Tobacco smoke exposure has been shown to dramatically alter the methylome of whole blood DNA, and we have previously observed that effects are very different among blood cell types. In the present work, we compare smoking effects on DNA methylation-based telomere length (DNAmTL) shortening, age acceleration (DNAmAA) of 6 DNAmAge clocks (Horvath, Hannum, Skin_Blood, PhenoAge, FitAge, GrimAge2), aging pace (DunedinPACE), and Stochastic Epigenetic Mitotic Timer of Cancer (stemTOC) in six major immune cell types isolated from the whole blood samples of the same individuals from 74 nonsmokers and 69 smokers. Telomere shortening in all cell types was significantly associated with smoking status, with smokers displaying greater telomere shortening. Examining the difference in DNAmTL shortening between smokers and nonsmokers across cell types, CD8+ T cells had the greatest DNAmTL shortening, while CD15+ granulocytes had the least shortening. Among six DNAmAge models tested, the strongest association between DNAmAge Acceleration (DNAmAA) and smoking status was observed for the GrimAge2 model, followed by the FitAge model and then the PhenoAge model, with smokers displaying greater age acceleration. For the GrimAge2 and FitAge models, as expected, smokers revealed greater age acceleration in whole blood samples. Across all cell types, the effect size in epigenetic age acceleration associated with smoking was significantly correlated with smoking effect as measured by mean AHRR cg05575921 demethylation values, with myeloid cell types showing the greatest effect and T cells least. However, the Hannum, Horvath, and Skin_Blood models (which were trained only by chronological age) were generally not significantly associated with smoking status in isolated cell types. We observed the DunedinPACE model was significantly associated with smoking status in all cell types and whole blood samples. Smokers had significantly higher aging pace in all cell types relative to nonsmokers. The stemTOC was not significantly associated with smoking status in isolated cell types. The present study demonstrates that tobacco smoking is significantly associated with methylation-based measures of telomere length shortening, biological age acceleration, and aging pace in six major immune cell types and whole blood. The effect of smoking on these outcomes differs among cell types, suggesting shifts in cell composition may play an important role in human aging as measured by DNA methylation models and epigenetic aging may play a role in smoking-related diseases.

We applied the TempO-LINC® platform to generate single-cell transcriptomic (SCTr) profiles of ∼40,000 HepaRG cells exposed to etoposide, brefeldin A, cycloheximide, rotenone, tBHQ, troglitazone, and tunicamycin at three concentrations for 24 hours. SCTr enabled a detailed analysis of adaptive stress response pathways (SRPs), including the unfolded protein response (UPR), oxidative stress response (OSR), heat shock response (HSR), and DNA damage response (DDR). Troglitazone upregulated lipid metabolism genes (PLIN2, ACOX1) along with HSR and UPR activation, with co-expression of DNAJA1, HSP90AA1, and DDIT3 in subsets of cells. Brefeldin A and tunicamycin strongly induced UPR markers (HSPA5, SYVN1, LMF2, PDIA4) in subsets of cells, with some also expressing apoptotic (DDIT3, CASP8) and autophagic (SQSTM1) genes, indicating diverse stress responses. Rotenone activated GDF15, TRIB3, and DDIT3 in a fraction of cells, accompanied by PLIN2 and mild UPR induction, reflecting heterogeneous mitochondrial stress responses. We scored individual cells using literature-derived SRP gene signatures to characterize overall stress phenotypes and clustered them using a generalized Jaccard metric. The clustering revealed five phenotypic groups spanning cell states associated with homeostasis, adaptive responses, terminal outcomes, autophagy, and apoptosis. By systematically analyzing the distributions of cells in different states across treatments, we visualized dynamic shifts in cellular subpopulations responding to chemicals, revealing early stress responses and potential transitions to cell death. Our findings suggest the utility of SCTr in decoding stress states that could provide possible insights into transitions between cellular adaptive and terminal transitions involved in toxicity.

In the USA, funding for rehabilitating Golden Eagles (Aquila chrysaetos) from injuries and diseases may serve to offset incidental take of the species as authorized by permit from the US Fish and Wildlife Service, e.g., for blade-strike fatalities at wind turbines. However, post-release survival of these eagles may differ from that of their wild conspecifics. Our objectives were to (1) estimate the survival rate of rehabilitated Golden Eagles tracked via satellite telemetry in western North America during their first post-release year; (2) compare the estimate to that of wild Golden Eagles; and (3) estimate how many rehabilitated individuals must be released such that one would be as likely to survive its first post-release year as a wild eagle would to survive any given year. During March 2013-June 2023, we tracked 27 Golden Eagles that had been admitted for rehabilitation when >= 1 yr (AFY) of age, mostly (>= 85.2%) due to anthropogenic factors; rehabilitation lasted 23-321 d (median = 125.0). Within 1 yr of release, 15 (55.6%) of the eagles died, nine (33.3%) remained alive, and fates of three (11.1%) were unknown (tracked 44 d, 134 d, and 294 d). The mean survival rate estimate from a multistate model was 0.31 (SD = 0.08), versus 0.87 (SD = 0.07) for wild AFY Golden Eagles. We estimated that 3.5 rehabilitated Golden Eagles must be released for one to be as likely to survive its first post-release year as a wild Golden Eagle would to survive any given year.

Exposures to pollutants rarely occur in isolation, often coexisting with other environmental stressors such as diet and may be particularly insidious in early life. The aim of this study was to examine effects of maternal exposure to cadmium (Cd) and consumption of a high-fructose diet (HFrD) on development of mouse offspring. Female CD-1 mice were administered either 0.5 or 5 ppm Cd in drinking water with or without an approximate 60% fructose diet for 3 weeks prior to mating. Dams were maintained on the same treatment until postnatal day (PND) 16. Cadmium concentrations in maternal, fetal, and neonatal liver increased in a concentration-dependent manner irrespective of diet. Endpoints known to be associated with Cd or HFrD adverse effects were assessed longitudinally in offspring from birth to young adulthood, including growth trajectory, pubertal development, body composition, glycemic tolerance and hepatic lipid accumulation. Maternal exposure to either Cd or HFrD alone significantly advanced onset of puberty, hypoglycemia, and reduced adiposity in adulthood. HFrD rarely exacerbated metal-initiated effects in most of the endpoints examined outside of pubertal timing. Because of chronic effects attributed to Cd or HFrD on metabolic function (e.g. glucose tolerance), transcriptomics and gene methylation analyses were performed on livers from neonatal and adult offspring. Data were largely consistent with phenotypic findings. In summary, maternal exposure to Cd or HFrD alone perturbed growth and development, producing long-lasting changes in metabolic function in adult offspring. HFrD did not appear to significantly exaggerate adverse outcomes attributed to metal exposure in the endpoints examined.

BackgroundThe aetiology of lung cancer among individuals who never smoked remains elusive, despite 15% of lung cancer cases in men and 53% in women worldwide being unrelated to smoking. Epigenetic alterations, particularly DNA methylation (DNAm) changes, have emerged as potential drivers. Yet, few prospective epigenome-wide association studies (EWAS), primarily focusing on peripheral blood DNAm with limited representation of never smokers, have been conducted.MethodsWe conducted a nested case-control study of 80 never-smoking incident lung cancer cases and 83 never-smoking controls within the Shanghai Women’s Health Study and Shanghai Men’s Health Study. DNAm was measured in prediagnostic oral rinse samples using Illumina MethylationEPIC array. Initially, we conducted an EWAS to identify differentially methylated positions (DMPs) associated with lung cancer in the discovery sample of 101 subjects. The top 50 DMPs were further evaluated in a replication sample of 62 subjects, and results were pooled using fixed-effect meta-analysis.ResultsOur study identified three DMPs significantly associated with lung cancer at the epigenome-wide significance level of p<8.22×10−8. These DMPs were identified as cg09198866 (MYH9;TXN2), cg01411366 (SLC9A10) and cg12787323. Furthermore, examination of the top 1000 DMPs indicated significant enrichment in epithelial regulatory regions and their involvement in small GTPase-mediated signal transduction pathways. Additionally, GrimAge acceleration was identified as a risk factor for lung cancer (OR=1.19 per year; 95% CI 1.06 to 1.34).ConclusionsWhile replication in a larger sample size is necessary, our findings suggest that DNAm patterns in prediagnostic oral rinse samples could provide novel insights into the underlying mechanisms of lung cancer in never smokers.

Bladder cancer, a common neoplasm, is primarily caused by tobacco smoking. Epigenetic alterations including DNA methylation have the potential to be used as prospective markers of increased risk, particularly in at-risk populations such as smokers. We aimed to investigate the potential of smoking-related white blood cell (WBC) methylation markers to contribute to an increase in bladder cancer risk prediction over classical questionnaire-based smoking metrics (i.e., duration, intensity, packyears) in a nested case–control study within the prospective prostate, lung, colorectal, and ovarian (PLCO) Cancer Screening Trial and the alpha-tocopherol, beta-carotene cancer (ATBC) Prevention Study (789 cases; 849 controls). We identified 200 differentially methylated sites associated with smoking status and 28 significantly associated (after correction for multiple testing) with bladder cancer risk among 2670 previously reported smoking-related cytosine–phosphate–guanines sites (CpGs). Similar patterns were observed across cohorts. Receiver operating characteristic (ROC) analyses indicated that cg05575921 (AHHR), the strongest smoking-related association we identified for bladder cancer risk, alone yielded similar predictive performance (AUC: 0.60) than classical smoking metrics (AUC: 0.59–0.62). Best prediction was achieved by including the first principal component (PC1) from the 200 smoking-related CpGs alongside smoking metrics (AUC: 0.63–0.65). Further, PC1 remained significantly associated with elevated bladder cancer risk after adjusting for smoking metrics. These findings suggest DNA methylation profiles reflect aspects of tobacco smoke exposure in addition to those captured by smoking duration, intensity and packyears, and/or individual susceptibility relevant to bladder cancer etiology, warranting further investigation.

Assessing impacts of wind farms on volant animals requires conducting fatality monitoring studies that incorporate integrated carcass detection trials to account for searcher detection probability and carcass persistence and to reduce biases in the estimated number of fatalities (Smallwood et al. 2018). These trials involve placing a wide range of volant animal carcasses that mimic the expected species composition and actual spatial and temporal patterns of fatalities deposited by the wind turbines and measuring rates of carcass detection (Smallwood et al. 2018). When combined with remote cameras, carcass placement trials can reveal the suite of local scavengers that affect carcass persistence. We report here on an incident of conspecific carcass removal by a Great Horned Owl (Bubo virginianus) documented during a study designed to assess scavenger removal rates of avian carcasses at a wind energy project (Smallwood et al. 2009, 2010). We conducted the scavenger removal study from 12 December 2006 to 28 September 2007 on a 250ha parcel of land administered by the East Bay Regional Park District in the Altamont Pass Wind Resource Area, California, USA. The study area contained 52 functional wind turbines situated in rows along ridgelines within a landscape dominated by annual grasses. Sixty-three avian carcasses representing diverse avian species and body sizes, including two Great Horned Owls, were used for this study. Carcasses were obtained fresh-dead as either road kills or from animal rehabilitation hospitals and were kept frozen until used. To avoid scavenger swamping, from one to five carcasses were placed each week at randomly chosen locations within 60-m radii of wind turbine bases throughout the facility. Infrared, motion-activated cameras (Reconyxt, Holmen, WI, USA) were attached to metal posts approximately 1 m above the ground and 1.5 m away from each carcass. When activated, the cameras were set to take five pictures in rapid succession, with a recovery phase of approximately 1 sec between firings. All scavenging trial locations were monitored with cameras for 21 d after carcass placement or until a carcass was removed by scavengers, whichever came first. In some instances, camera removals were delayed beyond 21 d due to field conditions. After cameras were removed, all trial locations with either partial carcasses or feathers continued to be monitored weekly by biologists until the end of the study. For more details on this study, see Smallwood et al. (2009). A Great Horned Owl carcass was placed on 19 December 2006 at 1500 H in front of a remote camera set-up. On 13 January 2007, at 1843 H, a Great Horned Owl was photographed landing on

PDF - 2930K, Timeline describing the collection of smoking information and methylation measurements for the study participants.

Supplementary Methods; Supplementary Figures S1-S5

Background Tobacco smoking alters the DNA methylation profiles of immune cells which may underpin some of the pathogenesis of smoking-associated diseases. To link smoking-driven epigenetic effects in specific immune cell types with disease risk, we isolated six leukocyte subtypes, CD14+ monocytes, CD15+ granulocytes, CD19+ B cells, CD4+ T cells, CD8+ T cells, and CD56+ natural killer cells, from whole blood of 67 healthy adult smokers and 74 nonsmokers for epigenome-wide association study (EWAS) using Illumina 450k and EPIC methylation arrays. Results Numbers of smoking-associated differentially methylated sites (smCpGs) at genome-wide significance ( p < 1.2 × 10 −7 ) varied widely across cell types, from 5 smCpGs in CD8+ T cells to 111 smCpGs in CD19+ B cells. We found unique smoking effects in each cell type, some of which were not apparent in whole blood. Methylation-based deconvolution to estimate B cell subtypes revealed that smokers had 7.2% ( p = 0.033) less naïve B cells. Adjusting for naïve and memory B cell proportions in EWAS and RNA-seq allowed the identification of genes enriched for B cell activation-related cytokine signaling pathways, Th1/Th2 responses, and hematopoietic cancers. Integrating with large-scale public datasets, 62 smCpGs were among CpGs associated with health-relevant EWASs. Furthermore, 74 smCpGs had reproducible methylation quantitative trait loci single nucleotide polymorphisms (SNPs) that were in complete linkage disequilibrium with genome-wide association study SNPs, associating with lung function, disease risks, and other traits. Conclusions We observed blood cell-type-specific smCpGs, a naïve-to-memory shift among B cells, and by integrating genome-wide datasets, we identified their potential links to disease risks and health traits.

PDF - 225K, Evaluation of the additional effect of grandmother smoking on DNA methylation at the 26 CpGs of interest, given the mother smoked in her pregnancy. Each point represents the association for each CpG in each category. *Significance threshold p<0.0019 (below the line is not statistically significant).

Past efforts to explain variation of invertebrate assemblages in freshwater wetlands have been less productive than anticipated. To explore why efforts are disappointing, we assembled large invertebrate data sets from North Dakota prairie potholes, California rock pools, and Georgia Carolina bay wetlands that addressed spatial (among wetlands) and temporal (among seasons and years) variation. We anticipated that these large data-set sizes would enable robust conclusions to be drawn, and each place had unique environmental conditions that might contribute to greater explanatory power. We used statistical techniques that partitioned variation in invertebrate assemblages into spatial and/or temporal components, and that also yielded a measure of the amount of unexplained variation; Permutational Multivariate Analysis of Variation and Principal Coordinates Analysis assessed whole assemblage variation, and Analysis of Variance or Analysis of Covariance assessed variation in taxon richness, total abundances, and abundances of wide-spread individual taxa. Across all locations, variation explained by spatial and temporal factors, and unexplained variation were of comparable magnitudes (i.e., similar R2 values of 50

PDF - 16K, Association between the combined mother and grandmother smoking categories and DNA methylation at 26 CpGs of interest. Each point represents the association for each CpG in each smoking category. *Significance threshold p<0.0019 (below the line is not statistically significant).

PDF - 80K, Association between the timing of mother smoking (before and during pregnancy) and DNA methylation at 26 CpGs of interest. Each point represents the association for each CpG in each smoking category. *Significance threshold p<0.0019 (below the line is not statistically significant).

Bronchopulmonary dysplasia (BPD) is a prevalent chronic lung disease of prematurity with limited treatment options. To uncover biomarkers of BPD risk, this study investigated epigenetic and transcriptomic signatures of prematurity at birth and during the neonatal period at day 14 and 28. Peripheral blood DNAs from preterm infants were applied to methylation arrays and cell-type composition was estimated by deconvolution. Covariate-adjusted robust linear regression elucidated BPD- and prolonged oxygen (≥ 14 days) exposure-associated CpGs. RNAs from cord and peripheral blood were sequenced, and differentially expressed genes (DEGs) for BPD or oxygen exposure were determined. Estimated neutrophil–lymphocyte ratios in peripheral blood at day 14 in BPD infants were significantly higher than nonBPD infants, suggesting an heightened inflammatory response in developing BPD. BPD-DEGs in cord blood indicated lymphopoiesis inhibition, altered Th1/Th2 responses, DNA damage, and organ degeneration. On day 14, BPD-associated CpGs were highly enriched in neutrophil activation, infection, and CD4 + T cell quantity, and BPD-DEGs were involved in DNA damage, cellular senescence, T cell homeostasis, and hyper-cytokinesis. On day 28, BPD-associated CpGs along with BPD-DEGs were enriched for phagocytosis, neurological disorder, and nucleotide metabolism. Oxygen supplementation markedly downregulated mitochondrial biogenesis genes and altered CpGs annotated to developmental genes. Prematurity-altered DNA methylation could cause abnormal lymphopoiesis, cellular assembly and cell cycle progression to increase BPD risk. Similar pathways between epigenome and transcriptome networks suggest coordination of the two in dysregulating leukopoiesis, adaptive immunity, and innate immunity. The results provide molecular insights into biomarkers for early detection and prevention of BPD.

Abstract In efforts to prevent extinction, resource managers are often tasked with increasing genetic diversity in a population of concern to prevent inbreeding depression or improve adaptive potential in a changing environment. The assumption that all small populations require measures to increase their genetic diversity may be unwarranted, and limited resources for conservation may be better utilized elsewhere. We test this assumption in a case study focused on the peregrine falcon (Falco peregrinus), a cosmopolitan circumpolar species with 19 named subspecies. We used whole‐genome resequencing to generate over two million single nucleotide polymorphisms (SNPs) from multiple individuals of all peregrine falcon subspecies. Our analyses revealed extensive variation among subspecies, with many island‐restricted and nonmigratory populations possessing lower overall genomic diversity, elevated inbreeding coefficients (FROH)—among the highest reported, and extensive runs of homozygosity (ROH) compared to mainland and migratory populations. Similarly, the majority of subspecies that are either nonmigratory or restricted to islands show a much longer history of low effective population size (Ne). While mutational load analyses indicated an increased proportion of homozygous‐derived deleterious variants (i.e., drift load) among nonmigrant and island populations compared to those that are migrant or reside on the mainland, no significant differences in the proportion of heterozygous deleterious variants (i.e., inbreeding load) was observed. Our results provide evidence that high levels of inbreeding may not be an existential threat for some populations or taxa. Additional factors such as the timing and severity of population declines are important to consider in management decisions about extinction potential.

PDF - 89K, Supplementary Table 1. Differential methylation in cord blood DNA in relation to the father's smoking prior to the mother's pregnancy. Supplementary Table 2. The combined effect of mother and grandmother smoking in pregnancy compared to only the mother smoking in pregnancy, on differential DNA methylation in cord blood. Legends for Supplementary Figures 1 through 4.