Epigenetics

BackgroundFathers adolescent smoking and overweight affect respiratory health in offspring, suggesting that paternal puberty exposures may influence offspring biological ageing through preconception epigenetic mechanisms. MethodsWe analyzed epigenetic age acceleration using four validated epigenetic clocks derived from blood DNA methylation in 892 RHINESSA offspring (mean age 27 years), linked to parental data on smoking and body shapes from RHINE/ECRHS. Linear regression examined parental smoking initiation ([≤]15 or >15 years) and overweight body shape (childhood/puberty or age 30) in relation to offspring epigenetic age acceleration, adjusting for offspring sex, age and parental socioeconomic status. Sensitivity analyses accounted for offspring smoking and BMI. ResultsPCHorvath ({beta} 1.53; 95% CI 0.02, 2.9), PCGrimAge (1.21; 0.03, 2.1), DunedinPACE (0.04; -0.001, 0.1) and PCPhenoAge (1.92; -0.3, 4.2) were accelerated in daughters of fathers who started smoking [≤]15 years. Likewise, PCHorvath (2.25; 1.2, 3.3), PCGrimAge (1.36; -0.2, 2.9), DunedinPACE (0.07; 0.01, 0.1) and PCPhenoAge (3.11; 1.8, 4.4) were accelerated in daughters and sons of fathers who had been overweight in childhood and puberty. These results remained largely unchanged after additional adjustments or stratification in sensitivity analyses. No associations were found for maternal smoking or overweight in puberty. ConclusionsEpigenetic ageing is accelerated in offspring of fathers who smoked or were overweight in puberty, independent of offspring lifestyle. These findings suggest that adolescent boys environment and lifestyle may be critical for next-generation health. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/26352444v1_fig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1eea189org.highwire.dtl.DTLVardef@1af41f4org.highwire.dtl.DTLVardef@1132932org.highwire.dtl.DTLVardef@f5ba2c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Graphical abstract Legend to graphical abstract Figure Fathers smoking or overweight during puberty was associated with accelerated epigenetic aging in offspring (n=892), independent of the offsprings own lifestyle. No such pattern was observed for maternal puberty exposures, or when paternal exposures occurred after puberty. Male puberty may be a critical window for next-generation health. C_FIG

Tobacco smoking induces DNA methylation (DNAm) changes in blood and other tissues, which may influence chronic health outcomes. However, the breadth of smoking-related DNAm changes remains unmapped, offering a space for employing novel technologies. To expand our understanding of smoking impacts on DNAm, we conducted an epigenome-wide association study (EWAS) comparing ever smokers to never smokers, using blood from a multiethnic U.S. study population (n=887). We employed the newly developed Illumina Methylation Screening Array (MSA) covering 269,094 unique sites, including 123,776 CpGs not assayed in previous EWAS. Trans-ethnic meta-analysis identified 152 differentially methylated positions (DMPs) associated with ever-smoking status (n=764); European-specific analysis yielded 129 DMPs (n=674), including 106 overlapping with trans-ethnic analysis. A separate, large-scale replication EWAS (n=2,190) confirmed 91 trans-ethnic and 77 European-specific DMPs. Among our findings, we identified 61 DMPs at CpGs novel to the MSA platform, including near both new and known smoking-associated genes. Most notably, we uncovered a dense cluster of 12 DMPs within a 1117 bp region of ECEL1P1, forming the most long-lasting, persistent smoking-associated DMR ever detected, even among former smokers who quit decades prior. We also detected new signals at AHRR, a well-known locus for smoking-related DNAm changes. eFORGE analysis revealed that detected smoking-associated DNAm changes are predominantly located in hematopoietic stem and progenitor cell (HSPC) DNase I hotspots, aligning with gene set enrichment analyses that highlighted pathways related to hematopoietic stem cell differentiation. Our findings suggest that HSPCs serve as a reservoir for an epigenetic memory of smoking. Additionally, we observed short-term cell-specific smoking-associated DNAm changes in myeloid cells. Our results demonstrate the utility of the MSA in expanding our knowledge of both transient and persistent environmental exposure-associated DNAm changes.

BackgroundDNA methylation-based biomarkers have been widely used to predict biological age; however, most blood-derived data have been used in most existing models, and whether cheek mucosa can serve as an alternative indicator for methylation-based estimation of aging-related and clinical phenotypes is unclear. MethodsDNA methylation profiles from cheek mucosa and whole blood of 186 Japanese adults were analyzed using Illumina Infinium Methylation Screening Array (MSA). Models were constructed to predict chronological age, phenotypic age, and clinical laboratory biomarkers from cheek mucosa- and blood-derived methylation data. In addition to applying the ordinary elastic net method, a two-stage residual learning method incorporating existing blood-based epigenetic clocks was applied for more accurate prediction of biological age. Sex-stratified analyses and comparisons of selected CpG features across sexes and tissues were performed. ResultsCheek mucosa-derived MSA methylation data enabled accurate prediction of chronological age (R = 0.965) and phenotypic age (R = 0.964) using the two-stage method. The performance gain achieved by the two-stage approach was greater for phenotypic age than for chronological age. Multiple clinical laboratory biomarkers could be predicted using cheek mucosa-derived methylation data, particularly after sex stratification, including inflammatory, metabolic, thyroid-related, and sex hormone-related markers. Most biomarkers that could be predicted using blood-derived methylation data were also predicted using cheek mucosa-derived methylation data. However, the CpG sites selected for prediction showed minimal overlap across sexes and tissues despite overlap in the corresponding predictable phenotypes. ConclusionsCheek mucosa-derived DNA methylation profiles measured using the MSA can predict chronological age, phenotypic age, and multiple clinically relevant laboratory biomarkers, supporting the utility of cheek mucosa as a less invasive alternative for methylation-based assessment of biological aging and systemic physiological state.

Vitamin D signaling has recognized roles in female reproductive physiology, but its effects at the chromatin level in endometrial stromal cells are still unclear. Here, we investigated how the active form of vitamin D, 1,25-dihydroxyvitamin D3, or calcitriol, influences the accessible chromatin landscape of human endometrial stromal cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed on T-HESCs treated with either a vehicle or 1,25(OH)2D3. Ligand treatment increased overall chromatin accessibility, shown by higher ATAC-seq signal intensity, while causing only minor changes in the total number of called peaks. Peak annotation revealed that accessible regions were spread across both promoter-proximal and distal genomic areas. Integrating this data with CUT&RUN and RNA sequencing showed that most vitamin D-responsive cistromic modifications and transcripts were linked to nearby open chromatin, though fewer were associated with regions that were significantly differentially accessible. These results suggest that 1,25(OH)2D3-dependent transcription mainly occurs within a permissive, pre-accessible chromatin environment. This study offers new evidence that active vitamin D influences the epigenomic landscape of human endometrial stromal cells, establishing the chromatin-based molecular response to a chemically-defined VDR ligand, 1,25(OH)2D3, relevant to stromal differentiation and preparation for decidualization. HighlightsO_LIFirst evidence suggesting the direct impact of active vitamin D, 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, enhanced the signal intensity of chromatin accessibility in human endometrial stromal cells C_LIO_LIMost accessible chromatin regions were shared between vehicle and ligand-treated human endometrial stromal cells C_LIO_LI1,25(OH)2D3-responsive transcription occurs largely within pre-accessible chromatin in human endometrial stromal cells C_LIO_LIAssay for transposase-accessible chromatin sequencing (ATAC-seq) defines a chromatin-level pharmacologic response to a chemically defined VDR ligand in human endometrial stromal cells C_LI

INTRODUCTION: MethylCog is a 29-CpG blood DNA methylation (DNAm) proxy for general cognitive ability (g). Its incremental association with blood biomarkers of Alzheimer's disease and related dementias (ADRD) and prospective cognitive ability remains unclear. METHODS: In the held-out test set from the original MethylCog study, we tested whether MethylCog explained baseline g beyond four ADRD blood biomarkers, and whether it predicted six-year follow-up g beyond baseline g and biomarkers. RESULTS: MethylCog showed a stronger age-adjusted association with baseline g than individual biomarkers (r=.368 vs absolute r=.083-.162). MethylCog added 10.0% variance beyond all four biomarkers cross-sectionally (p<.001) and predicted six-year follow-up g in the biomarker-adjusted model (beta=.108, p=.002). No individual ADRD biomarker independently predicted follow-up g. DISCUSSION: MethylCog may provide cognition-related DNAm information complementary to blood-based ADRD biomarkers.

Cartilage and bone that comprise craniofacial structures as well as neurons and glia of the peripheral nervous system are derived from a multipotent population of cranial neural crest cells, that respond to both cell intrinsic and extrinsic cues to differentiate into precise cell states. Both a genetic and epigenetic regulatory network are required for each step in the differentiation process, involving transcription factors, histone modifiers and chromatin remodelers. Here, we examined the direct transcriptional targets of two histone methyltransferases, Prdm3 and Prdm16 in zebrafish neural crest cells at 48 hours post fertilization in zebrafish. Using CUT&RUN, we examined both direct DNA binding and nucleosome association. At this stage of development, CUT&RUN fragment size analysis indicated that Prdm3 and Prdm16 are largely associated with nucleosomes. We further analyzed these nucleosome peak sets to identify 6 clusters where differential binding of Prdm3 and Prdm16 and differential enrichment of gene ontology terms for target genes was observed. We validated gene expression in each cluster by in situ hybridization chain reaction (HCR) at 48 hpf demonstrating that prdm3 and prdm16 mutants exhibit corresponding changes in gene expression of the putative gene targets identified. Finally, we performed CUT&RUN-qPCR in prdm3 and prdm16 mutant zebrafish embryos and demonstrated reduced binding at putative target loci. Together these data suggest that Prdm3 and Prdm16 regulate their transcriptional targets primarily by binding nucleosomes around their putative target loci to control downstream gene expression. HighlightsPrdm3 and Prdm16 associate with nucleosomes for regulation of gene expression Gene targets are altered in prdm3 and prdm16 mutant zebrafish Reduced binding is observed in respective mutants

Background Preterm birth (PTB) rates among Hispanic/Latina individuals in the United States have risen over the past decade. Data suggests this rise may be driven in part by psychosocial stress. Leukocyte telomere length (LTL), a marker of cumulative cellular aging that shortens under chronic stress, may capture stress-related biological vulnerability, but has not been examined as a potential population-level contributor to PTB in Hispanic/Latina pregnancies. Objective To examine the association between mid-pregnancy maternal LTL and PTB in a population-based Hispanic/Latina cohort. Methods In a case-control study nested within a California singleton birth cohort (n = 436 Hispanic/Latina individuals; 215 PTB, 221 term births), LTL was measured by quantitative PCR from biobank specimens collected from 15 to 20 weeks of gestation. Covariates from linked birth certificate and hospital discharge records were included. Logistic regression estimated ORs and 95% CIs of PTB by LTL examined continuously and by percentile category (<=10th, 11th-89th, >=90th) with and without adjustment for covariates. Results Mean and median LTL did not differ between PTB and term births. LTL at or below the 10th percentile was associated with elevated odds of PTB relative to full-term birth (12.6% versus 4.3%; ORc = 3.2, 95% CI 1.3-7.9), persisting after partial (ORadj1 = 3.2, 95% CI 1.3-8.3) and full covariate adjustment (ORadj2 = 3.4, 95% CI 1.3-9.3). Subgroup analyses showed consistent directional patterns across PTB subgroups and for early term birth (ORadj2 = 5.1, 95% CI 1.5-17.0). Conclusions Mid-pregnancy maternal LTL <=10th percentile was associated with more than three times the odds of PTB, with risk concentrated at the extreme low tail of the distribution. Consistent with a cumulative allostatic load model, markedly short LTL at mid-gestation may reflect elevated stress-related biological risk for preterm delivery. These findings support upstream investment in stress reduction and prospective LTL research in high-burden populations.

Background: It is well-established that males have a higher mortality risk than females. Immune cells and their function are known to undergo characteristic changes during aging, and immune cells are known to have sex differences. Immune cells and their function have been linked to mortality risk, but no studies have investigated to what degree, if at all, Immune Cell Biomarkers (ICBs) contribute to the known differences in mortality risk by sex. Methods: Using participant data from the Health and Retirement Study (n = 8,822), we applied multivariable linear regressions adjusting for age, cytomegalovirus (CMV) serostatus, sex, and race/ethnicity to identify differences by sex in 48 immune cell biomarker (ICB, e.g. T cells, B cells, Monocytes, etc.) percentages and counts (measured in 2016). We studied how the associations between ICBs and mortality risk differ by sex using stratified Cox Proportional Hazard (CPH) models. We estimated how inclusion of sex explained the relationship between ICBs and all-cause mortality, and conversely, how inclusion of individual and all ICBs combined explain the relationship between sex and all-cause mortality using multivariable modeling approaches. Results: Differences in ICBs by sex range between 2-38% (39/48 statistically significant). 9 ICBs were significantly associated with mortality risk in the entire sample. While different ICBs were significantly associated with mortality risk in the stratified analyses, particularly with respect to monocyte, B cell, and NK cell populations, adjusting for sex modestly influenced the hazard ratios of the ICBs (sex: 8 ICBs, percent change <5.4%). Furthermore, individual and cumulative contributions of ICBs in explaining the differences in mortality risk by sex were not significant.

Pregnancy requires tightly regulated immune adaptation, which may be altered by maternal adiposity and HIV infection. In high HIV prevalence settings with rising obesity rates, overlapping metabolic and infectious inflammation may shape gestational immune trajectories. We examined patterns of C-reactive (CRP), serum amyloid A (SAA), and interferon-gamma inducible protein-10 (IP-10) among 527 pregnant women living with HIV in Cape Town, South Africa. Immune markers were measured at up to four antenatal visits. Maternal body mass index (BMI) was categorised as normal, overweight or obese. Antiretroviral therapy (ART) exposure was classified as initiation before conception or during pregnancy. Mixed-effects models assessed associations, adjusting for baseline CD4 cell count and viral load. CRP concentrations remained elevated across pregnancy and were significantly higher among obese women independent of ART timing. In contrast, IP-10 concentrations were strongly associated with ART timing, with higher levels observed prior to ART initiation and declining thereafter; no independent association with BMI was observed. SAA concentrations were modestly lower among obese women and among those on preconception ART. These divergent patterns persisted after adjustment for HIV disease severity. These findings suggest that metabolic and HIV-related inflammatory pathways operate in parallel during pregnancy, with adiposity predominantly influencing acute-phase responses and HIV-related immune activation shaping interferon-associated chemokines trajectories.

Background: Despite the success of combination antiretroviral therapy (cART), vascular comorbidities, including cerebrovascular disease, are more prominent in people living with HIV (PLWH) compared to people without HIV (PWOH). However, quantitative assessments of cerebrovascular morphometry and their associations with cognitive outcomes in the context of HIV are still limited. In this study, we explore this missing link. Methods: Magnetic Resonance Angiography (MRA) data, blood markers, and neurocognitive assessments were collected from 73 PWOH subjects (male: 57, female: 16; age: 53 {+/-} 16) and 99 PLWH subjects (male: 66, female: 30, age: 53 {+/-} 11). Vessel morphometric features were quantified using intraCranial Artery Feature Extraction (iCafe) to investigate associations between vessel morphometry, markers of monocytes, endothelial cell activation, and cognitive performance. Results: HIV status predicted a lower total number of branches ({beta} = -0.224, p = 0.001, d = -0.517) and shorter total distal length ({beta} = -0.173, p = 0.021, d = -0.370) with a moderate effect size. Total branch number was found to be negatively associated with plasma levels of monocyte markers (sCD14: r = -0.167, p = 0.033; sCD163: r = -0.157, p = 0.045) and positively correlated with white matter cerebral blood flow (r = 0.550; p [&le;] 0.05). HIV status was the strongest predictor of overall cognitive performance in ANCOVA model ({beta} = -0.219, p = 0.006, d = -0.453). Conclusions: Our results suggest that cognitive impairment in PLWH is associated with vessel morphology metrics. Monocyte immune activation may contribute to changes in vessel morphology.

Background and Purpose: Ambient air pollution is an established risk factor for incident stroke, but whether post-discharge pollutant exposure influences stroke recurrence remains unknown. We investigated the association between post-discharge exposure to six ambient air pollutants and stroke recurrence in patients with acute ischemic stroke. Methods: We analyzed data from 27,346 patients in the CRCS-K-NIH nationwide multicenter registry of acute ischemic stroke patients (2014-2021) with confirmed ischemic stroke, residential address data, and matched air quality records. The primary exposure was the 3-month post-discharge average concentration of PM10, PM2.5, NO2, SO2, CO, and O2, assessed at the district level using inverse-distance weighted interpolation. The primary outcome was stroke recurrence from 3 to 15 months post-discharge. Cause-specific Cox proportional hazards models accounting for the multilevel data structure were used, with all-cause mortality as a competing risk. Restricted cubic splines assessed nonlinear dose-response relationships. Results: During follow-up (median 364.8 days), 765 patients experienced stroke recurrence and 471 died. Among the six pollutants, only SO2 showed a statistically significant association with recurrence (P for overall association in the restricted cubic spline analysis = 0.024). A potential threshold was identified at approximately 8.2 ppb, above which recurrence risk increased progressively (P for non-linearity = 0.095). The association was numerically stronger among older adults ([&ge;]75 years; P for interaction = 0.051) and women (P for interaction = 0.062). The highest SO2 concentrations were observed in harbor cities (Incheon, Ulsan, Busan), consistent with maritime shipping emissions. No significant associations were observed for the other five pollutants. Conclusions: Elevated post-discharge SO? exposure is associated with increased stroke recurrence risk, particularly in harbor regions and among older adults and women. These findings support incorporating ambient air quality monitoring into secondary stroke prevention strategies.

Assessing reproducibility across different molecular profiling studies is a persistent methodological challenge (Zhang et al., 2009; Sweeney et al., 2017; Ioannidis, 2005). Differences in platform technology, cohort composition, analytical pipelines, and feature definitions often make it difficult to interpret cross-study comparisons based solely on gene-identity overlap. In this study, we conducted a retrospective computational analysis of seven publicly available analytical datasets (including alternative analytical pipelines applied to the same cohort) derived from five biologically independent peripheral blood transcriptomic and DNA methylation cohorts, comprising 3,487 samples (1,824 Parkinsons disease cases and 1,663 controls). Reproducibility was evaluated using gene-identity overlap, enrichment-based comparisons, and a permutation-based framework to assess directional consistency of effect estimates across datasets. We also tested the robustness of results by varying false discovery rate thresholds and applying alternative probe-to-gene collapsing strategies. All analyses were performed using reproducible workflows implemented in R and Python with fixed random seeds. Across independent cohorts, gene-identity overlap was generally limited, with enrichment ratios close to one, especially when datasets were generated using different platforms. In several datasets, limited numbers of statistically significant features further constrained overlap-based comparisons. In contrast, directional consistency showed greater stability. High levels of directional consistency were observed across independent cohort comparisons when restricted to overlapping statistically significant features and remained stable across statistical thresholds (90.0% at FDR < 0.05 and 82.8% at FDR < 0.10). When evaluated across the full shared gene universe without conditioning on statistical significance, directional consistency was substantially lower ([~]30 to 32%) but remained significantly above permutation-based null expectations. Permutation testing confirmed that the observed directional consistency exceeded what would be expected by chance. A combined analysis including methodological replicates (n [&ge;] 3 datasets) showed 98.3% directional consistency; however, this estimate includes non-independent analytical pipelines applied to the same cohort and reflects analytical stability rather than independent biological replication. Rather than introducing a new statistical method, this study examines how commonly used reproducibility metrics behave under crossstudy heterogeneity and identifies their practical limitations and appropriate use boundaries.

Enhancers are key epigenetic regulatory elements that orchestrate spatiotemporal gene expression and are critical in mammalian development, gene regulation, and disease. Histone modifications such as H3K4me1 (a canonical enhancer mark) and H3K27ac (which distinguishes active enhancers) remain poorly characterized during early mammalian embryogenesis. Using low-input CUT&RUN (Cleavage Under Targets and Release Using Nuclease) with input as low as 50 cells, this study profiles genome-wide H3K4me1 and H3K27ac patterns in mouse oocytes and pre-implantation embryos. Both marks are enriched in distal regions and exhibit distinct sequence preferences and reprogramming dynamics in pre-implantation embryos. H3K27ac is reprogrammed at the 2-cell stage and marks active enhancers, while H3K4me1 is remodeled at the 4-cell stage and co-localizes with H3K27ac, overlapping with accessible chromatin regions. Interestingly, the co-localization of H3K4me1 and H3K27ac is also detected in promoter regions, where they exhibit a mutually exclusive pattern with H3K4me3. Three enhancer types-active (H3K4me1/H3K27ac), primed (H3K4me1), and poised (H3K4me1/H3K27me3)-are dynamically remodeled during maternal-to-zygotic transition (MZT), with active enhancers increasing significantly after zygotic genome activation. Furthermore, genome-wide super-enhancers are identified and mainly enriched in promoters. The differences in gene expression at different stages may be related to the specific motifs enriched by super-enhancers.

Cognitive impairment is a major source of disability in Parkinsonian disorders, yet biomarkers that distinguish cognitive status from cognitive decline remain limited. DNA methylation-based epigenetic aging measures capture complementary dimensions of biological aging, but it remains unclear whether they primarily reflect stable differences in cognitive vulnerability or longitudinal cognitive change. We examined associations between epigenetic aging measures and global cognition in the Parkinsons Progression Markers Initiative (PPMI) cohort. Seven epigenetic aging measures were derived from peripheral blood DNA methylation data, and cognition was assessed longitudinally using the Montreal Cognitive Assessment (MoCA). Linear mixed-effects models were applied in complementary frameworks, including baseline-plus-change-from-baseline models and within-person versus between-person decomposition models. Secondary analyses included baseline clock-by-time interaction models and a decline-focused sensitivity analysis. Across analyses, higher epigenetic aging was consistently associated with lower overall MoCA scores. In the baseline-plus-change-from-baseline models, the analytic baseline component showed the dominant signal, whereas the change-from-baseline terms were not significant after false discovery rate correction. In the within-person versus between-person decomposition models, associations were concentrated in the between-person component, while within-person deviation terms were not significant. Secondary analyses were consistent with this pattern. Together, these findings suggest that blood-based epigenetic aging measures may be more informative as biomarkers of cognitive status or vulnerability than as markers of short-term cognitive progression. Larger studies with longer follow-up and more detailed cognitive phenotyping are needed to clarify their longitudinal relevance.

Clonal hematopoiesis of indeterminate potential (CHIP) represents the age-related expansion of hematopoietic stem cells with preleukemic mutations. However, its association with all-cause and cause-specific mortality has not been well characterized in older adults. We aimed to evaluate whether CHIP is associated with all-cause and cause-specific mortality in a population of older women in the United States. Our study included 6,704 participants in the Women?s Health Initiative Long Life Study (WHI-LLS) without hematologic malignancy. The co-primary exposures were any CHIP (variant allele frequency [VAF] [&ge;] 2%) and large CHIP (VAF [&ge;] 10%), and the primary outcome was all-cause mortality. Multivariable-adjusted Cox proportional hazards models tested the associations of CHIP and CHIP subtypes with all-cause and cause-specific mortality. Any CHIP and large CHIP were independently associated with all-cause mortality, with multivariable-adjusted hazard ratios (aHRs) of 1.12 (95% confidence interval [CI] 1.04-1.21; P = 0.003) and 1.28 (95% CI 1.15-1.43; P < 0.001), respectively. In gene-specific analyses, non-DNMT3A CHIP was associated with all-cause mortality (aHR: 1.22 [95% CI: 1.12-1.34], P < 0.001), while DNMT3A CHIP was not (aHR: 1.07 [95% CI: 0.98-1.18], P = 0.13). Furthermore, large CHIP was associated with cardiovascular (aHR: 1.29 [95% CI: 1.08-1.55], P = 0.006), cancer (aHR: 1.49 [95% CI: 1.11-2.02], P = 0.009), and neurologic (aHR: 1.40 [95% CI: 1.07-1.84], P = 0.02) death. In this cohort of older women, CHIP, particularly large clones and non-DNMT3A CHIP, was associated with all-cause and cause-specific mortality. These findings suggest that clonal size and subtype may differentially influence mortality risk.

Objective: To characterize genome-wide DNA methylation patterns associated with sepsis using the Infinium Methylation EPIC v2.0 platform and to evaluate the feasibility of pooled methylation profiling in a pilot critical care cohort. Design: Single-center pilot epigenome-wide association study using pooled whole-blood genomic DNA and pool-level bioinformatic analysis. Setting: Academic medical center. Patients: Fifty critically ill adults enrolled within 48 hours of illness onset and 20 healthy controls. Interventions: None. Measurements and Main Results: Critically ill patients required mechanical ventilation and/or vasopressor support. Sepsis was defined according to Sepsis-3 criteria. Seventy individual samples were organized into 14 intended pools of 5 individuals each: 7 sepsis pools, 3 critically ill non-septic pools, and 4 healthy-control pools. One critically ill non-septic pool was excluded because of poor DNA quality, yielding 13 analyzable pools. For the primary pooled comparison, 7 sepsis pools were compared with 6 non-sepsis comparator pools comprising 2 critically ill non-septic and 4 healthy-control pools. After quality control and preprocessing with SeSAMe, 876,094 CpG sites were retained. The initial pool-level screen identified 170,897 candidate differentially methylated regions. Application of stringent secondary filters (false discovery rate <= 1%, absolute delta-beta >= 7.5%, and >= 5 CpGs per region) yielded a high-confidence subset with marked directional skewing, including 155 hypomethylated and 32 hypermethylated regions in sepsis. Differentially methylated region-associated genes were enriched in myeloid leukocyte activation, myeloid leukocyte-mediated immunity, defense response to bacterium, neutrophil granule biology, and hematopoietic cell lineage pathways. Additional signals involved microRNA-associated targets, ribosome biogenesis, RNA processing, long noncoding RNAs, and previously uncharacterized loci. Conclusions: In this pilot pooled EPIC v2.0 study, sepsis was associated with a biologically coherent, predominantly hypomethylated methylation signature enriched in myeloid and host-defense pathways. These findings support the feasibility of pooled methylation profiling for discovery-oriented sepsis biobank studies but should be interpreted as hypothesis-generating given the pool-level design, limited effective sample size, heterogeneous comparator group, and lack of direct validation against individual-level methylation profiles.

INTRODUCTION: Alzheimers disease (AD) is a multifactorial disorder, yet current research largely focuses on downstream biomarkers with limited attention to environmental contributors. Experimental studies suggest that per and polyfluoroalkyl substances (PFAS) may contribute to neuroimmune and neurodegenerative pathways relevant to AD. OBJECTIVE: To examine associations between PFAS exposure and neuroimmune and AD related plasma biomarkers in cognitively unimpaired rural adults. METHODS: In a cross sectional pilot study (n=48), serum concentrations of 33 PFAS were measured, including four legacy compounds (PFOS, PFHxS, PFOA, PFNA). Plasma neuroimmune related (ITGB2, SMOC1, TREM2, GFAP) and AD related biomarkers (Ab42/40, ptau217) were detected using proteomic analysis. RESULTS: PFOS showed moderate associations with ITGB2, SMOC1, and Ab42/40 in unadjusted analyses, which attenuated after adjustment for age. PFOA and PFNA demonstrated consistent inverse associations with TREM2 before and after adjustment. DISCUSSION: Findings suggest possible compound specific PFAS associations with immune and amyloid related biomarkers, supporting further investigation in longitudinal and PFAS mixture based studies.

Polycomb Repressive Complex 2 (PRC2) maintains repression of genes specific for other cell differentiation states. PRC2 binds RNA in vitro with a preference for G-rich sequences. UV-based crosslinking coupled with immunoprecipitation (CLIP) experiments have shown that PRC2 also binds RNA in cells. Recently, Guo et al reported that a stringent denaturing variant of CLIP called CLAP did not detect PRC2 RNA binding in cells. We present a reanalysis of CLAP data that supports direct interaction of PRC2 with RNA in cells. CLAP for Halo-tagged PRC2 subunits from mixed populations of human and mouse cells specifically enriched for RNA from the species in which the proteins were tagged. The lack of apparent PRC2 RNA binding in Guo and colleagues analysis stems from a scaling step that deflates enrichment scores for low-complexity CLAP samples. Our findings pave the way for studies seeking to determine the physiological roles of PRC2 RNA binding activity.

Understanding the immune response against HIV-1 and the natural resistance exhibited by HIV-exposed Seronegative Individuals (HESN) offers the possibility of proposing new control strategies. Several studies suggest an important role of HLA and KIR genes in protecting against HIV-1 infection. Moreover, there is an important gap in the knowledge of these genetic factors in seronegative Latin American men who have sex with men (MSM), a population largely underrepresented in HIV immunogenetic studies. This study aimed to identify HLA and KIR genetic profile associated with potential resistance to HIV-1 acquisition, in a cross-sectional study including a cohort of 60 HIV-1-seronegative Colombian MSM at low and high risk of HIV-1 infection. The high-risk group showed a higher frequency of the HLA-B*18 allele, and a lower frequency of the HLA*B35, which have been previously associated with protection and susceptibility to HIV-1 infection respectively. Likewise, the high-risk group exhibited a low frequency of Bx haplotypes, a higher frequency of one AA haplotype and differences in KIR gene profile, with a low frequency of the inhibitory KIR2DL5 and both activating KIR2DS1, KIR2DS2 and KIR2DS5 genes. These findings suggest that host immunogenetic factors may contribute to resistance to HIV-1 acquisition in highly exposed individuals.

BackgroundMetazoan adenosine-to-inosine (A-to-I) mRNA editing temporospatially diversifies the neuronal transcriptome and proteome. The limited read length from next-generation sequencing (NGS) constrains the quantification of the potentially differential editing levels across different splicing isoforms, restricting our understanding of the extent to which RNA editing contributes to molecular diversity and its interplay with splicing. MethodsWe employed reverse transcription nested PCR (RT-nPCR) and developed a novel interfering-Primer PCR (iPrimer PCR) technique to distinguish different transcripts of any gene. We selected multiple essential genes exhibiting RNA editing in coding sequences (CDSs) or untranslated regions (UTRs) for isoform-specific amplification and Sanger sequencing. ResultsNine different Adar isoforms together with pre-mRNA had distinct editing levels at the S>G auto-recoding site, which was predicted to have isoform-specific effects on catalytic activities. Although pre-mRNA editing might exert isoform-dependent promotion/suppression of splicing, closely located editing sites, such as those in neuronal genes qvr and stj, still exhibited high correlation in editing levels due to co-editing. iPrimer strategy further discovered differential recoding levels between the long/short 3UTR isoforms of gene jef. ConclusionsWe provide the first comprehensive solution for isoform-specific PCR amplification of any gene, enabling quantification of RNA editing level of different isoforms. Our results offer insights into how RNA editing interplays with splicing, and highlight its complicated role in expanding molecular diversity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/725286v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1ebc82org.highwire.dtl.DTLVardef@1ea365dorg.highwire.dtl.DTLVardef@1971aceorg.highwire.dtl.DTLVardef@160d053_HPS_FORMAT_FIGEXP M_FIG C_FIG We developed isoform-specific PCR followed by Sanger sequencing, and achieved the quantification of differential RNA editing levels in different transcripts of a gene.