Global analysis of cytosine and adenine DNA modifications across the tree of life.

Interpreting the function and metabolism of enzymatic DNA modifications requires both position-specific and global quantities. Sequencing-based techniques that deliver the former have become broadly accessible, but analytical methods for the global quantification of DNA modifications have thus far been applied mostly to individual problems. We established a mass spectrometric method for the sensitive and accurate quantification of multiple enzymatic DNA modifications. Then, we isolated DNA from 124 archean, bacterial, fungal, plant, and mammalian species, and several tissues and created a resource of global DNA modification quantities. Our dataset provides insights into the general nature of enzymatic DNA modifications, reveals unique biological cases, and provides complementary quantitative information to normalize and assess the accuracy of sequencing-based detection of DNA modifications. We report that only three of the studied DNA modifications, methylcytosine (5mdC), methyladenine (N6mdA) and hydroxymethylcytosine (5hmdC), were detected above a picomolar detection limit across species, and dominated in higher eukaryotes (5mdC), in bacteria (N6mdA), or the vertebrate central nervous systems (5hmdC). All three modifications were detected simultaneously in only one of the tested species, Raphanus sativus. In contrast, these modifications were either absent or detected only at trace quantities, across all yeasts and insect genomes studied. Further, we reveal interesting biological cases. For instance, in Allium cepa, Helianthus annuus, or Andropogon gerardi, more than 35% of cytosines were methylated. Additionally, next to the mammlian CNS, 5hmdC was also detected in plants like Lepidium sativum and was found on 8% of cytosines in the Garra barreimiae brain samples. Thus, identifying unexpected levels of DNA modifications in several wild species, our resource underscores the need to address biological diversity for studying DNA modifications.

N/O→B dative bonds supplemented by N-HN/HC hydrogen bonds make BN-cages an attractive candidate for DNA-nucleobase adsorption - an MP2 prediction.

The response of B12N12-nanocages towards DNA-nucleobases (adenine, guanine, cytosine, and thymine) is investigated using MP2 and DFT (M06-2X) levels of theory with the 6-311+G** basis set. Multiple BN-cage-nucleobase structures for each nucleobase emerged depending on the number of Lewis base centers of nucleobases. The main source of stability of these complexes is the N/O→B dative bond, where the N or O atom of nucleobases donates the lone-pair electron to one of the boron atoms of the nanocage. Nitrogen atoms of the BN-cage, adjacent to the B-site forming dative bond, act as a proton acceptor to form multiple (N-HN and N-HC) hydrogen bonds, where proton-donors NH and CH are part of nucleobases. MP2/6-311+G** adsorption energies are -43.1, -43.4 and -45.3 kcal mol-1 (B12N12-adenine), -37.1, -41.9 and -43.3 kcal mol-1 (B12N12-guanine), -41.3 and -43.4 (B12N12-cytosine), and -29.3 and -31.3 (B12N12-thymine). Similar adsorption energies were recorded for larger BN-fullerenes-nucleobases, namely B16N16 and B24N24. Changes in adsorption energies and structures of these nano-bio-hybrid materials in aqueous media are also discussed. Computationally cost-effective MP2 single point calculations at the M06-2X optimized geometries were found to be reliable in predicting adsorption energies. The effect of the BN-network and H-bonds on the adsorption process is assessed by comparing the results with simple BH3-nucleobase models. BSSE correction to the adsorption energy is not recommended.

Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups.

A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

Highly Enhanced Antitumor Immunity by a Three-Barreled Strategy of the l-Arginine-Promoted Nanovaccine and Gene-Mediated PD-L1 Blockade.

Weak T cell responses and immune checkpoints within tumors could be two key factors for limiting antitumor efficacy in the field of cancer immunotherapy. Thus, the combined strategy of tumor vaccines and immune checkpoint blockade has been widely studied and expected to boost antitumor immune responses. Herein, we first developed a two-barreled strategy to combine the nanovaccine with a gene-mediated PD-L1 blockade. On the one hand, polyethyleneimine (PEI) worked as a vaccine carrier to codeliver the antigen ovalbumin (OVA) and the adjuvant unmethylated cytosine-phosphate-guanine (CpG) to formulate the PEI/OVA/CpG nanovaccine through electrostatic binding, which realized both dendritic cell activation and antigen cross-presentation enhancement. On the other hand, the PD-L1 silence gene was loaded by PEI to form PEI/pshPD-L1 complexes, which were further in situ shielded by aldehyde-modified polyethylene glycol (OHC-PEG-CHO) via pH-responsive Schiff base bonds. The formed pshPD-L1@NPs could decrease PD-L1 expression on the tumor cells. However, such a combined two-barreled strategy improved feebly for tumor inhibition in comparison with monotherapy, exhibiting the antagonistic effect, which might be due to the limited T cell response enhancement in the tumor microenvironment. To solve this problem, we have further developed a three-barreled strategy to combine oral administration of l-arginine, which worked as an amplifier to induce robust T cell response enhancement, without causing the upregulation of other negative immune regulators. Superior antitumor behavior and tumor rechallenge protection were realized by the three-barreled strategy in B16F10-OVA (B16-OVA)-bearing mice. The unique three-barreled strategy we developed might offer a novel clinical therapeutic treatment.

A Cu9S5 nanoparticle-based CpG delivery system for synergistic photothermal-, photodynamic- and immunotherapy.

Despite its great potential in cancer therapy, phototherapy, including photothermal therapy (PTT) and photodynamic therapy (PDT), often cause metastasis of tumors. Immunotherapy has revolutionized the cancer treatment owing to the capability of activating immune system to eliminate tumors. However, the integration of phototherapy and immunotherapy in a single nanoagent for cancer therapy is still a challenging task. Here, we fabricated (Cu9S5@mSiO2-PpIX@MnO2@CpG (CSPM@CpG)) as a synergistic therapeutic model for phototherapy enhanced immunotherapy. The intracellular uptake of cytosine-phosphate-guanine (CpG) promoted the infiltration of cytotoxic T lymphocytes (CTLs) in tumor tissue, further stimulating the production of interferon gamma (IFN-γ) and remarkably elevating the immune response level. Excellent anti-tumor effects have been achieved by synergistic PTT/PDT/immunotherapy. The metastasis of tumors was effectively inhibited by the immune response of CpG. Thus, our proposed work provides a strategy to combine phototherapy with immunotherapy to enhance the therapeutic efficiency and further inhibit metastasis of tumors.

Enhanced Performance of DNA Methylation Markers by Simultaneous Measurement of Sense and Antisense DNA Strands after Cytosine Conversion.

BACKGROUND: Most existing DNA methylation-based methods for detection of circulating tumor DNA (ctDNA) are based on conversion of unmethylated cytosines to uracil. After conversion, the 2 DNA strands are no longer complementary; therefore, targeting only 1 DNA strand merely utilizes half of the available input DNA. We investigated whether the sensitivity of methylation-based ctDNA detection strategies could be increased by targeting both DNA strands after bisulfite conversion. METHODS: Dual-strand digital PCR assays were designed for the 3 colorectal cancer (CRC)-specific methylation markers KCNQ5, C9orf50, and CLIP4 and compared with previously reported single-strand assays. Performance was tested in tumor and leukocyte DNA, and the ability to detect ctDNA was investigated in plasma from 43 patients with CRC stages I to IV and 42 colonoscopy-confirmed healthy controls. RESULTS: Dual-strand assays quantified close to 100% of methylated control DNA input, whereas single-strand assays quantified approximately 50%. Furthermore, dual-strand assays showed a 2-fold increase in the number of methylated DNA copies detected when applied to DNA purified from tumor tissue and plasma from CRC patients. When the results of the 3 DNA methylation markers were combined into a ctDNA detection test and applied to plasma, the dual-strand assay format detected 86% of the cancers compared with 74% for the single-strand assay format. The specificity was 100% for both the dual- and single-strand test formats. CONCLUSION: Dual-strand assays enabled more sensitive detection of methylated ctDNA than single-strand assays.

Functional Systems Derived from Nucleobase Self-assembly.

Dynamic and reversible non-covalent interactions endow synthetic systems and materials with smart adaptive functions that allow them to response to diverse stimuli, interact with external agents, or repair structural defects. Inspired by the outstanding performance and selectivity of DNA in living systems, scientists are increasingly employing Watson-Crick nucleobase pairing to control the structure and properties of self-assembled materials. Two sets of complementary purine-pyrimidine pairs (guanine:cytosine and adenine:thymine(uracil)) are available that provide selective and directional H-bonding interactions, present multiple metal-coordination sites, and exhibit rich redox chemistry. In this review, we highlight several recent examples that profit from these features and employ nucleobase interactions in functional systems and materials, covering the fields of energy/electron transfer, charge transport, adaptive nanoparticles, porous materials, macromolecule self-assembly, or polymeric materials with adhesive or self-healing ability.

Enhanced Immunostimulatory Activity of a Cytosine-Phosphate-Guanosine Immunomodulator by the Assembly of Polymer DNA Wires and Spheres.

Unmethylated cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides are immunostimulatory nucleic acids wildly utilized as adjuvants or for vaccines to treat diseases. However, there is a lack of simple and efficient vectors for CpG oligodeoxynucleotide delivery with long-lasting immune stimulation. Herein, self-assembled polymer wires consisting of CpG motifs by hybridization chain reaction were constructed with excellent biocompatibility and immunostimulatory activity. The designed polymer DNA wires acted as programmable multivalent immunoadjuvants and triggered immune response, stimulated pro-inflammatory cytokine secretion, and induced the apoptosis of cancer cells. More strikingly, polymer nanospheres assembled from the polymer DNA wires and cationic poly-l-lysine further improved cellular uptake and continuously stimulate the lysosomal Toll-like receptor 9 of immune cells, thereby remarkably enhancing the activation of immune cells. These results demonstrated that self-assembled polymer DNA nanoassemblies with multivalent CpG could trigger strong immune response and further induce cancer cell death.

A cytosine-rich hairpin DNA loaded with silver nanoclusters as a fluorescent probe for uranium(IV) and mercury(II) ions.

A dually responsive fluorescent probe for determination of U(IV) and mercury(II) ions was synthesized. The probe consists of a cytosine-rich hairpin DNA loaded with silver nanoclusters (DNA-AgNCs). The fluorescence of the AgNCs is found to be quenched by UO2(II) at pH 5.0 and Hg(II) at pH 7.0 due to combined static and dynamic quenching. Under the optimal conditions, the green fluorescence of the DNA-AgNCs, best measured at excitation/emission wavelengths of 420/525 nm, decreases in the 4.0 to 75 pM UO2(II) concentration range, and in the 0.3 to 8.0 nM Hg(II) concentration range. The respective detection limits are as low as 1.8 pM and 0.1 nM. The method was successfully applied to the determination of UO2(II) and Hg(II) in (spiked) pond and taps waters and in soil extracts. Graphical abstract A label-free DNA was designed to synthesize green-fluorescent silver nanoclusters (AgNCs) and used for rapid dual detection of uranyl ions (UO2(II)) at pH 5.0 and of mercury ions (Hg(II)) at pH 7.0 in environmental samples.

DFT and MD Studies of Formaldehyde-Derived DNA Adducts: Molecular-Level Insights into the Differential Mispairing Potentials of the Adenine, Cytosine, and Guanine Lesions.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone is a potent nicotine-based carcinogen that generates many DNA lesions, including the HOCH2-C, HOCH2-G, and HOCH2-A hydroxymethyl adducts. Despite all lesions containing an altered exocyclic amino group, which allows the hydroxymethyl group to be directed away from the Watson-Crick binding face, only the most persistent adenine adduct is mutagenic. As a first step toward understanding this differential mutagenicity, density functional theory (DFT) and molecular dynamics (MD) simulations were used to gain atomic-level structural details of these DNA damage products. DFT calculations reveal that all three lesions exhibit conformational diversity. However, regardless of the hydroxymethyl-nucleobase orientation, both DFT and MD simulations highlight that HOCH2-C and HOCH2-G form pairs with the canonical complementary base (G and C, respectively) that are structural and energetically preferred over mispairs. In contrast, depending on the hydroxymethyl-nucleobase orientation, the Watson-Crick HOCH2-A:T pair can become significantly destabilized relative to undamaged A:T. As a result, HOCH2-A mispairs with G, C, and A are energetically accessible and maintain key geometrical features of canonical DNA. Overall, our data directly correlate with the reported differential mutagenicity of the hydroxylmethyl lesions and will encourage future studies to further uncover the cellular impact of the most persistent adenine lesion.

Nano-engineered flavonoids for cancer protection.

Diet and environment are two critical regulators that influence an individual's epigenetic profile. Besides the anterograde signaling, mitochondria act as a key regulator of epigenetic alterations in cancer either by controlling the concentration of the cofactors, activity of vital enzymes or by affecting the transcription of NF-kappaB and associated signaling molecules. As epigenetic modifications are the major drivers of aberrant gene expression, designing novel nutri-epigenomic strategies to modulate reversible epigenetic modifications will be important for effective cancer protection. In this regard, nutraceuticals such as flavonoids holds significant promise to modulate the epigenome through a network of interconnected anti-redox mechanisms. However, low solubility, rapid metabolism and poor absorption of flavonoids in gastrointestinal tract hinder their use in clinical settings. Therefore, it is imperative to develop nano-engineered systems which could considerably improve the targeted delivery of these bioactive compounds with better efficacy and pharmacokinetic properties. Concerted efforts in nano-engineering of flavonoids using polymer, lipid and complexation based approaches could provide successful bench-to-bedside translation of flavonoids as broad spectrum anti-cancer agents.

Resolution of the DNA methylation state of single CpG dyads using in silico strand annealing and WGBS data.

Whole-genome bisulfite sequencing (WGBS) has been widely used to quantify cytosine DNA methylation frequency in an expanding array of cell and tissue types. Because of the denaturing conditions used, this method ultimately leads to the measurement of methylation frequencies at single cytosines. Hence, the methylation frequency of CpG dyads (two complementary CG dinucleotides) can be only indirectly inferred by overlaying the methylation frequency of two cytosines measured independently. Furthermore, hemi-methylated CpGs (hemiCpGs) have not been previously analyzed in WGBS studies. We recently developed in silico strand annealing (iSA), a bioinformatics method applicable to WGBS data, to resolve the methylation status of CpG dyads into unmethylated, hemi-methylated, and methylated. HemiCpGs account for 4-20% of the DNA methylome in different cell types, and some can be inherited across cell divisions, suggesting a role as a stable epigenetic mark. Therefore, it is important to resolve hemiCpGs from fully methylated CpGs in WGBS studies. This protocol describes step-by-step commands to accomplish this task, including dividing alignments by strand, pairing alignments between strands, and extracting single-fragment methylation calls. The versatility of iSA enables its application downstream of other WGBS-related methods such as nasBS-seq (nascent DNA bisulfite sequencing), ChIP-BS-seq (ChIP followed by bisulfite sequencing), TAB-seq, oxBS-seq, and fCAB-seq. iSA is also tunable for analyzing the methylation status of cytosines in any sequence context. We exemplify this flexibility by uncovering the single-fragment non-CpG methylome. This protocol provides enough details for users with little experience in bioinformatic analysis and takes 2-7 h.

Separation-free single-base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites.

A separation-free single-base extension (SBE) assay utilizing fluorescence resonance energy transfer (FRET) was developed for rapid and convenient interrogation of DNA methylation status at specific cytosine and guanine dinucleotide sites. In this assay, the SBE was performed in a tube using an allele-specific oligonucleotide primer (i.e., extension primer) labeled with Cy3 as a FRET donor fluorophore at the 5'-end, a nucleotide terminator (dideoxynucleotide triphosphate) labeled with Cy5 as a FRET acceptor, a PCR amplicon derived from bisulfite-converted genomic DNA, and a DNA polymerase. A single base-extended primer (i.e., SBE product) that was 5'-Cy3- and 3'-Cy5-tagged was formed by incorporation of the Cy5-labeled terminator into the 3'-end of the extension primer, but only if the terminator added was complementary to the target nucleotide. The resulting SBE product brought the Cy3 donor and the Cy5 acceptor into close proximity. Illumination of the Cy3 donor resulted in successful FRET and excitation of the Cy5 acceptor, generating fluorescence emission from the acceptor. The capacity of the developed assay to discriminate as low as 10% methylation from a mixture of methylated and unmethylated DNA was demonstrated at multiple cytosine and guanine dinucleotide sites.

Study of Photochemical Cytosine to Uracil Transition via Ultrafast Photo-Cross-Linking Using Vinylcarbazole Derivatives in Duplex DNA.

Gene therapies, including genome editing, RNAi, anti-sense technology and chemical DNA editing are becoming major methods for the treatment of genetic disorders. Techniques like CRISPR-Cas9, zinc finger nuclease (ZFN) and transcription activator-like effector-based nuclease (TALEN) are a few such enzymatic techniques. Most enzymatic genome editing techniques have their disadvantages. Thus, non-enzymatic and non-invasive technologies for nucleic acid editing has been reported in this study which might possess some advantages over the older methods of DNA manipulation. 3-cyanovinyl carbazole (CNVK) based nucleic acid editing takes advantage of photo-cross-linking between a target pyrimidine and the CNVK to afford deamination of cytosine and convert it to uracil. This method previously required the use of high temperatures but, in this study, it has been optimized to take place at physiological conditions. Different counter bases (inosine, guanine and cytosine) complementary to the target cytosine were used, along with derivatives of CNVK (NH2VK and OHVK) to afford the deamination at physiological conditions.

Sonochemical incorporated of cytosine in Cu-H2bpdc as an antibacterial agent against standard and clinical strains of Proteus mirabilis with rsbA gene.

The cytosine embedded copper based metal-organic framework (Bio-MOF) was synthesized by facile one-step sonochemical method by simply mixing of 4-4, biphenyldicarboxylic, cytosine and copper nitrate (Bio-Cu-H2bpdc-Cy). The prepared bio-MOF was characterized by XRD, FTIR and FE-SEM techniques. The effect of Cu-H2bpdc-Cy on the expression of the rsbA gene was evaluated in the clinical and standard Proteus mirabilis and study of MIC of Cu-H2bpdc-Cy by microdilution against them that have the rsbA gene. According to different concentrations of MIC, MBC concentrations was cultured on blood agar culture medium. Regarding to the concentration of MIC, gene expression changes were obtained by real-time PCR. MIC for standard and clinical strains of Proteus mirabilis was 1.6 and 1.8 mg/ml, and also MBC was obtained to be 1.8 and 2.0 mg/ml, respectively. Finally, in the real time PCR method, expression of the rsbA gene in presences of bio-Cu-H2bpdc-Cy was reduced, but has no effect on the gene expression of the Housekeeping DNA Gyrase-B gene. Considering the effect of Cu-H2bpdc-Cy on the rsbA gene in Proteus mirabilis bacteria, it is possible to use of Cu-H2bpdc-Cy agent as a therapeutic supplement against this bacterium.

The origin of efficient triplet state population in sulfur-substituted nucleobases.

Elucidating the photophysical mechanisms in sulfur-substituted nucleobases (thiobases) is essential for designing prospective drugs for photo- and chemotherapeutic applications. Although it has long been established that the phototherapeutic activity of thiobases is intimately linked to efficient intersystem crossing into reactive triplet states, the molecular factors underlying this efficiency are poorly understood. Herein we combine femtosecond transient absorption experiments with quantum chemistry and nonadiabatic dynamics simulations to investigate 2-thiocytosine as a necessary step to unravel the electronic and structural elements that lead to ultrafast and near-unity triplet-state population in thiobases in general. We show that different parts of the potential energy surfaces are stabilized to different extents via thionation, quenching the intrinsic photostability of canonical DNA and RNA nucleobases. These findings satisfactorily explain why thiobases exhibit the fastest intersystem crossing lifetimes measured to date among bio-organic molecules and have near-unity triplet yields, whereas the triplet yields of canonical nucleobases are nearly zero.

SiO2 nanoparticles modified CPE as a biosensor for determination of i-motif DNA/Tamoxifen interaction.

Cytosine-rich DNA sequences can form a highly ordered structure known as i-motif in slightly acidic solutions. The stability of the folded i-motif structure is a good strategy to inhibit the telomerase reaction in cancer cells. The electrochemical biosensor was prepared by modifying carbon paste electrode with SiO2 nanoparticles to investigate drugs which can stabilize this structure. Tamoxifen (Tam), an antiestrogen hormonal agent for treatment of breast cancer, was chosen as the model ligand and its interaction with i-motif structure was examined. The interaction between i-motif DNA and Tam was studied in PBS buffer and [Fe(CN)6](3-) through the cyclic voltammetry and square wave voltammetry methods. The oxidation peak of Tam, due to the i-motif DNA/Tam interaction, was observed after i-motif immobilized on the surface of the electrode. The i-motif formation was investigated by circular dichroism spectroscopy and the results showed that this structure can certainly be made with pH around 4.5, but its stability reduced by going to the more alkaline pH. The selectivity which was studied in the presence of complementary strand demonstrated that i-motif structure could be stabilized in acidic pH even in the presence of its complementary strand.

The lipid moiety of brincidofovir is required for in vitro antiviral activity against Ebola virus.

Brincidofovir (BCV) is the 3-hexadecyloxy-1-propanol (HDP) lipid conjugate of the acyclic nucleoside phosphonate cidofovir (CDV). BCV has established broad-spectrum activity against double-stranded DNA (dsDNA) viruses; however, its activity against RNA viruses has been less thoroughly evaluated. Here, we report that BCV inhibited infection of Ebola virus in multiple human cell lines. Unlike the mechanism of action for BCV against cytomegalovirus and other dsDNA viruses, phosphorylation of CDV to the diphosphate form appeared unnecessary. Instead, antiviral activity required the lipid moiety and in vitro activity against EBOV was observed for several HDP-nucleotide conjugates.

Gold nanocluster-based vaccines for dual-delivery of antigens and immunostimulatory oligonucleotides.

We here report a facile one-pot synthesis of fluorescent gold nanoclusters (AuNCs) via the peptide biomineralization method, which can elicit specific immunological responses. The as-prepared peptide-protected AuNCs (peptide-AuNCs) display strong red fluorescence, and more importantly, as compared to the peptide alone, the immune stimulatory ability of the resulting peptide-AuNCs can not only be retained, but can also be efficaciously enhanced. Moreover, through a dual-delivery of antigen peptides and cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs), the as-prepared peptide-AuNC-CpG conjugates can also act as smart self-vaccines to assist in the generation of high immunostimulatory activity, and be applied as a probe for intracellular imaging. Both in vitro and in vivo studies provide strong evidence that the AuNC-based vaccines may be utilized as safe and efficient immunostimulatory agents that are able to prevent and/or treat a variety of ailments.

Lower methylation of glucocorticoid receptor gene promoter 1F in peripheral blood of veterans with posttraumatic stress disorder.

BACKGROUND: Enhanced glucocorticoid receptor (GR) sensitivity is present in people with posttraumatic stress disorder (PTSD), but the molecular mechanisms of GR sensitivity are not understood. Epigenetic factors have emerged as one potential mechanism that account for how trauma exposure leads to sustained PTSD symptoms given that PTSD develops in only a subset of trauma survivors. METHODS: Cytosine methylation of a relevant promoter of the GR gene (NR3C1-1F promoter) and three functional neuroendocrine markers of hypothalamic-pituitary-adrenal axis function were examined in a sample of 122 combat veterans. RESULTS: Lower NR3C1-1F promoter methylation in peripheral blood mononuclear cells (PBMCs) was observed in combat veterans with PTSD compared with combat-exposed veterans who did not develop PTSD. NR3C1-1F promoter methylation was also associated with three functional measures of glucocorticoid activity that have been associated with PTSD in combat veterans: PBMCs' lysozyme inhibition on the lysozyme suppression test, plasma cortisol decline on the low-dose (.50 mg) dexamethasone suppression test, and 24-hour urinary cortisol excretion. Finally, NR3C1-1F promoter methylation was inversely correlated with clinical markers and symptoms associated with PTSD. CONCLUSIONS: Alterations in NR3C1-1F promoter methylation may reflect enduring changes resulting from combat exposure that lead to functional neuroendocrine alterations. Because epigenetic measures are thought to reflect enduring effects of environmental exposures, they may be useful in distinguishing combat-exposed veterans who do or do not develop PTSD.