details on the use and execution of this protocol, please refer to Zambito et al. (2020).

Gradient radiation breeding and culture domestication of menaquinone producing strains.

By comparing the survival rate and positive mutation rate of the primary mutagenic strain and progeny mutagenic strain under different radiation doses, the results showed that the tolerance of the mutagenic strain to radiation dose increased with the increase of the mutagenic generations. We adopted an improved gradient radiation breeding strategy to improve the breeding efficiency. The strains were treated with radiation in four stages. The first stage was low energy N+ ion implantation (ion energy 15 keV, dose 80 × 2.6 × 1013 cm-2). In the second stage, the energy and dose of N+ ion reached to 20 keV, 90 × 2.6 × 1013 cm-2. In the third stage, 60Co-γ radiation (dose of 1.56 kGy) was used. In the fourth stage, the radiation dose of 60Co-γ increased to 1.82 kGy. After each stage of radiation, the MK (Menaquinone) precursor 1, 4-dihydroxy-2-naphthalate (DHNA) was used as the stress factor to domesticate the mutant strains. By gradually increasing the concentration of DHNA in the culture medium, the substrate tolerance of Flavobacterium sp. was effectively improved. By measuring SOD (superoxide dismutase) activity and malondialdehyde, it showed that the cell damage caused by radiation mutagenesis to the offspring mutant was less than that of the primary mutant. Changes in membrane permeability and membrane potential of the mutant strains were reflected in changes in fluorescence intensity of luciferin diacetate and rhodamine 123, which could explain the enhanced substrate tolerance of strain F-2. After gradient radiation breeding and culture acclimation, the biomass of mutant Strain F-2 was 6.59 g/L, and the MK yield was 9.59 mg/L.