Revisiting gas-chromatography/mass-spectrometry molar response factors for quantitative analysis (FID or TIC) of glycosidic linkages in polysaccharides produced by oral bacterial biofilms.

Methylation analysis was performed on methylated alditol acetate standards and Streptococcus mutans extracellular polymeric substances (EPS) produced from wild-type and Gtf knockout strains (∆GtfB, ∆GtfB, and ∆GtfD). The methylated alditol acetate standards were representative of glycosidic linkages found in S. mutans EPS and were used to calibrate the GC-MS system for an FID detector and MS (TIC) and produce molar response factor, a necessary step in quantitative analysis. FID response factors were consistent with literature values (Sweet et al., 1975) and found to be the superior option for quantitative results, although the TIC response factors now give researchers without access to an FID detector a needed option for molar response factor correction. The GC-MS analysis is then used to deliver the ratio of the linkage types within a biofilm.

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.

The potential use of glycosyl-transferase inhibitors for targeted reduction of S. mutans biofilms in dental materials.

Streptococcus mutans is the primary oral caries-forming bacteria, adept at producing "sticky" biofilms via the synthesis of insoluble extracellular polysaccharides (EPS), catalyzed by glucosyltransferases (GTFs). To circumvent the use of broad-spectrum antibiotics to combat these bacteria, this study sought to modify existing EPS-targeting small molecules with the ultimate goal of producing anti-biofilm polymer surfaces specifically targeting S. mutans. To achieve this, a known GTF inhibitor (G43) was modified with methoxy or tetraethyleneglycol substitutions in different positions (nine derivatives, tested at 50-µM) to pinpoint potential sites for future methacrylate functionalization, and then assessed against single-species S. mutans biofilms. As expected, the compounds did not diminish the bacterial viability. In general, the compounds with methoxy substitution were not effective in reducing EPS formation, whereas the tetraethyleneglycol substitution (G43-C3-TEG) led to a decrease in the concentration of insoluble EPS, although the effect is less pronounced than for the parent G43. This aligns with the reduced GTF-C activity observed at different concentrations of G43-C3-TEG, as well as the consequent decrease in EPS formation, and notable structural changes. In summary, this study determined that G43-C3-TEG is non-bactericidal and can selectively reduce the biofilm formation, by decreasing the production of EPS. This molecule will serve to functionalize surfaces of materials to be tested in future research.

Thiol Quantification Using Colorimetric Thiol-Disulfide Exchange in Nonaqueous Solvents.

A careful analysis of two (thiol-disulfide exchange) thiol quantification chromophores' behavior (Ellman's reagent and Aldrithiol-4) in nonaqueous solvents is presented. A wide range of kinetic profiles and response factors were measured to exhibit a large variance for nonaqueous systems. We report several robust benchtop and room-temperature methods using different organic solvents compared to aqueous conditions. Validation of analytical analyses in nonaqueous systems and quantification of the cysteine content of ovalbumin are also presented. This work serves as a treatise on the utilization of thiol-disulfide exchange chromophores under nonaqueous conditions for the quantification of thiols.

Effect of side-group methylation on the performance of methacrylamides and methacrylates for dentin hybridization.

The stability of the bond between polymeric adhesives to mineralized substrates is crucial in many biomedical applications. The objective of this study was to determine the effect of methyl substitution at the α- and ß-carbons on the kinetics of polymerization, monomer hydrolytic stability, and long-term bond strength to dentin for methacrylamide- and methacrylate-based crosslinked networks for dental adhesive applications. METHODS: Secondary methacrylamides (α-CH3 substituted=1-methyl HEMAM, ß-CH3 substituted=2-methyl HEMAM, and unsubstituted=HEMAM) and OH-terminated methacrylates (α- and ß-CH3 mixture=1-methyl HEMA and 2-methyl HEMA, and unsubstituted=HEMA) were copolymerized with urethane dimethacrylate. The kinetics of photopolymerization were followed in real-time using near-IR spectroscopy. Monomer hydrolysis kinetics were followed by NMR spectroscopy in water at pH 1 over 30 days. Solvated adhesives (40 vol% ethanol) were used to bond composite to dentin and microtensile bond strength (µTBS) measured after 24h and 6 months storage in water at 37°C. RESULTS: The rate of polymerization increased in the following order: OH-terminated methacrylates≥methacrylamides>NH2-terminated methacrylates, with minimal effect of the substitution. Final conversion ranged between 79% for 1-methyl AEMA and 94% for HEMA. 1-methyl-HEMAM showed the highest and most stable µTBS, while HEMA showed a 37% reduction after six months All groups showed measurable degradation after up to 4 days in pH 1, with the methacrylamides showing less degradation than the methacrylates. Additionally, transesterification products were observed in the methacrylamide groups. SIGNIFICANCE: Amide monomers were significantly more stable to hydrolysis than the analogous methacrylates. The addition of a α- or ß-CH3 groups increased the rate of hydrolysis, with the magnitude of the effect tracking with the expected base-catalyzed hydrolysis of esters or amides, but opposite in influence. The α-CH3 substituted secondary methacrylamide, 1-methyl HEMAM, showed the most stable adhesive interface. A side reaction was observed with transesterification of the monomers studied under ambient conditions, which was not expected under the relatively mild conditions used here, which warrants further investigation.

Synthesis of di- and triacrylamides with tertiary amine cores and their evaluation as monomers in dental adhesive interfaces.

PURPOSE/AIM: In an attempt to increase the service life of dental adhesive interfaces, more hydrolytically and enzymatically-stable methacrylate alternatives, such as methacrylamides, have been proposed. The aim of this study was to investigate polymerization behavior, as well as mechanical and biological properties of experimental adhesives containing multi-functional acrylamides. MATERIALS AND METHODS: Multi-functional acrylamides (N,N-Bis[(3-methylaminoacryl)propyl]methylamine - BMAAPMA, Tris[(2-methylaminoacryl)ethyl]amine - TMAAEA, N,N'-bis(acrylamido) 1,4-diazepane - BAADA, N,N-Diethyl-1,3-bis(acrylamido)propane - DEBAAP) or HEMA (2-Hydroxyethyl methacrylate - control) were added at 40 wt% to UDMA. 0.2 wt% DMPA and 0.4 wt% DPI-PF6 were used as initiators. Polymerization kinetics was followed in real-time in near-IR during photoactivation (320-500 nm, at 630 mW/cm2). Water sorption/solubility and flexural strength/modulus were measured according to ISO 4049. 1H NMR was used to assess monomer degradation kinetics. MTT assay was used to assess cytotoxicity against OD-21 and DPSC cells. Biofilm formation and adhesion were assessed by Luciferase Assay and Impingement technique, respectively. Solvated adhesives (40 vol% ethanol) were used to test interfacial adhesion strength. The results were analyzed by ANOVA/Tukey's test (α = 0.05). RESULTS: In general, the pure methacrylate mixture had higher rate of polymerization (Rpmax), degree of conversion (DC) at Rpmax, and final DC than the acrylamides. Flexural properties after water storage decreased between 11 and 65%, more markedly for acrylamides. Interfacial bond strength was greater and more stable long-term for the newly synthesized acrylamide formulations (less than 4% reduction at 6 months) compared to the methacrylate experimental control (42% reduction at 6 months). HEMA degraded by almost 90%, while the acrylamides showed no degradation in acidic conditions. Cytotoxicity and biofilm formation, in general, were similar for all groups. CONCLUSIONS: Despite demonstrating high water sorption, the acrylamide-containing materials had similar mechanical and biological properties and enhanced interfacial bond strength stability compared to the methacrylate control.

Methacrylamide-methacrylate hybrid monomers for dental applications.

OBJECTIVES: The susceptibility of methacrylates to hydrolytic and enzymatic degradation may be a contributing factor limiting the clinical lifespan of resin composite restorations. The elimination of labile ester bonds is a potential advantage of methacrylamides, which have been shown to produce more stable restorative interfaces. The rationale of this study is to design hydrolytically and enzymatically stable adhesive monomers, with the added benefit of being able to form crosslinked networks. The objective of this study was to synthesize difunctional, hybrid methacrylate-methacrylamide monomers, and evaluate them as potential monomers for dental adhesives. MATERIALS AND METHODS: HEMA, TEGDMA (controls) or secondary methacrylamides (HEMAM - commercially available, 2EM and 2dMM - newly synthesized) either bearing a hydroxyl group or a methacrylate functionality (Hybrids-Hy), were added at 40mass% to bisGMA. The photoinitiator system consisted of 2-dimethoxyphenyl acetophenone (DMPA) and diphenyl iodonium hexafluorophosphate (DPI-PF6) at 0.2 and 0.4mass%, respectively. Polymerization kinetics were followed in real-time by near-IR spectroscopy during light activation at 630mW/cm2 for 300s. Water sorption and solubility (WS, SL) were measured according to ISO 4049. Storage modulus in shear (G') for 300s was obtained by oscillatory rheometry. For the microtensile bond strength (µTBS), fully formulated adhesives containing 40vol% ethanol were used to restore caries-free human third molars. Bonded specimens with 1mm2 cross-sectional area were tested after 48h and 6 months storage in water at 37°C. Single bond (SB) was tested as a commercial control. Data were analysed with one-way ANOVA and Tukey's test and Student's t-test (α=0.05). RESULTS: In general, hybrid versions showed lower polymerization rate and degree of conversion, whereas the methacrylate controls, HEMA and TEGDMA, showed the highest values. The hybrid versions showed lower values of WS and SL than their monofunctional versions. HEMAM Hy showed the highest values of G' and TEGDMA, 2EM, and 2dMM-Hy the lowest. The µTBS values between 48h and 6 months were statistically reduced only for the HEMA and both 2dMM materials. The formulation containing the monofunctional methacrylamide (HEMAM) showed only 9% reduction in µTBS after 6 months of aging, while the other groups showed a decrease ranging between 18% and 33%. CONCLUSION: Overall, hybrid monomers showed lower reactivity than their analogous monofunctional versions, but had markedly lower water sorption. Shear storage modulus was affected differently by the addition of the second functionality. HEMAM-containing systems were able to maintain stable long-term dentin bond strength, which demonstrates that bonding stability is a result of the complex interplay among the factors studied. CLINICAL SIGNIFICANCE: The novel monomers showed here are potential alternatives to the current methacrylate adhesives, with selected formulations presenting greater bond stability.