Assessing individual responses to glucocorticoid drug therapies that compromise immune status and affect survival outcomes in neuro-oncology is a great challenge. Here we introduce a blood-based neutrophil dexamethasone methylation index (NDMI) that provides a measure of the epigenetic response of subjects to dexamethasone. This marker outperforms conventional approaches based on leukocyte composition as a marker of glucocorticoid response. The NDMI is associated with low CD4 T cells and the accumulation of monocytic myeloid-derived suppressor cells and also serves as prognostic factor in glioma survival. In a non-glioma population, the NDMI increases with a history of prednisone use. Therefore, it may also be informative in other conditions where glucocorticoids are employed. We conclude that DNA methylation remodeling within the peripheral immune compartment is a rich source of clinically relevant markers of glucocorticoid response. Glucocorticoids, such as dexamethasone, are used as anti-inflammatory and immunosuppressive drugs, however patients may exhibit resistance or side effects. Here the authors propose that a dexamethasone related neutrophil-specific DNA methylation index can be used as a marker of glucocorticoid exposure and response and as a prognostic factor in brain tumor survival. Because of their potent effects on immune cells, glucocorticoids have long been the most widely prescribed class of anti-inflammatory and immunosuppressive drugs2. However, glucocorticoid-treated patients may exhibit resistance or experience a wide range of adverse side effects. Our understanding of these risks is incomplete4. To account for individual variability, biomarkers have been developed that integrate variations in glucocorticoid exposure and response5. Several well-established safety concerns of glucocorticoids have been addressed with markers bridged to clinical outcomes, namely: adrenal suppression, insulin resistance, and accelerated bone turnover9. In the management of brain tumor patients, the synthetic glucocorticoid dexamethasone (DEX) is a mainstay for control of peritumoral edema, helping to alleviate symptoms due to high intracranial pressure or presence of tumor/edema in eloquent areas of brain10. No randomized controlled trials have specifically addressed DEX. However, its’ use in glioma patients has been associated with poor survival13 and compromised response to immunotherapy16, There are no established markers to assess these risks, although altered blood leukocyte composition has been used to mark glucocorticoid-related immunosuppression19. Depletion of circulating lymphocyte numbers23 and expansion of neutrophils and immunosuppressive myeloid cell populations25, including monocytic myeloid-derived suppressor cells (mMDSC)27, have been documented in DEX exposed brain tumor patients. Although blood leukocyte compositions are sensitive to glucocorticoid exposures, they lack specificity, as tumor inflammation and chemoradiation also directly affect them28. In our search to uncover more specific markers of glucocorticoid response, we turned to recent epigenetic studies showing that cell-specific chromatin modifications play a pivotal role in determining the genomic effects of glucocorticoids30. Cell-specific actions of glucocorticoids have been linked to highly accessible chromatin domains located preferentially within distal enhancer elements that are intrinsic to cell lineage31. Glucocorticoid alterations of DNA methylation may be integral to their genomic effects33. Because DNA methylation is a chemically stable endpoint and easy to access in peripheral blood, it is an attractive potential marker for glucocorticoid pathway activation. Previous studies of DNA methylation and glucocorticoids focused on a limited number of genes and cytosine guanine dinucleotide (CpG) loci in the context of neurodevelopmental and stress-related outcomes35. Furthermore, prior research did not take advantage of the cell-selective nature of glucocorticoid-mediated genomic effects. Comparative gene expression studies showed that, among different hematopoietic cell types, neutrophils exhibited the most pronounced transcriptional response to glucocorticoid treatments36. In severe COVID-19 subjects DEX induced profound transcriptional remodeling and induction of immunosuppressive neutrophil states37.Cortisol and synthetic glucocorticoid analogs activate intracellular glucocorticoid receptors (GR) and modulate many physiologic processes. Processes affected include carbohydrate and lipid metabolism, stress, and immune responsesIn this work, we explore DNA methylation as pharmacodynamic markers of DEX exposure in adult glioma patients who had and had not been given the drug as part of their routine care. We employ a bioinformatic approach to discover and assess DEX-related neutrophil-specific DNA methylation as a marker of in vivo glucocorticoid exposure and response, the neutrophil dexamethasone methylation index (NDMI). Our results indicate that blood DNA methylation can accurately discriminate DEX exposed and non-exposed subjects and that a methylation marker targeting blood neutrophils performs better than a marker based on altered blood leukocyte composition in characterizing individual glucocorticoid response. We also explore the association of NDMI scores with blood counts of CD4 T cells and mMDSC and show that neutrophil-specific methylation markers of DEX exposure serve as prognostic factors in brain tumor survival.p < 0.01) interactions with neutrophil proportions and one CpG that indicated significant interaction (p < 0.01) with B-cell proportions , whereas, in lymphocytic cell types , they were hypermethylated Fig. . Interes38 revealed a close correspondence of 21 differentially methylated genes with one co-expression module  or subjects using inhaled glucocorticoids (P = 0.03). NDMI scores of inhaled glucocorticoid users were not statistically significantly different from non-glucocorticoid users. Medical conditions recorded among the 18 non-glioma controls receiving oral prednisone included rheumatoid arthritis, solid organ transplants, kidney disease, lupus, scleroderma, multiple sclerosis, uveitis, hepatitis, and connective tissue disorder. The changes in immune cell proportion in controls taking prednisone were similar to those observed among glioma patients treated with DEX, Supplementary Table P = 0.008  and an extended case/control dataset (N = 602). In both, the ANOVA linear regression interaction terms of NDMI and DEX exposure were highly statistically significant (P < 0.0001). DEX treatments increased the slope gradient of NDMI regressed on neutrophil proportions among DEX exposed subjects  subjects. The performance of the GBM only NDMI  was greater than the leukocyte composition model but less than the NDMI created using all grades of glioma.We assessed the quality of our predictive models in an independent set of DEX exposed cases and non-glioma controls Fig. . Receiver2 = 0.64; p < 0.001) . A subset of subjects’ bloods was run in parallel using flow cytometry (FCM) and methylation deconvolution. Bland-Altman’s analysis of these paired data showed DNA methylation deconvolution to be highly accurate in estimating CD4 T cell concentrations  had CD4 counts <500 cells/μl and 16 (11.8%) had counts <200 cells/μl (Table p = 0.002) and concentrations (p = 0.005) of CD14+HLA-DRneg/low mMDSCs compared to non-exposed subjects. We further subdivided the mMDSC population by CD16 expression and found significant DEX-related increases in CD14+HLA-DRneg/lowCD16− cells/μl (p = 0.005). NDMI scores were positively correlated with total mMDSCs proportions  and concentrations  as well as CD16− mMDSC populations . The most robust model predicting mMDSC concentrations included monocyte counts. NDMI and monocyte counts together were positively associated with mMDSC levels in whole blood and together explained 76% of the variation in mMDSC levels and 77% of the variation of CD16− mMDSCs (p < 0.001). Average daily DEX exposure, although univariately associated with mMDSCs , was not significant in models containing monocytes and NDMI. Neither cumulative mg of DEX nor duration of use were significantly associated with variations in mMDSC levels (P > 0.05).We estimated blood mMDSC levels both as proportions of monocytes and as concentrations in 38 consecutive glioma volunteers using FCM Fig. . Subjectp = 0.001) and compared to paired DEX exposed monocytes from the same individuals (p = 0.03). Monocytes from DEX exposed subjects had higher NDMI scores compared to non-exposed monocytes (p = 0.002)  between exposed and non-exposed neutrophils. Of the 823 CpG, 683 exhibited the same direction of association (83%) as the CellDMC analysis. Of the 28 CpGs in the NDMI algorithm 9 (32%) exhibited a nominally statistically significant p value (p < 0.05) and a consistent direction of association as compared with the CellDMC analysis.To provide a reference for comparing NDMI scores in different immune subsets we compared DEX exposed cells and 12 different DEX naïve cell subtypes. In DEX naïve cells we observed significantly greater NDMI scores among myeloid-derived cells compared to lymphoid cells; Fig. 02) Fig. . To testned Fig. . We compIDH) wildtype GBM and IDH-wildtype astrocytoma patients, in each subtype survival times were statistically significantly different. Survival among patients with oligodendrogliomas was not significantly different by NDMI status. In a subset of 74 glioma subjects we evaluated blood NDMI, immune cell estimates and clinical variables in multivariate Cox proportional hazards survival models with extent of surgical resection with and without pre-operative tumor volume . The prevalence of both these cell types was best predicted by NDMI scores and not by either daily or cumulative drug doses.13, which have raised concerns about the widespread use of glucocorticoids. Early in diagnostic workup, even the suspicion of GBM can trigger the administration of high-dose DEX in routine clinical practice, regardless of neurological symptoms, tumor size, or extent of cerebral edema. Some of these exposures may be unnecessary and may lead to worsening outcomes. There is a lack of data and consensus in the field concerning optimal glucocorticoid dosing and schedules to guide clinical decision making. An ongoing trial (NCT04266977) is the first of its kind to formally test whether restrictive use of DEX in suspected GBM subjects is an acceptable and safe alternative to current practice, though in symptomatic subjects, DEX may be unavoidable. In these instances, our observation of increased survival risk associated with the NDMI score reinforces the need to titrate to the lowest dose possible14. In this regard, the NDMI response may provide a benchmark for calibrating the dose given to an individual patient with the dual goals of providing adequate clinical efficacy and minimizing toxicity, in particular if there is a need to re-start steroids during treatment. Such a precision approach has the potential to significantly reduce steroid-related morbidity in the GBM patient population.These results provide another datapoint that adds to historical observations16 the most important potential translational value of the NDMI score could be as a biomarker of response in immunotherapy trials. As an objective measure of extent of steroid exposure, the NDMI score could be utilized as a stratification factor in such trials to minimize confounding from steroid interference with therapies. Beyond these applications we observed that the NDMI score, but not DEX use, was a significant predictor of survival in IDH-wildtype GBM and astrocytoma, though not in oligodendroglioma. Thus, the NDMI score may be a more relevant prognostic factor in future studies of IDH-wildtype GBM.Given the convincing evidence that DEX compromises immunotherapies40, we did not observe altered NDMI scores in controls reporting inhaled glucocorticoid use. This is consistent with a recent study in pediatric asthma41 that failed to find consistent alterations in DNA methylation among users of inhaled glucocorticoids.Given the widespread availability of DNA methylation arrays and the open access Web-based tool that we have created, the NDMI approach is within reach for researchers wishing to assess clinical correlates with this measure. The small volumes of blood samples (0.5 mL) required, and the lack of any labor-intensive special processing needed for DNA methylation-based analyses make it an accessible technique for archival samples, and greatly enhances the value of samples in clinical biobanks. Finally, our observation of elevated NDMI scores among prednisone users in non-glioma controls highlights the applicability of this method to assessment of glucocorticoid use in a range of clinical indications beyond neuro-oncology . We also note that, consistent with the low systemic absorption associated with inhaled glucocorticoids43. We add to this knowledge that blood neutrophils dominate the leukocyte methylomic profile of DEX exposure. This result is reminiscent of earlier studies of glucocorticoid-induced gene transcription showing that the numbers of glucocorticoid-responsive genes were highest in hematopoietic compared to nonhematopoietic cells and that neutrophils had the strongest transcriptional response of all cell types studied36. Moreover, a heightened response to glucocorticoids by neutrophils is striking given their terminally differentiated state and short lifespan as well as their limited transcriptomic capacity compared with other leukocytes44. We found that DEX-responsive methylation in neutrophils was enriched in distal regulatory elements and glucocorticoid receptor binding sites. Consistent with previous work, we also showed that DEX-related differential methylation occurs at preexisting accessible chromatin locations as evidenced by their partially demethylated status in unexposed cells and occurring in a lineage-specific fashion. Among lymphocytes, the same sites were typically densely methylated. A robust association of differentially methylated genes encoding granule proteins and neutrophil activation functions was found in gene ontology analysis. Twenty-one of the neutrophil enriched genes overlapped with a previously described co-expression module in human bone marrow whose peak transcriptional expression occurs during the metamyelocyte and band neutrophil stages of myelopoiesis. In contrast, the group of 16 genes that comprise the NDMI predictor were distinct in that they did not belong to the same co-expression group and instead showed peak expression in the promyelocytic and PMN stages of myelopoiesis. Taken together, these results indicate functional heterogeneity among genes targeted by DEX differential methylation.The acute effects of DEX and other glucocorticoids on peripheral blood myeloid and lymphoid cells are well described and reflect the intimate linkage of the glucocorticoid pathway with hematopoiesis and immune regulation+ monocytes. We confirmed previously reported increased levels of mMDSCs among current DEX users and further those variations in mMDSCs were significantly correlated with individual NDMI scores. We also provide evidence that the monocyte count in conjunction with the NDMI score may improve our understanding of individual variations in mMDSCs. CD16− mMDSCs also demonstrated consistent associations with DEX exposure. Earlier studies reported that CD16− mMDSC populations display superior immune-suppressive activity compared with CD16+ fractions45. Significantly, of the three DEX exposure measures, only the average daily dose was associated with mMDSC concentrations. In models that contained monocytes and NDMI, daily DEX dose became insignificant, suggesting that the NDMI score captures individual variation in the epigenetic response to DEX treatments. Furthermore, the NDMI scores of isolated mMDSC and paired CD14+ HLA-DR+ monocytes from DEX exposed subjects were elevated compared to non-exposed monocytes. Based on these observations, we hypothesize that DEX influences both the neutrophil and monocyte cell methylomes, perhaps reflecting the shared DNA methylation landscape of DEX-responsive regions in both lineages. We have confirmed previously reported deficits in circulating CD4 T cell levels among glioma subjects prior to chemoradiation; 11.8% of our training population demonstrated <200 CD4 T cells/μl. A model incorporating an interaction between the NDMI, and total white cell count provided the strongest predictor of variations in CD4 levels. DEX exposure or clinicopathological variables did not improve model performance. Thus, although excess mMDSCs and low CD4 T cells often occur together in cancer patients, our data suggest that different features of the immune profile in conjunction with the NDMI can be useful to describe variation in these two important immunological parameters.Isolated neutrophils from DEX exposed subjects displayed the highest NDMI scores of all isolated cells and in each case the scores were elevated above paired whole-blood values from the same donors. These observations are consistent with our bioinformatic predictions. However, despite the lack of statistically significant interactions of methylation values and monocyte proportions, our methylation and FCM studies of mMDSCs also point to DEX-mediated epigenetic modification in subpopulations of CD1432. In rodent embryonic neural stem cells, Tet3, a member of the ten-eleven translocation family of methylcytosine dioxygenases, was implicated in mediating the effects of DEX on CpG methylation33. Whether similar mechanisms apply in adult human myelopoiesis is unknown. Finally, our studies have not assessed gene expression in DEX exposed cells. Recent studies in severe COVID-19 have shown that in vivo DEX exposures induce dramatic changes in peripheral blood neutrophil states leading to immunosuppressive cell subtypes not observed in unexposed subjects37. The relationship of different transcriptional states with DNA methylation could provide a mechanistic link between DEX exposures, immune profiles, and glioma survival. In conclusion, we find that alterations in DNA methylation of peripheral whole blood are sensitive indicators of DEX response and are potential pharmacodynamic markers of individual sensitivity to epigenetic modification that are bridged to glioma survival, elevated immunosuppressive myeloid cells and depletion of critical T cell populations.The current work has several limitations. First, our survival studies were not a randomized controlled study and in the biomarker development phase detailed longitudinal studies were not available to estimate the kinetics of change in DNA methylation during initiation of drug therapy or the recovery time following cessation. Our anectodical observations indicate that NDMI score can return to pre-therapy levels following ~7 days. Second, while the empirical associations of DNA methylation with DEX treatments are robust, a mechanistic understanding of these effects is lacking. At the tissue level, it is unknown whether DEX-related changes in DNA methylation arise through redistribution of preexisting subpopulations of cells in the bone marrow or marginated pools or instead reflect CpG level modification within stem/progenitor cells during myelopoiesis. At the molecular level, glucocorticoid ligand-triggered demethylation of single CpGs at de novo sites are rapid and replication independent, suggesting an active enzymatic mechanism46 Separate models were evaluated for each CpG probe and its interaction with CD4 T, CD8 T, B-cell, NK, monocyte, or neutrophil proportions. The training dataset was drawn from the UCSF Immune Profiles Study (IPS), a prospective neuro-oncology clinic-based collection of blood samples, imaging, and other clinical data from adult glioma patients. Presurgery blood samples were typically taken the day before surgery; none were obtained during or after exposure to anesthesia. Blood samples were transferred the same day as drawn for determining total cell counts of specific immune cell types, further processing, and FCM study. At time of blood draws, we also collected a presurgery questionnaire to document daily/cumulative DEX exposure. Additional post-surgery blood samples were collected on some subjects to explore changes in immune parameters following resection. The test dataset  and the survival evaluation set (n = 429 glioma cases) were drawn from the San Francisco Adult Glioma Study (AGS). AGS was a case-control study of glioma patients newly diagnosed between 1991 and 2012 who were residents of the San Francisco Bay Area or patients of the UCSF Neuro-oncology clinic and age and gender-matched controls without any history of glioma recruited through random digit dialing or from the UCSF blood draw lab47. Medical history and current medications were available for all the controls. Patients were selected who had both archival blood and tumor marker data47. AGS participants represent newly diagnosed glioma patients; no recurrent or secondary GBM cases are included. In both IPS and AGS cohorts, we classified cases according to the WHO 2016 classification scheme that delineates IDH-mutant and wildtype grade IV GBM, grades II and III astrocytoma, and IDH-mutant 1p/19q co-deleted oligodendrogliomas. In the AGS, blood samples were collected from patients a median of 100 days after they were histologically diagnosed. Clinical information was collected on patient treatments, including temozolomide chemotherapy, radiation therapy, the extent of surgery, and steroid use at the time of blood sampling. Information on current medication use at blood draw (including any steroid use and names of the types of steroid drugs taken) was obtained from an in-person blood draw questionnaire which collected information on treatments and medications taken at the time of blood draw; medication logs for DEX dosages were not available.These glioma studies were approved by the Institutional Review Board of the University of California, San Francisco, Human Research Protection Program in the Office of Ethics and Compliance under UCSF Federal wide Assurance 00000068 and met all relevant ethical regulations. Informed consent was obtained from all study participants. The study design utilized a training dataset to develop predictors of DEX exposure and a test dataset to evaluate their performance Fig. 48. All samples and array experiments were performed blinded to clinicopathologic variables. Approximately 200–500 ng of DNA was applied to Illumina DNA methylation EPIC 850K beach chip arrays. Preprocessing and quality control of fluorescence data were accomplished using the minfi Bioconductor package49. CpG loci having a sizable fraction (>25%) of detection p values above a predetermined threshold were filtered (detection P > 10E−5)50. A “noob” background correction method was used to account for background fluorescence and dye bias51. Beta-mixture quantile normalization (BMIQ) was performed to correct probe design bias52. The presence of technical sources of variability induced by plate or BeadChip was examined using principal components analysis (PCA), the top K principal components53 were examined in terms of their association with plate and BeadChip. If plate or BeadChip were found to be significantly associated with any of the top K principal components, we applied ComBat method54 for normalization using the sva Bioconductor package. The final dataset contained 830,277 probes.Frozen (−80 °C) anticoagulated whole blood or isolated cells were processed, DNA isolated, and bisulfite converted55. This immunomethylomic method provides the proportions of 6 major cell types  and the neutrophil/lymphocyte, CD4/CD8, and lymphocyte/monocyte ratios. DNA methylation-based immune profiles are highly accurate and reproducible56  we utilized 6 cell proportions and ratios of neutrophil/lymphocyte, CD4/CD8 T cell, and lymphocyte/monocytes. To discover in vivo DEX-responsive CpGs in the training dataset for predictors (ii) and (iii), we performed an epigenome-wide association study (EWAS) using the “lmfit” function in the limma package57 comparing methylation beta values for patients on DEX at the time of blood draw and patients not taking DEX at the time of blood draw. The EWAS yielded a list of probes nominally significantly differentially methylated p < 0.05 with an absolute delta beta value (≥0.10). CpGs identified in EWAS were further analyzed the CellDMC46 function of the EpiDISH package using R with the default threshold parameters (FDR ≤ 0.05)58. Neutrophil-specific CpG loci (N = 2621) and non-cell-specific CpG loci  were identified and used to develop the methylation DEX-responsive predictors. Elastic net regression was used to build the DEX exposure classifiers using (i) leukocyte composition, the (ii) neutrophil-specific, and (iii) non-cell-specific CpGs identified from CellDMC46. The R glmnet was used to build the elastic net models and cross-validation to identify the optimum lambda value59. Each of the three predictors was tested using an external dataset consisting of 99 DEX+ glioma samples and 453 DEX− control samples. An ROC curve was built to compare the predictive capability of the three models on the same test set60.We built three predictors of DEX exposure using elastic net regression procedures on the following data: (i) blood leukocyte composition, (ii) neutrophil-specific methylation, and iii) non-cell-specific methylation. Developing these markers required blood leukocyte composition, which we derived from a validated DNA methylation deconvolution methodp values are calculated to test for NR3C1 binding site enrichment in differentially methylated neutrophil-specific CpGs (n = 2621) using the total measured as background . Genomic information for transcription factor NR3C1 binding site is extracted from the ENCODE database “Transcription Factor ChIP-seq Clusters”, version wgEncodeRegTfbsClusteredV3. The Illumina HumanMethylationEPIC annotation file and the UCSC Genome Browser UCSC_hg19_refGene file were used to test neutrophil-specific CpGs (n = 2621) for genomic context enrichment among all tested CpGs . The relation to CpG islands and enhancers was identified from the Illumina HumanMethylationEPIC annotation file for each probe. To define the genomic regions as promoters, introns, exons, or intergenic for each probe, the annotateWithGeneParts function from the R-package genomation and the UCSC_hg19_refGene file were used to map the regions to all CpG loci on the Illumina HumanMethylationEPIC array. If a probe mapped to more than a single genomic region, the probe was assigned preferentially with the order promoters, exons, introns, and intergenic. Fisher’s exact tests of 2 × 2 tables were conducted to calculate odds ratios (ORs), p values, and 95% confidence interval for CpG island, enhancers, and genomic regions enrichment of neutrophil-specific CpGs compared to all other DEX differentially methylated loci. eFORGE 2.0 was used to assign histone chromatin marks to differentially methylated probes61 in the 28 CpG NDMI.Odds ratio and 63, substituting the myeloid marker CD64 for CD33. Relative and absolute quantities of total (CD14+CD64+CD11b+HLA-DRneg/low) and CD16 negative (CD14+CD64+CD11b+HLA-DRneg/lowCD16−) mMDSC populations were estimated. FCM was performed by the UCSF clinical immunology core using established clinical protocols and analytic-specific reagents. Blood samples (3 ml EDTA preserved whole blood) were collected, and total white blood cell (WBC) counts were recorded. Following red blood cell lysis, cells were mixed with a monoclonal antibody mixture including CD45, CD64, CD16, CD11b, HLA-DR  and were sampled soon after receiving high DEX doses .MDSCs are a heterogeneous population of immature myeloid cells, and the definition of these subsets has not yet reached a consensus. We defined mMDSC immunophenotype consistent with commonly employed markers−, CD19−, CD11b+, CD33+, CD14+, and CD15− cells were gated and plotted for HLA-DR expression. CD3 HLA-DR− neg cells and CD19 HLA-DR+ positive B cells were used to set the sorting gate for mMDSC cells lacking HLA-DR expression . HLA-DRhigh cells  were collected from the same individuals. For neutrophil isolations, a Histopaque two-step gradient (1.119 layered with 1.077) was used. Granulocytes were collected on the 1.119 layer above the red blood cells and below monocytes and lymphocytes; residual red cells were lysed with ammonium chloride. Cells were FcBlocked and stained with CD3, CD56, CD123, and CD49d in a negative depletion channel. Neutrophils were then sorted as CD19−, CD33−, and CD66b+ cells , the 1st and 3rd quartile of NDMI scores were estimated, and plots of the corresponding Kaplan–Meier curves were drawn.Overall survival is defined as the time from the initial diagnosis to death or last follow-up. Those patients without a date of death were censored at the date of the last follow-up. Multivariate survival models were initially built via regularized cox regressionWe conducted a series of simulation studies to assess the statistical power for detecting a difference in the mean value of the NDMI between DEX exposed and non-exposed individuals  to estimate the cell-specific means and standard deviations of the 28 CpGs that comprise the NDMI. The cell-specific means and standard deviations for the 28 NDMI CpGs were then used to calculate the two shape parameters of the beta-distribution using methods of moments estimation. Using the beta-distribution shape parameters, we next generated cell-specific methylation data, n0 non-exposed individuals by randomly sampling methylation beta values for the 28 NDMI CpGs using the previously estimated shape parameters. Here, j, and is a 28 × 6 matrix whose rows represent the 28 NDMI CpGs and whose columns are the 6 leukocyte subtypes. The same process was performed for generating cell-specific methylation for n1 DEX exposed individuals, n = 6 non-exposed individuals and n = 3 DEX exposed individuals. Using the cell-specific methylation data for the n0 non-exposed and n1 DEX exposed individuals, we next generated cell proportions for each of the samples α0) equal to 18, 73, and 127. Here, wj is a 1 × 6 vector representing the cell proportions for subject j and N = n0 + n1 is the total number of samples, which ranged from N = 20 to N = 80 in increments of 20. The total concentration parameter is related to the variability in the cellular landscape across samples wherein larger values are associated with less variability in the cellular landscape across samples and conversely, smaller values, more variability in the cellular landscape across samples  we next calculated the NDMI for each of the N samples and performed a two-sample t test to test the null hypothesis of no difference in the mean NDMI between DEX exposed and non-exposed individuals assuming a type 1-error rate of 5%. For a fixed proportion of neutrophils in whole blood, overall sample size N and the Dirichlet concentration parameter, the process described above was repeated 1000 times and statistical power was calculated as the proportion of times the null hypothesis was correctly rejected.To simulate in-silico whole-blood methylation data for DEX exposed and non-exposed individuals, we first used a publicly available dataset consisting of cell-specific DNA methylation data profiled using the Illumina HumanMethylationEPIC array for various isolated leukocyte subtypes, including: CD4 T cells, CD8T cells, B cells, natural killer cells, monocytes, and neutrophilsFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


Background: Pressure-strain loop (PSL) analysis provides a novel, less load-dependent non-invasive method to quantify myocardial work and demonstrates a significant correlation with the contractile reserve in adult athletes. We aim to validate PSL-derived markers in characterizing LV function in pre-adolescent basketball players by comparing results before and after the cardiopulmonary exercise test (CPX) and explore its association with CPX-derived aerobic capacity.Methods: Cardiac morphology and function in 20 pre-adolescent basketball players were assessed at 9.7 years old (9.7 ± 1.1 year) before and after cardiopulmonary exercise testing. Echocardiography was performed in all subjects, including two-dimensional speckle-tracking echocardiography (STE). Simultaneous brachial-cuff-measured blood pressure was recorded to perform PSL analysis.Results: Nineteen subjects were included in the final analysis. Exercise training in pre-adolescent males was associated with lower global work index (GWI) and global work efficiency (GWE) at rest. GWE at stress was significantly correlated with VO2max and peak O2 pulse . When indexed by body mass, GWI and GWE both significantly correlated with relative VO2max ; GWI and GWE at baseline and stress were all significantly correlated with peak O2 pulse .Conclusion: PSL-analysis-derived GWI and GWE at rest indexed by body mass are associated with cardiopulmonary exercise test-derived peak oxygen consumption and oxygen pulse in pre-adolescent athletes. Cardiac morphological and functional remodeling in response to long-term training includes increased left ventricular (LV) wall thickness, LV mass, increased LV and RV right ventricular (RV) chamber size  and low-2max) in adult athletes and were suggested to be good predictors of the contractile reserve under stress (2max and VO2max/kg).Pressure-strain loop (PSL) analysis provides a novel non-invasive method to quantify myocardial work, which takes into consideration the impact of afterload when evaluating LV mechanics  and thusr stress . The aimIn August 2019, 20 basketball players between 8 and 10 years old were recruited during their routine cardiopulmonary exercise test (CPX). They have been practicing basketball 5 times a week for 2 h each time for 18 months. They trained on a children’s basketball court, which was 22 m long and 12 m wide, with 2.6 m high baskets and a size five ball (22 cm diameter) for training. They train mainly with games and individual technical training with medium intensity. From Monday to Thursday afternoons are training and Friday afternoons are games, where there is no position differentiation between players on the court. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the “Science Research Ethics Committee at the Shanghai University of Sport.” All athletes’ parents were informed about the protocol of this study, which was outlined in an information letter. No data collection took place without parents’ consent.Participants underwent a complete two-dimensional echocardiographic study (2DE) at baseline and a focused scan immediately after CPX on Vivid iq  with a 2.5 MHz phased array transducer (M5Sc). Cuff-measured blood pressure was recorded concomitantly with the scanning session. Data were digitally stored for offline analysis . From 2DE, LV dimensions, ejection fraction by Simpson’s biplane method, and LV diastolic function parameters were assessed. Left atrial (LA) volume was measured using the biplane method. Right atrial (RA) area and RV function parameters were assessed in the RV-focused apical four-chamber view. Parameters were measured according to the latest recommendations . LV massLV myocardial work was calculated by integrating longitudinal strain and brachial-cuff-measured blood pressure, as previously described by Russel et al. . LV longGlobal myocardial work index (GWI): the total work within the area of the LV PSL between mitral valve closure and mitral valve opening.Constructive myocardial work (CW): shortening of the myocytes during systole adding lengthening of the myocytes during isovolumic relaxation.Wasted myocardial work (WW): lengthening of myocytes during systole (absorbing work done by other segments) adding shortening during the isovolumic relaxation phase (not contributing to ejection).Global cardiac work efficiency (GWE): Cardiac work efficiency was expressed as CW/(CW + WW) × 100% per segment and GWE as an average of all segmental values .2max) was determined by a ramp incremental exercise test . The exercise test was accepted as maximal if the majority of the following termination criteria were met: respiratory exchange ratio greater than 1.0, heart rate greater than 200 beats/minute, display of indicators of a maximal effort such as sweating and, despite strong verbal encouragement, the participant was unable or unwilling to continue.t-test or Wilcoxon signed rank test depending on data distribution. Z-test for single sample was performed to compare echocardiographic indices and relative VO2max of this cohort with reference data of normal children whose population mean and variance was known , and a value of p < 0.05 was considered statistically significant.The Shapiro-Wilk test for normality was performed on all data sets. Continuous data were expressed as mean ± SD or as median (interquartile range) according to data distribution. CPX data were compared with their predicted values through either paired as known . Specifias known . The conas known . The conas known . CompariAll participants completed echocardiographic scans and CPX but one was excluded from the final analysis due to poor image quality. The basic demographic characteristics of the athletes are summarized in 2max of the study population was significantly higher than their age-matched peers  (The relative VO0.00001) , which wAll participants reported more than 9 h of weekly exercise and were classified as active athletes without further subdivision. Data from 2DE and the Z-test results are summarized in p < 0.0001) due to increased heart rate, while peak systolic strain was not significantly different after CPX, which could be explained by the heterogeneous stress status each participant reached. For myocardial work indices, GWE rather than GWI changed significantly (p = 0.035).Exercise training in pre-adolescent males was associated with lower GWI and GWE at rest . In Figu2max  but ejection fraction, global longitudinal strain and fractional shortening were not . GWE at stress was significantly correlated with VO2max . When indexed by body mass, GWI and GWE both significantly correlated with relative VO2max .Among conventional LV functional parameters, only stroke volume (SV) and cardiac output (CO) were significantly correlated with VO2 pulse)  but ejection fraction, global longitudinal strain and fractional shortening were not . GWE at stress was significantly correlated with peak O2 pulse . When indexed by body mass, GWI and GWE at baseline and stress were all significantly correlated with peak O2 pulse .SV and CO were significantly correlated with peak oxygen pulse  GWI and GWE indexed by body mass correlates with relative VO2max in addition to SV, CO, age and BSA; 3) GWI and GWE indexed by body mass correlates with oxygen pulse in addition to SV and CO.The main findings of the present study can be summarized as follows: 1) only GWE at stress but no conventional LV systolic parameters or MW indices at rest correlates with VOThe morphological cardiac remodelling in pre-adolescent athletes has been well documented and confirmed by pooled analysis . HoweverAs the “pre-adolescent athletic heart” is guaranteed to transform into the “adult athletic heart” at certain time point if there is no cessation of training, the heterogeneity of study population could be one reason for the inconsistency. The limits of conventional echocardiographic parameters of LV systolic function or GLS could be another reason as they may not be sensitive enough to detect the functional change in pre-adolescent athletes. GLS remains load dependent , which m2max in pre-adolescent athletes. Previous studies in adult athletes have reported the association between either GWI or GWE at rest and relative VO2max (2max or relative VO2max but the indexed ones did. The indexed GWI is easy to understand as GWI is a measure of all the work generated by the heart and thus is closely related to the metabolic need of the body while indexing could address the difference of body mass among individuals which may be especially important in children. However, the indexed GWE is hard to understand as it is expressed as a ratio and thus should not be subject to the difference in body mass. The current study provides evidence for predicting contractile reserve at rest with MV indices in pre-adolescent athletes but future studies with larger sample size should be performed to validate the results.A major finding of the current study is that GWI and GWE indexed by body mass correlate with relative VOe VO2max . The cur2 pulse has been suggested to represent maximal working capacity (2 pulse as an important measure obtained from CPX (2 pulse stronger than with relative VO2max. As the calculation of GWI and GWE is based on single heartbeat, the better correlation with peak O2 pulse could be achieved after excluding the effect of heart rate. This finding also adds to the evidence for predicting aerobic capacity with MV indices in pre-adolescent athletes.Another important finding is the correlation of MW indices to the peak oxygen pulse. Oxygen pulse expresses the volume of oxygen ejected from the ventricles with each cardiac contraction and peak Ocapacity . Currentfrom CPX . In the Another interesting finding in the current study is that GWI of pre-adolescent males was lower than normal children. GWI has been demonstrated to be similar or higher in athletes than in sedentary controls in adults. Jain et al.  performeA major limitation of the current study is its limited sample size despite active recruitment performed by the researchers. Sensitivity tests were made by randomly excluding one subject and the major finding of the current study remained unchanged. However, it would be better validated with larger sample size.The current study is also subject to its narrow spectrum of sport disciplines. Thus, the generalizability of the current results needs to be clarified in a larger study population. The time interval between peak stress and echocardiographic scan may cause error in peak systolic strain measurement and thus affect PSL analysis. However, given the simultaneous measurement of blood pressure and the intrinsic heterogeneity of individual stress load, such error would not significantly change our current findings.PSL-analysis-derived GWI and GWE at rest indexed by body mass are associated with cardiopulmonary exercise test-derived peak oxygen consumption and oxygen pulse in pre-adolescent athletes. Our findings support the use of PSL analysis in the evaluation of pre-adolescent athletes’ heart.


In males only, insular CRF increased social investigation, and CRF1 and CB1 antagonists interfered with social interactions with stressed conspecifics. To investigate the molecular and cellular basis for the effect of CRF we examined insular CRF1 and CB1 mRNAs and found greater total insula CRF1 mRNA in females but greater CRF1 and CB1 mRNA colocalization in male insular cortex glutamatergic neurons that suggest complex, sex-specific organization of CRF and endocannabinoid systems. Together these results reveal a new mechanism by which stress and affect contribute to social affective behavior.Impairments in identifying and responding to the emotions of others manifest in a variety of psychopathologies. Therefore, elaborating the neurobiological mechanisms that underpin social responses to social emotions, or social affective behavior, is a translationally important goal. The insular cortex is consistently implicated in stress-related social and anxiety disorders, which are associated with diminished ability to make and use inferences about the emotions of others to guide behavior. We investigated how corticotropin-releasing factor (CRF), a neuromodulator evoked upon exposure to stressed conspecifics, influenced the insula. We hypothesized that social affective behavior requires CRF signaling in the insular cortex in order to detect stress in social interactions. In acute slices from male and female rats, CRF depolarized insular pyramidal neurons. In males, but not females, CRF suppressed presynaptic GABAergic inhibition leading to greater excitatory synaptic efficacy in a CRF receptor 1 (CRF Stressors and other salient emotional stimuli trigger a shift in attention and cognitive resources in order to orient attention and organize situationally adaptive behaviors. In the brain, the transition between resting and executive cognitive networks involves the insular cortex and the “salience network” , 2, whic1 and CRF2) that couple to a variety of G-proteins expressed in the brain region, cell type, and sex-specific ways [1 and CRF2 receptors are distributed throughout brain social behavioral networks making CRF an important contributor to many social behaviors [1 receptors and CRF immunoreactive fibers are expressed throughout the corticolimbic system, including in the insular cortex [Stress is a major precipitating factor for mental illness. Exposure to either a perceived threat to one’s own well-being (self stress), or to a social contact that is undergoing distress initiate the hypothalamic–pituitary–adrenocortical axis response by activation of corticotropin-releasing factor (CRF) neurons in the paraventricular hypothalamus (PVN) . The CRFfic ways , 14. CRFehaviors . CRF1 rer cortex –19. Obser cortex , 21. A fr cortex . CRF neur cortex . CRF mayr cortex .A and cannabinoid type 1 receptor (CB1). In social behavior, CRF augmented social investigation while a CRF1 antagonist interfered with social interactions with stressed conspecifics in male but not female rats. Because we observed sex-specific effects of CRF on physiology and behavior, we hypothesized that sex differences exist in CRF1 and CB1 at the cellular and molecular levels. We employed a combination of quantitative polymerase chain reaction (qPCR) and anatomical (in situ hybridization) analyses and found sex differences in CRF1 mRNA expression and differences in the cellular distribution of the transcripts in the insular cortex. Together, the data lead us to conclude that CRF, acting upon CRF1 receptors depolarizes pyramidal neurons triggering the release of endocannabinoids which suppress presynaptic inhibition. The result facilitated flow of information through the insula that appears to be necessary for coordinating social interactions with stressed conspecifics.Seeing CRF as a putative modulator of insula and considering the significant influence of stress on psychosocial processes, we investigated the effects of CRF on insular physiology and social affective behavior. In whole-cell recordings of insular pyramidal neurons, CRF depolarized the membrane potential. This translated to an increase of excitatory synaptic transmission, but only in recordings from male rats. The gain of synaptic efficacy appeared to be a case of CRF causing depolarization-induced suppression of inhibition (DSI ) as the For a complete description of the experimental methods please refer to the Male and Female Sprague-Dawley rats were obtained from Charles River Laboratories  at either age PN45 (test rats and adult conspecifics) or PN21 (juvenile conspecifics) and maintained in the same-sex groups of two to three. All procedures were conducted in accordance with the NIH Guide for the care and use of laboratory animals and approved by the Boston College Institutional Animal Care and Use Committee.A receptor antagonist SR95531 (2 µM) in the recording aCSF to block synaptic transmission. After achieving a whole-cell configuration, baseline recordings were made in aCSF until 10 min of stable baseline were observed, at which point 50 nM CRF  was added to the bath. The dose of 50 nM was selected after a pilot study using a range of doses from 50 to 300 nM, representative of the low [Whole-cell and extracellular field potential recordings were obtained from acute insular cortex slices from adult male and female rats with methods reported previously . Active  the low  and high the low  doses fo1 antagonist CP154526  were dissolved in water or DMSO and then diluted to their final concentration in aCSF and bath applied. Input/output curves were established at baseline (aCSF) and again after 10 min of drug application. Stimulations ranged from 0 to 5 V and occurred in biphasic (220 µs) 500 mV increments.Evoked field excitatory postsynaptic potentials (fEPSPs) were recorded on a perforated multielectrode array. CRF (50 or 300 nM), CRFA-mediated evoked inhibitory postsynaptic currents (eIPSCs), synaptic responses were evoked by a bipolar extracellular stimulating electrode in the presence of glutamatergic antagonists during voltage-clamp recordings of insular pyramidal neurons. eIPSCs were quantified as the peak amplitude observed in the 1 ms post stimulation and converted to z-scores using the mean and standard deviation of the baseline (aCSF).To determine the effect of CRF on GABA1 receptor [1 receptor antagonist CP154526 (Sigma) and CB1 receptor inverse agonist AM251 (Tocris) were dissolved in DMSO then diluted to 10 or 2 µM, respectively, in a vehicle of 10% DMSO and 0.9% saline.Bilateral guide cannula was implanted in the posterior insular cortex. The posterior insula was targeted because it is functionally connected to the social decision-making network , 27, necreceptor , 30, 31.1 antagonist and AM251 experiments, rats received 4 treatments  or , respectively, on consecutive days with treatment order counterbalanced in a Latin square design.One-on-one social interaction tests were completed in a quiet room as previously described , 32. The1 antagonist or CB1 antagonist as described above in a counterbalanced, within-subject design.The SAP tests allow for the quantification of social interactions initiated by a test rat toward either a stressed or unstressed conspecific, providing insight into the test animal’s discrimination of socioemotional affective cues; they were conducted exactly as previously described , 32, 33.1 (crhr1), CB1 (cnr1) CRF (crh), and CRF2 (crhr2) mRNA we performed quantitative Taq-man reverse transcriptase qPCR analysis on 1 mm dia, ~500-μm thick insular cortex punches, as previously described [To determine the relative expression of CRFescribed .1 , CB1 , vesicular glutamate transporter 1  and DAPI. The total number of nuclei (DAPI) and glutamate cells (Vglut+DAPI), and cells colocalized with CRF1, CB1 or both were determined. Effects of sex on cell counts were assessed in two-way ANOVAs with sex as a between-group factor and side as a within-subject factor. The mean of left and right hemisphere counts are shown.RNAScope was performed on insular cortex sections according to the vendor’s instructions (ACDBio). Briefly, tissue was thawed, fixed and treated with a RNAScope cocktail including probes for CRFt-tests, final sample size and post hoc test results are presented in the figure legends.All analyses were performed in Prism  and results of analysis of variance, 1 receptor, which is the primary CRF receptor in insula [1 antagonist appeared to prevent those changes suggesting that the CRF1 receptor is the primary target of CRF in the insular cortex.Active and passive intrinsic properties  and dampening  modulation. To better understand how CRF might alter insular cortex information throughput, we investigated CRF effects on synaptic transmission Fig. . fEPSP iPSP Fig. . In summA. We tested this by pretreating male insular slices with GABAA antagonist SR95531 (2 μM) and then applied CRF (300 nM) and repeated fEPSP input/output characterization. In the presence of the GABAA antagonist, CRF had no apparent effect . In a direct comparison, we found both CRF1 antagonist and the GABAA antagonist blocked CRF based increases in insular excitatory synaptic transmission . Surprisingly, CRF had no effect on either measure or the AMPA/NMDA ratio , depolarization leads to the reduction in GABA release and the phenomenon of DSI [1 in mediating effects of CRF. We utilized AM251 (2 μM), a CB1 inverse agonist [1 receptors on both principle neurons and inhibitory interneurons resulting in a net excitatory modulation. AM251  and CRF type 2 receptor (crhr2) mRNAs were not detectable, which is consistent with prior reports [vGlut1), CRF1 (crhr1) and CB1 (cnr1) in insula sections from male and female adult rats . However, in males we found more glutamate neurons colocalized with CB1 and more glutamate neurons colocalized with both CRF1 and CB1 mRNAs than females.The pattern of sex-specific effects of CRF on physiology and social behavior led us to hypothesize that sex differences exist in the amount or distribution of insular CRFles Fig. . CRF , th1 antagonist prevented this pattern suggesting that activation of the CRF acute stress response, as reported by Sterely et al., likely occurs in the experimental rat during the SAP test. The framework is that the decision to approach or avoid another involves assessment of the subject’s core affect  and social affective information about the social target . Avoidance occurs when the targets are adults, possibly because the subject experiences greater internal distress/danger as the adult targets could be communicating imminent environmental threat or pose a threat  themselves. Alternatively, when the targets are juveniles, there is less (or none) social threat and approach occurs, possibly because the signals given off by stressed juveniles are social attractants [Detecting socioemotional cues and using this information to inform whether to approach or avoid others is a vital aspect of social behavior. CRF receptors are distributed across many structures critical to social behavior  and the ractants  or thereractants . As thisractants  likely rractants . Third, ractants . Consistractants . The cur1 and CB1 mRNAs. Interestingly, qPCR revealed a greater relative amount of insular CRF1 mRNA in females compared to males and no difference in relative CB1 expression between males and females, indicating that the synaptic and behavioral sex differences found here are not likely due to less available CRF1. These results contrast some prior reports that find greater CRF1 mRNA in prefrontal cortex of males [1 receptor binding varies in sex-specific ways with female adult rats tending to have more binding than males [1 and CB1 mRNA containing nuclei and glutamatergic neurons in males compared to females. While it is important to note that these measures of CRF1 and CB1 mRNAs are nuclear and may not reflect the actual expression of the receptors on synaptic terminals arriving from sources afferent to the insula, the distribution does illuminate a mechanism for sex-specific effects. First, the male insula contained more CRF1 expressing glutamatergic neurons than females, providing a larger population of neurons to be depolarized. Notably, both males and females exhibited CRF-induced changes in intrinsic neuronal physiology suggesting that female insula neurons express sufficient CRF1 for modulation of intrinsic excitability. Sex differences in physiology emerged at the synaptic level and the combination of mRNA distribution and pharmacology results suggest that this is due to much higher CB1 expression in males (Fig. 1-dependent release of eCBs that modulate presynaptic inhibition and behavior via CB1 in males. Females may not have sufficient insular CB1 to complete this cascade of signals and so CRF does not alter synaptic efficacy or behavior. It remains possible that there are other sex differences including the distribution of these receptors within the cortical interneuron subtypes or the cellular signaling cascades involved, both of which are exciting and clinically relevant directions for future research.The CRF system is complex, comprising of many sex, brain region and cell type-specific differences and functional contributions to behavior. As such, while the finding that CRF in the insula acts in a sex-specific manner to augment social behavior and drive social affective behavior in males is novel, the fact that a sex difference exists is not surprising. Here we began to understand the molecular basis for the sex difference by quantifying the relative expression and cellular distribution of CRFof males  but it ian males . RegardiHuman neuroimaging studies implicate the insula in an impressive range of cognitive processes , 8, 56, Supplementary Methods and resultsSupplementary Table 1


Spatial and temporal scales of indoor constituents are modulated by rates of chemical reactions, surface interactions and building ventilation, providing critical insights for better assessments of human exposure to hazardous pollutants, as well as the transport of indoor chemicals outdoors.Historically air constituents have been assumed to be well mixed in indoor environments, with single point measurements and box modeling representing a room or a house. Here we demonstrate that this fundamental assumption needs to be revisited through advanced model simulations and extensive measurements of bleach cleaning. We show that inorganic chlorinated products, such as hypochlorous acid and chloramines generated via multiphase reactions, exhibit spatial and vertical concentration gradients in a room, with short-lived  Concentration of air pollutants has generally been considered uniform in indoor environments. Here, the authors show that chlorinated cleaning products generate concentration gradients of pollutants due to photochemical reactions in sunlit areas, requiring a finer assessment of human exposure to these contaminants. Hypochlorite bleach is an effective disinfectant that kills a wide variety of microorganisms and is increasingly used to control infectious disease spread in various indoor locations including schools, hospitals, and residential buildings4. Following bleach use, a number of chlorinated compounds including hypochlorous acid (HOCl) and molecular chlorine gas (Cl2) can be released6, which are hazardous by causing skin lipid oxidation7 and cytotoxic injury in the respiratory tract4. The House Observations of Microbial and Environmental Chemistry (HOMEChem) campaign8 has revealed that a series of multiphase reactions involving nitrite (NO2−) and ammonia (NH3) in the applied bleach onto a floor can lead to the formation of nitryl chloride (ClNO2) and chloramines 5, which have strong irritation effects with the potential to damage tissues4. Bleach cleaning chemistry also produces several toxic compounds including isocyanates, cyanogen chloride, and chlorocarbons9. As such, bleach can pose an increased risk of respiratory infections and symptoms such as wheezing and asthma10.People spend on average 90% of their time indoors and even longer especially in their homes during the current COVID-19 pandemic. Concentrations of indoor gaseous compounds and aerosol particles are often much higher compared to outdoors owing to indoor emission sources including human activities such as cleaning and cooking11. Hence, indoor measurements are mostly conducted at a single location in a room and at a fixed height and there have been only a few measurements of spatial and vertical distributions of gas pollutants and particulate matter13. While computational fluid dynamics (CFD) simulations have been applied to resolve indoor air flows and spatial distributions of non-reactive indoor species14, indoor chemistry models often employ a box model with the concept of deposition velocity assuming that there is a well-mixed core region separated from indoor surfaces by boundary layers11. However, this assumption may not be warranted for reactive and short-lived species such as radicals and bleach cleaning products. To better quantify human exposure to indoor pollutants, it is essential to evaluate spatial distributions and temporal scales of emitted compounds, which are currently poorly understood.Historically indoor air constituents have been assumed to become well mixed and homogeneously distributed after being introduced into ventilated indoor environments15 to simulate extensive measurements of a bleach cleaning event from HOMEChem. We show that ∙OH radicals and bleach cleaning products exhibit spatial and vertical concentration gradients in a room as modulated by rates of chemical reactions, surface interactions, and ventilation.To elucidate the spatial and temporal scales of variability of indoor air pollutants, in this paper we go far beyond earlier studies by integrating multiple indoor models including gas-phase chemistry modeling, multiphase kinetic modeling, and computational fluid dynamics (CFD) simulations5. It treats outdoor–indoor air exchange, gas-phase reactions, photolysis, wall loss, heterogeneous reactions at indoor surfaces and particles, and aqueous reactions in the aqueous bleach, while assuming that species would be mixed homogeneously in the room where the bleach was applied5  with the Master Chemical Mechanism, was used to quantify the radical production rates and refine the predicted radical concentrations17  that is largely present on indoor surfaces as a reservoir of HONO18, to generate ClNO2 which can partition into the gas phase. NH3, emitted by human occupants and off-gassing from building materials and indoor surfaces19, partitions into the aqueous bleach to participate in a series of reactions with HOCl to generate NCl35, leading to an increase of NCl3 and a decrease of NH3 in the gas phase. Afterwards, the bleach products decayed faster than the air exchange rate, which is also captured very well by accounting for deposition to indoor surfaces.By resolving spatial heterogeneity, the CFD model reproduces the dynamic concentration changes at the sampling points remarkably well Fig. . After t∙OH radicals during the bleach cleaning event can be mainly explained by a cascade of reactions initiated via Cl2 photolysis: the formed Cl radicals react with volatile organic compounds (VOCs) to generate peroxy and alkoxy radicals, which propagate to HO2 and then ∙OH through reactions involving NO. Gas-phase model simulations indicate that this process accounts for >90% of ∙OH production, while ∙OH radicals can also be generated via photolysis of HOCl and HONO5. The generated ∙OH radicals react rapidly with a number of indoor gas-phase species including NOx and VOCs with an estimated ∙OH reactivity of 65 s−1 during the cleaning event  because of the non-uniform indoor airflow, showing 30–50% higher concentrations than at other points. Cl2 also exhibits similar spatial distributions , O3 (4 ppb), NOx (7 ppb), and VOCs (100 ppb) as well as typical photolysis and surface deposition rates  exhibit sharp spatial gradients and their temporal scales are determined mainly by reaction rates, and only marginally affected by deposition and ventilation rates  such as Cl, NOates see .3, NO, Cl2, and O3 would exhibit spatial gradients within a room. The temporal and spatial distributions of these species are controlled by both chemical processes and indoor air flow conditions. For NH3 and semi-volatile organic compounds (SVOCs) that may undergo reversible partitioning to indoor surface reservoirs18, the true spatial gradients are likely reduced from the model predictions due to surface emissions.Room scale: Processes that exceed the microscale but still occur within a room (~0.1–10 m). Moderately long-lived species  such as NH2, and CO2 are mostly well mixed within the indoor space. Their temporal scales are mainly controlled by ventilation rates. During HOMEChem, HONO was measured in two different locations (P2 and P7), showing very similar concentrations , possibly affecting other rooms and the entire building by circulation and even being transported outdoors. Long-lived species such as VOCs, NOSimilar to the atmosphere23. The widely applied concept of deposition velocity, which expresses the species flux density to the surface divided by its concentration in the uniformly mixed core region, may need to be revisited11 for simulating short-lived and moderately long-lived species. Note that this analysis of temporal and spatial scales implicitly assumes that spatial gradients are driven by a perturbation such as cleaning, cooking, and other activities at steady-state conditions; there will not be the same gradients in the absence of a perturbation. Spatial heterogeneity in photon fluxes also leads to spatial gradients of photoactive species. Resolving mass transport and chemical reactions in the boundary layer16 and on indoor surfaces would be required for an accurate description of deposition processes24.A better understanding of spatial distributions of indoor species is highly critical for accurate assessments of human exposure to indoor oxidants and SVOCs including toxic chlorinated and nitrogenated VOCs20. Because of relatively low air exchange rates in residences, non-reactive gas-phase species remain indoors for 3–4 h. At higher air exchange rates that are often deployed in industrial buildings with mechanical ventilation, the temporal and spatial scales of moderately long-lived and long-lived species would both decrease as the species are transported to the ambient atmosphere at a faster rate , a cavity ring-down spectrometer and a laser-induced fluorescence using the fluorescence assay with gas expansion technique instrument (LIF-FAGE). Two separate TOF-CIMS instruments were deployed to sample air from the kitchen (P2): one with utilizing iodide chemical ionization to measure HOCl, Cl2, ClNO2, and NCl3 and another utilizing acetate chemical ionization to measure HONO. The cavity ring-down measured NH3 sampled in the kitchen, while the LIF-FAGE instrument measured ∙OH, HO2∙, and HONO next to the living room window. Spectrally resolved solar irradiance was measured with a hand-held spectrometer collocated with the LIF-FAGE instrument. The air exchange rate during the period of the bleaching experiment on the 8th June was controlled by a heating ventilation and air conditioning (HVAC) system and was measured as ∼0.7 h−1.The House Observations of Microbial and Environmental Chemistry (HOMEChem) campaign and the bleach experiments that occurred during the campaign have previously been described in detail2, chloramines, and NH3 directly above the bleach surface at different times. The CFD model geometry was designed by mimicking air flow and emission conditions of the bleach products observed in the measurement campaign. The CFD model resolves a total of 11 chemical reactions 17, a near-explicit photochemical box model constructed based on a comprehensive chemical mechanism. INDCM also treats exchange with outdoors, internal emissions, photolysis, and deposition to surfaces (The multiphase kinetic model treats various processes including air exchange, uptake to particulate matter and indoor surfaces, photolysis, gas-phase reactions and reactions in the aqueous bleach, and transport of semi-volatile species through a boundary layer above the floor Fig. . The kinsurfaces .Supplementary information.Peer Review File.


GPR151 expression is restricted to the habenula. In a previous report, we created conditional ChrnB4-Cre × Oprm1fl/fl  mice, where MORs are deleted specifically in Chrnb4-positive neurons restricted to the habenula, and shown a role for these receptors in naloxone aversion. Here we characterized the implication of habenular MORs in social behaviors. B4MOR−/− mice and B4MOR/++ mice were compared in several social behavior measures, including the chronic social stress defeat (CSDS) paradigm, the social preference (SP) test and social conditioned place preference (sCPP). In the CSDS, B4MOR−/− mice showed lower preference for the social target (unfamiliar mouse of a different strain) at baseline, providing a first indication of deficient social interactions in mice lacking habenular MORs. In the SP test, B4MOR−/− mice further showed reduced sociability for an unfamiliar conspecific mouse. In the sCPP, B4MOR−/− mice also showed impaired place preference for their previous familiar littermates after social isolation. We next created and tested Gpr151−/− mice in the SP test, and also found reduced social preference compared to Gpr151/++ mice. Altogether our results support the underexplored notion that the habenula regulates social behaviors. Also, our data suggest that the inhibitory habenular MOR and GPR151 receptors normally promote social reward, possibly by dampening the aversive habenula activity.The mu opioid receptor (MOR) and the orphan GPR151 receptor are inhibitory G protein coupled receptors that are enriched in the habenula, a small brain region involved in aversion processing, addiction and mood disorders. While MOR expression in the brain is widespread,  Social interactions are crucial in individual’s health: while positive social interactions  improve health and well-being throughout life, negative social interactions  can lead to pathological situations like health-damaging behaviors, stress, depression or suicide3. Reduced social reward valuation and/or increased reactivity to social rejection can be associated with different psychopathologies including depression6.Social interactions  can either have benefits or costs10, and these effects extend to local injections in the nucleus accumbens—a central reward structure11. This was interpreted as an increased/decreased rewarding value of social play induced by MOR activation/blockade, respectively12. Using positron emission tomography in healthy human volunteers, social rejection was found to activate the MOR system in mood- and motivation-regulating brain sites , and this activation did not reach significance in patients suffering from major depressive disorder (MDD14). MOR activation in the nucleus accumbens also correlated with motivation for social interactions in healthy controls but not in MDD patients14. Thus, these data emphasize the MOR system as an important integrator of social information and MOR manipulation could be used to model social psychopathologies in animals. Indeed, mice lacking MORs display social attachment deficits (juveniles16) and an autistic-like behavior (adults20). MORs therefore influence different aspects of social behaviors, however circuit mechanisms and brain sites other than the nucleus accumbens have been little explored.Mu opioid receptors (MORs) contribute to both positive and negative social behaviors. Social play behavior is either increased or decreased by morphine or naloxone, respectively21), an epithalamic structure implicated in mood-related dysfunctions24. Animal models of depression lead to an increase in metabolic activity in both nuclei of the habenula  and the MHb has been shown to be implicated in fear-28, aversion-30, or anxiety-32 responses. Emerging evidence also suggests a role for the MHb in social behaviors. Mice lacking the β4-subunit of the nicotine acetylcholine receptor, which is expressed almost exclusively in the MHb33, showed deficits in both nicotinic- and non-nicotinic reinforcing responses35 and, intriguingly, also showed increased heart rate in response to social isolation36. Further, mice lacking the Ca+ activated Cl-channel TMEM16A showed altered levels of anxiety/fear responses and social interactions31. Thus, concurrent with aversion processing, the MHb seems to modulate social behaviors. Whether the highly abundant MORs of the MHb are involved in social behaviors, has not been tested.MORs are enriched in the medial subnucleus of the habenula (MHb−/− mice), leading to a 50% reduction of total habenular MORs. We found that mutant mice are less sensitive to naloxone aversion without evident reward processing deficiencies29, suggesting that MORs (inhibitory receptors) may act as a brake to reduce the well-established aversive activity of habenula. Here we investigated whether MOR activity at this brain site also influences social responses. To do so, we evaluated sociability behaviors in B4MOR mice and our results show reduced response in all the tests. Because the MOR is an inhibitory G protein coupled receptor (GPCR), we also tested whether another inhibitory GPCR would play a similar role. We found social deficits in mice lacking the orphan GPCR GPR151, which is almost exclusively expressed in the MHb41. Altogether our data demonstrate for the first time that habenular receptors MOR and GPR151 contribute to facilitate social behaviors and suggest that the tonic control of MHb activity by inhibitory GPCRs is essential to maintain healthy social interactions.Recently we deleted MORs in ChrnB4-positive neurons  mice lacking MORs in nicotinic acetylcholine receptor beta4 subunit-expressing neurons, primarily expressed in the medial habenula. The genetic mouse line was described earlier by our laboratory and maintained (at least ten generations) on a c57bl6:sv129 (50:50) background to produce experimental B4MOR−/− mice and their B4MOR+/+ littermates as in43. Homozygous Oprm1−/− mice  were created in our laboratory and are available at Jackson laboratories (B6.129S2-Oprm1tm1Kff/J Strain #:00755). Gpr151−/− mice (c57bl6 background) were created using a knock-in strategy , so that the fluorescent reporter eGFP is produced instead of the GPR151 receptor in homozygous null mutants  were used as aggressors for the defeat. Commercial c57bl6:sv129 mice were used to judge the atypical behavior of B4MOR−/− versus B4MOR+/+ mice, as well as Oprm1−/− versus Oprm1+/+ mice in the reverse three chamber social test. Commercial c57bl/6J mice (Charles River) were used as interactors for Gpr151−/− and Gpr151/++ mice in the social preference test.Commercial c57bl6:sv129 mice (Jackson Laboratories) were used as interactors for B4MORAll procedures in this report were conducted in accordance with ARRIVE guidelines, the guidelines set forth by the Canadian Council of Animal Care and by the Animal Care Committees of McGill University/Douglas Mental Health University Institute and were also approved by the Regional Committee of Ethic in Animals Experiment of Strasbourg .Descriptions of housing and behavioral procedures are in the Suppl Methods. This includes the description of chronic social defeat stress (CSDS) followed by a social interaction test (SIT), the social preference (SP) test, the social conditioned place preference test (sCPP), the food pellets self-administration paradigm, the reverse three chamber social test and the real time place preference testing.P’s ≤ 0.05) were followed by Holm-sidak’s multiple comparisons tests. Refer to Suppl Table 1 for description of all statistical tests.Data are presented as means ± SEM. Social behavior tests  were represented across time  and were analyzed using RM ANOVAs. Significant main effects or interaction effects  would trigger enhanced social avoidance in B4MOR−/− mice. We tested B4MOR−/− and B4MOR+/+ mice in a chronic social defeat stress paradigm46 for 10 consecutive days. Control mice were not exposed to physical aggressions. Twenty-four hours after the procedure, sociability for a novel CD1 mouse was tested in an open field  B4MOR+/+ mice , the previous social stress exposure, and the genotype  = 17.75, p = 0.04; Fig. +/+ controls: removing this group from the analysis made the association non-significant (χ2(6) = 7.88, p = 0.25; Fig. −/− mice—which was intriguing as this group was previously not exposed to chronic social stress. This finding is another indication that B4MOR−/− mice display reduced sociability at baseline.We then clustered individual mice during the social interaction (time spent in the CD1 zone) based on their sociability index using the WARD method Fig. . We foun−/− mice, we compared B4MOR−/− with B4MOR+/+ mice in the three-chamber social test, to assess social preference for an unfamiliar mouse. In this test, mice have free choice to spend time in the three chambers of the apparatus during a 10-min habituation period followed by a 5-min social test  paradigm in B4MORice Fig. A. Reward−/− mice deficits to value social interactions as rewarding could be the consequence of learning and/or memory deficits. We then tested whether low reward valuation also applied to palatable food. B4MOR−/− and B4MOR+/+ mice self-administered similar amounts of chocolate pellets under FR1, FR5 and PR , the flexibility to learn the new task was similar for B4MOR−/− and B4MOR+/+ mice. Of note, our previous work showed that B4MOR−/− mice also show intact Morphine CPP29. Altogether, these data suggest that no generalized learning and/or memory deficit seems to impair the social reward-context association in B4MOR−/− mice.In this test, B4MOR PR Fig.  suggestieGFP expression upon deletion of the Gpr151 gene coding sequence  confirmed the absence of Gpr151 mRNA in the habenular complex of Gpr151−/− mice , suggesting a social interaction deficit. We next show that these mutant mice show reduced sociability in the three-chamber social preference test, as well as reduced place preference to a social context, confirming the alteration of social behaviors. We finally show reduced sociability in the social preference test in mice lacking GPR151, an orphan GPCR enriched in the MHb. Altogether, these data support a role for Gi/o coupled GPCRs of the MHb in promoting social behaviors, likely through the tonic inhibition of MHb activity.In this report, we first show that mice lacking 36. Indeed, habenula function is mainly studied in the context of mood disorders. Habenular activity was reported to be increased in MDD patients49. Consistent with this finding, animal models of depression show increased metabolic activity in both the medial (MHb) and the lateral (LHb) nuclei of the habenula while neural activity is generally lowered in other brain areas26. The function of the LHb in depressive-like states has been well characterized50 and reduced LHb activity parallels reduced depressive-like symptoms52. Neural activity in the MHb and the interpeduncular nucleus (IPN)—its main projection site—is also increased in animal models of depression25. MORs have their strongest expression in the MHb21 and the MOR system is already known to regulate depressive-like states54. We therefore here evaluated the role of habenular MORs29 using a model of depression, the chronic social defeat paradigm56, with the assumption that MOR activity may alleviate the aversive consequences of stress, and limit social avoidance in this test. Unexpectedly, while no genotype effect was observed in the stress response, mice lacking habenular MORs showed a social deficit at baseline. This was a first indication for a role of the MHb in the regulation of social behaviors, which we further confirmed through several social testing paradigms and the genetic deletion of another inhibitory GPCR specific to the MHb.Only few studies have proposed a role of the MHb in social behaviors58, and was also shown to extend to the social aspects of behavior3. In the literature, the alteration of MOR function was measured in different mental health disorders with social deficiencies, including depression61. Thus, constitutive MOR knockout mice (Oprm1−/− mice) present a large range of social deficiencies, including a decrease in social attachment, reciprocal direct social interactions, social preference, social conditioned place preference, ultrasound vocalizations (USVs) emission in response to social cues and social exploratory activation to USVs20. The pharmacology also indicates that MOR activation/blockade increases/decreases social responses, respectively12. MOR activity at the level of the mesolimbic pathway partly contributes to these effects11 but nothing is known at the level of the habenula, where MOR density is extremely high21.The implication of MORs in reward- and pain-related processes is well characterized62) in two different tasks. In the first task , undefeated B4MOR−/− mice behaved like defeated B4MOR+/+ mice with a reduced social exploration , mice were group housed and social preference for an unfamiliar peer from the same strain was tested. Under these different conditions, B4MOR−/− mice did not dissociate between the social and the empty compartment . Thus, abnormal/atypical social behavior does not seem to contribute to deficits in social interactions behavior in B4MOR−/− as it is likely the case for constitutive Oprm1−/− mice61.It has recently been shown that mice lacking MORs constitutively demonstrate an “atypical” social behavior leading judge mice to decrease their interaction time with themMHb Fig. , suggest41, and this highly restricted expression pattern in the brain makes this particular GPCR a highly valuable potential target for brain disease. Little is known about the function of GPR151, but an implication of GPR151 in nicotine intake was shown, supposing a role for this receptor in drug addiction and reward processes40. We hypothesized that, as for MHb MORs, GPR151 may act as a brake on the habenulo-interpeduncular pathway. The mechanism by which these receptors would be endogenously active is still to be determined, but if this is the case, we would expect Gpr151−/− mice to show impaired sociability. Indeed, while Gpr151/++ mice showed sociability for a peer , Gpr151−/− mice were indifferent to a social partner . Finally, a next step could be the identification of circuit mechanisms underlying the roles of habenular MOR and GPR151 in the regulation of social behaviors. We recently showed that optostimulation of habenular MOR-positive neurons projecting to the IPN is aversive45, an activity that could partly contribute to the impaired sociability observed in mice lacking habenular MORs. However similar optostimulation parameters applied to habenular GPR151-positive neurons projecting to the IPN did not seem to be aversive (Fig. Altogether, our results suggest that habenular Gi/o receptors participate to the attribution of the hedonic value of social interactions. Whether this function is related to hedonic or motivational aspects of social interactions is unknown and could be determined using operant social self-administration modelsors Fig. . Thus, iive Fig. . Althoug69 and depression3, therefore targeting habenula function may benefit to this particular dimension of psychiatric conditions. In particular, the development of GPR151 agonists may deliver highly specific drugs with minimal adverse effects, in high demand in the area of mental health.In summary, our findings reveal the importance of MHb activity, and receptors modulating this activity, in the control of social behaviors. This study opens new therapeutic perspectives, because social interactions deficits are associated with several psychopathologies including addictionSupplementary Table 1.Supplementary Figures.Supplementary Information.


JCI, Tian et al. describe the use of bicistronic CAR T cells that target multiple antigens expressed in neuroblastoma to overcome antigenic heterogeneity. Combining this approach with interventions that enhance T cell trafficking and prevent acquired dysfunction in the TME may lead to a long-awaited breakthrough in the clinical implementation of CAR T cells for the treatment of solid tumors.Chimeric antigen receptor (CAR) T cells have demonstrated success in treating select hematological malignancies, but their activity in solid tumors has been comparably modest. Challenges specific to treating solid tumors include trafficking and distribution throughout the tumor site, overcoming the immunosuppressive tumor microenvironment (TME), and identifying antigenic targets that are widely expressed and indispensable to tumor biology. In this issue of the  Chimeric antigen receptor (CAR) T cells are generated by harvesting a patient’s T cells and engineering them to express synthetic receptors composed of 3 key regulatory elements: (a) an extracellular single chain variable fragment (scFv) that confers extracellular target-recognition specificity, derived from the target-binding domain of antibodies, (b) a hinge domain and transmembrane spacer that provides flexibility and stability, and (c) an intracellular domain that triggers a signaling cascade that promotes T cell survival, proliferation, and cytotoxicity . First gImpediments to CAR T cell activity in solid tumors can be attributed to several key characteristics: trafficking into the tumor, an immunosuppressive environment, heterogenous antigen expression, intracellular antigens, and immune exhaustion . While CTian and colleagues screened for potent CARs against neuroblastoma tumor antigens glypican-2  and CD276  using methods to quantify and characterize surface proteins, including digital droplet PCR, pooled competitive optimization of CAR by cellular indexing of transcriptomes and epitopes–sequencing , and cytotoxicity assays. Having identified the most effective CARs against each of the 2 TAAs, the authors engineered BiCisCAR T cells to express CARs against both GPC2 and CD276. Since combined expression of GPC2 and CD276 is observed in 95% of neuroblastoma samples, these BiCisCAR T cells have specificity for a greater proportion of cancer cells compared to single target CAR T cells. Compared with single target CAR T cells, BiCisCAR T cells not only had comparable in vitro cytotoxicity against neuroblastoma cells coexpressing GPC2 and CD276, but they also secreted greater amounts of cytokines, such as IFN-γ and TNF-α. Thus, an advantage of BiCisCAR T cells over single target CAR T cells is their ability to initiate a robust, local inflammatory immune response, promoting recruitment and activation of other arms of the immune system, such as macrophages and dendritic cells .+ and GPC2+ cells respectively, whereas the BiCisCAR T cells were able to completely eradicate leukemia and prevent recurrence (Since tumor escape due to immunoediting is a key challenge to implementing CAR T cells in solid tumors, Tian and colleagues examined the ability of BiCisCAR T cells to overcome heterogenous antigen expression by using neuroblastoma cell lines that had CRISPR mediated ablation of GPC2 or CD276. While GPC2 CARs lose cytotoxicity in the context of GPC2 ablation and CD276 lose cytotoxicity in the context of CD276 ablation, BiCisCARs retained cytotoxicity with both GPC2KO and CD276KO neuroblastoma cells. A particularly striking example of BiCisCAR T cells’ ability to prevent tumor escape was demonstrated with a heterogeneous metastatic model, where NALM6 leukemia cells expressing either GPC2 or CD276 were mixed in a 1:1 ratio and injected into mice. Both GPC2 and CD276 CAR T cells failed to control tumor growth, due to the dominance of CD276Effective CAR T cell therapy depends on cytotoxicity as well as long-term persistence in the body while maintaining resistance to induction of exhaustion programs. BiCisCARs were superior to single target CARs due to greater persistence in the spleen and reduced expression of PD-1, LAG-3 and TIM-3 on the CAR T cell surface 21 days after infusion. As the authors demonstrate, the higher degree of persistence may be due to a more central memory phenotype .PDCD1 locus, while differentiation into memory cells is associated with remethylation (The findings reported by Tian and colleagues provide important insights into how effective CAR T cell therapies can be developed for solid tumors . Greaterhylation . It is lIn contrast to animal models in which tumors are often eradicated quickly, limiting the opportunity for antigenic shift and tumor escape, clinical tumor regression is often a slower process. As a result, antigen-persistence could expose BiCisCAR T cells to similar exhaustion pathways observed in clinical trials using CAR T cells , 13. Sin


Climatic, acidic deposition, and forest disturbance datasets were developed to elucidate the influence of these factors on terrestrial N availability through time. We hypothesized that spatial and temporal variation of terrestrial N availability, for which tree-ring δ15N records serve as a proxy, affects the variation of stream nitrate concentration across space and time. Across space at the Blue Ridge study sites, stream nitrate concentration increased linearly with increasing catchment mean wood δ15N. Over time, stream nitrate concentrations decreased with decreasing wood δ15N in five of the six catchments. Wood δ15N showed a significant negative relationship with disturbance and acidic deposition. Disturbance likely exacerbated N limitation by inducing nitrate leaching and ultimately enhancing vegetative uptake. As observed elsewhere, lower rates of acidic deposition and subsequent deacidification of soils may increase terrestrial N availability. Despite the ephemeral modifications of terrestrial N availability by these two drivers and climate, long-term declines in terrestrial N availability were robust and have likely driven much of the declines in stream nitrate concentration throughout the central Appalachians.A limitation to understanding drivers of long-term trends in terrestrial nitrogen (N) availability in forests and its subsequent influence on stream nitrate export is a general lack of integrated analyses using long-term data on terrestrial and aquatic N cycling at comparable spatial scales. Here we analyze relationships between stream nitrate concentrations and wood  On the other hand, greater N demand resulting from factors such as longer growing seasons or elevated atmospheric CO2 concentrations could reduce N availability . The predominantly deciduous forests of UBR and PR lie on poorly buffered, infertile soils and are likely experiencing deacidification as the soils regain base cations and limit toxic aluminum mobilization after decades of elevated rates of acidic deposition [et al 2019]. As soils continue to recover from past acidic deposition, increased ammonification/nitrification and subsequent denitrification, an emission pathway that greatly enriches soil 15N [δ15N and terrestrial N availability. Ultimately, the magnitude and duration of this release of legacy N and its impact on terrestrial N availability and stream nitrate loss is likely conditional on the proportion of unprocessed atmospheric nitrate reaching streams [2 [Despite the aforementioned limitations, the significant coefficient estimates offer some insights into forest- wide drivers of terrestrial N availability and parallel observations from other studies using different methods to track changes in terrestrial N and carbon cycling After removing the long-term trend of declining S and N deposition and declining wood ood δ15N , suggestsoil 15N , may be reams [2 .δ15N also declined in response to disturbance, suggesting that ephemeral disturbances [δ15N data from forests of the Pacific Northwest [δ15N and terrestrial N availability described above is that increased nitrification rates spurred by decreased vegetative uptake may temporarily reduce δ15N signature of nitrate in the soil. This decline in the isotopic signature of plant available nitrogen in the soil is a transient situation where the system can have a simultaneous increase in N availability yet have a reduction in the δ15N signature of plant available nitrate. The interplay of short and long-term impacts of disturbance on δ15N in soils and plants and more importantly on terrestrial N availability in ecosystems should be further explored.Wood urbances , the losδ15N.Declines in terrestrial N availability over the past 30 years drove reductions in stream nitrate concentration and loss at our study sites, consistent with reported declines in nitrate export from forested catchments throughout the mid-Atlantic and the Northeast over a similar time period [Sup. Material