Methylation Pattern of the CD14 and TLR2 Genes in Human Dental Pulp

Introduction

Pattern recognition receptors, such as toll-like receptor 2 (TLR-2) and TLR-4, participate in the activation of immune cells by microorganisms in dental pulp. However, the expression levels of pattern recognition receptors can be modulated by epigenetic factors, especially DNA methylation. In this study, the methylation status of the TLR-2 and CD14 (TLR4 co-receptor) genes in healthy and inflamed human dental pulp was examined.

Methods

The Methyl-Profiler DNA Methylation qPCR Assay was used to verify the DNA methylation patterns.

Results

No differences in the methylation patterns were observed between the 2 groups. Most DNA was unmethylated in both groups.

Conclusions

The hypomethylation of TLR2 and CD14 genes is a usual feature in human dental pulp.     

Cellular and Molecular Tissue Response to Triple Antibiotic Intracanal Dressing

Introduction

The aim of this study was to characterize the response of mouse subcutaneous tissue to triple antibiotic paste (TAP) using conventional light microscopy and real-time PCR (qRT-PCR).

Methods

Polyethylene tubes containing TAP or calcium hydroxide (CH) (ie, the control group) were implanted in mouse subcutaneous tissue. Animals that received empty tubes or no tubes were used as additional controls. After periods of 7, 21, and 63 days postimplantation, the specimens were removed and subjected to histologic processing. The number of inflammatory cells and vessels, vessel areas, vascular density, and relative percentage of collagen were evaluated. Gene expression of proinflammatory (interleukin-1 beta, tumor necrosis factor alpha, and interleukin 17) and anti-inflammatory (transforming growth factor beta) cytokines and angiogenic factors (vascular endothelial growth factor and hypoxia-inducible factor-1 alpha) was quantified by 7 and 21 days postimplantation. Results were analyzed using the Student t test, analysis of variance, and the Tukey test (α = 0.05).

Results

TAP induced an exuberant inflammatory and angiogenic response, with higher numbers of inflammatory cells, higher vascular area and density, and lower relative percentage of collagen compared with CH. In general, the expression of genes involved in inflammation and angiogenesis was higher in the TAP group compared with animals that received CH or empty tubes.

Conclusions

The response of mouse subcutaneous tissue to TAP was characterized by exuberant and persistent inflammatory and angiogenic responses with no repair and high gene expression of biomarkers associated with inflammation and angiogenesis.     

Micromorphology of the Dental Pulp Is Highly Preserved in Cancer Patients Who Underwent Head and Neck Radiotherapy

Introduction

Teeth are often included in the radiation field during head and neck radiotherapy, and recent clinical evidence suggests that dental pulp is negatively affected by the direct effects of radiation, leading to impaired sensitivity of the dental pulp. Therefore, this study aimed to investigate the direct effects of radiation on the microvasculature, innervation, and extracellular matrix of the dental pulp of patients who have undergone head and neck radiotherapy.

Methods

Twenty-three samples of dental pulp from patients who finished head and neck radiotherapy were analyzed. Samples were histologically processed and stained with hematoxylin-eosin for morphologic evaluation of the microvasculature, innervation, and extracellular matrix. Subsequently, immunohistochemical analysis of proteins related to vascularization (CD34 and smooth muscle actin), innervation (S-100, NCAM/CD56, and neurofilament), and extracellular matrix (vimentin) of the dental pulp was performed.

Results

The morphologic study identified preservation of the microvasculature, nerve bundles, and components of the extracellular matrix in all studied samples. The immunohistochemical analysis confirmed the morphologic findings and showed a normal pattern of expression for the studied proteins in all samples.

Conclusions

Direct effects of radiotherapy are not able to generate morphologic changes in the microvasculature, innervation, and extracellular matrix components of the dental pulp in head and neck cancer patients.     

Synthesis of natural 1-O-alkylglycerols: a study on the chemoselective opening of the epoxide ring by onium quaternary salts (N and P) and ionic liquids

A chemoselective route for the synthesis of 1-O-alkylglycerols chimyl (1), batyl (2), and selachyl (3) is reported. These compounds can be naturally isolated from shark liver oil and the skin of animals such as stingrays and chimeras and exhibit potential anti-fouling activity. The synthetic approach developed in this work included two distinct methods of preparation. The first was based on solvent-free reactions catalyzed by onium quaternary salts (N and P) and ionic liquids; the second methodology was based on a series of one-pot reactions.

A chemoselective route for the synthesis of 1-O-alkylglycerols chimyl (1), batyl (2), and selachyl (3) is reported.     

Deuterium equilibrium isotope effects in a supramolecular receptor for the hydrochalcogenide and halide anions

We highlight a convenient synthesis to selectively deuterate an aryl C–H hydrogen bond donor in an arylethynyl bisurea supramolecular anion receptor and use the Perrin method of competitive titrations to study the deuterium equilibrium isotope effects (DEIE) of anion binding for HS⁻, Cl⁻, and Br⁻. This work highlights the utility and also challenges in using this method to determine EIE with highly reactive and/or weakly binding anions.

We highlight a convenient synthesis to selectively deuterate an aryl C–H hydrogen bond donor in a supramolecular anion receptor and use competitive titrations to study the deuterium equilibrium isotope effects (DEIE) in binding HS⁻, Cl⁻, and Br⁻.

Molecular recognition and host–guest binding in both biological and synthetic systems are often driven by a mixture of competitive and additive primarily non-covalent interactions. Understanding the role of each of these forces in a host–guest system can reveal insights into the driving forces behind binding and help inform on the molecular design of future hosts.^1–3 Equilibrium isotope effects (EIE), also referred to as binding isotope effects (BIE) in structural molecular biology,⁴ measure the effect of isotopic substitution on supramolecular interactions through changes in the vibrational energy of the substituted bond. These studies can be used to elucidate the complex non-covalent forces involved in host conformational changes and host–guest binding.^5–8Examples from structural molecular biology have demonstrated that EIEs can reveal mechanistic information in enzyme–ligand binding events.^4,9 Isotopic substitution in synthetic supramolecular systems has been used both for labelling purposes and for studying individual non-covalent interactions. For example, Bergman, Raymond, and coworkers used deuterium equilibrium isotope effects (DEIE) to study benzylphosphonium cation guest binding in a self-assembled supramolecular complex in aqueous solution.¹⁰ From these DEIE studies, the authors found that attractive cation⋯π interactions in the interior of the host were important for promoting guest binding, and that C–H⋯π and π⋯π interactions were relatively small contributors. In another example, Shimizu and coworkers studied the DEIE on the strength of C–H⋯π interactions in their molecular balances.¹¹ Both computational and experimental results showed that the strength of C–H⋯π and C–D⋯π interactions were about equal, settling the debate on which interaction is stronger and easing concerns about using deuteration for spectroscopic and labelling applications.Previously, we used DEIE to study Cl⁻ binding with the arylethynyl bisurea anion receptor 1^H/D (Fig. 1) in DMSO-d₆.¹² We found an experimental DEIE of 1.019 ± 0.010, which matched the computationally-predicted DEIE of 1.020. Further computational analysis determined that the DEIE was due to a distorted N–H⋯Cl⁻ hydrogen bond geometry, which resulted in changes in the C–H/D bond vibrational energy in the host–guest complex. In addition, Paneth and coworkers performed a computational study with 1^H and other hydrogen bonding supramolecular Cl⁻ receptors to determine the EIE of ^35/37Cl⁻ binding in these hosts.¹³ Because isotope effects, both equilibrium and kinetic, originate solely from changes in the vibrational energy of the isotopically labelled bond, the EIE arising from this study came from changes in the vibrational energies of the bonds in the supramolecular hosts when participating in hydrogen bonding with Cl⁻ isotopes. Indeed, a linear relationship was observed between the hydrogen bond donor (D) D–H bond lengths in the host–guest complex and the computed ^35/37Cl EIE.Open in a separate windowFig. 1Arylethynyl bisurea receptors 1^H and 1^D used in our previous DEIE study of Cl⁻ binding. Related receptors 2^H and 2^D are used in this study to avoid reaction of the nitro group with HS⁻.Previous EIE studies with receptor 1^H/D have focused on Cl⁻ binding; however, to the best of our knowledge, no work has yet investigated the EIE of hydrosulfide (HS⁻) binding in this or other systems. HS⁻ is a highly reactive anion that plays crucial roles in biology. At physiological pH, HS⁻ is favored in solution by a 3 : 1 ratio over its conjugate acid, hydrogen sulfide (H₂S). H₂S has been identified as the third physiological gasotransmitter alongside CO and NO and plays essential roles in physiological systems.¹⁵ Despite its high nucleophilicity and redox activity, HS⁻ has been observed to be bound through non-covalent interactions in the protein crystal structure of a bacterial ion channel¹⁶ and in the turn-over state of vanadium-containing nitrogenase.¹⁷ The supramolecular chemistry of HS⁻ is under-studied in synthetic supramolecular receptors, likely due to the inherent high reactivity of HS⁻. Indeed, we are aware of only three families of receptors that have been shown to reversibly bind HS⁻.^18–21Recently, we used a series of arylethynyl bisurea anion receptors to investigate and demonstrate a linear free energy relationship between the polarity of a non-traditional C–H hydrogen bond donor and the solution binding energy of HS⁻, HSe⁻, Cl⁻ and Br⁻.¹⁴ A major and unexpected finding of this study was that HS⁻ demonstrated a significant increase in sensitivity towards the polarity of the C–H hydrogen donor over HSe⁻, Cl⁻ and Br⁻. Although increasing the polarity of the C–H hydrogen bond donor did not lead to changes in selectivity between Cl⁻, Br⁻, and HSe⁻, we observed a 9-fold increase in selectivity for HS⁻ over Cl⁻, suggesting a fresh approach to selective HS⁻ recognition using non-covalent interactions. In this current study, we label the C–H hydrogen bond donor in an arylethynyl bisurea receptor with a deuterium atom (2^H/D, Fig. 1) to further investigate this apparent preference of polar C–H H-bond donors for HS⁻ over Cl⁻ and Br⁻ through DEIE.Receptor 2^H is a previously reported anion receptor for HS⁻, Cl⁻, and Br⁻ and was prepared by established methods.¹⁴ Deuterium labelling of the isotopologue 2^D was achieved by selective monodeuteration of intermediates through methods similar to those reported in the literature (Scheme 1).²² The diazonium salt 3 was synthesized in a 71% yield from 2,6-diiodo-4-trifluoromethylaniline.²³ Dediazonation in DMF-d₇ is catalyzed by FeSO₄ and allows for selective synthesis of monodeuterated intermediate 4. The deuteration step proceeds through a radical pathway that uses DMF-d₇ as the deuterium source. This deuteration reaction provides efficient deuterium incorporation even with up to 50% by volume H₂O in the reaction solution due to the differential bond strengths in DMF and H₂O.²² Sonogashira cross-coupling reaction of 4 and 4-t-butyl-2-ethynylaniline²⁴ afforded 5 in 45% yield. Subsequent addition with 4-methoxyphenyl isocyanate gave 2^D in 34% yield. Compound 2^D and intermediates were characterized through ¹H, ²H, ¹³C{¹H}, and ¹⁹F NMR spectroscopy and high-resolution mass spectrometry (see ESI^†).Open in a separate windowScheme 1Synthetic route for the selective deuteration of anion receptor 2^D.Previous work on the DEIE of Cl⁻ binding with 1^H/D in DMSO revealed an experimental isotope effect of 1.019 ± 0.010. Therefore, we expected similar small DEIEs for HS⁻, Cl⁻, and Br⁻ binding with 2^H/D. Typical methods to determine binding constants (K_a) in supramolecular systems use non-linear regression fitting of titration data. Results from this method can be affected by small errors in the known initial host and guest concentration, quality of the titration isotherm, and subsequent data fitting, which when taken together often results in 2–15% errors in K_a. To increase the precision in K^H_a/K^D_a data for this study, we used the Perrin method of competitive titrations,²⁵ which has been shown previously to reduce errors in EIE values significantly with errors as small as 0.0004.²⁶ In this method, a linearized plot of the chemical shifts of 2^H (δ_H) and 2^D (δ_D) in fast exchange with an anionic guest is fit by linear regression to eqn (1):(δ⁰_H − δ_H)(δ_D − δ^f_D) = DEIE(δ⁰_D − δ_D)(δ_H − δ^f_H)1The slope of the linear regression is equal to the DEIE of the system. Because the linear regression only relies on chemical shift values and is independent of host and guest concentration, the precision of the method is limited to the precision of the NMR instrument and quality of data fitting.In addition, ¹³C NMR spectroscopy is sensitive to isotopic labelling and can show changes in chemical shifts between isotopomers. We were able to differentiate between the ¹³C NMR signals for C^ab, C¹ and C² for free and bound 2^H and 2^D (Fig. 2a) in 10% DMSO-d₆/CD₃CN, which were similar to those reported for 1^H/D in DMSO-d₆.¹² Competitive ¹³C NMR spectroscopy titrations were performed in anaerobic and anhydrous 10% DMSO-d₆/CD₃CN at 25 °C with mixtures of 2^H and 2^D in combined concentrations between 5.71 and 13.46 mM. Aliquots of the tetrabutylammonium (TBA) salts of HS⁻, Cl⁻, and Br⁻ were added until the system had reached saturation (Titration method A in ESI^†). In an effort to decrease reactivity of HS⁻ with 2^H/D and DMSO over long periods of time and decrease oxygen and water contaminations, some titrations with HS⁻ were performed by splitting the host solution of 2^H/D between four J-young NMR tubes. For each point in the competitive titration, TBASH was added to a new solution of 2^H/D inside an N₂-filled glovebox shortly before obtaining a ¹³C NMR spectra (Titration method B in ESI^†). The C^ab, C¹ and C^{2 13}C NMR signals were tracked for 2^H and 2^D in each titration for each anion. A representative competitive titration and linearized plots for Cl⁻ binding is shown in Fig. 2.Open in a separate windowFig. 2(a) Representation of the host–guest equilibrium between 2^H/D and Cl⁻. (b) Differences in the chemical shifts between the 2^H and 2^D isotopologues are observed in the ¹³C NMR signals for the C^ab, C¹, and C² carbons. ¹³C NMR signals for the C^ab, C¹, and C² carbons in 2^H and 2^D are tracked throughout a titration. (c–e) Linearized plots from fitting the chemical shifts of the C^ab, C¹, and C² throughout a titration to eqn (1).The DEIE data calculated from tracking the chemical shifts of the C^ab, C¹ and C^{2 13}C NMR signals from Cl⁻ and Br⁻ binding are summarized in eqn (1) through linear regression is included in parentheses

Addition of obliquely crossed spherocylindrical lenses

This paper presents a three-step method for the addition of obliquely crossed spherocylinders. Contrary to other methods it does not require algorithms or complicated abstract concepts to secure the consistency of the results.     

Chemokine and inflammatory cytokine changes during chronic wound healing.

Wound healing is a complex process resulting from an interplay of processes including coagulation, inflammation, angiogenesis, and epithelialization. The chemokine family has been shown to contain members that are potent regulators of many of these pathways. Because we have previously shown that chemokines "pool" in biologic wound dressings, we studied the levels of CXC and CC chemokines, along with key inflammatory mediators, serially from a group of patients undergoing therapy for chronic venous leg ulcers. After 8 weeks, all patients had marked clinical healing of their ulcers (median 63.3% reduction in size) with two of 10 completely healed. Wound fluids extracted from dressings showed high levels of platelet factor-4 and interferon-gamma-inducible protein, with a trend toward increases in the ratio of the sums of the angiogenic versus angiostatic CXC chemokines (p = 0.082) in the tissues collected from the center of the ulcers during wound closure. Neutrophil-activating peptide-2 and interleukin-8 accounted for the most changes in wound fluid angiogenic chemokines, with significant differences both as compared with baseline levels and with patients' plasma level noted at various time points between weeks 0 and 8. The level of angiostatic chemokines, interferon-y inducible protein 10 and platelet-activating-4, fell most significantly between weeks 0 and 3 as compared with plasma levels. The observed shift toward angiogenic CXC chemokines suggests that effective healing in chronic venous insufficiency ulcers appears to "move" the ulcer bed toward a state more conducive to epithelialization,characteristic of the proliferative phase of wound healing. CC chemokines were also elevated at baseline in the wound fluid samples as compared with the patients' plasma levels. Macrophage inflammatory protein-1 (3 and regulated on activation, normal T expressed and secreted (RANTES) levels decreased with healing, whereas there were significant increases in the tissue levels of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 a over the first 4 weeks of therapy (p< or = 0.05 for both). Coincident with these changes was a steady increase in the ratio of interleukin-1 R/interleukin-1 receptor antagonist protein in the ulcer center tissues, which also correlated with healing (p < 0 .05) as compared with a decreasing ratio at the ulcer edge, and a biphasic response in the wound fluids. These findings suggest that advanced wound care techniques help move the ulcer from a chronic inflammatory state into one more characteristic of the late inflammatory/early proliferative phase of wound healing. Chemokines may play a critical role in the pathogenesis of chronic venous ulcers through their effects on angiogenesis and/or the progression of inflammatory reactions at the site of injury.