Intelligent salivary biosensors for periodontitis: in vitro simulation of oral oxidative stress conditions.

ASTRACT: One of the most common oral diseases affecting millions of people worldwide is periodontitis. Usually, proteins in body fluids are used as biomarkers of diseases. This study focused on hydrogen peroxide, lipopolysaccharide (LPS), and lactic acid as salivary non-protein biomarkers for oxidative stress conditions of periodontitis. Electrochemical analysis of artificial saliva was done using Gamry with increasing hydrogen peroxide, bLPS, and lactic acid concentrations. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were conducted. From EIS data, change in capacitance and CV plot area were calculated for each test condition. Hydrogen peroxide groups had a decrease in CV area and an increase in percentage change in capacitance, lipopolysaccharide groups had a decrease in CV area and a decrease in percentage change in capacitance, and lactic acid groups had an increase of CV area and an increase in percentage change in capacitance with increasing concentrations. These data showed a unique combination of electrochemical properties for the three biomarkers. Scanning electron microscopy (SEM) with energy dispersive spectroscopy (EDS) employed to observe the change in the electrode surface and elemental composition data present on the sensor surface also showed a unique trend of elemental weight percentages. Machine learning models using hydrogen peroxide, LPS, and lactic acid electrochemical data were applied for the prediction of risk levels of periodontitis. The detection of hydrogen peroxide, LPS, and lactic acid by electrochemical biosensors indicates the potential to use these molecules as electrochemical biomarkers and use the data for ML-driven prediction tool for the periodontitis risk levels.

Nanoceria Aggregate Formulation Promotes Buffer Stability, Cell Clustering, and Reduction of Adherent Biofilm in Streptococcus mutans.

Streptococcus mutans is one of the key etiological factors in tooth-borne biofilm development that leads to dental caries in the presence of fermentable sugars. We previously reported on the ability of acid-stabilized nanoceria (CeO2-NP) produced by the hydrolysis of ceric salts to limit biofilm adherence of S. mutans via non-bactericidal mechanism(s). Herein, we report a chondroitin sulfate A (CSA) formulation (CeO2-NP-CSA) comprising nanoceria aggregates that promotes resistance to bulk precipitation under a range of conditions with retention of the biofilm-inhibiting activity, allowing for a more thorough mechanistic study of its bioactivity. The principal mechanism of reduced in vitro biofilm adherence of S. mutans by CeO2-NP-CSA is the production of nonadherent cell clusters. Additionally, dose-dependent in vitro human cell toxicity studies demonstrated no additional toxicity beyond that of equimolar doses of sodium fluoride, currently utilized in many oral health products. This study represents a unique approach and use of a nanoceria aggregate formulation with implications for promoting oral health and dental caries prevention as an adjunctive treatment.

A Review on Saliva-Based Health Diagnostics: Biomarker Selection and Future Directions.

The human body has a unique way of saying when something is wrong with it. The molecules in the body fluids can be helpful in the early detection of diseases by enabling health and preventing disease progression. These biomarkers enabling better healthcare are becoming an extensive area of research interest. Biosensors that detect these biomarkers are becoming the future, especially Point Of Care (POC) biosensors that remove the need to be physically present in the hospital. Detection of complex and systemic diseases using biosensors has a long way to go. Saliva-based biosensors are gaining attention among body fluids due to their non-invasive collection and ability to detect periodontal disease and identify systemic diseases. The possibility of saliva-based diagnostic biosensors has gained much publicity, with companies sending home kits for ancestry prediction. Saliva-based testing for covid 19 has revealed effective clinical use and relevance of the economic collection. Based on universal biomarkers, the detection of systemic diseases is a booming research arena. Lots of research on saliva-based biosensors is available, but it still poses challenges and limitations as POC devices. This review paper talks about the relevance of saliva and its usefulness as a biosensor. Also, it has recommendations that need to be considered to enable it as a possible diagnostic tool.

Identification of Small Molecule Inhibitors against Staphylococcus aureus Dihydroorotase via HTS.

Drug-resistant Staphylococcus aureus is an imminent threat to public health, increasing the importance of drug discovery utilizing unexplored bacterial pathways and enzyme targets. De novo pyrimidine biosynthesis is a specialized, highly conserved pathway implicated in both the survival and virulence of several clinically relevant pathogens. Class I dihydroorotase (DHOase) is a separate and distinct enzyme present in gram positive bacteria (i.e., S. aureus, B. anthracis) that converts carbamoyl-aspartate (Ca-asp) to dihydroorotate (DHO)-an integral step in the de novo pyrimidine biosynthesis pathway. This study sets forth a high-throughput screening (HTS) of 3000 fragment compounds by a colorimetry-based enzymatic assay as a primary screen, identifying small molecule inhibitors of S. aureus DHOase (SaDHOase), followed by hit validation with a direct binding analysis using surface plasmon resonance (SPR). Competition SPR studies of six hit compounds and eight additional analogs with the substrate Ca-asp determined the best compound to be a competitive inhibitor with a KD value of 11 µM, which is 10-fold tighter than Ca-asp. Preliminary structure-activity relationship (SAR) provides the foundation for further structure-based antimicrobial inhibitor design against S. aureus.

Hydrolyzed Ce(IV) salts limit sucrose-dependent biofilm formation by Streptococcus mutans.

Several studies have focused on the antimicrobial effects of cerium oxide nanoparticles (CeO2-NP) but few have focused on their effects on bacteria under initial biofilm formation conditions. Streptococcus mutans is a prolific biofilm former contributing to dental caries in the presence of fermentable carbohydrates and is a recognized target for therapeutic intervention. CeO2-NP derived solely from Ce(IV) salt hydrolysis were found to reduce adherent bacteria by approximately 40% while commercial dispersions of "bare" CeO2-NP (e.g., 3 nm, 10-20 nm, 30 nm diameter) and Ce(NO3)3·6H2O were either inactive or observed to slightly increase biofilm formation under similar in vitro conditions. Planktonic growth and dispersal assays support a non-bactericidal mode of biofilm inhibition active in the initial phases of S. mutans biofilm production. Human cell proliferation assays suggest only minor effects of hydrolyzed Ce(IV) salts on cellular metabolism at concentrations up to 1 mM Ce, with less observed toxicity compared to equimolar concentrations of AgNO3. The results presented herein have implications in clinical dentistry.

Identification and design of novel small molecule inhibitors against MERS-CoV papain-like protease via high-throughput screening and molecular modeling.

The development of new therapeutic agents against the coronavirus causing Middle East Respiratory Syndrome (MERS) is a continuing imperative. The initial MERS-CoV epidemic was contained entirely through public health measures, but episodic cases continue, as there are currently no therapeutic agents effective in the treatment of MERS-CoV, although multiple strategies have been proposed. In this study, we screened 30,000 compounds from three different compound libraries against one of the essential proteases, the papain-like protease (PLpro), using a fluorescence-based enzymatic assay followed by surface plasmon resonance (SPR) direct binding analysis for hit confirmation. Mode of inhibition assays and competition SPR studies revealed two compounds to be competitive inhibitors. To improve upon the inhibitory activity of the best hit compounds, a small fragment library consisting of 352 fragments was screened in the presence of each hit compound, resulting in one fragment that enhanced the IC50 value of the best hit compound by 3-fold. Molecular docking and MM/PBSA binding energy calculations were used to predict potential binding sites, providing insight for design and synthesis of next-generation compounds.

Hit-to-Lead: Hit Validation and Assessment.

High-throughput screening assays have become nearly ubiquitous in the search for small compounds or peptides that can modulate biological processes for therapeutic purposes. While many assays have become quite robust, with well-established protocols, the subsequent steps of validating the hits and choosing the best ones to take forward into leads for further chemical development are less established. In this chapter, we describe a variety of approaches, including chemical assessment, the use of various computational approaches, a variety of counter-screens, and "orthogonal" biophysical assays using nuclear magnetic resonance, surface plasmon resonance, isothermal titration calorimetry or thermal shift assays as methods for validating and assessing the quality of hits.

Stabilization of reduced molybdenum-iron-sulfur single- and double-cubane clusters by cyanide ligation.

Recent work has shown that cyanide ligation increases the redox potentials of Fe(4)S(4) clusters, enabling the isolation of [Fe(4)S(4)(CN)4]4-, the first synthetic Fe(4)S(4) cluster obtained in the all-ferrous oxidation state (Scott, T. A.; Berlinguette, C. P.; Holm, R. H.; Zhou, H.-C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9741). The generality of reduced cluster stabilization has been examined with MoFe(3)S(4) clusters. Reaction of single-cubane [(Tp)MoFe(3)S(4)(PEt(3))3]1+ and edge-bridged double-cubane [(Tp)2Mo(2)Fe(6)S(8)(PEt(3))4] with cyanide in acetonitrile affords [(Tp)MoFe(3)S(4)(CN)3]2- (2) and [(Tp)2Mo(2)Fe(6)S(8)(CN)4]4- (5), respectively. Reduction of 2 with KC(14)H(10) yields [(Tp)MoFe(3)S(4)(CN)3]3- (3). Clusters were isolated in approximately 70-90% yields as Et(4)N+ or Bu(4)N+ salts; clusters 3 and 5 contain all-ferrous cores, and 3 is the first [MoFe(3)S(4)]1+ cluster isolated in substance. The structures of 2 and 3 are very similar; the volume of the reduced cluster core is slightly larger (2.5%), a usual effect upon reduction of cubane-type Fe(4)S(4) and MFe(3)S(4) clusters. Redox potentials and 57Fe isomer shifts of [(Tp)MoFe(3)S(4)L3]2-,3- and [(Tp)2Mo(2)Fe(6)S(8)L(4)]4-,3- clusters with L = CN-, PhS-, halide, and PEt3 are compared. Clusters with pi-donor ligands (L = halide, PhS) exhibit larger isomer shifts and lower (more negative) redox potentials, while pi-acceptor ligands (L = CN, PEt3) induce smaller isomer shifts and higher (less-negative) redox potentials. When the potentials of 3/2 and [(Tp)MoFe(3)S(4)(SPh)3]3-/2- are compared, cyanide stabilizes 3 by 270 mV versus the reduced thiolate cluster, commensurate with the 310 mV stabilization of [Fe(4)S(4)(CN)4]4- versus [Fe(4)S(4)(SPh)4]4- where four ligands differ. These results demonstrate the efficacy of cyanide stabilization of lower cluster oxidation states. (Tp = hydrotris(pyrazolyl)borate(1-)).

Model studies of the Cu(B) site of cytochrome c oxidase utilizing a Zn(II) complex containing an imidazole-phenol cross-linked ligand.

Cytochrome c oxidase, the enzyme complex responsible for the four-electron reduction of O2 to H2O, contains an unusual histidine-tyrosine cross-link in its bimetallic heme a3-CuB active site. We have synthesised an unhindered, tripodal chelating ligand, BPAIP, containing the unusual ortho-imidazole-phenol linkage, which mimics the coordination environment of the CuB center. The ligand was used to investigate the physicochemical (pKa, oxidation potential) and coordination properties of the imidazole-phenol linkage when bound to a dication. Zn(II) coordination lowers the pKa of the phenol by 0.6 log units, and increases the potential of the phenolate/phenoxyl radical couple by approximately 50 mV. These results are consistent with inductive withdrawal of electron density from the phenolic ring. Spectroscopic data and theoretical calculations (DFT) were used to establish that the cationic complex [Zn(BPAIP)Br]+ has an axially distorted trigonal bipyramidal structure, with three coordinating nitrogen ligands (two pyridine and one imidazole) occupying the equatorial plane and the bromide and the tertiary amine nitrogen of the tripod in the axial positions. Interestingly, the Zn-Namine bonding interaction is weak or absent in [Zn(BPAIP)Br]+ and the complex gains stability in basic solutions, as indicated by 1H NMR spectroscopy. These observations are supported by theoretical calculations (DFT), which suggest that the electron-donating capacity of the equatorial imidazole ligand can be varied by modulation of the protonation and/or redox state of the cross-linked phenol. Deprotonation of the phenol makes the equatorial imidazole a stronger sigma-donor, resulting in an increased Zn-Nimd interaction and thereby leading to distortion of the axial ligand axis toward a more tetrahedral geometry.

Model studies of the histidine-tyrosine cross-link in cytochrome C oxidase reveal the flexible substituent effect of the imidazole moiety.

[reaction: see text] Experimental and theoretical studies were carried out to interrogate the effect of an imidazole substituent in each of the ortho, meta, and para positions on the pK(a), E degrees , and O-H BDE of phenol. The results reveal that imidazole substitution lowers the pK(a) of phenol and increases the E degrees of phenoxide due to its sigma-electron withdrawing ability (sigma(p)(-) = +0.21, sigma(m)(-) = +0.45) but decreases the O-H BDE and E degrees of phenol due to its pi-electron-donating ability (sigma(p)(+) = -0.45).

Vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans: FTIR and electrochemical studies on Tyr-D4-labeled and on Tyr280His and Tyr35Phe mutant enzymes.

A combined electrochemical and FTIR spectroscopic approach was used to identify the vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans which change upon electron transfer and coupled proton transfer. Electrochemically induced FTIR difference spectra of the Tyr-D4-labeled cytochrome c oxidase reveal that only small contributions arise from the tyrosines. Contributions between 1600 and 1560 cm(-1) are attributed to nu8a/8b(CC) ring modes. The nu19(CC) ring mode for the protonated form of tyrosines is proposed to absorb with an uncommonly small signal at 1525-1518 cm(-1) and for the deprotonated form at 1496-1486 cm(-1), accompanied by the increase of the nu19(CC) ring mode of the Tyr-D(4)-labeled oxidase at approximately 1434 cm(-1). A signal at 1270 cm(-1) can be tentatively attributed to the nu7'a(CO) and delta(COH) mode of a protonated tyrosine. Uncommon absorptions, like the mode at 1524 cm(-1), indicate the involvement of Tyr280 in the spectra. Tyr280 is a crucial residue close to the binuclear center and is covalently bonded to His276. The possible changes of the spectral properties are discussed together with the absorbance spectra of tyrosine bound to histidine. The vibrational modes of Tyr280 are further analyzed in combination with the mutation to histidine, which is assumed to abolish the covalent bonding. The electrochemically induced FTIR difference spectra of the Tyr280His mutant point to a change in protonation state in the environment of the binuclear center. Together with an observed decrease of a signal at 1736 cm(-1), previously assigned to Glu278, a possible functional coupling is reflected. In direct comparison to the FTIR difference spectra of the D4-labeled compound and comparing the spectra at pH 7 and 4.8, the protonation state of Tyr280 is discussed. Furthermore, a detailed analysis of the mutant is presented, the FTIR spectra of the CO adduct revealing a partial loss of Cu(B). Electrochemical redox titrations reflect a downshift of the heme a3 midpoint potential by 95 +/- 10 mV. Another tyrosine identified to show redox dependent changes upon electron transfer is Tyr35, a residue in the proposed D-pathway of the cytochrome c oxidase.