Probe Sites Research Articles

Elucidating the interaction of propofol and serum albumin by spectroscopic and docking methods

The interaction of human serum albumin (HSA) with propofol has been studied by fluorescence spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy and docking methods. A gradual decrease in Stern–Volmer quenching constants with the increase in temperature showed the static mode of fluorescence quenching. The obtained binding constant (KA) was 8.18×105M−1. Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes were calculated, which revealed that the reaction is spontaneous, exothermic and hydrophobic force driven. FT-IR test revealed conformational changes of the protein and destruction of H-bonding upon interaction. Moreover, propofol induced a decrease in α-helical contents probably with increment of random coils or/and β-sheets of HSA, as observed from the far-UV CD spectra. Molecular docking and site probing study depicted that propofol fits into the hydrophobic pocket close to Sudlow site I in domain IIA of HSA. The present study will be helpful in understanding the binding mechanism of propofol and associated stability and conformational changes.

Probing the binding of two 19-nortestosterone derivatives to human serum albumin: insights into the interactions of steroid hormone drugs with functional biomacromolecule.

Norethindrone acetate (NETA) is a fatty acid ester of norethindrone (NET) that can convert to its more active parent compound NET when orally administered. To study the interactions of NETA and NET with human serum albumin (HSA), we applied fluorescence spectroscopy, circular dichroism (CD), and molecular docking. The effects of metal ions on the HSA-NETA/NET system were also explored. Fluorescence data showed that the quenching mechanism of HSA by NETA and NET was consistent with a static model and that the binding constant of NETA was higher than that of NET. Thermodynamic parameters indicated that hydrogen bonds and van der Waals forces were the main forces maintaining the stability of the HSA-NETA/NET complex. Molecular modeling studies revealed that NETA and NET were bound within subdomain IIA of HSA, in accordance with the site probe results. Synchronous fluorescence spectroscopy, CD, and three-dimensional fluorescence spectroscopy further confirmed that the binding of NETA/NET to HSA changed the secondary structure of the protein. All other metal ions, except for Ca(2+) , decreased the K value of the HSA-NETA/NET system with enhancement of the maximum effectiveness of NETA/NET. Three commercially available steroid hormone drugs influenced the binding ability of NETA on HSA to different extents. This study provides novel insights into the interactions between HSA and NETA/NET, as well as a solid foundation for future research on drug pharmacokinetics and pharmacodynamics. Copyright © 2016 John Wiley & Sons, Ltd.

Single-Molecule Fluorescence Imaging of Interfacial DNA Hybridization Kinetics at Selective Capture Surfaces.

Accurate knowledge of the kinetics of complementary oligonucleotide hybridization is integral to the design and understanding of DNA-based biosensors. In this work, single-molecule fluorescence imaging is applied to measuring rates of hybridization between fluorescently labeled target ssDNA and unlabeled probe ssDNA immobilized on glass surfaces. In the absence of probe site labeling, the capture surface must be highly selective to avoid the influence of nonspecific adsorption on the interpretation of single-molecule imaging results. This is accomplished by increasing the probe molecule site densities by a factor of ∼100 compared to optically resolvable sites so that nonspecific interactions compete with a much greater number of capture sites and by immobilizing sulfonate groups to passivate the surface between probe strands. The resulting substrates exhibit very low nonspecific adsorption, and the selectivity for binding a complementary target sequence exceeds that of a scrambled sequence by nearly 3 orders of magnitude. The population of immobilized DNA probe sites is quantified by counting individual DNA duplexes at low target concentrations, and those results are used to calibrate fluorescence intensities on the same sample at much higher target concentrations to measure a full binding isotherm. Dissociation rates are determined from interfacial residence times of individual DNA duplexes. Equilibrium and rate constants of hybridization, K(a) = 38 (±1) μM(-1), k(on) = 1.64 (±0.06) × 10(6) M(-1) s(-1), and k(off) = 4.3 (±0.1) × 10(-2) s(-1), were found not to change with surface density of immobilized probe DNA, indicating that hybridization events at neighboring probe sites are independent. To test the influence of probe-strand immobilization on hybridization, the kinetics of the probe target reaction at the surface were compared with the same reaction in free solution, and the equilibrium constants and dissociation and association rates were found to be nearly equivalent. The selectivity of these capture surfaces should facilitate sensitive investigations of DNA hybridization at the limit of counting molecules. Because the immobilized probe DNA on these surfaces is unlabeled, photobleaching of a probe label is not an issue, allowing capture substrates to be used for long periods of time or even reused in multiple experiments.

On the dynamical nature of the active center in a single-site photocatalyst visualized by 4D ultrafast electron microscopy

Understanding the dynamical nature of the catalytic active site embedded in complex systems at the atomic level is critical to developing efficient photocatalytic materials. Here, we report, using 4D ultrafast electron microscopy, the spatiotemporal behaviors of titanium and oxygen in a titanosilicate catalytic material. The observed changes in Bragg diffraction intensity with time at the specific lattice planes, and with a tilted geometry, provide the relaxation pathway: the Ti(4+)=O(2-) double bond transformation to a Ti(3+)-O(1-) single bond via the individual atomic displacements of the titanium and the apical oxygen. The dilation of the double bond is up to 0.8 Å and occurs on the femtosecond time scale. These findings suggest the direct catalytic involvement of the Ti(3+)-O(1-) local structure, the significance of nonthermal processes at the reactive site, and the efficient photo-induced electron transfer that plays a pivotal role in many photocatalytic reactions.

Interactions of cephalexin with bovine serum albumin: displacement reaction and molecular docking.

Introduction: The drug-plasma protein interaction is a fundamental issue in guessing and checking the serious drug side effects related with other drugs. The purpose of this research was to study the interaction of cephalexin with bovine serum albumin (BSA) and displacement reaction using site probes. Methods: The interaction mechanism concerning cephalexin (CPL) with BSA was investigated using various spectroscopic methods and molecular modeling method. The binding sites number, n, apparent binding constant, K, and thermodynamic parameters, ΔG0, ΔH0, and ΔS0 were considered at different temperatures. To evaluate the experimental results, molecular docking modeling was calculated. Results: The distance, r=1.156 nm between BSA and CPL were found in accordance with the Forster theory of non-radiation energy transfer (FRET) indicating energy transfer occurs between BSA and CPL. According to the binding parameters and ΔG0= negative values and ΔS0= 28.275 j mol-1K-1, a static quenching process is effective in the CPL-BSA interaction spontaneously. ΔG0 for the CPL-BSA complex obtained from the docking simulation is -28.99 kj mol-1, which is close to experimental ΔG of binding, -21.349 kj mol-1 that indicates a good agreement between the results of docking methods and experimental data. Conclusion: The outcomes of spectroscopic methods revealed that the conformation of BSA changed during drug-BSA interaction. The results of FRET propose that CPL quenches the fluorescence of BSA by static quenching and FRET. The displacement study showed that phenylbutazon and ketoprofen displaced CPL, indicating that its binding site on albumin is site I and Gentamicin cannot be displaced from the binding site of CPL. All results of molecular docking method agreed with the results of experimental data.

Agreement between lower esophageal and nasopharyngeal temperatures in children ventilated with an endotracheal tube with leak.

A temperature probe placed in the lower third of the esophagus accurately reflects core temperature in anesthetized children. Temperature probes are commonly placed in the nasopharynx in children, but when utilizing an uncuffed endotracheal tube (ETT) with a softly audible leak, ventilated gases from the trachea can escape upwards toward the nasopharynx, thereby potentially causing a cooling effect in the nasopharynx. We sought to establish if nasopharyngeal and lower esophageal temperatures are in agreement in children undergoing general anesthesia, both in scenarios of ventilation with a cuffed ETT that has minimal or no leak (cuff up), as well as an ETT with leak (cuff down). A prospective, crossover agreement study was performed on anesthetized children. Children were intubated with a MicroCuff(®) ETT and had temperature probes inserted into both the nasopharynx and lower esophagus. Under standardized ventilator and gas flow settings, temperatures were recorded with the ETT cuff inflated, and with the cuff deflated. Bland-Altman plots were utilized to assess agreement of temperatures. Fifty patients successfully completed this study. The mean difference between esophageal and nasopharyngeal temperature was found to be -0.03°C in the presence of minimal or no leak around the ETT (cuff up), with 95% limits of agreement (LOA) of -0.22 to 0.15°C. The mean difference between esophageal and nasopharyngeal temperature was found to be 0.1°C when a larger leak existed around the ETT (cuff down), with LOA of -0.31 to 0.51°C. Nasopharyngeal temperature accurately reflects lower esophageal temperature when there is minimal or no ETT leak. When a larger ETT leak is present, nasopharyngeal temperature is on average 0.1°C cooler than lower esophageal temperature. As the nasopharyngeal temperature probe site confers the advantage of simplicity of accurate placement compared to its esophageal counterpart, our findings support the use of nasopharyngeal temperature probes in children ventilated with both cuffed and uncuffed ETTs.

Evaluation of the binding interaction between bovine serum albumin and dimethyl fumarate, an anti-inflammatory drug by multispectroscopic methods

The information of the quenching reaction of bovine serum albumin with dimethyl fumarate is obtained by multi-spectroscopic methods. The number of binding sites, n and binding constants, KA were determined at different temperatures. The effect of increasing temperature on Stern–Volmer quenching constants (KD) indicates that a dynamic quenching mechanism is involved in the interaction. The analysis of thermodynamic quantities namely, ∆H° and ∆S° suggested hydrophobic forces playing a major role in the interaction between dimethyl fumarate and bovine serum albumin. The binding site of dimethyl fumarate on bovine serum albumin was determined by displacement studies, using the site probes viz., warfarin, ibuprofen and digitoxin. The determination of magnitude of the distance of approach for molecular interactions between dimethyl fumarate and bovine serum albumin is calculated according to the theory of Förster energy transfer. The CD, 3D fluorescence spectra, synchronous fluorescence measurements and FT-IR spectral results were indicative of the change in secondary structure of the protein. The influence of some of the metal ions on the binding interaction was also studied.

Quantum mechanical calculation of electric fields and vibrational Stark shifts at active site of human aldose reductase.

Recent advance in biophysics has made it possible to directly measure site-specific electric field at internal sites of proteins using molecular probes with C = O or C≡N groups in the context of vibrational Stark effect. These measurements directly probe changes of electric field at specific protein sites due to, e.g., mutation and are very useful in protein design. Computational simulation of the Stark effect based on force fields such as AMBER and OPLS, while providing good insight, shows large errors in comparison to experimental measurement due to inherent difficulties associated with point charge based representation of force fields. In this study, quantum mechanical calculation of protein's internal electrostatic properties and vibrational Stark shifts was carried out by using electrostatically embedded generalized molecular fractionation with conjugate caps method. Quantum calculated change of mutation-induced electric field and vibrational Stark shift is reported at the internal probing site of enzyme human aldose reductase. The quantum result is in much better agreement with experimental data than those predicted by force fields, underscoring the deficiency of traditional point charge models describing intra-protein electrostatic properties.

Chromatographic analysis of the effects of fatty acids and glycation on binding by probes for Sudlow sites I and II to human serum albumin.

The primary endogenous ligands of human serum albumin (HSA) are non-esterified fatty acids, with 0.1-2mol of fatty acids normally being bound to HSA. In type II diabetes, fatty acid levels in serum are often elevated, and the presence of high glucose results in an increase in the non-enzymatic glycation of HSA. High-performance affinity chromatography (HPAC) was used to examine the combined effects of glycation and the presence of long chain fatty acids on the binding of HSA with R-warfarin and l-tryptophan (i.e., probes for Sudlow sites I and II, the major sites for drugs on this protein). Zonal elution competition studies were used to examine the interactions of myristic acid, palmitic acid and stearic acid with these probes on HSA. It was found that all these fatty acids had direct competition with R-warfarin at Sudlow site I of normal HSA and glycated HSA, with the glycated HSA typically having stronger binding for the fatty acids at this site. At Sudlow site II, direct competition was observed for all the fatty acids with l-tryptophan when using normal HSA, while glycated HSA gave no competition or positive allosteric interactions between these fatty acids and l-tryptophan. These data indicated that glycation can alter the interactions of drugs and fatty acids at specific binding sites on HSA. The results of this study should lead to a better understanding of how these interactions may change during diabetes and demonstrate how HPAC can be used to examine drug/solute-protein interactions in complex systems.

Estimating DNA methylation levels by joint modeling of multiple methylation profiles from microarray data.

DNA methylation studies have been revolutionized by the recent development of high throughput array-based platforms. Most of the existing methods analyze microarray methylation data on a probe-by-probe basis, ignoring probe-specific effects and correlations among methylation levels at neighboring genomic locations. These methods can potentially miss functionally relevant findings associated with genomic regions. In this article, we propose a statistical model that allows us to pool information on the same probe across multiple samples to estimate the probe affinity effect, and to borrow strength from the neighboring probe sites to better estimate the methylation values. Using a simulation study, we demonstrate that our method can provide accurate model-based estimates. We further use the proposed method to develop a new procedure for detecting differentially methylated regions, and compare it with a state-of-the-art approach via a data application.

Electrode modifications to lower electrode impedance and improve neural signal recording sensitivity

Objective. Although electrode size should be miniaturized to provide higher selectivity for neural signal recording and to avoid tissue damage, small sized electrodes induce high impedance, which decreases recording quality. In this work, the electrode surface was modified to increase the effective surface area to lower the electrode impedance and to improve the neural signal detection quality by optimizing plasma conditions. Approach. A tetrafluoromethane (CF4) plasma was used to increase the effective surface area of gold electrode sites of polyimide-based neural probes. In vitro electrode impedance and in vivo neural signal recording and stimulation were characterized. Main results. For 15 μm diameter (dia.) electrode size, the average surface roughness could be increased from 1.7 to 22 nm after plasma treatment, and the electrode impedance was decreased by 98%. Averaged background noise power in the range of 1 to 1000 Hz was decreased to −106 dB after the 30 μm dia. electrodes were plasma modified—lower than the noise level of −86 dB without plasma treatment. Neural probes with plasma-modified electrode sites of 15 and 30 μm dia. were implanted to the anterior cingulate cortex (ACC) region for acute recording of spontaneous and electrical evoked local field potential (LFP) of neural signals. Spontaneous LFP recorded in vivo by the plasma-modified electrodes of 30 μm dia. was two times higher compared to electrodes without treatment. For a stimulation current of 400 μA, electrically evoked LFP recorded by the plasma-modified electrodes was seven times higher than those without plasma exposure. Significance. A controllable technology was developed to increase the effective surface area of electrodes using a CF4 plasma. Plasma-modified electrodes improved the quality of the neural probe recording and more sensitive to record spontaneous and evoked LFP in the ACC region.

Macroscopic alignment of poly(3‐hexylthiophene) for enhanced long‐range collection of photogenerated carriers

ABSTRACTAn epitaxy‐directing solvent additive 1,3,5‐trichlorobenzene is combined with an off‐center spin‐casting technique to produce poly(3‐hexylthiophene) (P3HT) fibers with uniaxial in‐plane alignment on the centimeter scale. Photoconductive atomic force microscopy (pc‐AFM) is used to characterize planar heterojunction devices assembled from phenyl‐C61 butyric acid methyl ester (PCBM) acceptor and both aligned and unaligned P3HT donor. By varying the relative positions of the laser spot (site of carrier generation) and probe (site of hole extraction), it is found that devices with aligned P3HT exhibit anisotropic and greatly enhanced long‐range photocarrier transport, with nearly 10% of original photocurrent measured 400 µm from the laser spot along the direction parallel to the alignment. Complementary thin film transistor (TFT) measurements reveal a factor of ∼3.5 difference in the hole mobilities parallel and perpendicular to the direction of alignment. Together, these findings highlight the importance of macroscopic alignment as a strategy to overcome the low mobilities of disordered polymer semiconductors.1 © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2016, 54, 180–188

Identification of Novel D-Aspartate Oxidase Inhibitors by in Silico Screening and Their Functional and Structural Characterization in Vitro.

D-Aspartate oxidase (DDO) is a degradative enzyme that is stereospecific for acidic D-amino acids, including D-aspartate, a potential agonist of the N-methyl-D-aspartate (NMDA) receptor. Dysfunction of NMDA receptor-mediated neurotransmission has been implicated in the onset of various mental disorders, such as schizophrenia. Hence, a DDO inhibitor that increases the brain levels of D-aspartate and thereby activates NMDA receptor function is expected to be a useful compound. To search for potent DDO inhibitor(s), a large number of compounds were screened in silico, and several compounds were identified as candidates. They were then characterized and evaluated as novel DDO inhibitors in vitro (e.g., the inhibitor constant value of 5-aminonicotinic acid for human DDO was 3.80 μM). The present results indicate that some of these compounds may serve as lead compounds for the development of a clinically useful DDO inhibitor and as active site probes to elucidate the structure-function relationships of DDO.

Epigenome-wide association study reveals decreased average methylation levels years before breast cancer diagnosis.

BackgroundInterest in the potential of DNA methylation in peripheral blood as a biomarker of cancer risk is increasing. We aimed to assess whether epigenome-wide DNA methylation measured in peripheral blood samples obtained before onset of the disease is associated with increased risk of breast cancer. We report on three independent prospective nested case-control studies from the European Prospective Investigation into Cancer and Nutrition (EPIC-Italy; n = 162 matched case-control pairs), the Norwegian Women and Cancer study (NOWAC; n = 168 matched pairs), and the Breakthrough Generations Study (BGS; n = 548 matched pairs). We used the Illumina 450k array to measure methylation in the EPIC and NOWAC cohorts. Whole-genome bisulphite sequencing (WGBS) was performed on the BGS cohort using pooled DNA samples, combined to reach 50× coverage across ~16 million CpG sites in the genome including 450k array CpG sites. Mean β values over all probes were calculated as a measurement for epigenome-wide methylation.ResultsIn EPIC, we found that high epigenome-wide methylation was associated with lower risk of breast cancer (odds ratio (OR) per 1 SD = 0.61, 95 % confidence interval (CI) 0.47–0.80; −0.2 % average difference in epigenome-wide methylation for cases and controls). Specifically, this was observed in gene bodies (OR = 0.51, 95 % CI 0.38–0.69) but not in gene promoters (OR = 0.92, 95 % CI 0.64–1.32). The association was not replicated in NOWAC (OR = 1.03 95 % CI 0.81–1.30). The reasons for heterogeneity across studies are unclear. However, data from the BGS cohort was consistent with epigenome-wide hypomethylation in breast cancer cases across the overlapping 450k probe sites (difference in average epigenome-wide methylation in case and control DNA pools = −0.2 %).ConclusionsWe conclude that epigenome-wide hypomethylation of DNA from pre-diagnostic blood samples may be predictive of breast cancer risk and may thus be useful as a clinical biomarker.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0104-2) contains supplementary material, which is available to authorized users.

Investigation into the interaction of methylparaben and erythromycin with human serum albumin using multispectroscopic methods.

In this paper, the interaction of methylparaben and erythromycin with human serum albumin (HSA) was studied for the first time using spectroscopic methods including Fourier transform infrared (FTIR) spectroscopy and UV absorption spectroscopy in combination with fluorescence quenching under physiological conditions. The binding parameters were evaluated using a fluorescence quenching method. Based on Förster's theory of non-radiation energy transfer, the binding average distance, r between the donor (HSA) and the acceptor (methylparaben and erythromycin) was evaluated. UV/vis absorption, FTIR, synchronous and 3D spectral results showed that the conformation of HSA was changed in the presence of methylparaben and erythromycin. The thermodynamic parameters were calculated according to the van't Hoff equation and are discussed. The effect of some biological metal ions and site probes on the binding of methylparaben and erythromycin to HSA were further examined.

Molecular modeling and multispectroscopic studies of the interaction of hepatitis B drug, adefovir dipivoxil with human serum albumin

The interaction of hepatitis B drug, adefovir dipivoxil with human serum albumin (HSA) was studied by using UV–vis, fluorometric, circular dichroism (CD) and molecular docking techniques. The results indicated that the binding of the drug to HSA caused fluorescence quenching through static quenching mechanism with binding constant of 1.3×103M−1. The thermodynamic parameters indicated that the hydrophobic force contacts are the major forces in the stability of protein-drug complex (ΔH>0 and ΔS>0). The displacement experiments using the site probes viz., warfarin and ibuprofen showed that adefovir dipivoxil could bind to the site III of HSA. The results of CD and UV–vis spectroscopy indicated that the binding of the drug induced some conformational changes in HSA. Furthermore, the study of molecular docking also confirmed binding of adefovir dipivoxil to the site III of HSA by hydrophobic interaction.

Assessment of copy number variations in the brain genome of schizophrenia patients.

BackgroundCytogenomic mutations and chromosomal abnormality are implicated in the neuropathology of several brain diseases. Cell heterogeneity of brain tissues makes their detection and validation difficult, however. In the present study, we analyzed gene dosage alterations in brain DNA of schizophrenia patients and compared those with the copy number variations (CNVs) identified in schizophrenia patients as well as with those in Asian lymphocyte DNA and attempted to obtain hints at the pathological contribution of cytogenomic instability to schizophrenia.ResultsBrain DNA was extracted from postmortem striatum of schizophrenia patients and control subjects (n = 48 each) and subjected to the direct two color microarray analysis that limits technical data variations. Disease-associated biases of relative DNA doses were statistically analyzed with Bonferroni’s compensation on the premise of brain cell mosaicism. We found that the relative gene dosage of 85 regions significantly varied among a million of probe sites. In the candidate CNV regions, 26 regions had no overlaps with the common CNVs found in Asian populations and included the genes (i.e., ANTXRL, CHST9, DNM3, NDST3, SDK1, STRC, SKY) that are associated with schizophrenia and/or other psychiatric diseases. The majority of these candidate CNVs exhibited high statistical probabilities but their signal differences in gene dosage were less than 1.5-fold. For test evaluation, we rather selected the 10 candidate CNV regions that exhibited higher aberration scores or larger global effects and were thus confirmable by PCR. Quantitative PCR verified the loss of gene dosage at two loci (1p36.21 and 1p13.3) and confirmed the global variation of the copy number distributions at two loci (11p15.4 and 13q21.1), both indicating the utility of the present strategy. These test loci, however, exhibited the same somatic CNV patterns in the other brain region.ConclusionsThe present study lists the candidate regions potentially representing cytogenomic CNVs in the brain of schizophrenia patients, although the significant but modest alterations in their brain genome doses largely remain to be characterized further.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-015-0144-5) contains supplementary material, which is available to authorized users.

Retrospective analysis of 26 complete-arch implant-supported monolithic zirconia prostheses with feldspathic porcelain veneering limited to the facial surface

Statement of problemMonolithic zirconia prostheses on teeth or implants have been proposed in recent years as a potential treatment. To date, limited data regarding the outcomes of these prostheses have been presented and are mainly based on limited sample size and short-term follow-up. Data on complete-arch monolithic zirconia prostheses are relatively scarce. PurposeThe purpose of this retrospective study was to evaluate the clinical performances of 26 implant-supported, complete-arch, monolithic zirconia restorations with facial feldspathic porcelain veneers for the rehabilitation of completely edentulous patients. Material and methodsAll patients’ charts from 2 private practices from 2010 to 2013 were reviewed. Patients rehabilitated with a complete-arch implant-supported monolithic zirconia prostheses were included in the study. Several parameters were recorded so as to evaluate the outcome of these rehabilitations: implant survival and success rates, prosthesis survival rate, interproximal bone loss, periimplant probing depth, and bleeding on probing. The number and type of prosthetic complications were also recorded. Data were analyzed with descriptive statistics. ResultsEighteen patients were treated with a total of 26 complete-arch fixed prostheses. The mean follow-up time was 20.9 months (SD 13.6; range, 10 to 36 months). In total, 154 implants were placed supporting 309 retainers and pontics. The implant survival rate was 100% and the success rate was 94.8%. Mean bone loss was 0.66 mm (SD 0.59 mm). Mean probing depth was 3.4 mm (SD 0.92 mm). Bleeding on probing was positive in 19% of probing sites. The prosthesis survival rate was 100%. ConclusionsThe results of this retrospective evaluation showed that monolithic zirconia restorations with facial porcelain veneer provided satisfactory clinical performance and suggest that these rehabilitations are a viable treatment option for completely edentulous patients.

Carprofen pharmacokinetics in plasma and in control and inflamed canine tissue fluid using in vivo ultrafiltration.

Measurement of unbound drug concentrations at their sites of action is necessary for accurate PK/PD modeling. The objective of this study was to determine the unbound concentration of carprofen in canine interstitial fluid (ISF) using in vivo ultrafiltration and to compare pharmacokinetic parameters of free carprofen concentrations between inflamed and control tissue sites. We hypothesized that active concentrations of carprofen would exhibit different dispositions in ISF between inflamed vs. normal tissues. Bilateral ultrafiltration probes were placed subcutaneously in six healthy Beagle dogs 12 h prior to induction of inflammation. Two milliliters of either 2% carrageenan or saline control was injected subcutaneously at each probe site, 12 h prior to intravenous carprofen (4 mg/kg) administration. Plasma and ISF samples were collected at regular intervals for 72 h, and carprofen concentrations were determined using HPLC. Prostaglandin E2 (PGE2 ) concentrations were quantified in ISF using ELISA. Unbound carprofen concentrations were higher in ISF compared with predicted unbound plasma drug concentrations. Concentrations were not significantly higher in inflamed ISF compared with control ISF. Compartmental modeling was used to generate pharmacokinetic parameter estimates, which were not significantly different between sites. Terminal half-life (T½) was longer in the ISF compared with plasma. PGE2 in ISF decreased following administration of carprofen. In vivo ultrafiltration is a reliable method to determine unbound carprofen in ISF, and that disposition of unbound drug into tissue is much higher than predicted from unbound drug concentration in plasma. However, concentrations and pharmacokinetic parameter estimates are not significantly different in inflamed vs. un-inflamed tissues.

Body mass index associated with genome-wide methylation in breast tissue.

Gene expression studies indicate that body mass index (BMI) is associated with molecular pathways involved in inflammation, insulin-like growth factor activation, and other carcinogenic processes in breast tissue. The goal of this study was to determine whether BMI is associated with gene methylation in breast tissue and to identify pathways that are commonly methylated in association with high BMI. Epigenome-wide methylation profiles were determined using the Illumina HumanMethylation450 BeadChip array in the non-diseased breast tissue of 81 women undergoing breast surgery between 2009 and 2013 at the University of North Carolina Hospitals. Multivariable, robust linear regression was performed to identify methylation sites associated with BMI at a false discovery rate q value <0.05. Gene expression microarray data was used to identify which of the BMI-associated methylation sites also showed correlation with gene expression. Gene set enrichment analysis was conducted to assess which pathways were enriched among the BMI-associated methylation sites. Of the 431,568 methylation sites analyzed, 2573 were associated with BMI (q value <0.05), 57 % of which showed an inverse correlation with BMI. Pathways enriched among the 2573 probe sites included those involved in inflammation, insulin receptor signaling, and leptin signaling. We were able to map 1251 of the BMI-associated methylation sites to gene expression data, and, of these, 226 (18 %) showed substantial correlations with gene expression. Our results suggest that BMI is associated with genome-wide methylation in non-diseased breast tissue and may influence epigenetic pathways involved in inflammatory and other carcinogenic processes.