Cytotoxicity of dilutions of bioceramic materials in stem cells of human exfoliated deciduous teeth.

OBJECTIVE: Several materials have been developed to preserve pulp vitality. They should have ideal cytocompatibility characteristics to promote the activity of stem cells of human exfoliated deciduous teeth (SHED) and thus heal pulp tissue. OBJECTIVE: To evaluate the cytotoxicity of different dilutions of bioceramic material extracts in SHED. METHODOLOGY: SHED were immersed in αMEM + the material extract according to the following experimental groups: Group 1 (G1) -BBio membrane, Group 2 (G2) - Bio-C Repair, Group 3 (G3) - MTA Repair HP, Group 4 (G4) - TheraCal LC, and Group 5 (G5) - Biodentine. Positive and negative control groups were maintained respectively in αMEM + 10% FBS and Milli-Q Water. The methods to analyze cell viability and proliferation involved MTT and Alamar Blue assays at 24, 48, and 72H after the contact of the SHED with bioceramic extracts at 1:1 and 1:2 dilutions. Data were analyzed by the three-way ANOVA, followed by Tukey's test (p<0.05). RESULTS: At 1:1 dilution, SHED in contact with the MTA HP Repair extract showed statistically higher cell viability than the other experimental groups and the negative control (p<0.05), except for TheraCal LC (p> 0.05). At 1:2 dilution, BBio Membrane and Bio-C showed statistically higher values in intra- and intergroup comparisons (p<0.05). BBio Membrane, Bio-C Repair, and Biodentine extracts at 1:1 dilution showed greater cytotoxicity than 1:2 dilution in all periods (p<0.05). CONCLUSION: MTA HP Repair showed the lowest cytotoxicity even at a 1:1 dilution. At a 1:2 dilution, the SHED in contact with the BBio membrane extract showed high cell viability. Thus, the BBio membrane would be a new non-cytotoxic biomaterial for SHED. Results offer possibilities of biomaterials that can be indicated for use in clinical regenerative procedures of the dentin-pulp complex.

Asymmetric total synthesis of polycyclic xanthenes and discovery of a WalK activator active against MRSA.

The development of new antibiotics continues to pose challenges, particularly considering the growing threat of multidrug-resistant Staphylococcus aureus. Structurally diverse natural products provide a promising source of antibiotics. Herein, we outline a concise approach for the collective asymmetric total synthesis of polycyclic xanthene myrtucommulone D and five related congeners. The strategy involves rapid assembly of the challenging benzopyrano[2,3-a]xanthene core, highly diastereoselective establishment of three contiguous stereocenters through a retro-hemiketalization/double Michael cascade reaction, and a Mitsunobu-mediated chiral resolution approach with high optical purity and broad substrate scope. Quantum mechanical calculations provide insight into stereoselective construction mechanism of the three contiguous stereocenters. Additionally, this work leads to the discovery of an antibacterial agent against both drug-sensitive and drug-resistant S. aureus. This compound operates through a unique mechanism that promotes bacterial autolysis by activating the two-component sensory histidine kinase WalK. Our research holds potential for future antibacterial drug development.

High-throughput combination assay for studying biofilm formation of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli, the most common cause for urinary tract infections, forms biofilm enhancing its antibiotic resistance. To assess the effects of compounds on biofilm formation of uropathogenic Escherichia coli UMN026 strain, a high-throughput combination assay using resazurin followed by crystal violet staining was optimized for 384-well microplate. Optimized assay parameters included, for example, resazurin and crystal violet concentrations, and incubation time for readouts. For the assay validation, quality parameters Z' factor, coefficient of variation, signal-to-noise, and signal-to-background were calculated. Microplate uniformity, signal variability, edge well effects, and fold shift were also assessed. Finally, a screening with known antibacterial compounds was conducted to evaluate the assay performance. The best conditions found were achieved by using 12 µg/mL resazurin for 150 min and 0.023% crystal violet. This assay was able to detect compounds displaying antibiofilm activity against UMN026 strain at sub-inhibitory concentrations, in terms of metabolic activity and/or biomass.

Characterizing Modulators of Protease-activated Receptors with a Calcium Mobilization Assay Using a Plate Reader.

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of Gαq signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.

Ultra-pH-sensitive nanoparticle of gambogenic acid for tumor targeting therapy via anti-vascular strategy plus immunotherapy.

Although the combination of anti-vascular strategy plus immunotherapy has emerged as the optimal first-line treatment of hepatocellular carcinoma, lack of tumor targeting leads to low antitumor efficacy and serious side effect. Here, we report an ultra-pH-sensitive nanoparticle of gambogenic acid (GNA) encapsulated by poly(ethylene glycol)-poly(2-azepane ethyl methacrylate) (PEG-PAEMA) for tumor-targeting combined therapy of anti-vascular strategy plus immunotherapy. PEG-PAEMA-GNA nanoparticle was quite stable at pH 7.4 for 30 d. In contrast, it exerted size shrinkage, charge reversal and the release of GNA at pH 6.7 within 24 h. Moreover, PEG-PAEMA-GNA significantly enhanced the anti-vascular activity, membrane-disruptive capability and pro-apoptosis when pH changed from 7.4 to 6.7. Western blot analysis exhibits that PEG-PAEMA and its GNA nanoparticle facilitated the phosphorylation of STING protein. In vivo assays show that PEG-PAEMA-GNA not only displayed much higher tumor inhibition of 92 % than 37 % of free GNA, but also inhibited tumor vasculature, promoted the maturation of dendritic cells and recruited more cytotoxic t-lymphocytes for sufficient anti-vascular therapy and immunotherapy. All these results demonstrate that PEG-PAEMA-GNA displayed tumor-targeting combined treatment of anti-vascular therapy and immunotherapy. This study offers a simple and novel method for the combination of anti-vascular therapy and immunotherapy with high selectivity towards tumor.

Discovery of Di(het)arylmethane and Dibenzoxanthene Derivatives as Potential Anticancer Agents.

A family of bifunctional dihetarylmethanes and dibenzoxanthenes is assembled via a reaction of acetals containing a 2-chloroacetamide moiety with phenols and related oxygen-containing heterocycles. These compounds demonstrated selective antitumor activity associated with the induction of cell apoptosis and inhibition of the process of glycolysis. In particular, bis(heteroaryl)methane containing two 4-hydroxy-6-methyl-2H-pyran-2-one moieties combine excellent in vitro antitumor efficacy with an IC50 of 1.7 µM in HuTu-80 human duodenal adenocarcinoma models with a high selectivity index of 73. Overall, this work highlights the therapeutic potential of dimeric compounds assembled from functionalized acetals and builds a starting point for the development of a new family of anticancer agents.

Tetrahydroxanthene-1,3(2H)-diones and Oxidized Hexadiene Derivatives from Uvaria leptopoda and Their Biological Activities.

The first phytochemical investigation of the twig extract of Uvaria leptopoda resulted in the isolation and identification of three new tetrahydroxanthene-1,3(2H)-diones, uvarialeptones A-C, two new oxidized hexadiene derivatives, uvarialeptols A and B, together with ten known compounds. Their structures were elucidated by spectroscopic techniques and mass spectrometry. Uvarialeptones A and B were unprecedented tetrahydroxanthene-1,3(2H)-dione dimers which exhibited a cyclobutane ring via [2 + 2] cycloaddition from uvarialeptone C and 9a-O-methyloxymitrone, respectively. The structure of uvarialeptone A was confirmed by X-ray diffraction analysis using Mo Kα radiation. Compound 3 inhibited NO production at an IC50 value of 6.7 ± 0.1 µM.

Developing NIR xanthene-chalcone fluorophores with large Stokes shifts for fluorescence imaging.

A series of novel near-infrared (NIR) xanthene-chalcone fluorophores were constructed through a modular synthesis with the electron-donating xanthene moiety and the electron-withdrawing chalcone moiety. These fluorophores are convenient for fluorescence imaging in living cells, benefiting from their NIR emissions (650-710 nm), large Stokes shifts (>100 nm), moderate quantum yields and low cytotoxicity. The substituted hydroxyl group of the xanthene-chalcone fluorophore HCA-E facilitates the development of multifunctional fluorescent probes. As an example, a highly sensitive and selective probe N-HCA-E for glutathione (GSH) detection was developed based on the fluorophore HCA-E. A 4-nitrobenzenesulfonyl (4-Ns) group was introduced to cage the hydroxyl group of HCA-E, which was used as a selective recognition site for the thiol of GSH and an effective fluorescence quencher. Probe N-HCA-E revealed NIR "turn-on" fluorescence (709 nm) for endogenous and exogenous GSH detection in lysosomes with a large Stokes shift (129 nm) and high anti-interference ability.

Dihydroxanthene-based monoamine oxidase A-activated photosensitizers for photodynamic/photothermal therapy of tumors.

Small molecule photosensitizers for combined in vivo tailored cancer diagnostics and photodynamic/photothermal therapy are desperately needed. Monoamine oxidase A (MAO-A)-activated therapeutic and diagnostic compounds provide great selectivity because MAO-A can be employed as a biomarker for associated Tumors. In order to screen photosensitizers with photodynamic therapeutic potential, we have created a range of near-infrared fluorescent molecules in this work by combining dihydroxanthene parent with various heterocyclic fluorescent dyes. The NIR fluorescent diagnostic probe, DHMQ, was created by combining the screened fluorescent dye matrices with the propylamino group, which is the recognition moiety of MAO-A, based on the oxidative deamination mechanism of the enzyme. This probe has a low toxicity level and can identify MAO-A precisely. It has the ability to use fluorescence imaging on mice and cells to track MAO-A activity in real-time. It has strong phototoxicity and can produce singlet oxygen when exposed to laser light. The temperature used in photothermal imaging can get up to 50 °C, which can harm tumor cells permanently and have a positive phototherapeutic impact on tumors grown from SH-SY5Y xenograft mice. The concept of using MAO-A effectively in diseases is expanded by the MAO-A-activated diagnostic-integrated photosensitizers, which offer a new platform for in vivo cancer diagnostics and targeted anticancer treatment.

Xanthene-Based Dyes for Photoacoustic Imaging and their Use as Analyte-Responsive Probes.

Developing imaging tools that can report on the presence of disease-relevant analytes in multicellular organisms can provide insight into fundamental disease mechanisms as well as provide diagnostic tools for the clinic. Photoacoustic imaging (PAI) is a light-in, sound-out imaging technique that allows for high resolution, deep-tissue imaging with applications in pre-clinical and point-of-care settings. The continued development of near-infrared (NIR) absorbing small-molecule dyes promises to improve the capabilities of this emerging imaging modality. For example, new dye scaffolds bearing chemoselective functionalities are enabling the detection and quantification of disease-relevant analytes through activity-based sensing (ABS) approaches. Recently described strategies to engineer NIR absorbing xanthenes have enabled development of analyte-responsive PAI probes using this classic dye scaffold. Herein, we present current strategies for red-shifting the spectral properties of xanthenes via bridging heteroatom or auxochrome modifications. Additionally, we explore how these strategies, coupled with chemoselective spiroring-opening approaches, have been employed to create ABS probes for in vivo detection of hypochlorous acid, nitric oxide, copper (II), human NAD(P)H: quinone oxidoreductase isozyme 1, and carbon monoxide. Given the versatility of the xanthene scaffold, we anticipate continued growth and development of analyte-responsive PAI imaging probes based on this dye class.

Design, synthesis, and biological evaluation of gambogenic acid derivatives: Unraveling their anti-cancer effects by inducing pyroptosis.

Gambogenic acid (GNA), a caged xanthone derived from Garcinia hanburyi, exhibits a wide range of anti-cancer properties. The caged skeleton of GNA serves as the fundamental pharmacophore responsible for its antitumor effects. However, limited exploration has focused on the structural modifications of GNA. This study endeavors to diversify the structure of GNA and enhance its anti-cancer efficacy. Sulfoximines, recognized as pivotal motifs in medicinal chemistry due to their outstanding properties, have featured in several anti-cancer drugs undergoing clinical trials. Accordingly, a series of 33 GNA derivatives combined with sulfoximines were synthesized and evaluated for their anti-cancer effects against MIAPaCa2, MDA-MB-231, and A549 cells in vitro. The activity screening led to the identification of compound 12k, which exhibited the most potent anti-cancer effect. Mechanistic studies revealed that 12k primarily induced pyroptosis in MIAPaCa2 and MDA-MB-231 cells by activating the caspase-3/gasdermin E (GSDME) pathway. These findings suggested that 12k is a promising drug candidate in cancer therapy and highlighted the potential of sulfoximines as a valuable functional group in drug discovery.

Rational Design and Biological Application of Hybrid Fluorophores.

Fluorophores are considered powerful tools for not only enabling the visualization of cell structures, substructures, and biological processes, but also making for the quantitative and qualitative measurement of various analytes in living systems. However, most fluorophores do not meet the diverse requirements for biological applications in terms of their photophysical and biological properties. Hybridization is an important strategy in molecular engineering that provides fluorophores with complementarity and multifunctionality. This review summarizes the basic strategies of hybridization with four classes of fluorophores, including xanthene, cyanine, coumarin, and BODIPY with a focus on their structure-property relationship (SPR) and biological applications. This review aims to provide rational hybrid ideas for expanding the reservoir of knowledge regarding fluorophores and promoting the development of newly produced fluorophores for applications in the field of life sciences.

THQ-Xanthene: An Emerging Strategy to Create Next-Generation NIR-I/II Fluorophores.

Near-infrared fluorescence imaging is vital for exploring the biological world. The short emissions (<650 nm) and small Stokes shifts (<30 nm) of current xanthene dyes obstruct their biological applications since a long time. Recently, a potent and universal THQ structural modification technique that shifts emission to the NIR-I/II range and enables a substantial Stokes shift (>100 nm) for THQ-modified xanthene dyes is established. Thus, a timely discussion of THQ-xanthene and its applications is extensive. Hence, the advent, working principles, development trajectory, and biological applications of THQ-xanthene dyes, especially in the fields of fluorescence probe-based sensing and imaging, cancer theranostics, and super-resolution imaging, are introduced. It is envisioned that the THQ modification tactic is a simple yet exceptional approach to upgrade the performance of conventional xanthene dyes. THQ-xanthene will advance the strides of xanthene-based potentials in early fluorescent diagnosis of diseases, cancer theranostics, and imaging-guided surgery.

Resazurin Reduction-Based Assays Revisited: Guidelines for Accurate Reporting of Relative Differences on Metabolic Status.

Cell viability and metabolic activity are ubiquitous parameters used in biochemistry, molecular biology, and biotechnological studies. Virtually all toxicology and pharmacological projects include at some point the evaluation of cell viability and/or metabolic activity. Among the methods used to address cell metabolic activity, resazurin reduction is probably the most common. At variance with resazurin, resorufin is intrinsically fluorescent, which simplifies its detection. Resazurin conversion to resorufin in the presence of cells is used as a reporter of metabolic activity of cells and can be detected by a simple fluorometric assay. UV-Vis absorbance is an alternative technique but is not as sensitive. In contrast to its wide empirical "black box" use, the chemical and cell biology fundamentals of the resazurin assay are underexplored. Resorufin is further converted to other species, which jeopardizes the linearity of the assays, and the interference of extracellular processes has to be accounted for when quantitative bioassays are aimed at. In this work, we revisit the fundamentals of metabolic activity assays based on the reduction of resazurin. Deviation to linearity both in calibration and kinetics, as well as the existence of competing reactions for resazurin and resorufin and their impact on the outcome of the assay, are addressed. In brief, fluorometric ratio assays using low resazurin concentrations obtained from data collected at short time intervals are proposed to ensure reliable conclusions.

Common xanthene fluorescent dyes are visible-light activatable CO-releasing molecules.

Fluorescein, eosin Y, and rose bengal are dyes used in clinical medicine and considered (photo-)chemically stable. Upon extensive irradiation with visible light in aqueous solutions, we found that these compounds release carbon monoxide (CO) - a bioactive gasotransmitter - in 40-100% yields along with the production of low-mass secondary photoproducts, such as phthalic and formic acids, in a multistep degradation process. Such photochemistry should be considered in applications of these dyes, and they could also be utilized as visible-light activatable CO-releasing molecules (photoCORMs) with biological implications.

Synthesis of Cyano-Benzylidene Xanthene Synthons Using a Diprotic Brønsted Acid Catalyst, and Their Application as Efficient Inhibitors of Aluminum Corrosion in Alkaline Solutions.

Novel cyano-benzylidene xanthene derivatives were synthesized using one-pot and condensation reactions. A diprotic Brønsted acid (i.e., oxalic acid) was used as an effective catalyst for the promotion of the synthesis process of the new starting xanthene-aldehyde compound. Different xanthene concentrations (ca. 0.1-2.0 mM) were applied as corrosion inhibitors to control the alkaline uniform corrosion of aluminum. Measurements were conducted in 1.0 M NaOH solution using Tafel extrapolation and linear polarization resistance (LPR) methods. The investigated xanthenes acted as mixed-type inhibitors that primarily affect the anodic process. Their inhibition efficiency values were enhanced with inhibitor concentration, and varied according to their chemical structures. At a concentration of 2.0 mM, the best-performing studied xanthene derivative recorded maximum inhibition efficiency values of 98.9% (calculated via the Tafel extrapolation method) and 98.4% (estimated via the LPR method). Scanning electron microscopy (SEM) was used to examine the morphology of the corroded and inhibited aluminum surfaces, revealing strong inhibitory action of each studied compound. High-resolution X-ray photoelectron spectroscopy (XPS) profiles validated the inhibitor compounds' adsorption on the Al surface. Density functional theory (DFT) and Monte Carlo simulations were applied to investigate the distinction of the anticorrosive behavior among the studied xanthenes toward the Al (111) surface. The non-planarity of xanthenes and the presence of the nitrile group were the key players in the adsorption process. A match between the experimental and theoretical findings was evidenced.

BioMOF-Mn: An Antimicrobial Agent and an Efficient Nanocatalyst for Domino One-Pot Preparation of Xanthene Derivatives.

In this paper, a new Mn-based metal-organic framework [UoB-6] was obtained via a one-step ultrasonic irradiation method with the ligand (H2bdda: 4,4'-(1,4-phenylenebis(azaneylylidene))bis(methaneylylidene))dibenzoic acid. The structural integrity of the synthesized BioMOF-Mn was corroborated by FT-IR, EDX, ICP, XRD, TEM, DLS, FESEM, and BET-BJH analyses. The aerobic oxidative domino reaction of benzyl alcohols or aldehydes with dimedone derivatives was performed in the presence of the UoB-6 catalyst to produce xanthene derivatives in good yields. Hot filtration and Hg poisoning tests proved the heterogeneous nature of the catalyst. Novel synthesized xanthene-based bis-aldehydes were introduced as potent HDAC1 inhibitors according to molecular docking calculations. Finally, the inhibitory activities of Mn-MOF nanoparticles were evaluated on Escherichia coli and Candida albicans. The MIC, MBC, and MFC values were determined from 2048 to 4096 µg·mL-1 according to antimicrobial susceptibility testing methods. The inhibitory effects of antimicrobial agents can be exacerbated when loaded on BioMOFs.

Controlled Synthesis of Luminescent Xanthene Dyes and Use of Ionic Liquid in Thermochromic Reaction.

In this study, we demonstrate six novel xanthene derivatives and their spectroscopic and chemical properties. The presented synthesis examination allowed us to obtain two different compounds during one step, with open and closed lactone rings substituted with different length alkyl chains. Increasing the reaction efficiency to 77% was obtained using the microwave-assisted method. Moreover, the modification of O-alkylation synthesis in an ecofriendly way using a ball mill led to achieving exclusively one opened ring product. All of the synthesized compounds showed different spectroscopic behaviors in comparison with the different organic dyes; the typical concentration quenching of luminescence was not observed. The relationship between the length of the alkyl chain and the time of luminescence decay is presented. Synthetized closed forms of dyes turned out to be promising leuco dyes. For the first time, an ionic liquid was used as a developer of synthesized xanthene derivatives (as leuco dyes), which led to obtaining an irreversible thermochromic marker.

Molecular structure perspective on Temperature-Sensitive properties of rhodamine aqueous solutions.

As one of the most commonly used organic fluorescent dyes, recently rhodamines have been successfully employed in temperature sensing. However, few works have been reported on their temperature-sensitive properties, which inevitably limiting their further applications. In order to solve such problem, we investigated temperature-sensitive properties of rhodamine 110, 123, 19, 6G, B and 3B focusing on their fluorescence emission spectra; and analyzed them in the molecular structure perspective. It is demonstrated that the fluorescence emission intensities of all studied rhodamines decreased with higher temperature, which inevitably enhances the probability of collisions among molecules, thus definitely leads to energy loss in fluorescence emission. While these rhodamines still have various temperature sensitivities mainly due to the substitutes: the substitute on the benzene carboxylate has little effect; the amino substituents of the three-ring xanthene enhance the temperature sensitivity due to their rotation weakening the rigidity of the three-ring xanthene; and the methyl substituents on the three-ring xanthene reduce the temperature sensitivity by enhancing the rigidity and stability of the three-ring xanthene as well as hindering the rotation of ethylamino. These findings can also be extended to other organic fluorescent dyes proved by coumarins comparable to rhodamines. The results provided by this work can be useful reference and guidance to further develop organic fluorescent dyes especially for temperature sensing.

A novel, dual action chimera comprising DNA methylating agent and near-IR xanthene-cyanine photosensitizer for combined anticancer therapy.

A facile synthesis, biological evaluation and photodynamic properties of novel activatable anticancer molecular hybrids (chimeras) Ch and I-Ch are described. The chimeras consist of DNA methylating methyl triazene moiety and fluorogenic xanthene-cyanine (XCy) or iodinated xanthene-cyanine (I-XCy) photosensitizer. These two anticancer core structures are bound by means of a self-immolative 4-aminobenzyl alcohol linker. The hydrolytic cleavage of the carbamate protecting group promotes activation of both DNA methylating monomethyl triazene and phototoxic xanthene-cyanine dye providing, in addition, a near-IR emission signal for detection of the drug activation events. Preliminary antiproliferative assay demonstrates that the developed chimeras exhibit higher antitumor activity in the breast cancer cell line upon near-IR light irradiation compared to their structural constituents, xanthene-cyanine photosensitizer and monomethyl triazene substance.