Exertional heat illness encompasses a continuum from heat exhaustion (EHE) to heat stroke (EHS), yet the molecular mechanisms remain poorly understood. DNA methylation offers a stable epigenetic signature linking environmental stress to gene regulation and long-term physiological outcomes. We profiled genome-wide DNA methylation in blood from active-duty service members hospitalized for EHE (n = 36), heat injury (EHI; n = 18), or EHS (n = 50). Blood was collected longitudinally and analyzed using the Illumina 850 K array, with normalization and time-course clustering (TCseq) to identify co-regulated CpG networks. Ingenuity Pathway Analysis was applied differentially methylated probes (p < 0.01, Δβ > 0.04). At diagnosis, both EHI and EHS shared hypomethylation in pathways regulating heat sensing, oxidative defense, and vascular tone. Over time, EHI exhibited adaptive methylation changes supporting neuronal repair, glucocorticoid regulation, and cytoskeletal stability. In contrast, EHS demonstrated sustained downregulation of metabolic and cardiovascular regulators, persistent inflammasome activation, and oxidative imbalance. Early patterns were shared between EHI and EHS, reflecting a common acute stress response, but later responses diverged: EHI showed partial epigenetic recovery, while EHS exhibited sustained metabolic suppression and inflammasome activation. These time-dependent methylation signatures identify potential molecular targets for promoting recovery and preventing long-term complications of heat illnesses.

Static blood-based biomarkers provide limited insight into psychiatric illness and recovery, largely because they capture only a single, unperturbed snapshot of inherently dynamic biological systems. In this study, we applied a dynamic, longitudinal dual-state approach to characterize peripheral blood mononuclear cell (PBMC) transcriptional trajectories associated with psychotherapy outcomes in post-traumatic stress disorder (PTSD). PBMC transcriptional profiles were generated for veterans with PTSD (n = 46, 21, and 17, respectively) and controls (n = 26, 26, and 23) at pre-treatment, post-treatment, and follow-up. The PTSD cohort with profiles across all three timepoints was divided into responders (n = 6) and non-responders (n = 11) based on their Clinician-Administered PTSD Scale for DSM-5 (CAPS-5) trajectories. All subjects were profiled in both unstimulated and dexamethasone-stimulated states. At pre-treatment, unstimulated responders and non-responders exhibited pronounced transcriptional differences exceeding those observed between PTSD and controls. Longitudinally, unstimulated responders showed a marked shift toward control-like profiles, whereas non-responders retained persistent divergence. Pathway analyses identified a prominent temporal signal within Class C/3 G-protein-coupled receptor pathways driven largely by bitter taste receptors (TAS2Rs), along with subgroup-specific regulation of GABAB, opioid, and NMDA receptor signaling. Dexamethasone-induced responses further differentiated subgroups and showed opposing associations with recovery trajectories, implicating glucocorticoid sensitivity as a potential predictor of treatment outcomes. These findings indicate that biological responsiveness itself constitutes a measurable phenotype of recovery, and that dynamic functional profiling of living immune cells can reveal clinically relevant trajectories and candidate mechanisms not accessible through static biomarkers.

Background: Exertional heat stress induces a spectrum of illnesses, from mild heat exhaustion (EHE) to life-threatening heat stroke (EHS). While an epigenetic basis for heat acclimation "memory" is proposed, the dynamic molecular changes defining the severity and progression of human exertional heat illness remain poorly understood. Study objective: The primary objective of this study was to characterize and compare the longitudinal dynamics of whole-blood DNA methylation across the clinical spectrum of exertional heat illness. Hypothesis. We hypothesized that the severity of exertional heat illness would be encoded by distinct and persistent epigenetic signatures in peripheral blood. Methods: We conducted a longitudinal analysis of whole-blood DNA methylation in a human cohort diagnosed with EHE (n=32), Exertional Heat Injury (EHI, n=16), or EHS (n=39). Methylation profiles were assessed at initial diagnosis (T0) and across four subsequent time-bins representing acute, sub-acute, and later recovery phases ( >8 days). Data: We employed differential methylation analysis, time-course clustering, and pathway enrichment to define the epigenetic landscape of each condition. Summary of Results: At diagnosis, EHS and EHI displayed thousands of differentially methylated probes (DMPs) compared to EHE, with largely distinct signatures (67% and 15% unique DMPs, respectively), indicating severity-specific initial responses. Longitudinal analysis revealed that while EHE profiles normalized rapidly, EHS and EHI exhibited dramatic and persistent methylation changes over time. Temporal clustering identified divergent regulatory programs. EHI was characterized by transient upregulation of pathways related to neuronal protection (BDNF), stress regulation (FKBP5), and adaptive immunity, followed by sustained downregulation of synaptic and T-cell signaling pathways, suggesting a shift toward a resource-conserving recovery state. In contrast, EHS showed a delayed and sustained upregulation of maladaptive pathways, including inflammasome activation (CARD8, GSDMD), proteotoxic stress (HSP90AB1), and oxidative strain (TXNRD1), reflecting a progression toward systemic immune and cellular collapse. Conclusions: Exertional heat stress engraves divergent, severity-dependent epigenetic trajectories in peripheral blood. EHI is marked by adaptive methylation programs geared toward repair and recovery, while EHS triggers a persistent, pathological signature of systemic inflammation. These findings provide a molecular framework for understanding heat illness progression, offer a rich source for potential biomarkers to predict clinical outcomes, and highlight novel therapeutic targets to mitigate the long-term consequences of severe heat injury. Funding sources- This study was funded by US Army Medical Research and Development Command (USAMRDC).Disclaimer Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. The investigators have adhered to the policies for protection of human subjects as prescribed in AR 70–25/DoD Instruction (DoDI) 3216.02. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

IntroductionAcute mountain sickness (AMS) is a common altitude illness that occurs when individuals rapidly ascend to altitudes ≥2,500 m without proper acclimatization. Genetic and genomic factors can contribute to the development of AMS or predispose individuals to susceptibility. This study aimed to investigate differential gene regulation and biological pathways to diagnose AMS from high-altitude (HA; 4,300 m) blood samples and predict AMS-susceptible (AMS+) and AMS-resistant (AMS─) individuals from sea-level (SL; 50 m) blood samples.MethodsTwo independent cohorts were used to ensure the robustness of the findings. Blood samples were collected from participants at SL and HA. RNA sequencing was employed to profile gene expression. Differential expression analysis and pathway enrichment were performed to uncover transcriptomic signatures associated with AMS. Biomarker panels were developed for diagnostic and predictive purposes.ResultsAt HA, hemoglobin-related genes (HBA1, HBA2, and HBB) and phosphodiesterase 5A (PDE5A) emerged as key differentiators between AMS+ and AMS− individuals. The cAMP response element-binding protein (CREB) pathway exhibited contrasting regulatory patterns at SL and HA, reflecting potential adaptation mechanisms to hypoxic conditions. Diagnostic and predictive biomarker panels were proposed based on the identified transcriptomic signatures, demonstrating strong potential for distinguishing AMS+ from AMS− individuals.DiscussionThe findings highlight the importance of hemoglobin-related genes and the CREB pathway in AMS susceptibility and adaptation to hypoxia. The differential regulation of these pathways provides novel insights into the biological mechanisms underlying AMS. The proposed biomarker panels offer promising avenues for the early diagnosis and prediction of AMS risk, which could enhance preventive and therapeutic strategies.

Background: DNA methylation has emerged as a potential epigenetic mechanism influencing adaptations to heat illness in mice, yet methylation patterns that occur with heat illness in humans is unknown. Purpose: The purpose of this study was to assess methylation patterns induced by heat exhaustion, heat injury, or exertional heat stroke (EHS) in humans, representing increasing severity of heat illness, respectively. Hypothesis: Our hypothesis was that methylation sites related to inflammation and organ injury would be hypomethylated due to heat illness. Methods: Genomic DNA was extracted using the PAXgene Blood DNA Kit (Qiagen, Germantown MD, USA) from multiple timepoints from individuals who had heat exhaustion (n = 17), heat injury (n = 6), or EHS (n = 26). Genomic DNA (500 ng) was treated with sodium bisulfite using the Zymo EZ96 DNA Methylation Kit (Zymo Research, Orange CA, USA), and genome-wide DNA methylation patterns were profiled using the Infinium HumanMethylation450 BeadChip (850K) Kit (Illumina, Inc., San Diego CA, USA). All samples passed the log median intensity quality control of methylated and unmethylated channels assessed using the R minfi package v1.30.049. BMIQ normalization was applied on 99 EHS longitudinal samples. We utilized the ChAMP package for data normalization, filtering, and processing. The Short Time Series Expression Miner (STEM) was used to analyze patterns across three time points for EHS patients. STEM determines which profiles are statistically significant by comparing the actual number of genes assigned to a profile with the expected number of genes based on random chance. Profiles with more genes than expected are considered statistically significant, indicating that the expression pattern in those profiles is biologically meaningful. Ingenuity Pathway Analysis® was used to categorize pathways. Results: Epigenetic profiling of blood samples from individuals after collapse from heat illness revealed significant and distinct methylation changes, which were particularly pronounced in those suffering from EHS. In EHS samples, we identified 1,389 unique probes belonging to seven separate STEM profiles. Specifically, we found hypermethylation patterns related to detoxification of ROS, adipocyte signaling, insulin secretion signaling, and the WNT/B-Catenin pathway. We found hypomethylation patterns in the hepatic fibrosis pathway, serotonin pathway, and cytoskeleton signaling pathways, with the largest amount of hypomethylation appearing in Rho Family signaling. Genes such as RAB11FIP3, TTN, STAT5B, SAMSN1, PRG, LTBP1, PPARG, GPX3, ARHGEF3, and ITGAM, demonstrated hypermethylation, implicating disruptions in pathways responsible for maintaining structural integrity, metabolic homeostasis, and immune regulation. Additionally, hypermethylation of FAM109A and GNAZ, genes involved in neuronal signaling, suggests broader impacts affecting neural communication which is consistent with the altered mental status that is a hallmark of EHS at the time of collapse. Conversely, ZFP36L1, CDH11, and CH25H, exhibited hypomethylation, with roles in RNA stability, lipid metabolism, and extracellular matrix remodeling. Conclusion: Our results identify known and novel methylation patterns that may underpin the physiological and molecular responses to heat illness, particularly EHS. These insights provide a foundation for future research into the role of DNA methylation in mediating long-term health outcomes associated with heat injuries. Funded by USAMRDC; author views not representative of the Army or DoD. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Post-traumatic stress disorder (PTSD) is a complex, debilitating condition prevalent among military personnel exposed to traumatic events, necessitating biomarkers for early detection and intervention. Using data from the Millennium Cohort Study, the largest and longest-running military health study initiated in 2001, our objective was to identify specific microRNA (miRNA) expression patterns associated with distinct PTSD symptom trajectories among service members and veterans and assess their potential for predicting resilience and symptom severity. We analyzed 1052 serum samples obtained from the Department of Defense Serum Repository and linked with survey data collected at baseline and across three follow-up waves (2001-2011), using miRNA sequencing and statistical modeling. Our analysis identified five PTSD trajectories-resilient, pre-existing, new-onset moderate, new-onset severe, and adaptive-and revealed significant dysregulation of three key miRNAs (miR-182-5p, miR-9-5p, miR-204-5p) in participants with PTSD compared to resilient individuals. These miRNAs, which inhibit brain-derived neurotrophic factor (BDNF) and target pathways like NFκB, Notch, and TGF-alpha, were associated with neuronal plasticity, inflammation, and tissue repair, reflecting PTSD pathophysiology. These findings suggest that miRNA profiles could serve as biomarkers for early identification of PTSD risk and resilience, guiding targeted interventions to improve long-term health outcomes for military personnel.

IntroductionBlast injury has been implicated as the major cause of traumatic brain injury (TBI) and ocular system injury, in military operations in Iraq and Afghanistan. Soldiers exposed to traumatic stress also have undiagnosed, chronic vision problems. Here we hypothesize that excessive intake of ω-6 fatty acid linoleic acid (LA) and insufficiency of dietary long chain ω-3 polyunsaturated fatty acids (PUFAs, e.g., docosahexaenoic acid; DHA) would dysregulate endocannabinoid-mediated neuronal plasticity and immune response. The study objective was to determine the effect of blast-TBI and traumatic stress on retinal gene expression and assess the role of dietary deficiency of long chain ω-3 PUFAs on the vulnerability to these injury models.MethodsLinoleic acid was used as an independent variable to reflect the dietary increase in LA from 1 percent of energy (en%) to 8 en% present in the current western diets, and these custom LA diets were also devoid of long chain ω-3 PUFAs. Animals were exposed to a simulated blast overpressure wave followed by a weight drop head-concussion to induce TBI. A Separate group of rats were subjected to traumatic stress by a forced immersion underwater.ResultsOur findings showed that blast-TBI exposure, post 14 days, produced significant neuropathological changes such as axonal degeneration in the brain optic tracts from all the three diet groups, especially in rats fed the DHA-deprived 1 en% LA diet. Transcriptomic analysis showed that presence of DHA in the house chow diet prevented blast-induced disruption of neuronal plasticity by activating molecular networks like SNARE signaling, endocannabinoid pathway, and synaptic long-term depression when compared to DHA-deprived 8 en% LA diet group. Under traumatic stress, retinal synaptic function, neurovascular coupling, and opioid signaling mechanisms were dysregulated in rodents fed DHA-deficient diets (i.e., 8 en% LA and 1 en% LA), where reducing the levels of ω-6 linoleic acid from 8 en% to 1 en% was associated with increased neuronal plasticity and suppressed immune signaling.ConclusionThe findings of our study suggest that deprivation of long chain ω-3 PUFAs in the diet affects endocannabinoid-mediated neuronal plasticity, vascular function and inflammatory response that could influence the resistance of veterans to TBI and psychological trauma.

Background Approximately 40% of Service Members deployed in support of Operation Enduring Freedom (OEF) and Operation Iraqi Freedom (OIF) and an astounding 80% of Veterans overall report experiencing pain. Currently, drugs that adequately treat pain may result in addiction and substance abuse or negative side effects such as nausea, vomiting, renal and cardiovascular issues, among other physiological and cognitive problems. Inadequate acute pain management can lead to the development of chronic pain. Combat and non-combat injuries, acute and chronic pain all have the potential to impact return-to-duty rates/decisions, thereby negatively affecting the Fighting Force. To develop more effective pain therapeutics, the molecular mechanisms contributing to the development of neuropathic pain are under intense investigation and further research is needed to fully understand neuropathic pain induction and maintenance. The overarching objective of this study is to identify microRNA (miRNA) changes in key brain regions during the onset and progression of neuropathic pain in a rodent model. Results Changes in miRNA expression were observed at day 15 post-SNL in the amygdala and thalamus. The majority of changes were observed in the left side of the brain, contralateral to the right-sided SNL injury. The DE miRNAs identified mainly in the amygdala and thalamus did not overlap between brain regions. The altered miRNAs regulate key signaling pathways and genes important in pain development. Discussion The majority of epigenetic studies investigating altered miRNA expression in the pain field have explored the peripheral nervous system. Very few studies have evaluated miRNA dynamics in the brain following neuropathic pain development. This study provides key insights into changes occurring in the brain following peripheral nerve injury. Our lab has previously identified circulating extracellular vesicle (EV) miRNAs that are altered in the blood post-SNL. There is some overlap between the blood and brain miRNAs that may serve as key biomarkers in prognosis and/or diagnosis of a peripheral nerve injury and the development of chronic pain.

Clinical mental health researchers may understandably struggle with how to incorporate biological assessments in clinical research. The options are numerous and are described in a vast and complex body of literature. Here we provide guidelines to assist mental health researchers seeking to include biological measures in their studies. Apart from a focus on behavioral outcomes as measured via interviews or questionnaires, we advocate for a focus on biological pathways in clinical trials and epidemiological studies that may help clarify pathophysiology and mechanisms of action, delineate biological subgroups of participants, mediate treatment effects, and inform personalized treatment strategies. With this paper we aim to bridge the gap between clinical and biological mental health research by (1) discussing the clinical relevance, measurement reliability, and feasibility of relevant peripheral biomarkers; (2) addressing five types of biological tissues, namely blood, saliva, urine, stool and hair; and (3) providing information on how to control sources of measurement variability.

Using a model of combat and operational stress reaction (COSR), our lab recently showed that exposure to an unpredictable combat stress (UPCS) procedure prior to a thermal injury increases pain sensitivity in male rats. Additionally, our lab has recently shown that circulating extracellular vesicle-microRNAs (EV-miRNAs), which normally function to suppress inflammation, were down-regulated in a male rat model of neuropathic pain. In this current study, male and female rats ex-posed to UPCS, followed by thermal injury, were evaluated for changes in circulating EV-miRNAs. Adult female and male Sprague Dawley rats were exposed to a UPCS procedure for either 2 or 4 weeks. Groups consisted of the following: nonstress (NS), stress (S), NS + thermal injury (TI), and S + TI. Mechanical sensitivity was measured, and plasma was collected at baseline, throughout the UPCS exposure, and post-thermal injury. EV-miRNA isolation was performed, followed by small RNA sequencing and subsequent data analysis. UPCS exposure alone resulted in mechanical allodynia in both male and female rats at specific time points. Thermal-injury induction occurring at peak UPCS resulted in increased mechanical allodynia in the injured hind paw compared to thermal injury alone. Differential expression of the EV-miRNAs was observed between the NS and S groups as well as between NS + TI and S + TI groups. Consistent differences in EV-miRNAs are detectable in both COSR as well as during the development of mechanical sensitivity and potentially serve as key regulators, biomarkers, and targets in the treatment of COSR and thermal-injury induced mechanical sensitivity. Perspective: This article presents the effects of unpredictable combat stress and thermal injury on EV-contained microRNAs in an animal model. These same mechanisms may exist in clinical patients and could be future prognostic and diagnostic biomarkers.(R) 2023 Published by Elsevier Inc. on behalf of United States Association for the Study of Pain, Inc All rights reserved.

Metabolomics, proteomics and DNA methylome assays, when done in tandem from the same blood sample and analyzed together, offer an opportunity to evaluate the molecular basis of post-traumatic stress disorder (PTSD) course and pathogenesis. We performed separate metabolomics, proteomics, and DNA methylome assays on blood samples from two well-characterized cohorts of 159 active duty male participants with relatively recent onset PTSD (<1.5 years) and 300 male veterans with chronic PTSD (>7 years). Analyses of the multi-omics datasets from these two independent cohorts were used to identify convergent and distinct molecular profiles that might constitute potential signatures of severity and progression of PTSD and its comorbid conditions. Molecular signatures indicative of homeostatic processes such as signaling and metabolic pathways involved in cellular remodeling, neurogenesis, molecular safeguards against oxidative stress, metabolism of polyunsaturated fatty acids, regulation of normal immune response, post-transcriptional regulation, cellular maintenance and markers of longevity were significantly activated in the active duty participants with recent PTSD. In contrast, we observed significantly altered multimodal molecular signatures associated with chronic inflammation, neurodegeneration, cardiovascular and metabolic disorders, and cellular attritions in the veterans with chronic PTSD. Activation status of signaling and metabolic pathways at the early and late timepoints of PTSD demonstrated the differential molecular changes related to homeostatic processes at its recent and multi-system syndromes at its chronic phase. Molecular alterations in the recent PTSD seem to indicate some sort of recalibration or compensatory response, possibly directed in mitigating the pathological trajectory of the disorder.

A clinical incident is typically manifested by several molecular events; therefore, it seems logical that a successful diagnosis, prognosis, or stratification of a clinical landmark require multiple biomarkers. In this report, we presented a machine learning pipeline, namely "Biomarker discovery process at binomial decision point" (2BDP) that took an integrative approach in systematically curating independent variables (e.g., multiple molecular markers) to explain an output variable (e.g., clinical landmark) of binary in nature. In a logical sequence, 2BDP includes feature selection, unsupervised model development and cross validation. In the present work, the efficiency of 2BDP was demonstrated by finding three biomarker panels that independently explained three stages of Alzheimer's disease (AD) marked as Braak stages I, II and III, respectively. We designed three assortments from the entire cohort based on these Braak stages; subsequently, each assortment was split into two populations at Braak score I, II or III. 2BDP systematically integrated random forest and logistic regression fitting model to find biomarker panels with minimum features that explained these three assortments, e.g., significantly differentiated two populations segregated by Braak stage I, II or III, respectively. Thereafter, the efficacies of these panels were measured by the area under the curve (AUC) values of the receiver operating characteristic (ROC) plot. The AUCROC was calculated by two cross-validation methods. Final set of gene markers was a mix of novel and a priori established AD signatures. These markers were weighted by unique coefficients and linearly connected in a group of 2-10 to explain Braak stage I, II or III by AUC >= 0.8. Small sample size and a lack of distinctly recruited Training and Test sets were the limitations of the present undertaking; yet 2BDP demonstrated its capability to curate a panel of optimum numbers of biomarkers to describe the outcome variable with high efficacy.

Posttraumatic stress disorder (PTSD) is a heritable (h2 = 24–71%) psychiatric illness. Copy number variation (CNV) is a form of rare genetic variation that has been implicated in the etiology of psychiatric disorders, but no large-scale investigation of CNV in PTSD has been performed. We present an association study of CNV burden and PTSD symptoms in a sample of 114,383 participants (13,036 cases and 101,347 controls) of European ancestry. CNVs were called using two calling algorithms and intersected to a consensus set. Quality control was performed to remove strong outlier samples. CNVs were examined for association with PTSD within each cohort using linear or logistic regression analysis adjusted for population structure and CNV quality metrics, then inverse variance weighted meta-analyzed across cohorts. We examined the genome-wide total span of CNVs, enrichment of CNVs within specified gene-sets, and CNVs overlapping individual genes and implicated neurodevelopmental regions. The total distance covered by deletions crossing over known neurodevelopmental CNV regions was significant (beta = 0.029, SE = 0.005, P = 6.3 × 10−8). The genome-wide neurodevelopmental CNV burden identified explains 0.034% of the variation in PTSD symptoms. The 15q11.2 BP1-BP2 microdeletion region was significantly associated with PTSD (beta = 0.0206, SE = 0.0056, P = 0.0002). No individual significant genes interrupted by CNV were identified. 22 gene pathways related to the function of the nervous system and brain were significant in pathway analysis (FDR q < 0.05), but these associations were not significant once NDD regions were removed. A larger sample size, better detection methods, and annotated resources of CNV are needed to explore this relationship further.