Cysteamine Chemisorption at Mercury-Solution Interfaces in the Context of Redox and Microdissociation Equilibria.

The redox behavior and chemisorption of cysteamine (CA) at a charged mercury surface are described, with an emphasis on its acid-base properties supported by molecular dynamics and quantum mechanical calculations. It was found that CA forms chemisorbed layers on the surface of the mercury electrode. The formation of Hg-CA complexes is connected to mercury disproportionation, as reflected in peaks SII and SI at potentials higher than the electrode potential of zero charge (p.z.c.). Both the process of chemisorption of CA and its consequent redox transformation are proton-dependent. Also, depending on the protonation of CA, the formation of typical populations of chemisorbed conformers can be observed. In addition, cystamine (CA disulfide dimer) can be reduced on the mercury surface. Between the potentials of this reduction and peak SI, the p.z.c. of the electrode used can be found. Furthermore, CA can serve as an LMW catalyst for hydrogen evolution. The mechanistic insights presented here can be used for follow-up research on CA chemisorption and targeted modification of other metallic surfaces.

Phytocannabinoids and gingival inflammation: Preclinical findings and a placebo-controlled double-blind randomized clinical trial with cannabidiol.

OBJECTIVE: The aim of this study was to: (1) evaluate the anti-inflammatory effects of cannabidiol (CBD) on primary cultures of human gingival fibroblasts (HGFs) and (2) to clinically monitor the effect of CBD in subjects with periodontitis. BACKGROUND: The use of phytocannabinoids is a new approach in the treatment of widely prevalent periodontal disease. MATERIALS AND METHODS: Cannabinoid receptors were analyzed by western blot and interleukin production detected using enzyme immunoassay. Activation of the Nrf2 pathway was studied via monitoring the mRNA level of heme oxygenase-1. Antimicrobial effects were determined by standard microdilution and 16S rRNA screening. In the clinical part, a placebo-control double-blind randomized study was conducted (56 days) in three groups (n = 90) using dental gel without CBD (group A) and with 1% (w/w) CBD (group B) and corresponding toothpaste (group A - no CBD, group B - with CBD) for home use to maintain oral health. Group C used dental gel containing 1% chlorhexidine digluconate (active comparator) and toothpaste without CBD. RESULTS: Human gingival fibroblasts were confirmed to express the cannabinoid receptor CB2. Lipopolysaccharide-induced cells exhibited increased production of pro-inflammatory IL-6 and IL-8, with deceasing levels upon exposure to CBD. CBD also exhibited antimicrobial activities against Porphyromonas gingivalis, with an MIC of 1.5 µg/mL. Activation of the Nrf2 pathway was also demonstrated. In the clinical part, statistically significant improvement was found for the gingival, gingival bleeding, and modified gingival indices between placebo group A and CBD group B after 56 days. CONCLUSIONS: Cannabidiol reduced inflammation and the growth of selected periodontal pathogenic bacteria. The clinical trial demonstrated a statistically significant improvement after CBD application. No adverse effects of CBD were reported by patients or observed upon clinical examination during the study. The results are a promising basis for a more comprehensive investigation of the application of non-psychotropic cannabinoids in dentistry.

Local and cooperative structural transitions of double-stranded DNA in choline-based deep eutectic solvents.

The possibility of using deep eutectic solvents (DESs) as co-solvents for stabilizing and preserving the native structure of DNA provides an attractive opportunity in the field of DNA biotechnology. The rationale of this work is a systematic investigation of the effect of hydrated choline-based DES on the structural stability of a 30-base-pair double-stranded DNA model via a combination of spectroscopic experiments and MD simulations. UV absorption and CD experiments provide evidence of a significant contribution of DESs to the stabilization of the double-stranded canonical (B-form) DNA structure. Multi-wavelength synchrotron UV Resonance Raman (UVRR) measurements indicate that the hydration shell of adenine-thymine pairs is strongly perturbed in the presence of DESs and that the preferential interaction between H-bond sites of guanine residues and DESs is significantly involved in the stabilization of the dsDNA. Finally, MD calculations show that the minor groove of DNA is significantly selective for the choline part of the investigated DESs compared to the major groove. This finding is likely to have a significant impact not only in terms of thermal stability but also in the modulation of ligand-DNA interactions.

Electrochemistry in sensing of molecular interactions of proteins and their behavior in an electric field.

Electrochemical methods can be used not only for the sensitive analysis of proteins but also for deeper research into their structure, transport functions (transfer of electrons and protons), and sensing their interactions with soft and solid surfaces. Last but not least, electrochemical tools are useful for investigating the effect of an electric field on protein structure, the direct application of electrochemical methods for controlling protein function, or the micromanipulation of supramolecular protein structures. There are many experimental arrangements (modalities), from the classic configuration that works with an electrochemical cell to miniaturized electrochemical sensors and microchip platforms. The support of computational chemistry methods which appropriately complement the interpretation framework of experimental results is also important. This text describes recent directions in electrochemical methods for the determination of proteins and briefly summarizes available methodologies for the selective labeling of proteins using redox-active probes. Attention is also paid to the theoretical aspects of electron transport and the effect of an external electric field on the structure of selected proteins. Instead of providing a comprehensive overview, we aim to highlight areas of interest that have not been summarized recently, but, at the same time, represent current trends in the field.

A Systematic Review of Dynamic Forces and Kinematic Indicators of Front and Roundhouse Kicks across Varied Conditions and Participant Experience.

Impact force and maximum velocity are important indicators of kick efficiency. Therefore, this systematic review compared the front kick (FK) and roundhouse kick (RK), including their impact force, maximum velocity, angular velocity, and execution time, considering various target types and experience levels. Following PRISMA guidelines, the Web of Science, SportDiscus, and PubMed were systematically searched for articles published from January 1982 to May 2022. Normalized kicking values were compared using one-way ANOVA. Eighteen articles included FKs (sample: 113 elite men, 109 sub-elite men, and 46 novices), and twenty-five articles included RKs (sample: 238 elite men, 143 sub-elite men, and 27 novice men). The results indicate that the impact force of the FK were 47% (p < 0.01), 92% (p < 0.01), and 120% (p < 0.01) higher than those of the RK across novice, sub-elite, and elite groups, respectively. Moreover, the maximum foot velocity of the RK was 44% (p < 0.01) and 48% (p < 0.01) higher than that of the FK for the sub-elite and elite groups, respectively. Furthermore, the elite group had 65% (p < 0.01) higher knee extension angular velocity with the RK than with the FK and 138% (p < 0.01) higher hip extension angular velocity with the FK than with the RK. In summary, the findings suggest that the FK is more effective in generating forceful kicks, while the RK has the potential for rapid execution.

Hepatic biotransformation of non-psychotropic phytocannabinoids and activity screening on cytochromes P450 and UDP-glucuronosyltransferases.

This study examined the biotransformation of phytocannabinoids in human hepatocytes. The susceptibility of the tested compounds to transformations in hepatocytes exhibited the following hierarchy: cannabinol (CBN) > cannabigerol (CBG) > cannabichromene (CBC) > cannabidiol (CBD). Biotransformation included hydroxylation, oxidation to a carboxylic acid, dehydrogenation, hydrogenation, dehydration, loss/shortening of alkyl, glucuronidation and sulfation. CBN was primarily metabolized by oxidation of a methyl to a carboxylic acid group, while CBD, CBG and CBC were preferentially metabolized by direct glucuronidation. The study also screened for the activity of recombinant human cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs), which could catalyze the hydroxylation and glucuronidation of the tested compounds, respectively. We found that CBD was hydroxylated mainly by CYPs 2C8, 2C19, 2D6; CBN by 1A2, 2C9, 2C19 and 2D6; and CBG by 2B6, 2C9, 2C19 and 2D6. CBC exhibited higher susceptibility to CYP-mediated transformation than the other tested compounds, mainly with CYPs 1A2, 2B6, 2C8, 2C19, 2D6 and 3A4 being involved. Further, CBD was primarily glucuronidated by UGTs 1A3, 1A7, 1A8, 1A9 and 2B7; CBN by 1A7, 1A8, 1A9 and 2B7; CBG by 1A3, 1A7, 1A8, 1A9, 2B4, 2B7 and 2B17; and the glucuronidation of CBC was catalyzed by UGTs 1A1, 1A8, 1A9 and 2B7.

Hexahelicene DNA-binding: Minor groove selectivity, semi-intercalation and chiral recognition.

In this contribution, we focused on a fundamental study targeting the interaction of water-soluble [6]helicene derivative 1 (1-butyl-3-(2-methyl[6]helicenyl)-imidazolium bromide) with double-stranded (ds) DNA. A synthetic 30-base pair duplex, plasmid, chromosomal calf thymus and salmon DNA were investigated using electrochemistry, electrophoresis and spectroscopic tools supported by molecular dynamics (MD) and quantum mechanical approaches. Both experimental and theoretical work revealed the minor groove binding of 1 to the dsDNA. Both the positively charged imidazole ring and hydrophobic part of the side chain contributed to the accommodation of 1 into the dsDNA structure. Neither intercalation into the duplex DNA nor the stable binding of 1 to single-stranded DNA were found in topoisomerase relaxation experiments with structural components of 1, i.e. [6]helicene (2) and 1-butyl-3-methylimidazolium bromide (3), nor by theoretical calculations. Finally, the binding of optically pure enantiomers (P)-1 and (M)-1 was studied using circular dichroism spectroscopy, isothermal titration calorimetry and UV Resonance Raman (UVRR) methods. Using MD and quantum mechanical methods, minor groove and semi-intercalation were proposed for compound 1 as the predominant binding modes. From the UVRR findings, we also can conclude that 1 tends to preferentially interact with adenine and guanine residues in the structure of dsDNA.

Lipid-based liquid crystalline materials in electrochemical sensing and nanocarrier technology.

Some biologically active substances are unstable and poorly soluble in aqueous media, at the same time exhibiting low bioavailability. The incorporation of these biologically active compounds into the structure of a lipid-based lyotropic liquid crystalline phase or nanoparticles can increase or improve their stability and transport properties, subsequent bioavailability, and applicability in general. The aim of this short overview is (1) to clarify the principle of self-assembly of lipidic amphiphilic molecules in an aqueous environment and (2) to present lipidic bicontinuous cubic and hexagonal phases and their current biosensing (with a focus on electrochemical protocols) and biomedical applications.

Safety assessment and redox status in rats after chronic exposure to cannabidiol and cannabigerol.

Cannabidiol (CBD) and cannabigerol (CBG) are the two main non-psychotropic phytocannabinoids with high application potential in drug development. Both substances are redox-active and are intensively investigated for their cytoprotective and antioxidant action in vitro. In this study, we focused on an in vivo safety evaluation and the effect of CBD and CBG on the redox status in rats in a 90-d experiment. The substances were administered orogastrically in a dose of 0.66 mg synthetic CBD or 0.66 mg/1.33 mg CBG/kg/day. CBD produced no changes in the red or white blood count or biochemical blood parameters in comparison to the control. No deviations in the morphology or histology of the gastrointestinal tract and liver were observed. After 90 d of CBD exposure, a significant improvement in redox status was found in the blood plasma and liver. The concentration of malondialdehyde and carbonylated proteins was reduced compared to the control. In contrast to CBD, total oxidative stress was significantly increased and this was accompanied by an elevated level of malondialdehyde and carbonylated proteins in CBG-treated animals. Hepatotoxic (regressive changes) manifestations, disruption in white cell count, and alterations in the ALT activity, level of creatinine and ionized calcium were also found in CBG-treated animals. Based on liquid chromatography-mass spectrometry analysis, CBD/CBG accumulated in rat tissues (in the liver, brain, muscle, heart, kidney and skin) at a low ng level per gram. Both CBD and CBG molecular structures include a resorcinol moiety. In CBG, there is an extra dimethyloctadienyl structural pattern, which is most likely responsible for the disruption to the redox status and hepatic environment. The results are valuable to further investigation of the effects of CBD on redox status and should contribute towards opening up critical discussion on the applicability of other non-psychotropic cannabinoids.

Cannabidiol and periodontal inflammatory disease: A critical assessment.

Cannabidiol (CBD), a non-psychotropic cannabinoid produced by the genus Cannabis, is a phytoceutical that activates the endocannabinoid system (ECS) through binding to CB1 and CB2 receptors. The ECS is involved in cellular homeostasis and regulates metabolic processes in virtually all mammalian tissues. Published studies on CBD focus, inter alia, on its use in prophylaxis and as an anti-inflammatory agent. Here the authors present a critical assessment of the effects of CBD on inflammatory periodontal diseases caused by bacterial virulence factors, and evaluate critically the possible benefits and drawbacks of CBD use in dentistry. Particular attention is paid to the interaction of CBD with microbially colonized oral tissues, the inflammatory response in relation to the immune response, and the destruction/regeneration of hard and soft tissues of the periodontium.

Serum albumin as a primary non-covalent binding protein for nitro-oleic acid.

This work explores the interaction of 9/10-nitro-oleic acid (NO2-OA) with human serum albumin (HSA). The molecular mechanism of the biological action of NO2-OA is to our knowledge based on a reversible covalent reaction-Michael addition of nucleophilic amino acid residues of proteins. Since HSA is an important fatty acid transporter, a key question is whether NO2-OA can bind covalently or non-covalently to HSA, similarly to oleic acid (OA), which can interact with the FA1-FA7 binding sites of the HSA molecule. 1H NMR studies and competition analysis with OA and the drugs ibuprofen and warfarin were used to investigate a potential non-covalent binding mode. NO2-OA/HSA binding was confirmed to compete with warfarin for FA-7 with significantly higher affinity. NO2-OA competes with ibuprofen for FA-3 and FA-6, however, in contrast to the situation with warfarin, the binding affinities are not significantly different. The described interactions are based exclusively on non-covalent binding. No covalent binding of NO2-OA to HSA was detected by MS/MS. More detailed studies based on MALDI-TOF-MS and Ellman's assay indicated that HSA can be covalently modified in the presence of NO2-OA to a very limited extent. It was also shown that NO2-OA has a higher affinity to HSA than that of OA.

Cubosomal lipid formulation of nitroalkene fatty acids: Preparation, stability and biological effects.

Lipid nitroalkenes - nitro-fatty acids (NO2-FAs) are formed in vivo via the interaction of reactive nitrogen species with unsaturated fatty acids. The resulting electrophilic NO2-FAs play an important role in redox homeostasis and cellular stress response. This study investigated the physicochemical properties and reactivity of two NO2-FAs: 9/10-nitrooleic acid (1) and its newly prepared 1-monoacyl ester, (E)-2,3-hydroxypropyl 9/10-nitrooctadec-9-enoate (2), both synthesized by a direct radical nitration approach. Compounds 1 and 2 were investigated in an aqueous medium and after incorporation into lipid nanoparticles prepared from 1-monoolein, cubosomes 1@CUB and 2@CUB. Using an electrochemical analysis and LC-MS, free 1 and 2 were found to be unstable under acidic conditions, and their degradation occurred in an aqueous environment within a few minutes or hours. This degradation was associated with the production of the NO radical, as confirmed by fluorescence assay. In contrast, preparations 1@CUB and 2@CUB exhibited a significant increase in the stability of the loaded 1 and 2 up to several days to weeks. In addition to experimental data, density functional theory-based calculation results on the electronic structure and structural variability (open and closed configuration) of 1 and 2 were obtained. Finally, experiments with a human HaCaT keratinocyte cell line demonstrated the ability of 1@CUB and 2@CUB to penetrate through the cytoplasmic membrane and modulate cellular pathways, which was exemplified by the Keap1 protein level monitoring. Free 1 and 2 and the cubosomes prepared from them showed cytotoxic effect on HaCaT cells with IC50 values ranging from 1 to 8 µM after 24 h. The further development of cubosomal preparations with embedded electrophilic NO2-FAs may not only contribute to the field of fundamental research, but also to their application using an optimized lipid delivery vehicle.

Flavin-Helicene Amphiphilic Hybrids: Synthesis, Characterization, and Preparation of Surface-Supported Films.

This work reports on the preparation and structural characterization of flavo[7]helicene 1 (flavin-[7]helicene conjugate), which was subsequently characterized at the molecular level in either an aqueous environment or an organic phase, at the supramolecular level in the form of polymeric layers, and also embedded in a lipidic mesophase environment to study the resulting properties of such a hybrid relative to its parent molecules. The flavin benzo[g]pteridin-2,4-dione (isoalloxazine) was selected for conjugation because of its photoactivity and reversible redox behavior. Compound 1 was prepared from 2-nitroso[6]helicene and 6-methylamino-3-methyluracil, and characterized using common structural and spectroscopic tools: circular dichroism (CD), circularly polarized luminescence (CPL) spectroscopy, cyclic voltammetry (CV), and DFT quantum calculations. In addition, a methodology that allows the loading of 1 enantiomers into an internally nanostructured lipid (1-monoolein) matrix was developed.

Antioxidant function of phytocannabinoids: Molecular basis of their stability and cytoprotective properties under UV-irradiation.

In this contribution, a comprehensive study of the redox transformation, electronic structure, stability and photoprotective properties of phytocannabinoids is presented. The non-psychotropic cannabidiol (CBD), cannabigerol (CBG), cannabinol (CBN), cannabichromene (CBC), and psychotropic tetrahydrocannabinol (THC) isomers and iso-THC were included in the study. The results show that under aqueous ambient conditions at pH 7.4, non-psychotropic cannabinoids are slight or moderate electron-donors and they are relatively stable, in the following order: CBD > CBG ≥ CBN > CBC. In contrast, psychotropic Δ9-THC degrades approximately one order of magnitude faster than CBD. The degradation (oxidation) is associated with the transformation of OH groups and changes in the double-bond system of the investigated molecules. The satisfactory stability of cannabinoids is associated with the fact that their OH groups are fully protonated at pH 7.4 (pKa is ≥ 9). The instability of CBN and CBC was accelerated after exposure to UVA radiation, with CBD (or CBG) being stable for up to 24 h. To support their topical applications, an in vitro dermatological comparative study of cytotoxic, phototoxic and UVA or UVB photoprotective effects using normal human dermal fibroblasts (NHDF) and keratinocytes (HaCaT) was done. NHDF are approx. twice as sensitive to the cannabinoids' toxicity as HaCaT. Specifically, toxicity IC50 values for CBD after 24 h of incubation are 7.1 and 12.8 µM for NHDF and HaCaT, respectively. None of the studied cannabinoids were phototoxic. Extensive testing has shown that CBD is the most effective protectant against UVA radiation of the studied cannabinoids. For UVB radiation, CBN was the most effective. The results acquired could be used for further redox biology studies on phytocannabinoids and evaluations of their mechanism of action at the molecular level. Furthermore, the UVA and UVB photoprotectivity of phytocannabinoids could also be utilized in the development of new cannabinoid-based topical preparations.

Cysteamine assay for the evaluation of bioactive electrophiles.

Covalent modifications of thiol and amine groups may control the function of proteins involved in the regulatory and signaling pathways of the cell. In this study, we developed a simple cysteamine assay which can be used to study the reactivity of electrophilic compounds towards primary amine and thiol groups in an aqueous environment. The detection principle is based on the electrochemical, photometrical and mass spectrometric analyses of cysteamine (2-aminoethanethiol) as the molecular probe. This technique is useful for studying the reaction kinetics of electrophiles with thiol (SH) and amino (NH2) groups. The decrease in analytical responses of cysteamine was monitored to evaluate the reactivity of three electrophilic activators of the Nrf2 pathway, which mediates the cellular stress response. The SH-reactivity under cell-free conditions of the tested electrophiles decreased in the following order: 4-hydroxy-2-nonenal ≥ nitro-oleic acid > sulforaphane. However, as shown in RAW264.7 cells, the tested compounds activated Nrf2-dependent gene expression in the opposite order: sulforaphane > nitro-oleic acid ≥ 4-hydroxy-2-nonenal. Although other factors in addition to chemical reactivity play a role in biological systems, we conclude that this cysteamine assay is a useful tool for screening potentially bioactive electrophiles and for studying their reactivity at a molecular level.

Electrophilic characteristics and aqueous behavior of fatty acid nitroalkenes.

Fatty acid nitroalkenes (NO2-FA) are endogenously-generated products of the reaction of metabolic and inflammatory-derived nitrogen dioxide (.NO2) with unsaturated fatty acids. These species mediate signaling actions and induce adaptive responses in preclinical models of inflammatory and metabolic diseases. The nitroalkene substituent possesses an electrophilic nature, resulting in rapid and reversible reactions with biological nucleophiles such as cysteine, thus supporting post-translational modifications (PTM) of proteins having susceptible nucleophilic centers. These reactions contribute to enzyme regulation, modulation of inflammation and cell proliferation and the regulation of gene expression responses. Herein, focus is placed on the reduction-oxidation (redox) characteristics and stability of specific NO2-FA regioisomers having biological and clinical relevance; nitro-oleic acid (NO2-OA), bis-allylic nitro-linoleic acid (NO2-LA) and the conjugated diene-containing nitro-conjugated linoleic acid (NO2-cLA). Cyclic and alternating-current voltammetry and chronopotentiometry were used to the study of reduction potentials of these NO2-FA. R-NO2 reduction was observed around -0.8 V (vs. Ag/AgCl/3 M KCl) and is related to relative NO2-FA electrophilicity. This reduction process could be utilized for the evaluation of NO2-FA stability in aqueous milieu, shown herein to be pH dependent. In addition, electron paramagnetic resonance (EPR) spectroscopy was used to define the stability of the nitroalkene moiety under aqueous conditions, specifically under conditions where nitric oxide (.NO) release could be detected. The experimental data were supported by density functional theory calculations using 6-311++G (d,p) basis set and B3LYP functional. Based on experimental and computational approaches, the relative electrophilicities of these NO2-FA are NO2-cLA >> NO2-LA > NO2-OA. Micellarization and vesiculation largely define these biophysical characteristics in aqueous, nucleophile-free conditions. At concentrations below the critical micellar concentration (CMC), monomeric NO2-FA predominate, while at greater concentrations a micellar phase consisting of self-assembled lipid structures predominates. The CMC, determined by dynamic light scattering in 0.1 M phosphate buffer (pH 7.4) at 25 °C, was 6.9 (NO2-LA) 10.6 (NO2-OA) and 42.3 µM (NO2-cLA), respectively. In aggregate, this study provides new insight into the biophysical properties of NO2-FA that are important for better understanding the cell signaling and pharmacological potential of this class of mediators.

Sensors and microarrays in protein biomarker monitoring: an electrochemical perspective spots.

The development of clinically applicable portable sensors and multiplex protein biomarker assays is one of the most important goals of laboratory medicine today. Sensing strategies based on electrochemical devices are discussed in this overview, with special emphasis on detection principles derived from voltammetry, electrogenerated chemiluminescence, bipolar electrochemistry and impedance-based measurements. Up-to-date examples of electrochemical methods in biomedical research and development are highlighted here, including critical evaluation and future directions of the analysis, development and validation of new protein biomarkers.

Chiral Electrochemistry: Anodic Deposition of Enantiopure Helical Molecules.

Chirality is a fascinating phenomenon for recent electrochemistry and materials research, allowing for the preparation of detection platforms based on analyte enantiodiscrimination and the development of advanced chiroptical devices and chiral electrodes. In this Viewpoint, we highlight new directions in the field of chiral helical polyaromatic molecules (mainly helicenes) that are useful for the preparation of optically and redox-active polymers and/or self-assembled thin layers, nanostructures and functional electrode surfaces. Instead of the previously reported chiral materials prepared by molecular imprinting, a concept based on the preparation of inherently chiral helicene-based materials with (opto)electrochemical applicability is presented. A short overview of well-established electrochemical methods for the research of chiral molecules is also outlined.

Analytical techniques for multiplex analysis of protein biomarkers.

INTRODUCTION: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. AREAS COVERED: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. EXPERT COMMENTARY: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine.