Duchenne muscular dystrophy (DMD) is a neuromuscular disease characterized by progressive weakness of the skeletal and cardiac muscles. This X-linked disorder is caused by open reading frame disrupting mutations in the DMD gene, resulting in strong reduction or complete absence of dystrophin protein. In order to use dystrophin as a supportive or even surrogate biomarker in clinical studies on investigational drugs aiming at correcting the primary cause of the disease, the ability to reliably quantify dystrophin expression in muscle biopsies of DMD patients pre- and post-treatment is essential. Here we demonstrate the application of the ProteinSimple capillary immunoassay (Wes) method, a gel- and blot-free method requiring less sample, antibody and time to run than conventional Western blot assay. We optimized dystrophin quantification by Wes using 2 different antibodies and found it to be highly sensitive, reproducible and quantitative over a large dynamic range. Using a healthy control muscle sample as a reference and α-actinin as a protein loading/muscle content control, a panel of skeletal muscle samples consisting of 31 healthy controls, 25 Becker Muscle dystrophy (BMD) and 17 DMD samples was subjected to Wes analysis. In healthy controls dystrophin levels varied 3 to 5-fold between the highest and lowest muscle samples, with the reference sample representing the average of all 31 samples. In BMD muscle samples dystrophin levels ranged from 10% to 90%, with an average of 33% of the healthy muscle average, while for the DMD samples the average dystrophin level was 1.3%, ranging from 0.7% to 7% of the healthy muscle average. In conclusion, Wes is a suitable, efficient and reliable method for quantification of dystrophin expression as a biomarker in DMD clinical drug development. Duchenne muscular dystrophy (DMD) is a neuromuscular disease that affects 1 in 5000–6000 newborn boys  and is cCurrently, several potential therapies are being explored and developed to restore dystrophin in muscle of DMD patients, including gene therapy, stop codon read through, exon skip inducing antisense oligonucleotides (AONs) and CRISPR/Cas9 technology. To be able to monitor the efficiency of these therapies, reliable quantification of dystrophin expression as a supportive biomarker is essential. The use of dystrophin as a surrogate biomarker is under debate as it remains unresolved how much dystrophin is required for a clinically relevant improvement of muscle function. Several attempts have been made to correlate dystrophin levels in BMD patients to clinical severity, but this is hampered by the highly variable nature of the mutations in BMD, affecting different functional domains of dystrophin. BMD mutations shortening the central rod domain of dystrophin are generally correlated with a less severe phenotype, despite giving rise to a wide variety in dystrophin levels, while in-frame deletions affecting the N-terminal actin-binding domain generally result in a more severe disease course, indicating that not just levels of dystrophin but also the composition of the mutant dystrophin protein in BMD affect the clinical outcome  method for dystrophin detection. Wes has been shown to sensitively and reproducibly detect proteins of various sizes, while needing only small amounts of sample and taking much less time than WB ,24. In Whttp://asterand.com/Asterand/about/ethics.htm; control19), Myobank AFM , Vrije Universiteit Medical Center  and Leiden University Medical Center . All BMD biopsies were obtained from Leiden University Medical Center . DMD biopsies were obtained from patients participating in BioMarin clinical studies (NCT02958202 (BMN044); NCT01826474 (BMN045); NCT01957059 (BMN053); DMD1-12), University Hospital of Leuven (DMD15-17) and Myobank AFM .Prior to initiation and commencement of this study written informed consent for research use was obtained from all subjects or their legal representatives and all skeletal muscle samples were approved for research use by the local institutional research ethics review boards or medical ethics committees of the University Medical Centres, University Hospitals or Institutes listed below. Human control muscle samples were obtained from a commercial tissue bank  is given in Skeletal muscle biopsies were frozen in 2-methylbutane cooled in liquid nitrogen  or were frozen directly in liquid nitrogen and stored at -80°C or in liquid nitrogen until further use. Protein lysates from muscle tissue were prepared by sectioning or cutting off small pieces of in total ~ 5–10 μg, placing them in a Magnalyser vial without beads  and adding 100 μl Protein Lysis Buffer /8 ml; 5% β-Mercaptoethanol). After briefly spinning down, approximately 15 beads were added to each tube. The samples were then homogenized by 2–4 cycles  in the MagNA Lyser Instrument (Roche 03358976001) and spun down for 5 min at 13000 rpm. The supernatant was supplemented with glycerol  and then samples were stored at -80°C until further use.To measure total protein concentration, 20x dilutions of the lysates were precipitated using the Compat-Able Protein Assay Preparation Reagent kit (Thermo Scientific 23215) to eliminate interfering substances from protein assay samples and make them compatible with the BCA assay. Concentrations were measured using the Pierce BCA Protein assay kit (Thermo Scientific 23227), according to the manufacturer’s instructions.Wes analysis was performed on a Wes system  according to the manufacturer’s instructions using a 66–440 kDa Separation Module (ProteinSimple SM-W006 or SM-W008) and either the Anti-Rabbit Detection Module (ProteinSimple DM-001) or the Anti-Mouse Detection Module (ProteinSimple DM-002), depending on the primary antibody used. In brief, skeletal muscle samples were diluted to an appropriate concentration  in sample buffer (100x diluted ‘10x Sample Buffer 2’ from the Separation Module), then mixed with Fluorescent Master Mix and heated at 95°C for 5 min. The samples, blocking reagent (antibody diluent), primary antibodies (in antibody diluent), HRP-conjugated secondary antibodies and chemiluminescent substrate were pipetted into the plate (part of Separation Module). Instrument default settings were used: stacking and separation at 475 V for 30 min; blocking reagent for 5 min, primary and secondary antibody both for 30 min; Luminol/peroxide chemiluminescence detection for ~15 min (exposures of 1-2-4-8-16-32-64-128-512s). The resulting electropherograms were inspected to check whether automatic peak detection required any manual correction. Identification of specific peaks becomes more difficult when dystrophin levels are close to background. In particular in DMD samples the background is not only higher due to loading of 10-fold more protein, but also due to the presence of various dystrophin isoforms, and revertant and trace dystrophin with variable size. The following criteria were used to discriminate low dystrophin signals from background: The peak signal-to-noise (S/N) ratio given by the software must be ≥10, and the peak height/baseline ratio  must be ≥3.2 > 0.99, based on 4–6 points in a relevant concentration range.To control for differences in signal between experiments, a 6-point calibration curve of a healthy muscle reference sample was routinely included, usually ranging from 0.008–0.250 μg or 0.004–0.125 μg, depending on the samples analysed. This reference sample was selected from a panel of healthy controls based on it displaying average dystrophin levels with both ab154168 and Mandys106. The following criteria were applied to each calibration curve: the 0.125 μg point must have a chemiluminescence peak of >150.000 arbitrary units (to be determined for each Wes system), and the linearity of the curve must have an RIn addition, to control for muscle content, the sample dilutions used for dystrophin detection were further diluted to a concentration of 2.5 μg/ml total protein (0.0125 μg loaded) and were subsequently analysed for α-actinin levels. For this α-actinin analysis a calibration curve was also included, ranging from 0.0008–0.025 μg of the same healthy control as used for dystrophin analysis. The final muscle content-corrected dystrophin values were generated by first expressing both the dystrophin and α-actinin signals as percentage of control  using the following formula:An overview of the workflow for Wes is depicted in Four different anti-dystrophin antibodies were tested for their signal in Wes, recognising different epitopes . AntibodSamples including a healthy muscle reference sample were diluted to an appropriate concentration with milli-Q, then 4x Laemli Sample Buffer (Biorad) was added (ratio 3:1) and the samples were denatured at 95°C for 5 min. SDS-PAGE was performed in a cold room (4°C). The samples were loaded onto a 3–8% gradient Tris acetate poly-acrylamide gel (Bio-rad Criterion XT) and run for 4 hours at 100V (~50 mA) in Invitrogen 20x Novex Tris-Acetate SDS Running Buffer. The proteins were then blotted on to a 0.2M nitrocellulose membrane, using a Turbo Transfer pack in combination with the Trans Blot Turbo Transfer System (Bio-rad), for 15 min at 25V. The following incubations were performed, with TBST wash-steps in between: 1h blocking at RT (TBS-5% milk powder)—overnight primary antibody incubation in cold room (1/2000 for ab154168 in TBST and 1/125 for Mandys106 in TBST)– 15 min blocking at RT– 1h secondary antibody incubation at RT (1/5000 anti-rabbit-IRDye 800CW for ab154168 and 1/5000 anti-mouse-IRDye 800CW for Mandys106 in TBST). Membranes were scanned on the Odyssey system (Li-COR).To control for loading and muscle content, the blots were re-probed for α-actinin. After dystrophin detection, the membranes were incubated overnight in a cold room with an anti-α-actinin antibody  at a 1/3000 dilution, followed by 1hr at RT with secondary antibody anti-rabbit-IRDye 800CW (Li-Cor 925–32211) at a 1/5000 dilution. Protein bands were visualized with Odyssey. As this was a mostly qualitative experiment, no calculations were made .To bridge from traditional WB analysis to the more state of the art Wes analysis, skeletal muscle samples from a healthy control, a BMD patient and 3 DMD patients with different mutations were analysed using both methods. Using WB the full length dystrophin band was visible in the healthy control at the expected size of 427 kDa for antibodies Mandys106 and ab154168 . The dysNext the same samples were subjected to Wes analysis. Wes analysis was much more sensitive, requiring only a fraction of the amount of sample to give a dystrophin signal. A sharply defined chemiluminescent dystrophin signal was quantifiable by calculation of the area under the curve (AUC) in the healthy control and BMD sample, and in 2 of the 3 DMD samples using ab154168 and only ~1 μg of total protein , which iThe dystrophin signal detected by Wes displayed a molecular weight of approximately 300 kDa, which is lower than the predicted 427 kDa for the full-length (muscle) dystrophin isoform. This size discrepancy can be explained by the molecular weight ladder used in Wes, which underestimates molecular weights above 280 kDa . The speWes analysis also allowed for identification of dystrophin isoforms, including the Dp71 isoform which was clearly visible and quantifiable in all 3 of the DMD samples using ab154168 . Using MIn conclusion, Wes analysis is more sensitive than WB and allows for dystrophin quantification in 1 μg of loaded protein from skeletal muscle samples derived from healthy controls, BMD and DMD patients, which is 20-200x less material than required for WB. Obtained chemiluminescent signals are well above background and easily quantifiable by calculation of the AUC.To set up Wes as a new standard for dystrophin quantification, we optimized and validated: 1) the optimal antibody dilution to be used to achieve a maximum dystrophin signal at a fixed amount of protein loaded and 2) the range of protein to be loaded at the optimal antibody dilution to ensure linearity of the dystrophin signal. Two antibodies were selected: rabbit monoclonal ab154168 because of its C-terminal epitope and strong signal with very little background, and Mandys106 because of its epitope in the central rod domain and specificity for human dystrophin. The antibodies were titrated using a healthy control sample and 2 different DMD samples with either low or (relatively) high dystrophin levels. The optimal antibody concentration was considered to be a saturating dilution in which addition of more antibody would not further increase the signal, implying coverage of all free epitopes. For Mandys106 a 1/50 dilution was required while for ab154168 an antibody dilution of 1/1000 was enough to reach a dystrophin signal plateau . This waNext, the optimal amount of protein to be loaded was determined, using the selected antibody dilutions. In order to define the linear range of detection, a total protein concentration dilution series of the same healthy control and DMD samples was loaded. This experiment showed that Wes is capable of detecting dystrophin at very low concentrations. For the healthy control, chemiluminescence increased linearly with protein concentration from 0.002 μg up to 1.25 μg for Mandys106  and fromSince the amount of protein to be loaded for DMD samples analysis is relatively high for Wes , we investigated whether this may cause aspecific background signal or interfere with sample running efficiency in the capillary. To test the limits of detection while keeping the total protein amount loaded constant, different ratios of a healthy control skeletal muscle protein lysate were spiked into lysate from a DMD patient with low trace dystrophin. When loading 0.125 μg total protein, dystrophin amounts ranging from 1.25% -100% of healthy control could be linearly detected using ab154168  . To see Similar experiments with Mandys106 showed more aspecific background staining, leading to less sensitivity at low dystrophin concentrations. Loading of 0.125 μg gave a linear response for DMD samples spiked with 5%-100% of healthy control . Below tTo determine the reproducibility (precision) of the method, an experiment was set up to address several sources of variability. An important source of intra-assay variation is the pipetting (dilution) error, while inter-assay variation can be caused by inter-operator variation, differences between antibody batches, small differences in Wes performance  and differences between different Wes systems. To test this, 3 different dilutions of the healthy control were made and aliquoted , and designated low, medium or high based on the amount of dystrophin. Three independent dilutions of the low, medium and high samples were prepared per experiment and quantified in duplicate by 2 different operators for both antibodies. These experiments showed that the reproducibility of the assay is high but may be influenced by the (primary) antibody used. The raw chemiluminescence values were higher for Mandys106 than for ab154168, especially for the low sample measurements. The reproducibility was better for ab154168 than for Mandys106 . For ab1For Mandys106 the average duplicate well CV based on chemiluminescence was 3%-15%, with outliers of up to 32% . After cFor dystrophin detection by Western blot, a muscle-specific protein unaffected by DMD is often used to correct for differences in sample loading or in muscle content of the samples. Alpha-actinin is a cytoskeletal actin-binding protein, localizing to the Z-disks in striated, cardiac and smooth muscle cells, where it stabilizes the muscle contractile apparatus . It is nTo allow use of α-actinin as a normalisation protein in Wes, α-actinin antibody ab68167 was optimized similarly to the dystrophin antibodies described above . Since eTo use dystrophin as a biomarker in drug development programs, a standard healthy control sample with a representative (average) dystrophin expression is required to define 100% levels. In a pilot experiment on 14 healthy human muscle biopsies we selected a sample that had average control dystrophin levels and of which sufficient material was available for numerous Wes analyses. Using our optimized Wes protocol we determined the range of dystrophin expression in an extended panel of 31 healthy human muscle biopsies. These biopsies were mostly derived from quadriceps or tibialis muscle and were obtained from both male and female donors ranging from age 22 to 75 years . DystropDystrophin levels were also determined in a panel of 25 BMD tibialis muscle samples with different dystrophin mutations. Dystrophin levels ranged from 10% to 90% of CTRL, with individuals with a deletion in the actin binding domain (exons 2–8) displaying the lowest dystrophin levels . AlthougFinally, a total of 17 DMD skeletal muscle samples were analysed by Wes . These iThe Wes-quantified levels of dystrophin in healthy controls, BMD and DMD samples were subsequently ranked from low to high . While tTo support the use of dystrophin as biomarker in clinical studies on investigational drugs aiming at dystrophin restoration in DMD patients, we have developed a quantitative method based on the ProteinSimple capillary immunoassay (Wes). The assay is highly sensitive and reproducible, linear over a wide dynamic range and with a LLOQ low enough to quantify trace dystrophin levels present in skeletal muscle of DMD patients.The high assay sensitivity was demonstrated by LLOQs well below 1% of a healthy control, as well as the ability to detect differences in dystrophin levels as small as 0.125% of healthy control. The reproducibility for detecting dystrophin is in line with the supplier’s claim of a CV of 15% for intra-assay and 20% for inter-assay variation. While chemiluminescence results may vary between runs, the use of a calibration curve derived from a standard healthy control sample for quantification reduced overall variation to ≤16% (ab154168). This enables sensitive analysis of samples across almost the whole range of dystrophin levels from DMD (if above LLOQ) to healthy control within one experiment.To control for differences in capillary loading and/or muscle content of samples in Wes, a housekeeping protein is essential, preferably one that has a constant expression in muscle cells regardless of disease status. Several potential candidate housekeeping proteins for muscle were selected for this purpose including α-actinin, vinculin and spectrin. For spectrin several antibodies were tested but none gave a specific signal on Wes. Compared to vinculin the α-actinin antibody gave the most clear signal with the strongest, cleanest peaks and, moreover, its expression appeared to be highly comparable between the BMD and DMD samples in this study, suggesting that its expression is not strongly affected by disease status. Therefore, we selected α-actinin for normalization. Both vinculin and α-actinin were expressed at too high levels to allow detection at the same protein concentration as dystrophin, requiring further dilution of the samples used for dystrophin detection, and therefore had to be run in separate capillaries. This is not preferred since it does not allow for correction for what exactly is loaded per capillary. However, any upstream error will be reflected in the α-actinin measurement, including errors in protein concentration measurement, calculation and pipetting errors. An improved approach that allows detection of both dystrophin and α-actinin at the same protein concentration (but not in the same capillary) is to dilute the secondary HRP-conjugated antibody used for α-actinin detection with unconjugated antibody (data not shown). An advantage of this is further reduction of variation introduced by pipetting errors during the dilution step (which would no longer be necessary).The reproducibility of dystrophin quantification by Wes is in line with the FDA/EMA regulations for quantitative biochemical analysis: CVs between replicate measurements have to be below 20% or 25% (at LLOQ). For repeated/incurred sample analysis 2/3 of the results should be within 20% for small molecules and 30% for large molecules . Inter-assay variation per operator was well below 20% for both dystrophin antibodies tested. The inter-operator variation for Mandys106 was slightly higher (26% for the low dystrophin sample and 21% for the high dystrophin sample), which relates more to this specific antibody than to the Wes methodology itself.So far Western blot (WB) in conjunction with immunofluorescence analysis (IFA) have been the most widely used methods to quantify dystrophin levels in biopsies from patients in clinical trials on dystrophin-restoring therapies. WB has a much smaller dynamic range for dystrophin quantification than Wes, since 10-fold more protein lysate of a (high) DMD sample needed to be loaded than of a healthy control muscle sample to allow detection of (trace) dystrophin. This is in contrast to Wes where the same amount of loaded protein was sufficient for dystrophin quantification, both in healthy control samples as well as in DMD samples . The requirement for >100-fold less sample material is a big advantage of Wes, since patient biopsies can be very small  and therefore limit the amount and type of analyses that can be performed. Quantification of dystrophin by Wes also has a much higher reproducibility compared to WB. In this study the overall assay variation, calculated over all 18 measurements for each sample (3 dilutions x 3 experiments x 2 operators) was 8%-16%, which is much lower than for WB: A multiple-laboratory study investigating the feasibility of using WB to measure dystrophin as a biomarker in DMD clinical trials showed a high assay variation, especially for low dystrophin expressing samples  [We previously developed a semi-automated, objective and reproducible immunofluorescence analysis method optimized for assessing dystrophin levels per individual muscle fiber in an entire fiber population in muscle biopsies from BMD or DMD patients . In this2 = 0.10), which is likely a combined consequence of differences in assay performance and the use of a different dystrophin antibody. The BMD samples with the lowest dystrophin levels (10% CTRL) were relatively close to the DMD samples with highest dystrophin (7% CTRL), which, being speculative, may suggest that dystrophin levels starting from 10% CTRL upwards would be the minimal amount to be aimed for in dystrophin-restoring therapies. It must however be noted that the skeletal muscle samples analysed here are only a relatively small subset, do not represent the full array of deletions in BMD and DMD, and more importantly were not selected to investigate the correlation between dystrophin levels and functional phenotype. In addition, the 31 healthy control samples we analysed were all derived from adults (age 22–75), and we cannot exclude that dystrophin levels in younger healthy male controls more similar in age to DMD boys participating in dystrophin-restoring clinical trials may be different to the spectrum of dystrophin levels characterised here in healthy control adults. In addition, different skeletal muscles may display differences in dystrophin levels, although we did not analyse sufficient numbers of the different skeletal muscle types to draw any conclusions on this. Having purified dystrophin protein available as a reference standard would be preferable. However, production of purified, stable full length dystrophin protein remains technically challenging and is not available at this time. The standard practice of expressing results as a percentage of a healthy control makes results easier to interpret, but may cause bias when comparing different studies, if different reference controls are used. We recommend to select a healthy control skeletal muscle sample as a reference standard that has mean dystrophin levels in a panel of healthy control samples and of which there is sufficient material available to allow it to be taken along in a large number of Wes analyses.DMD patients were historically diagnosed by absence of dystrophin as determined by qualitative assessment of muscle sections using immunofluorescence (IFA) . HoweverIn conclusion, the Wes method for dystrophin detection is specific, sensitive and reproducible. It can detect trace dystrophin levels in DMD as well as the whole dystrophin range covered by BMD and healthy control muscle samples, and is thus suitable for quantification of dystrophin expression as a biomarker in DMD clinical drug development.S1 Fig1: HiMark pre-stained marker (Invitrogen); 2: Wes ladder + streptavidin-IRDye800CW. The ladders do not completely line up in the high molecular weight region. The 440 band of Wes ladder migrated slower than 460 band of HiMark ladder, which can lead to underestimation of high molecular weights >280 kDa on the Wes.(TIF)Click here for additional data file.S2 FigBoth antibodies show good linearity from 0%-100% of healthy control when loading 1.25 μg of healthy control. Experiments were performed in triplicate.(TIF)Click here for additional data file.S3 FigA) Antibody dilution response curve on 25 μg/ml healthy human muscle sample. B) Total protein loading response curve , using 1/100 ab68167. The red arrows indicate the dilution/concentrations selected for further experiments.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Haplotype phasing of genetic variants is important for clinical interpretation of the genome, population genetic analysis and functional genomic analysis of allelic activity. Here we present phASER, an accurate approach for phasing variants that are overlapped by sequencing reads, including those from RNA sequencing (RNA-seq), which often span multiple exons due to splicing. Using diverse RNA-seq data we demonstrate that this provides more accurate phasing of rare variants compared with population-based phasing and allows phasing of variants in the same gene up to hundreds of kilobases away that cannot be obtained from DNA sequencing (DNA-seq) reads. We show that in the context of medical genetic studies this improves the resolution of compound heterozygotes. Additionally, phASER provides measures of haplotypic expression that increase power and accuracy in studies of allelic expression. In summary, phasing using RNA-seq and phASER is accurate and improves studies where rare variant haplotypes or allelic expression is needed. Genome interpretation and analysis of allelic activity requires appropriate haplotype phasing. Here the authors present phASER, a fast and accurate method for variant phrasing from RNA-seq and genome sequencing data. Existing methods to phase variants include phasing by transmission2de novo variants, phasing by sequencing long genomic fragments489The phasing of rare and In this work we thoroughly benchmark phASER, showing that our method for haplotype assembly is accurate by comparison to other commonly used read backed phasing methods using gold standard datasets. We show that through enhanced quality control measures RNA-seq can be used to accurately phase variants over much larger distances than DNA-seq, and that the addition of RNA-seq significantly increases the number of rare variants that can be phased. To demonstrate this we apply phASER to genetic studies and show that the inclusion of RNA-seq improves the resolution of compound heterozygotes, and propose an example workflow for the incorporation of expression and phase information in medical genetic studies. Finally, we show that haplotypic expression generated by phASER improves allelic expression studies by increasing power and accuracy.n variants), and selects the configuration with the most support. To prevent an exponential increase in haplotype search space while maintaining accuracy phASER splits large haplotypes into sub-blocks at points spanned by the fewest edges , and found that with default settings phASER identified the correct phasing for 98% of the variants phased. Unlike population based phasing, read backed phasing across sequencing assays performed well for low frequency variants (As a gold standard we compared phASER used with high coverage RNA-seq data generated from a lymphoblastoid cell line (LCL)variants . Finallyvariants . Both phvariants .To evaluate the increase in phasing distance facilitated by RNA-seq reads, we compared phASER results between WES, WGS and two read lengths of paired-end (PE) RNA-seq (75 bp and 250 bp) from 4 Genotype-Tissue Expression (GTEx) individuals where matched libraries were availableWhen population phased data are available, haplotype blocks are phased relative to each other, producing a single genome wide phase through a method we call phase anchoring. Phase anchoring uses the population phase of each variant in a block weighted by their allele frequencies to assign a genome wide phase, since common variants are more likely to have correct population phasing . A similWe next sought to benchmark phASER when used with RNA-seq data in the context of genetic studies. First we used GTEx data to demonstrate the number of coding variants that could be phased as a function of the number of tissues for which RNA sequencing data is available. We began with whole blood, and progressively added libraries from up to 14 other distinct tissues. With joint phasing using 14 tissues, almost 50% of all heterozygous coding variants could be phased with at least one other variant . When usWe next applied phASER to assess its ability to identify cases of compound heterozygosity for damaging variants using WES and RNA-seq data from 345 1,000 Genomes individuals14trans), while assigning a lower priority to a median of 44 alleles, where the alleles were on the same haplotype (cis)  . The incOutside of medical genetics, variant phasing is important for allelic expression (AE) analysis, which aims to quantify the relative expression of one allele versus anotherIn summary, phASER provides scalable and high confidence variant phasing, incorporating RNA-seq and DNA-seq data with population phasing, allowing phasing over longer distances than previous read based methods. We have demonstrated that this method has direct applications in medical genetics, where improved resolution of compound heterozygotes can lead to changes in their interpretation. Furthermore, phASER improves the accuracy of haplotypic expression when integrating allelic counts across variants by reducing false positives. Our approach will complement the existing repertoire of phasing methodsP<0.01) are removed. It should be noted that this test addresses instances where due to sequencing error individual bases on a read have been misread, and it does not address errors with read mapping, which can happen at sites with genetic variation. For a discussion of methods to address allelic read mapping issues please see Castel et al.n), however runtime is prevented from exponentially increasing by splitting haplotypes into sub-blocks at positions spanned by the fewest number of edges  are mapped to heterozygous variants, and for each heterozygous variant a hashed set of all overlapping reads is produced, which allows for quick comparison of overlapping reads between variants. Connections between variants are established whenever a read (or read mate) overlaps more than one variant. For each pair of connected variants, edges between alleles are defined by determining the phase that has the most read support, and a test is performed to determine if there is significant evidence of a conflicting phase (see below) . Those eof edges . These sof edges . The maxof edges . Read vaof edges .P value of <0.01  could be observed by chance from sequencing noise alone. In this test, significance indicates more conflicting reads than would be expected from noise alone, and thus suggests that there may be an error in the phase selected by phASER. A conservative approach is to filter out any variant connections with a nominal of <0.01 . Less st1|a2 and b1|b2 with a haplotype structure a1b1| a2b2. Let a1:b1 denote a read spanning alleles a1 and b1. Reads supporting the true haplotype in this case are a1:b1, and a2:b2, and any other configurations correspond to conflicting evidence. Let us only consider reads generated from the first haplotype. The probability of observing a read supporting the correct haplotype structures, ps, is (1−3ɛ)2+ɛ2, where the first term corresponds to the probability of observing a1:b1 (the case in which either of the sites being affected by noise in the read), and the second term is the probability of observing a2:b2 . Binomial distribution is used to evaluate the probability of observing equal or less supporting reads for two given SNP sites:The test is based on a uniform error model in which a true allele nucleotide can be substituted randomly to any other nucleotide. All pairwise substitutions in this model are assumed to be equally probable. We denote this pairwise substitution probability with ɛ. Let us assume a pair of SNPs ans and n are the number of reads supporting the chosen haplotype structure and total number of reads respectively. The pair-wise substitution rate ɛ is calculated from over all SNP sites as:where where mismatches correspond to all cases that a nucleotide other than the reference or the alternative allele where observed at the site. Only variants where >50% of the reads come from the reference and alternative alleles are used to calculate the substitution rate to avoid inflation of noise estimates as a result of genotyping error.When population phased data are available phASER will attempt to determine the genome wide phase for each haplotype block using phase anchoring. Phase anchoring operates on the assumption that common variants are more likely to be phased correctly using population data, so for each variant within a haplotype the genome wide phase determined by population phasing is weighted by the variant allele frequency. The genome wide phase across all variants with the most support after weighting is selected for each haplotype block.For each haplotype block:α (phase support for configuration 1)=Σ(MAF of variants supporting configuration 1)β (phase support for configuration 2)=Σ(MAF of variants supporting configuration 2)if α>β genome wide phase=configuration 1if α<β genome wide phase=configuration 2Anchor Phase Confidence=max/(α+β)The following settings were used unless otherwise noted. For all libraries: alignment score quantile cutoff of 0.05, BASEQ of 10, and conflicting configuration threshold of 0.01, indels ignored, and maximum block size of 15. For RNA-seq libraries: no maximum insert size, MAPQ of 255 (indicates unique in STAR). For exome-seq libraries: 500 bp maximum insert size, MAPQ of 40. For whole genome sequencing libraries: 1,000 bp maximum insert size, MAPQ of 40. All variants in HLA genes were filtered.https://github.com/secastel/phaser). In addition to the phASER core software we provide two scripts: one which given an input VCF that has been phased using phASER will identify all interactions between alleles (phASER Annotate), and retrieve information such as allele frequency and predicted variant effect if supplied with the appropriate files, and second, a script which will use haplotypic counts produced by phASER in combination with population phasing to produce gene level haplotypic read counts for use in allelic expression studies (phASER Gene AE).Source code and complete documentation for phASER and its associated tools are available through GitHub , 1,000 (WGS), 1e6 (PE 75 RNA), mbq=10, mmq=40 (WES and WGS), mmq=255 (PE 75 RNA). phASER was run with default settings (see phASER settings). Simulated PE 75 WGS data was produced with ART Chocolate Cherry Cake 03-19-2015 (ref. http://www.1000genomes.org). Raw (FASTQ) RNA sequencing data from 1,000 Genomes individual derived LCLs was downloaded from the European Nucleotide Archive (ERP001942), and aligned with STAR to hg19. For comparison of phase statistics between sequencing assays the following GTEx individuals were used: S32W, T5JC, T6MN, WFON. Both short and long read RNA-seq was obtained for whole blood and LCLs, and aligned using STAR to hg19. WES reads were aligned using Bowtie 2 to hg19. GTEx data is available through dbGaP for authorized users (phs000424.v6.p1). For rare variant phasing comparison, RNA-seq from whole blood, fibroblasts, sun exposed skin, and adipose were used, alongside WES and WGS libraries, from GTEx individuals X4EO, XUW1, U8XE, XOTO. GTEx individual ZAB4 was used for comparison of number phased variants versus number of RNA-seq tissues used. For comparison to transmission phasing the following data from the 1,000 Genomes individual NA12878 was used: exome-seq downloaded from 1,000 Genomes website, whole genome sequencing data (NCBI SRA ERS179577), RNA-seq from a LCL (NCBI GEO GSM1372331), transmission phased genotypes . Whole genome sequencing libraries were down sampled to 5x to increase speed of analyses and ensure comparable read depths across sequencing assays.For analyses involving 1,000 Genomes individuals, phase 3 genotypes and population phasing where were used with hg19 aligned exome-seq data, both downloaded from the 1,000 Genomes website (http://www.1000genomes.org), the European Nucleotide Archive (ERP001942), dbGaP (phs000424.v6.p1), NCBI Sequence Read Archive (ERS179577), NCBI Gene Expression Omnibus (GSM1372331), and Illumina Platinum Genome, http://www.illumina.com/platinumgenomes/).Data used in this study was retrieved from the 1,000 Genomes website .Supplementary Figures 1-7


These nanophotonic aperiodic lattices have easily tailorable supercell tiles with well-defined and discrete lattice basis elements and they show rich Fourier spectra. The presented nanophotonic lattices are designed functionally akin to two-dimensional aperiodic composite lattices with unconventional flexibility- comprising periodic photonic crystals and/or in-plane photonic quasicrystals as pattern design subsystems. The fabricated composite lattice-structured Si nanostructures are comparatively analyzed with a range of nanophotonic structures with conventional lattice geometries of periodic, disordered random as well as in-plane quasicrystalline photonic lattices with comparable lattice parameters. As a proof of concept of compatibility with advanced bottom-up liquid phase crystallized (LPC) Si thin film fabrication, the experimental structural analysis is further extended to double-side-textured deterministic aperiodic lattice-structured 10 μm thick large area LPC Si film on nanoimprinted substrates.Exotic manipulation of the flow of photons in nanoengineered materials with an aperiodic distribution of nanostructures plays a key role in efficiency-enhanced broadband photonic and plasmonic technologies for spectrally tailorable integrated biosensing, nanostructured thin film solarcells, white light emitting diodes, novel plasmonic ensembles etc. Through a generic deterministic nanotechnological route here we show subwavelength-scale silicon (Si) nanostructures on nanoimprinted glass substrate in large area (4 cm The dense Fourier spectra of such aperiodic lattice-embedded nanostructured materials could strongly modulate and enable flexible tailoring of the light-matter interaction for broad band nanophotonic applications in comparison to unstructured bulk materials1232678deterministic aperiodic composite photonic lattices’, from now on called for brevity as ‘composite lattices’. They are functionally embedded with designed characteristics of multiple lattice periodicities of conventional photonic crystals and/or in-plane higher order rotational symmetries of common photonic quasicrystals as subsystems in pattern design, hence the denomination ‘composite lattices’. By means of advanced nanoengineering, the presented large area composite lattices are deterministically designed and realized functionally similar to conventional two dimensional composite aperiodic lattices, while surpassing the dense distribution of lattice points in real space proportional to the increase in sets of intergrowth elements2 with advanced functional features of aperiodic composite nanophotonic lattices which are easily tailorable and scalable for the spectral region of interest and application. Though the presented nanostructuring approach is applicable to any semiconductor materials in principle, it is particularly important to silicon, an indirect semiconductor but widely used in integrated broad band applications. As one of the tests of broadband performance of the fabricated Si nanophotonic composite lattices, they are comparatively analyzed with a range of nanophotonic structures with conventional lattice geometries of periodic, disordered random or in-plane quasicrystalline photonic lattices with comparable parameters. The chosen nanoimprinted structures are comparable in terms of fill fraction and spatial reciprocal space relation for an objective reference and scalability. We use a generic Fourier reconstruction method involving selective superposition of components to generate easily tailorable irradiance profiles of composite lattices. By a subsequent thresholding and discretization of lattice points within a chosen mesoscopic supercell with boundary side length typically of 10 to 25 μm, the possible fabrication limitations of the otherwise dense and possibly overlapping spatial distribution of superlattice nanostructures in real space is also surpassed. It is pertinent to be noted that if adopted for substrate structuring using large throughput fabrication processes like nanoimprint lithography, earlier investigated large area deterministic nanophotonic aperiodic lattices with mesoscopic supercell comprising of nanostructures with distributed volumetric basis shapes consisting of very fine and sharp grooves might face a technological challenge. Those structurally fine-featured supercells may get drastically modified during overlaying multi-layered photonic device fabrication processes of standard bottom-up approach7The seemingly counter intuitive impact of panels, slow light integrated chips, structured thin film photovoltaics, novel surface-enhanced Raman scattering nanoplasmonic building blocks, artificially engineered tailorable photonic bandgap materials and efficient nanophotonic test beds for nonlinear light-matter interactions like Anderson localization, and multiple wavelength higher harmonic generation234811121314151617181920s ≥ 2 sets and subsequent discretization (see Methods). Each set has a characteristic length scale and a rotational symmetry qm (where m is from 1 to s). In qm is same in all sets, starting with a lattice embedded with ten-fold rotational symmetry (qm = 5), a hexagonal PPC comprised of three sets (qm = 6) and a PPC with dodecahedral rotational symmetry (qm = 12). The last row  images of experimentally realized nanoimprinted glass substrates and their respective far field diffraction pattern intensity distributions are shown in the second and third columns. In principle any practically viable arbitrary number of rotational symmetries can be embedded given the complexity involved in the generated aperiodic pattern. For brevity, conventional periodic and in-plane qauasicrystalline photonic lattices can be considered here as subgroups within this broad class of photonic lattices when s = 1 and the respective number q point to their fundamental rotational symmetry. To picturize the details of our approach we closely consider one of the above cases, PPChexa , where s = 3 and q1 = q2 = q3 = 6  of diverse composite photonic lattices, in-plane quasicrystals, periodic lattices and the disordered random lattices involves state of art high resolution e-beam lithography for the one-time master wafer fabrication and subsequent large throughput as well as cost effective nanoimprint lithography for highly resolved large area nanostructures (22see Methods). In hexa geometry is given. Further we show, after a polysilicon wet etch process (see Methods), the subsequently fabricated PPChexa and 12-fold rotational symmetry NCNH-embedded Si thin films with 300 nm Si deposition thickness  as summarized in jsc,max is estimated for artificially engineered Si nanostructures with the lattice geometry of PPChexa compared to the corresponding value of the periodic hexagonal lattice under investigation and the same trend of enhancement is observed in comparison to the investigated disordered random lattice too. Whereas these enhancements in comparison to a planar reference were respectively +175.3% and +189.8%. For the aperiodic in-plane 12-fold symmetry quasicrystal structured samples with NH and NCNH respectively these enhancements in jsc,max were +10.5% and +14.28% in comparison to hexagonal lattice. It can be observed from the optical absorption spectra . In x intermediate layer on nanoimprinted glass substrate. The structural characterizations verify the material structural quality of the deterministic aperiodic structured 10 μm thick LPC Si films with well-maintained basis structural features. If needed the lattice basis structuring can be even further tailored with ease from columnar to smoothed hills in order to tune the electrical material quality of c-Si thin film for varied applications without compromising the designed aperiodic geometrical features. Moreover, our generic approach for nanoengineered deterministic aperiodic lattices with rich Fourier spectra offers a great flexibility of optimization through tailoring their lattice geometry, basis shape, and spectral response range to match to the desired different thin film thicknesses, material of interest and for intended nanophotonic applications. The industrial application-viable and standard bottom-up fabrication technologies-compatible approach which we have demonstrated is envisaged to suit well for broadband integrated nanophotonic devices which demands large throughput and high resolution structured semiconductor materials with advanced tailorable features in nanoscale1234Extending further, as a proof of concept of compatibility with advanced bottom up liquid phase crystallized (LPC) Si thin film fabrication, we fabricate and do the detailed structural analysis of deterministic aperiodic double-side textured 10 μm thick Si films with enhanced material quality via laser beam crystallization. The geometrical as well as discrete motifs of the deterministic aperiodic lattice with subwavelength features is well maintained even after multiple layer depositions  azimuthally equidistant components in the kx-ky Fourier plane. The components in a given set are radially equidistant from the origin designed as per the desired neighbor distance of a constituent lattice structure to be embedded within the composite lattice. The km,n component in k-vector diagrams  and the nickel mask was subsequently removed in hydrochloric acid.Structured silicon master wafers were prepared by electron beam lithography, mask inversion by a lift-off process and final reactive ion etching. Each of the presented photonic lattice pattern was exposed by a 100 kV Vistec EBPG5000+ES onto an area of 2.0 cm × 2.1 cm on a silicon substrate coated with a 160 nm thick layer of positive electron beam resist ZEP520A (ZEON Corp.). After development in hexyl acetate and rinsing in isopropyl alcohol, the structured resist was coated with a 30 nm thick nickel layer, which served as etch mask after resist removal in a lift-off procedure in dimethyl formamide. Finally, the silicon was etched 370 nm deep in a highly anisotropic reactive ion etching process  trichlorosilane short F13-TCS  in a vacuum oven. As per the requirement, for high resolution nanoimprint process we prepared composite poly-(dimethyl) siloxane (PDMS) stamps or else conventional soft PDMS stamps were used. Composite PDMS (c-PDMS) stamps are composed of two layers: a thin hard PDMS (h-PDMS) layer supported by a thick soft PDMS (s-PDMS) layer. For the c-PDMS stamp preparation2 atmosphere results in solid phase crystallization of the silicon film aside from the columnar substrate features. The still amorphous Si parts around the columns are removed by wet-chemical etching in an etch solution consisting of concentrated HNO3 , concentrated H3PO4 , HF  and H2O (43 parts) resulting in nanocone-nanoholesFor the fabrication of nanocone-nanoholes (NCNH) and nanoholes (NH) structured Si thin films, a Si layer of the desired thickness is deposited by electron-beam evaporation in amorphous phase on the cleaned nanoimprinted glass substrates at a substrate-temperature of 300 °C. Subsequent thermal annealing at 600 °C for several hours in a tube furnace within an Nx diffusion barrier layer of around 100 nm thickness is deposited on the cleaned nanoimprinted glass substrates by physical vapour deposition using a reactive magnetron sputtering system. Subsequently the desired thickness of nominally intrinsic nanocrystalline Si was deposited by e-beam evaporation at a substrate-temperature of 600 °C. Subsequently, a 250 nm thick SiOx capping layer was deposited on top of the layer stack by reactive magnetron sputtering. This capping layer prevents the surface of the Si melt from levelling out during crystallization and thus allows maintaining the initial topography of the deposited layer akin to that of the nanoimprinted substrate texturing2. Crystallization was performed at a substrate temperature of 600 °C and by moving the samples under the laser beam with a velocity of 11 mm/s. Further, the SiOx capping layer at the surface was removed using a 5% HF solution as etchant in a wet etching process resulting in a double side textured Si thin film with well-maintained structural features of the nanoimprinted substrate.For the fabrication of the presented double-side textured liquid phase crystallized Si thin films, first a SiOjsc,max is calculated by,The absorption spectra of the nano-patterned crystalline silicon thin films were measured inside an integrating sphere of a PerkinElmer1050 optical spectrometer with wavelength resolution of 2 nm. Samples were tilted by 8 degrees to ensure that the specularly reflected light does not escape through the entrance port. The maximum achievable short circuit current density e, absorption A(λ) and ΦAM1.5g (λ) the photon flux of the AM1.5 g solar irradiance spectrum. It represents the upper limit of the current density of photovoltaic devices at zero voltage at illumination with the global solar irradiance spectrum AM1.5 g and serves as figure of merit for integrated absorption. Here, it is assumed that each photon absorbed in the spectral range from 300 to 1100 nm in the nanostructured silicon film contributes to the solar cell current and no electrical losses occur, e.g. by recombination.with elementary charge How to cite this article: Xavier, J. et al. Deterministic composite nanophotonic lattices in large area for broadband applications. Sci. Rep.6, 38744; doi: 10.1038/srep38744 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Supplemental Digital Content is Available in the Text. There is a great need for new therapeutics for the treatment of pain. A possible avenue to development of such therapeutics is to interfere with signaling pathways engaged in peripheral nociceptors that cause these neurons to become hyperexcitable. There is strong evidence that mitogen-activated protein kinases and phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin signaling pathways are key modulators of nociceptor excitability in vitro and in vivo. Activation of adenosine monophosphate-activated protein kinase (AMPK) can inhibit signaling in both of these pathways, and AMPK activators have been shown to inhibit nociceptor excitability and pain hypersensitivity in rodents. R419 is one of, if not the most potent AMPK activator described to date. We tested whether R419 activates AMPK in dorsal root ganglion (DRG) neurons and if this leads to decreased pain hypersensitivity in mice. We find that R419 activates AMPK in DRG neurons resulting in decreased mitogen-activated protein kinase signaling, decreased nascent protein synthesis, and enhanced P body formation. R419 attenuates nerve growth factor (NGF)-induced changes in excitability in DRG neurons and blocks NGF-induced mechanical pain amplification in vivo. Moreover, locally applied R419 attenuates pain hypersensitivity in a model of postsurgical pain and blocks the development of hyperalgesic priming in response to both NGF and incision. We conclude that R419 is a promising lead candidate compound for the development of potent and specific AMPK activation to inhibit pain hypersensitivity as a result of injury. These signaling pathways can lead to the phosphorylation of voltage-gated channels59 and an increase in protein synthesis via convergent signaling to the eukaryotic initiation factor (eIF) proteins.45 Both of these events are linked to increased excitability in nociceptors but they can be difficult to attenuate by using kinase or enzyme inhibitors that target single signaling factor because feedback signaling is a frequent consequence of PI3K/mTOR- or MAPK-specific antagonists.41Endogenous pro-nociceptive factors are released in response to injury and act via their cognate receptors expressed by nociceptive sensory neurons in the dorsal root ganglion (DRG) to alter the excitability of these neurons.43 Previous studies in rodents have shown that AMPK activators attenuate inflammatory pain,50 incision-evoked pain,56 trauma-induced neuropathic pain,35 diabetic neuropathic pain,28 bone cancer pain,53 and chemotherapy-induced peripheral neuropathic pain.30 Direct application of AMPK activators to nociceptive neurons decreases their excitability and inhibits signaling in PI3K/mTOR and MAPK signaling pathways.56 Adenosine monophosphate-activated protein kinase can be activated indirectly by inhibiting mitochondrial complex I activity that raises intracellular AMP/ATP ratios, indirectly through changes in intracellular calcium, or directly through positive allosteric modulators that interact with sites on the heterotrimeric enzyme complex.16 The antidiabetic drug metformin activates AMPK at least in part by inhibition of mitochondrial complex I,52 but metformin does this only at high μM/mM concentrations and requires an organic cation transporter to enter cells.9 Despite these limitations, metformin is effective in many of the preclinical models listed above, and retrospective studies in humans55 suggest a beneficial effect on pain.A possible solution to this problem is to target the adenosine monophosphate-activated protein kinase (AMPK) that attenuates PI3K/mTOR and/or MAPK signaling and short-circuits feedback signaling.20 making it one of the most potent AMPK activators yet described. We find that R419 activates AMPK in DRG neurons, leading to inhibition of MAPK signaling and a robust decrease in cellular excitability. Moreover, R419 blocks nerve growth factor (NGF)- and incision-evoked mechanical hypersensitivity and hyperalgesic priming via local injection. Our findings support the further development of regionally applied AMPK activators for the beneficial modification of injury-induced nociceptive plasticity.As a first step toward the development of more potent and specific complex I inhibitors that activate AMPK for the treatment of pain, we have examined the effect of R419 on nociceptive neurons in vitro and in vivo. R419 activates AMPK at nM concentrations,All procedures that involved use of animals were approved by the Institutional Animal Care and Use Committee of The University of Texas at Dallas and were in accordance with International Association for the Study of Pain guidelines. All behavioral studies were conducted using male Swiss Webster (Taconic Laboratories) mice weighing between 20 and 25 g. Mice were used in behavioral experiments starting 1 week after arrival at the animal facility at University of Texas at Dallas. Animals were housed with a 12-hour light/dark cycle and had food and water available ad libitum.40 and modified by Banik et al.4 We made incisions in the skin and underlying muscle prior to suturing the wound as described by Banik et al.4 Animals that had plantar incision were briefly anesthetized with isoflurane during the procedure. Calibrated von Frey filaments (Stoelting) were used for mechanical stimulation of the plantar surface of the left hind paw, and withdrawal thresholds were calculated using the up-down method . R419  was made as a DMSO stock solution and then diluted into dosing vehicles. Dosing was carried out in a volume of 25 μL sterile saline for intraplantar injections or in 0.5% hydroxypropyl methylcellulose plus 0.1% polysorbate-80  in ultrapure H2O in a total volume of 200 μL for oral gavage. The experimenters measuring mechanical withdrawal thresholds were always blinded to the experimental conditions. Mice were randomized to groups by a blinded experimenter, and mice of individual groups were never housed together .Mice were placed in acrylic boxes with wire mesh floors and allowed to habituate for 1 hour. After pretreatment mechanical thresholds were recorded, animals received intraplantar injections in a volume of 25 μL sterile saline. Other mice had plantar incision surgery, using a model first described by Pogatzki and Rajad-lysine  for Western blot experiments or on 12-mm glass coverslips  in a 24-well tissue culture plate  at 37°C with 95% air and 5% CO2. Harvested tissues were homogenized in lysis buffer, protease inhibitor cocktail , and phosphatase inhibitor cocktail 2/3  and prepared for standard Western blotting with polyvinylidene difluoride (PVDF) membranes . Densitometric analyses were performed on immunoblots with ImageJ software . Antibodies used are shown in Table Dorsal root ganglia (DRGs) were extracted aseptically from Swiss Webster mice for each Western blot or cellular imaging experiment and placed in Hank's Buffered Salt Solution  on ice. Following DRG neuron dissociation, neurons were cultured for 7 days on 12-well plates coated with poly-1 and protein puromycelation. For the puromycelation, surface sensing of translation (SUnSET) assay,51 puromycin  was applied during the last 15 minutes of the 1-hour incubation with drug. Immediately following the puromycin incubation, cells were washed in chilled Hank's Buffered Salt Solution containing 0.00036% digitonin (Sigma) for 2 minutes prior to fixation for the removal of background puromycin. Images were taken on an Olympus Fluoview FV1200 laser scanning confocal microscope and analyzed using the colocalization tool within Olympus FV software.For immunocytochemistry, treatments were performed to assess acetyl-CoA carboxylase (ACC) phosphorylation, P body formation,Whole cell patch-clamp experiments were performed on isolated DRG neurons in vitro using a MultiClamp 700B  patch-clamp amplifier and PClamp 9 acquisition software (Axon Instruments). Recordings were sampled at 20 kHz and filtered at 1 kHz. Series resistance was typically <7 MΩ and was compensated 60% to 80%. Data were analyzed using Clampfit 10  and Origin 8 . Action potentials were elicited by injecting slow ramp currents from 0.1 to 0.7 nA with Δ = 0.2 nA over 1 second to mimic slow depolarization.http://links.lww.com/PR9/A0).Detailed methods are available in Supplemental Methods  Prism version 6 for PC or Mac. Statistical tests used are described in figure legends. The a priori level of significance was set at 95%.56 One-hour treatment with both 100 μM resveratrol and 300 nM R419 led to increased phosphorylation of AMPK at threonine 172 . Consistent with previous studies, we observed an increase in P body formation in DRG neurons , suggesting that R419 has excellent specificity at nM concentrations. From these findings, we conclude that R419 activates AMPK in DRG neurons and that this leads to a decrease in ERK activity, enhanced P body formation, and decreased mRNA translation.Next, we sought to examine cellular effects in DRG neurons with AMPK activation through R419. Because DRG cultures contain neurons, fibroblasts, Schwann cells, satellite glial cells, and other cell types, it is important to demonstrate that R419 is capable of stimulating AMPK-mediated actions in DRG neurons. We first looked at ACC phosphorylation on serine 79 using a phospho-specific antibody characterized for immunocytochemistry. Using corrected total cellular fluorescence on neurons labeled with βIII tubulin, we observed a significant increase in p-ACC with R419 (300 nM) treatment for 1 hour Fig. A. We havons Fig. B and a dons Fig. C upon tr32 Nerve growth factor signals through MAPK and PI3K/mTOR pathways to evoke changes in nociceptor excitability and induce mechanical hypersensitivity in vivo.45 We tested whether R419 could block NGF-induced changes in behavior in vivo or in cellular excitability in vitro. Mice received intraplantar injections of NGF with or without R419, and mechanical hypersensitivity was assessed by the von Frey test. R419 dose-dependently inhibited mechanical hypersensitivity with a peak effect at 10 μg dose  overnight. We treated DRG neurons with vehicle or 2 concentrations of R419 (300 and 1000 nM) for 1 hour prior to performing current clamp recordings on small-diameter DRG neurons. We assessed ramp current-evoked spiking and measured the number of evoked action potentials and the time to first action potential peak with each ramp current. R419 inhibited the number of action potentials evoked by ramp currents in a concentration-dependent fashion  injection, we observed the inhibition of incision-evoked mechanical hypersensitivity  R419 is a potent, specific inhibitor of mitochondrial complex I leading to activation of AMPK in DRG neurons, (2) R419-mediated AMPK activation results in inhibition of ERK signaling but does not disrupt PI3K/mTOR signaling, (3) R419 blocks nascent protein synthesis in DRG neurons and increases P body formation, (4) R419 inhibits NGF-evoked mechanical hypersensitivity, hyperalgesic priming, and neuronal hyperexcitability, and (5) multiple, local dosing with R419 blocks postsurgical pain and hyperalgesic priming produced by incision. These findings add to a growing body of evidence, suggesting that AMPK activation can lead to inhibition of pain amplification mechanisms in the peripheral nervous system.53), on natural products that are generally regarded as safe , or on tool compounds that activate AMPK but lack potent actions that are desirable in clinical candidate compounds . While all of these compounds have shown efficacy in several preclinical models of persistent inflammatory pain, neuropathic pain or postsurgical pain, A769662 is the most potent of these molecules with an EC50 for AMPK activation in the 50 to 200 μM range depending on the cellular assay.10 Importantly, R419 is more than 100 times more potent than these other tool compounds with previously reported EC50s in the 100 to 300 nM range,20 a potency which we have confirmed here on DRG neurons using several different assays.Previous work assessing AMPK activators for the treatment of pain has focused largely on drugs that are already clinically available  have been shown to be effective against chemotherapy-induced peripheral neuropathic pain in rodents.62 Metformin, which also inhibits complex I to activate AMPK,39 was recently shown to prevent the development of chemotherapy-induced peripheral neuropathic pain in mice.30 This suggests that a possible endpoint for complex I inhibitors for the attenuation of pain is the activation of AMPK. Metformin has activity at other targets, such as inhibition of AMP deaminase,38 inhibition of cyclic AMP signaling,37 and inhibition of mitochondrial glycerophosphate dehydrogenase29; therefore, it is difficult to reach firm conclusions about mechanisms of action with metformin. It is also clear that clinical concentrations of metformin58 may not align with the concentrations of drug that are achieved in preclinical models,60 creating further uncertainty about how mechanism of action relates to clinical or preclinical phenotype. We believe that our work with R419 brings clarity to this issue. R419 is a potent and specific complex I inhibitor that robustly activates AMPK at nM concentrations. Our work supports a role for complex I inhibition as a mechanism for AMPK activation in sensory neurons and suggests that previously observed pain-modifying effects of other complex I inhibitors may be attributable to AMPK activation. This is particularly important in light of the recent findings that AMPK activation is required to link mitochondrial fragmentation with mitochondrial fission and mitophagy to eliminate and replace damaged mitochondria.57The mechanism of action of R419 is mitochondrial complex I inhibition, an effect which raises AMP levels in cells causing AMPK activation.63 (and other pro-nociceptive factors) at the site of incision, whereas injection of NGF only introduces the factor exogenously for a short period of time. Nevertheless, it is remarkable that even with this short tissue half-life, R419 is capable of producing a long-lasting modification of pain plasticity with limited local dosing. We propose that this creates a promising therapeutic approach for AMPK activators. Local application of drugs is increasingly being used in therapeutics because it allows for spatial control of drug concentrations without risk of systemic toxicities or other side effects . A similar complex I inhibitor that activates AMPK was recently shown to produce lactic acidosis with systemic administration in humans.27 Our work suggests that local application of AMPK activators at sites of surgery can reduce postsurgical pain and prevent the development of long-lasting nociceptive plasticity that may influence the transition to a chronic pain state48 while avoiding important negative systemic effects in patients.27 Insofar, as chronic pain after surgery remains a major clinical problem,25 AMPK activators like R419 may be used advantageously via local application in this setting.R419 was more efficacious when given locally than when administered systemically. While we did not perform dosing titers to determine equivalent drug concentrations with local vs oral administration, we did measure drug concentration in the hind paw following local injection. Our findings demonstrate a tissue half-life of ∼1 hour for R419. R419 effectively inhibited NGF-induced mechanical hypersensitivity and hyperalgesic priming with a single dose and required 2 doses for efficacy against incision-evoked mechanical hypersensitivity and hyperalgesic priming. This is likely because incision leads to long-lasting increases in NGFWe conclude that R419 activates AMPK in sensory neurons leading to inhibition of ERK signaling, decreased nascent protein synthesis, attenuated nociceptor excitability, and locally mediated inhibition of pain plasticity in vivo. Our work shows that very potent, indirect activators of AMPK can modify pain responses in vivo even with relatively brief drug exposure. This creates opportunities for further development of AMPK activators as analgesics with efficacy via a local mechanism of action.The authors have no conflicts of interest to declare.This work was supported by NIH grants: R01GM102575 (T.J.P. and G.D.), R01NS065926 (T.J.P.) and The University of Texas STARS program research support grant (T.J.P. and G.D.) and by an unrestricted grant from Rigel Pharmaceuticals (T.J.P. and G.D.). Y. Hitoshi is an employee of Rigel Pharmaceuticals.


We have previously reported levels of myeloid cells in the periphery and in the tumor microenvironment (TME) of patients with primary breast cancer (PBC) and colorectal cancer (CRC). We found that both PBC and CRC patients have significantly higher levels of granulocytic and immature myeloid cells in the TME. Additionally, we reported an expansion of circulating granulocytic myeloid cells in CRC patients, but not in PBC patients. In this report, we compared levels of myeloid cells between these two common cancers and have added data from more cancer patients. We also investigated associations between clinical stage/histological grade of tumors and levels of myeloid cells in cancer patients. We found that although granulocytic myeloid cells were expanded in the TME of both PBC and CRC patients, the levels of these cells were significantly higher in the TME of CRC patients. Moreover, our results indicate that increased levels of circulating granulocytic myeloid cells are associated with poorly differentiated tumors in CRC patients. Taken together, this work suggests that CRC patients may benefit more from the development of therapeutic agents to promote myeloid cell differentiation or inhibition for the reversal of immune suppression. Immunosuppressive cells are known to impair antitumor immune responses primarily by inhibiting host T cell responses against tumor antigens. Myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) are suppressive cells, which can dampen immune responses and are found elevated in periphery and the tumor microenvironment (TME) of various human cancers to ultimately facilitate tumor progression. Numerous studies have aimed to investigate the levels of these cells in cancer patients in order to target these cells to provoke antitumor immunity and inhibit tumor progression.Colorectal and breast cancers are two of the most common human cancers worldwide resulting in a combined total of up to 1.4 million deaths globally , 3. HoweRecent decades have seen great advances in developing novel therapeutic approaches of cancer immunotherapy. Better understanding of the immune profile of the TME and periphery of cancer patients can help in identifying key components of host immune response that may be targeted to revert immunosuppression. We have also recently shown an expansion of highly suppressive infiltrating Treg in the TME of CRC and PBC patients , 7. TherWe found that PBC patients have significantly higher levels of myeloid cells with granulocytic morphology  and immature myeloid cells (IMC) in the TME. Interestingly, this expansion was not reflected in peripheral blood of PBC patients, as the levels of circulating myeloid cells were similar to HD. In contrast, we reported an expansion of GMC in both peripheral blood and the TME of CRC patients. IMC were also expanded in the TME of CRC patients but not in peripheral blood. In this study, we compared levels of myeloid cells in circulation and the TME of patients with two of the most common cancers but presenting with distinct clinical and pathological features.n = 10 and CRC n = 11). Written consent forms were signed by all patients and donors prior to sample collection, under ethics approved by Al Ain ethics committee UAEU, UAE (13/23-CRD 244/13).Peripheral blood samples were collected from 21 HD, 30 PBC patients, and 20 CRC patients who did not receive any treatment prior to surgery at Tawam Hospital, Al Ain, UAE, and Al Noor Hospital, Abu Dhabi, UAE. Tumor and paired, adjacent normal tissue samples were also collected from cancer patients (PBC n = 15) were compared with patients with stage II and III cancers (n = 15), while CRC patients with stage I and II (n = 9) were compared with CRC patients with stage III and IV (n = 11). Additionally, patients with tumor histological grade I and II (PBC n = 15 and CRC n = 16) representing well-to-moderately defined tumor cells were compared with patients with III tumors, indicating poorly defined tumor cells (PBC: n = 15 and CRC: n = 4). Patients were divided and compared based on the TNM staging and tumor histological grade. PBC patients with stage I , and 30 IU/ml of deoxyribonuclease I (Sigma-Aldrich), and placed on a roll-over mixer at 37°C for 60 minutes. The cell suspension was then passed through a 100 μm BD Falcon cell strainer  to remove debris and aggregates. Cells were then resuspended in RPMI-1640 enriched with 10% FCS and 1% Penicillin/Streptomycin after washing with RPMI-1640.Enzymatic digestion of fresh tumor tissues (TT) and paired, adjacent, nontumor normal tissues (NT) from breast and colorectal cancer patients was performed as described previously , 5. BrieFresh whole blood and isolated cells from freshly resected NT and TT were stained for phenotypical analyses as described previously , 5. BrieImmune cells extracted from ED were first blocked for Fc receptor using FcR Blocker , and 7AAD viability dye (eBioscience) was used to gate live cells. Cells were then stained with myeloid markers, and the flow cytometric data were analyzed on BD FACSuite software (BD Biosciences).p value of ≤0.05 was considered statistically significant. The data are presented as means ± SEM.Each subpopulation of cells identified from flow cytometric analyses was compared within the three study groups by comparing the relative percentage of each subpopulation from the respective parent cell population and also by comparing the calculated percentages of each subpopulation. Statistical analyses were performed using GraphPad Prism 5.0 software . One-way ANOVA/Kruskal-Wallis tests followed by paired/Wilcoxon matched-pairs signed-rank test or unpaired/Mann–Whitney tests were used to examine the differences within groups or between groups, respectively. n = 30 and myeloid cells in TME; n = 9) and CRC patients  and compared the levels between cancer patients and also with circulating myeloid cell levels in HD (n = 21).We have previously reported levels of circulating myeloid cells in 23 PBC and 20 CRC patients and the levels of infiltrating myeloid cells in 7 PBC and 9 CRC patients , 5 using+ myeloid cells between the three study groups . We found that CRC patients have significantly higher levels of CD33+CD11b+HLA-DR−/low cells than HD and PBC . CRC patients showed significantly higher levels of GMC in peripheral blood compared to HD  and PBC  as shown in We then analyzed CD15 and CD14 expression within the CD33p = 0.004). Levels of ARG1-expressing GMC cells were significantly higher in the peripheral blood of CRC patients than in HD (CRC: 63.1 ± 2.9 versus HD: 43.2 ± 4.1) and PBC patients  expression, indicative of their suppressive potential. There were no significant differences in the levels of ARG1-expressing MMC in the three study groups  were compared with tumor stage III and IV patients (n = 11), while PBC patients with stage I (n = 15) were compared with stage II and III cancer patients (n = 15). We found that CRC patients with advanced stage disease have significantly higher levels of circulating GMC than PBC with advanced disease . For peripheral blood, CRC patients with tumor stage I and II ( disease . There w disease .n = 4) had significantly higher levels of circulating GMC levels compared to patients with grade I and II tumors , Tie2-expressing monocytes, dendritic cells (DC), tumor-associated macrophages, IMC, and MDSC . ExpansiWe have previously reported an expansion of infiltrating myeloid cells in breast and colorectal cancers compared to normal tissue milieu , 5. Thes+ GMC in CRC compared to PBC in the present study shows the potential immunosuppressive role of myeloid cells in circulation in CRC patients.The close phenotypical and functional proximity of tumor-associated neutrophils (TAN) with G-MDSC  and idenin vivo is a novel approach [in vivo through APC to reintroduce in cancer patients to elicit potent immune response [+ APC are not expanded in the peripheral blood of CRC and PBC, they are higher in the TME of both cancers. Interestingly, we found that levels of APC in normal colon tissue are higher than in normal breast tissue. Intestinal APC which include DC and macrophage are an integral part of the innate mucosal system maintaining tolerance against food antigens and intestinal microbiota [APC have a pivotal role in antitumor immune response. Immunotherapeutic approach through the introduction of a tumor-associated antigen with an adjuvant to activate T cells approach . Sipuleuapproach . Adoptivresponse . These mresponse . In our crobiota . Moreovecrobiota .Tumor histological grade and disease stage based on TNM staging criteria are well-established tools to predict the clinical outcome in various cancers . In a stIn conclusion, further to our previous findings of elevated levels of GMC and IMC in circulation and in the TME of CRC patients  and an e


Interindividual variation in the blood DNA methylome was described using principal components (PC) scores derived from principal component analysis and showed that the NFP program (PC10: p = 0.029) and a history of abuse/neglect  significantly associated with DNA methylome variation at 27 years of age independent of gender, ancestry, cellular heterogeneity, and a polygenic risk index for major psychiatric disorders. The magnitude of the association between child maltreatment and DNA methylation was reduced when accounting for lifestyle factors, including smoking. These findings reflect the sustained impact of both childhood adversity as well as intervention programs that target such adversity on the epigenome but highlight the need for prospective longitudinal studies of DNA methylome variation in the context of early intervention programs.This study reveals the influence of child maltreatment on DNA methylation across the genome and provides the first evidence that a psychosocial intervention program, the Nurse Family Partnership (NFP), which targets mothers at risk for abusive parenting, associates with variation in the DNA methylome in adult offspring. The 188 participants were born to women randomly assigned to control ( Childhood adversity also associates with chemically stable, epigenetic modifications, such as DNA methylation4, which may then maintain the environmental effect on genomic function and health3. Genome-wide DNA methylation (the DNA methylome) is dynamic across development6 and altered by early social experience, as shown in peripheral cells as well as in postmortem brain samples8. These findings suggest the DNA methylome may help identify those affected by early life adversity and facilitate precision medicine initiatives in psychiatry.Childhood adversity increases the risk for most forms of non-communicable chronic diseases10. However, data from programs of early intervention, with establishing clinical efficacy, are lacking. Such evidence is required to evaluate the clinical utility of the DNA methylome for personalized approaches in the fields of perinatal psychiatry and child/adolescent mental health.One study to date has provided some evidence that a psychosocial intervention may moderate the effects of parental depression on features of the DNA methylome such as the epigenetic age11, on genome-wide DNA methylation. The NFP reduces CAN and improves neurodevelopmental outcomes13. We hypothesized that variation in genome-wide DNA methylation would associate with both CAN and the NFP intervention.This report describes the results of a unique 27-year longitudinal study examining the influence of child abuse/neglect (CAN) and maternal participation in the Nurse Family Partnership (NFP) program, a psychosocial intervention program that specifically targets maternal health and mother–child interactionsN = 400) conducted with a primarily Caucasian sample (89%), which has been described in detail previously13. Primiparous women were recruited with one or more of the following risk factors: young age (<19 years), unmarried, or low socioeconomic status (SES). Women were randomly assigned to control or nurse-visited groups. Treatment groups received nurse visits during pregnancy or to age 2 years postpartum. Nurses promoted maternal health behaviors, parental care, and personal development. All nurse-visited women were combined to form a single intervention group given comparable program effects on adolescent antisocial behavior through age 15 years13. Approval for this study was obtained from the Institutional Review Board (University of Colorado) protocol #04-0002.We used the original NFP trial sample (n = 99) and intervention (n = 89) groups provided blood samples and completed the computerized diagnostic interview schedule14 to assess (1) major depression, (2) generalized anxiety, (3) PTSD, (4) antisocial personality/conduct disorder and (5) substance abuse. We created a summary variable reflecting the total number of psychiatric diagnoses. Participants' history of CAN from birth to age 15 years was detailed using substantiated reports from Child Protective Services records and converted to a binary variable .Offspring  of women from the control  was extracted from blood16, bisulfite converted using the EZ-DNA Methylation Kit (Zymo Research), and hybridized on the Illumina Infinium HumanMethylation450K BeadChip (450K array) according to the manufacturers' protocol. The experiment was run in three batches with samples randomized across chip and technical replicates (n = 19) included for quality control of chip and batch effects. We identified a chip and batch effect, which was corrected using ComBat17. One sample failed quality control, e.g., low call rate18 (n = 1) and was removed, resulting in a final pool of 187  samples. The 450K array provides quantitative data on DNA methylation at >480,000 CpGs across the genome. We focused our analysis on autosomal CpGs showing interindividual variation in DNA methylation (range of ≥10%), thus denoted as variably methylated CpGs . vCpGs were subjected to principal component analysis (PCA) within Array Studio (OmicSoft). PCA is a data reduction strategy that describes the variability in a dataset using discrete, independent components19. Principal component 1 (PC1) is the linear function that describes the greatest amount of variability in the data, followed by PC2, etc.Biosamples from the adult offspring of mothers enrolled in the NFP and DNA methylation was described using a genome-wide platform20 for gene ontology and transcription factor interaction analysis. P-values for transcription factor interaction analyses were determined using hypergeometric intersection (Metacore) or a modified Fisher’s exact test (Enrichr).We used MetaCore+MetaDrug v6.23.67496 (GeneGo) and Enrichrp < 0.05) with CAN-associated vCpGs or NFP treatment group. We performed Enrichr analyses on the resultant vCpGs associated with a PC score and a predictor of interest .We extracted PC loadings for each vCpG and each of the top 10 PC scores. We identified vCpGs (1) most strongly associated with a given PC score (defined as loading score within top 10%) and (2) nominally associated  using a genome-wide platform  according to the manufacturers' guidelines. We removed SNPs with a low call rate (<95%) and minor allele frequency (<5%) and used PCA to describe population structure within the NFP cohort22. Two PCs best described the population structure within this cohort  for each participant within our dataset using a custom script implemented in Python and Apache Spark  informed by findings from the large independent dataset from the Psychiatric Genomics Consortium (PGC) encompassing five major psychiatric disorders25. We selected SNPs that associated with a range of mental disorders in the PGC ‘discovery dataset’ at a variety of p-value thresholds: p < 0.5–0.001 and generated corresponding PRS as previously described25.We performed imputation using the Sanger Imputation Service26. We also excluded all non-specific and SNP-disrupted probes27 in our regression models to identify vCpGs associated with a history of CAN. We used a two-sample one-tailed Kolmogorov–Smirnov goodness of fit hypothesis test (pkstest) to examine p-value distributions, as test of the null (unskewed/flat) distribution. A distribution with pkstest <0.001 was considered a significantly skewed distribution.All multivariate models of PC scores included gender, age, number of psychiatric diagnoses, maternal education (as an index of early life socioeconomic status), and estimates of the proportions of major cell types found in whole blood derived from methylation data using a well-established reference-based algorithmp = 0.460). Those providing biosamples experienced more mental health problems than the remainder of the Elmira cohort at age 27 years (p = 0.005). There was no difference in the total number of diagnoses at age 27 years between the nurse-visited group and controls (p = 0.401) in the biosample cohort. Likewise, there was no difference in psychiatric diagnoses between those reporting a history of maltreatment and the remainder of the group (p = 0.457).There were no significant group differences in maternal characteristics at baseline, participant gender, or self-reported ethnicity between those included in our analysis at age 27 years and the remainder of the Elmira NFP cohort Table . ParticiWe used PCA of vCpGs to generate PC scores for the first 10 PCs for each participant. The first 10 PCs accounted for 26% of the variance in DNA methylation. Figure p = 0.029; Fig. p for interaction = 0.174). Enrichr analysis of CpGs that associated with PC10 and maternal participation in the NFP revealed enrichment for biological processes related to synaptic function, neurogenesis, and peripheral nervous system development  to test the association between the maternal NFP treatment group and DNA methylome variation. Maternal treatment group (control vs. nurse-visited) showed a significant association with PC10, which accounted for 1.0% of the variance in DNA methylation. The association between PC10 and NFP treatment group survived adjustment for early life SES, gender, population stratification, age at time of biosampling, cell type, psychiatric diagnoses, and maltreatment history p = 0.09; Fig. 2p = 0.029) and PC2 (p = 0.009), which accounted for 10.3 and 3.3% of the variance in DNA methylation, respectively. Enrichr analysis of vCpGs associated with PC1 and maltreatment history revealed a trend-level enrichment for biological processes related to protein phosphorylation and immune cell activation/differentiation. CpGs that associated with PC2 and maltreatment history revealed significant enrichment for processes related to transcription factor regulation and transcription  associated with CAN, with two vCpGs passing Benjamini–Hochberg False Discovery Rate correction . AHRR DNA hypomethylation (including cg21161138) consistently associates with smoking29. Likewise DNA methylation at cg03440944, located in C7orf40, associates with smoking31. We tested whether participants with a history of maltreatment reported greater smoking at time of biosample collection. Indeed, we found a significant association between CAN and current smoking that is consistent with previous reports linking child maltreatment to increased health-risk behaviors32. Thus 75% of participants with a history of CAN were current smokers compared with 41% of pariticipants from the non-abused group . The association between smoking and CAN with AHRR DNA methylation was confirmed using an independent analysis of DNA methylation by pyrosequencing , providing an additional source of biological variance in DNA methylation. Nevertheless, controlling for smoking in our regression model marginally reduced the number of CAN-associated vCpGs  but significantly reduced the effect size (Cohen’s d = 0.82–1.00 to d = 0.58–0.70) of CAN on DNA methylation of vCpGs within AHRR C7orf40  but controlling for smoking did not substantively change the association between CAN and PC2 (p = 0.011). Likewise, the association between maternal participation in the NFP and PC10 was relatively unchanged (p = 0.038) following adjustment for smoking.Next we tested whether current smoking confounded our ANOVA findings. The association between CAN and PC1 was reduced to trend level (NR3C1), estrogen receptor alpha (ESR1), and estrogen receptor beta  that associated with the number of current psychiatric diagnoses in the NFP biosample cohort . There was no significant between-group differences in Cross-PRS0.2 as a function of child maltreatment (p = 0.605) or maternal participation in the NFP program (p = 0.716).We identified a cross-disorder PRS at a 0.2 was significantly associated with multiple PC scores , while CAN was associated with PC1 (p = 0.079) and PC2 (p = 0.009) after adjusting for the Cross-PRS0.2.Unadjusted bivariate analyses showed that Cross-PRSsee Fig. . Linear r = 0.37, p = 2.80E-07). We found no significant association between measures of epigenetic age acceleration and history of child maltreatment (p = 0.530), maternal participation in the NFP program (p = 0.491), or the number of mental disorders recorded at age 27 years (p = 0.152).Chronological age and estimated epigenetic age were moderately correlated in this cohort  is somewhat comparable to the error within the epigenetic clock predictor (3.6 years)10. In line with our data, Brody et al. report no direct association between a program designed to reduce harsh parenting and epigenetic age acceleration in a large sample of young adults9. However, the authors do note a moderating role of the intervention on parental depression-associated epigenetic age acceleration, with significantly greater age acceleration in the control group. Taken together with the results of our PCA, such findings suggest that intervention programs may influence features of the DNA methylome and could be of use for precision medicine efforts in child/adolescent health but further work is needed.Maternal NFP participation or history of CAN did not predict epigenetic age acceleration in this cohort. This is in line with a recent study which found that cumulative lifetime stress, rather than childhood trauma alone, predicted epigenetic age acceleration40. Likewise, our analyses are based on those participants who provided biosamples at age 27 years, approximately half of the original Elmira NFP cohort, who were more likely to have experienced abuse and psychiatric disorder. It is possible that our findings were affected by the selective attrition evident at the 27-year follow-up  and thus may underestimate NFP program effects on DNA methylome variation.We do note that the association between maternal NFP program participation and offspring DNA methylation was small, accounting for <1% of the variance in the DNA methylome. However, the 450K array describes <3% of the DNA methylome and provides limited coverage of enhancer regions, which play an important role in transcriptional regulation and mental health41. Likewise, our study benefits from the inclusion of paired genetic data as an important determinant of variation in DNA methylation. We used a broad index of genetic risk for multiple psychiatric disorders and report not a significant association with both the number of current psychiatric diagnoses and variation in DNA methylation. However, our PRS analyses provided little evidence of overlap between PRS- and child maltreatment-associated vCpGs. These findings are somewhat in line with those of Hannon et al., who report little overlap between CpGs associated with the presence of a diagnosis of schizophrenia and a PRS for the disorder42.Our study benefits from the randomized controlled trial and longitudinal design of the NFP study and an objective measure of CAN (substantiated cases from Child Protective Services records). Indeed our findings may also underestimate the true effect of CAN on methylome variation due to unreported instances of maltreatment and the likelihood of at least some CAN contamination within our control group26 and considered cell type in all of our analyses. However, we cannot rule out residual confounding by cell types not estimated by this method. Likewise, due to the unique nature of this 27-year study our sample size was relatively small and a comparable replication cohort was not available. The primary objective of this study was to examine how the early social environment shapes variation in the DNA methylome. We did not set out to perform an epigenome-wide association study of NFP program effects on DNA methylation and were not powered to do so (see Supplemental Table Limitations of our study include the lack of a complete blood count at the time of sample collection, which we addressed using a well-established computational method to ascertain cell type based on DNA methylation profiles43.Another limitation is the cross-sectional nature of biological sample collection and the time from the termination of the NFP intervention at age 2 years to the sampling at 27 years. Our biosampling strategy also precluded an analysis of gene expression or other features of the epigenome, which limits our understanding of the functional consequence of variation in DNA methylation. As such, a causal analysis of the effects of the NFP or CAN on DNA methylation is not possible and these findings require replication, especially in light of our finding that child maltreatment associates with health-risk behaviors, which in turn predict variation in DNA methylation. A longitudinal, prospective study of the epigenome is needed to directly address these issues and is justified by our findings. These considerations notwithstanding, our analyses reveal a significant association between the maternal-focused NFP intervention and variation in genome-wide DNA methylation in the adult offspring. We initially hypothesized such a relationship based on the strong effects of the NFP intervention on the health and behavior of the offspringThe results reported here further document the impact of the social environment on the epigenetic signals that regulate genomic function and provide preliminary evidence for the impact of psychosocial early interventions on the epigenome. Prospectively designed cohorts incorporating longitudinal biosampling will clarify the relationship between early intervention and the DNA methylome and its predictive value for mental health outcomes.Supplementary Tables and Figures