Microbiome imaging goes à la carte: Incorporating click chemistry into the fluorescence-activating and absorption-shifting tag (FAST) imaging platform.

The human microbiome is predominantly composed of facultative and obligate anaerobic bacteria that live in hypoxic/anoxic polymicrobial biofilm communities. Given the oxidative sensitivity of large fractions of the human microbiota, green fluorescent protein (GFP) and related genetically-encoded fluorophores only offer limited utility for live cell imaging due the oxygen requirement for chromophore maturation. Consequently, new fluorescent imaging modalities are needed to study polymicrobial interactions and microbiome-host interactions within anaerobic environments. The fluorescence-activating and absorption shifting tag (FAST) is a rapidly developing genetically-encoded fluorescent imaging technology that exhibits tremendous potential to address this need. In the FAST system, fluorescence only occurs when the FAST protein is complexed with one of a suite of cognate small molecule fluorogens. To expand the utility of FAST imaging, we sought to develop a modular platform (Click-FAST) to democratize fluorogen engineering for personalized use cases. Using Click-FAST, investigators can quickly and affordably sample a vast chemical space of compounds, potentially imparting a broad range of desired functionalities to the parental fluorogen. In this work, we demonstrate the utility of the Click-FAST platform using a novel fluorogen, PLBlaze-alkyne, which incorporates the widely available small molecule ethylvanillin as the hydroxybenzylidine head group. Different azido reagents were clicked onto PLBlaze-alkyne and shown to impart useful characteristics to the fluorogen, such as selective bacterial labeling in mixed populations as well as fluorescent signal enhancement. Conjugation of an 80 Å PEG molecule to PLBlaze-alkyne illustrates the broad size range of functional fluorogen chimeras that can be employed. This PEGylated fluorogen also functions as an exquisitely selective membrane permeability marker capable of outperforming propidium iodide as a fluorescent marker of cell viability.

Antibacterial, ester-free monomers: Polymerization kinetics, mechanical properties, biocompatibility and anti-biofilm activity.

OBJECTIVES: Quaternary ammonium (QA) methacrylate monomers have been extensively investigated and demonstrate excellent antibacterial properties. However, the presence of ester bonds makes them prone to degradation in the oral cavity. In this study, ester-free QA monomers based on meth-acrylamides were synthesized and screened for polymerization kinetics, mechanical properties and antibacterial effects. MATERIALS AND METHODS: Tertiary quaternary ammonium acrylamides (AM) and methacrylamides (MAM) with alkyl side chain lengths of 9 and 14 carbons (C9 and C14) were synthesized and incorporated at 10 wt% into experimental composites based on BisGMA:TEGDMA (1:1), camphorquinone/ethyl-4-dimethylaminobenzoate (0.2/0.8 wt%) and 70 wt% barium glass fillers. Analogous methacrylate versions (MA) were used as controls. Degree of conversion (DC) and rate of polymerization (RP) during photoactivation (800 mW/cm2) were followed in real-time with near-IR. Flexural Strength (FS) and Modulus (E) were measured on 2 × 2 × 25 mm bars in 3-point bending after 24 h dry storage and 7-day storage in water at 37 °C. Antimicrobial properties and biofilm adhesion (fouling) were evaluated by bioluminescence (Luciferase Assay) and biofilm removal by water spray microjet impingement test, respectively. Cytotoxicity was assessed by MTT assay on dental pulp stem cells (DPSC). Data were analyzed with one-way ANOVA/Tukey's test (α = 0.05). RESULTS: DC was similar for all groups tested (∼70%). Both MAMs and C14-AM presented significantly lower RP. Under dry conditions, FS (110-120 MPa) and E (8-9 GPa) were similar for all groups. After water storage, all materials presented FS/E similar to the control, except for C14-AM (for FS) and C14-MAM (for E), which were lower. All C14 versions were strongly antibacterial, decreasing the titer counts of biofilm by more than two orders of magnitude in comparison to the control. C9 monomers did not present significant antibacterial nor antifouling properties. And biofilms had approximately equivalent adhesion on the C9 composites as on the control. Cytotoxicity did not show significant differences between the MA and AM versions and the control group. CONCLUSIONS: C14-QA monomers based on methacrylates and meth-acrylamides present strong antibacterial properties, and in general, similar conversion/mechanical properties compared to the methacrylate control. STATEMENT OF SIGNIFICANCE: This work demonstrates the viability of methacrylamides and acrylamides as potential components in dental restorative materials with antimicrobial properties. The use of ester-free polymerizable functionalities has the potential of improving the degradation resistance of these materials long-term. The use of (meth)acrylamides did not interfere with the antimicrobial potential of quaternary ammonium-based materials.