Systems genetic analysis of lignin biosynthesis in Populus tremula.

The genetic control underlying natural variation in lignin content and composition in trees is not fully understood. We performed a systems genetic analysis to uncover the genetic regulation of lignin biosynthesis in a natural 'SwAsp' population of aspen (Populus tremula) trees. We analyzed gene expression by RNA sequencing (RNA-seq) in differentiating xylem tissues, and lignin content and composition using Pyrolysis-GC-MS in mature wood of 268 trees from 99 genotypes. Abundant variation was observed for lignin content and composition, and genome-wide association study identified proteins in the pentose phosphate pathway and arabinogalactan protein glycosylation among the top-ranked genes that are associated with these traits. Variation in gene expression and the associated genetic polymorphism was revealed through the identification of 312 705 local and 292 003 distant expression quantitative trait loci (eQTL). A co-expression network analysis suggested modularization of lignin biosynthesis and novel functions for the lignin-biosynthetic CINNAMYL ALCOHOL DEHYDROGENASE 2 and CAFFEOYL-CoA O-METHYLTRANSFERASE 3. PHENYLALANINE AMMONIA LYASE 3 was co-expressed with HOMEOBOX PROTEIN 5 (HB5), and the role of HB5 in stimulating lignification was demonstrated in transgenic trees. The systems genetic approach allowed linking natural variation in lignin biosynthesis to trees´ responses to external cues such as mechanical stimulus and nutrient availability.

Real-time performance improvement optimizes damage control resuscitation best practice adherence: Results of a pilot prospective observational study.

BACKGROUND: Maintaining balanced blood product ratios during damage control resuscitation (DCR) is independently associated with improved survival. We hypothesized that real-time performance improvement (RT-PI) would increase adherence to DCR best practice. STUDY DESIGN AND METHODS: From December 2020-August 2021, we prospectively used a bedside RT-PI tool to guide DCR in severely injured patients surviving at least 30 min. RT-PI study patients were compared to contemporary control patients at our institution and historic PROMMTT study patients. A subset of patients transfused ≥6 U red blood cells (RBC) in 6 h (MT+) was also identified. The primary endpoint was percentage time in a high ratio range (≥3:4) of plasma (PLAS):RBC and platelet (PLT):RBC over 6 h. Secondary endpoints included time to massive transfusion protocol activation, time to calcium and tranexamic acid (TXA) dosing, and cumulative 6-h ratios. RESULTS: Included patients (n = 772) were 35 (24-51) years old with an Injury Severity Score of 27 (17-38) and 42% had penetrating injuries. RT-PI (n = 10) patients spent 96% of the 6-h resuscitation in a high PLAS:RBC range, no different versus CONTROL (n = 87) (96%) but more than PROMMTT (n = 675) (25%, p < .001). In the MT+ subgroup, optimal PLAS:RBC and PLT:RBC were maintained for the entire 6 h in RT-PI (n = 4) versus PROMMTT (n = 391) patients for both PLAS (p < .001) and PLT ratios (p < .001). Time to TXA also improved significantly in RT-PI versus CONTROL patients (27 min [22-31] vs. 51 min [29-98], p = .035). CONCLUSION: In this prospective study, RT-PI was associated with optimized DCR. Multicenter validation of this novel approach to optimizing DCR implementation is warranted.

Utica/Point Pleasant brine isotopic compositions (δ7Li, δ11B, δ138Ba) elucidate mechanisms of lithium enrichment in the Appalachian Basin.

Global Li production will require a ∼500 % increase to meet 2050 projected energy storage demands. One potential source is oil and gas wastewater (i.e., produced water or brine), which naturally has high total dissolved solids (TDS) concentrations, that can also be enriched in Li (>100 mg/L). Understanding the sources and mechanisms responsible for high naturally-occurring Li concentrations can aid in efficient targeting of these brines. The isotopic composition (δ7Li, δ11B, δ138Ba) of produced water and core samples from the Utica Shale and Point Pleasant Formation (UPP) in the Appalachian Basin, USA indicates that depth-dependent thermal maturity and water-rock interaction, including diagenetic clay mineral transformations, likely control Li concentrations. A survey of Li content in produced waters throughout the USA indicates that Appalachian Basin brines from the Marcellus Shale to the UPP have the potential for economic resource recovery.

Treating young adult cannabis use disorder with text message-delivered peer network counseling.

BACKGROUND: Approximately 16.5% of U.S. young adults have a cannabis use disorder (CUD) and are at risk for negative outcomes. Treatment can reduce cannabis use, but young adults are less likely to seek help than older adults. Peer Network Counseling-txt (PNC-txt) is a brief, text-delivered, Motivational Interviewing-informed substance use intervention focusing on peer relations and activity spaces as mechanisms for behavioral change. PNC-txt has shown evidence of reducing tobacco and cannabis use with adolescents and young adults, but it has not been tested in the context of legal cannabis use. The current randomized controlled trial sought to expand the evidence regarding the context of PNC-txt effects, comparing one state in which cannabis is legal (Colorado) and one state in which it is not (Tennessee). We hypothesized that participants randomized to PNC-txt would show significant reductions in cannabis use compared to controls, with larger reductions for females and those in Colorado, and that peer relations and activity space would mediate effects. METHODS: One thousand, seventy eight 18-25 year olds (CO: 551; TN: 527) who met screening criteria for CUD and biologically-verified cannabis use were randomly assigned to PNC-txt or waitlist control condition. Every other day for 4 weeks, participants assigned to PNC-txt received pre-programmed text conversations, tailored via data from the baseline assessment. Self-report and biological indicators of cannabis use were measured at 1-, 3-, and 6-months. DISCUSSION: Data analysis is underway. Results will provide evidence regarding whether, and how, PNC-txt reduces cannabis use in young adults with CUD. TRIAL REGISTRATION: This trial was prospectively registered on September 28, 2020 with ClinicalTrials.gov (NCT04567394).

Cannabis Co-Use Among Black Individuals with Chronic Pain Who Use Opioids: Associations with Other Substance Use and Pain Related Outcomes.

Background Black individuals with chronic musculoskeletal (MSK) pain tend to experience worse pain and opioid use-related outcomes, including other substance co-use, compared with non-Hispanic White individuals. Co-using cannabis with opioids could instigate a cascade of pain-related vulnerabilities and poor outcomes. Here, we test associations between cannabis/opioid co-use and pain-related outcomes among Black individuals with chronic MSK pain. Methods Black adults with chronic MSK pain who use opioids (N=401; 51.62% female, Mage=35.90, SD=11.03) completed online measures of pain intensity/interference, emotional distress, opioid dependence, and risky use of other substances. Results Compared with opioid use alone, opioid and cannabis co-use was associated with elevated anxiety and depression symptoms, opioid dependence, and risky substance use, but not pain. Conclusions Black individuals with chronic MSK pain who co-use opioids and cannabis warrant targeted interventions that address their needs. Tailored interventions could help address disparities in pain-related outcomes and opioid morbidity and mortality rates.

Working memory limitations constrain visual episodic long-term memory at both specific and gist levels of representation.

Limitations in one's capacity to encode information in working memory (WM) constrain later access to that information in long-term memory (LTM). The present study examined whether these WM constraints on episodic LTM are limited to specific representations of past episodes or also extend to gist representations. Across three experiments, young adult participants (n = 40 per experiment) studied objects in set sizes of two or six items, either sequentially (Experiments 1a and 1b) or simultaneously (Experiment 2). They then completed old/new recognition tests immediately after each sequence (WM tests). After a long study phase, participants completed LTM conjoint recognition tests, featuring old but untested items from the WM phase, lures that were similar to studied items at gist but not specific levels of representation, and new items unrelated to studied items at both specific and gist levels of representation. Results showed that LTM estimates of specific and gist memory representations from a multinomial-processing-tree model were reduced for items encoded under supra-capacity set sizes (six items) relative to within-capacity set sizes (two items). These results suggest that WM encoding capacity limitations constrain episodic LTM at both specific and gist levels of representation, at least for visual objects. The ability to retrieve from LTM each type of representation for a visual item is contingent on the degree to which the item could be encoded in WM.

Durable Objective Response to Lurbinectedin in Small Cell Bladder Cancer with TP53 Mutation: A Molecular-Directed Strategy.

Small cell bladder cancer (SCBC) is a rare and aggressive disease, often treated with platinum/etoposide-based chemotherapy. Key molecular drivers include the inactivation of onco-suppressor genes (TP53, RB1) and amplifications in proto-oncogenes (MYC). We report a patient with SCBC who achieved an objective and prolonged response to lurbinectedin, which has been approved for metastatic small cell lung cancer, after developing disease progression on cisplatin/etoposide and nivolumab/ipilimumab. A genomic analysis of a metastatic biopsy prior to lurbinectedin initiation revealed a TP53 mutation and amplification of the cell cycle regulators E2F3 and MYCL. A repeat biopsy following the development of lurbinectedin resistance showed a new actionable ERBB2 alteration without significant change in the tumor mutation burden (six mutations/Mb). The present report suggests that lurbinectedin may be active and should be further explored in SCBC harboring TP53 mutations and amplifications in E2F3 and MYC family complexes.

Beyond Coronary Artery Disease: Assessing the Microcirculation.

Ischemic heart disease (IHD) affects more than 20 million adults in the United States. Although classically attributed to atherosclerosis of the epicardial coronary arteries, nearly half of patients with stable angina and IHD who undergo invasive coronary angiography do not have obstructive epicardial coronary artery disease. Ischemia with nonobstructive coronary arteries is frequently caused by microvascular angina with underlying coronary microvascular dysfunction (CMD). Greater understanding the pathophysiology, diagnosis, and treatment of CMD holds promise to improve clinical outcomes of patients with ischemic heart disease.

Association Between Utilization of Services and Perioperative Outcomes for Lung Cancer Resection.

INTRODUCTION: Lung cancer resection has largely focused on perioperative outcomes (eg, mortality) to benchmark performance. While variations in perioperative outcomes and in utilization of services (eg, ambulatory procedures, hospitalization) have been independently demonstrated, there has been limited evaluation of associations between these outcomes. We evaluated the association between perioperative outcomes and utilization of services to evaluate provider performance across a broader context of care. PATIENTS AND METHODS: This was a retrospective cohort study of patients undergoing lung cancer resection in 2017 to 2019. We utilized hierarchical logistic regression models to determine risk- and reliability-adjusted mortality and risk-adjusted utilization of services, at the hospital-level. We then evaluated utilization of services across quartiles of perioperative mortality. RESULTS: A total of 15,168 patients across 297 hospitals underwent lung cancer resection. Mean risk- and reliability-adjusted 90-day mortality varied between 1.58% (95% CI, 1.54%-1.62%) and 2.74% (95% CI, 2.59%-2.90%) across quartiles. Risk-adjusted utilization of all ambulatory procedures was highest in the best performing (lowest mortality) quartile at 37.7% (95% CI, 33.6%-41.8%). Additionally, risk-adjusted inpatient utilization prior to and after surgery was lowest in the best performing quartile at 15% (95% CI, 13.7%-16.3%) and 19.3% (95% CI, 17.5%-21.0%), respectively. CONCLUSIONS: Hospitals with the lowest perioperative mortality demonstrated trends towards using more outpatient resources prior to surgery, but fewer inpatient services surrounding lung cancer resection. This correlation highlights the importance of incorporating utilization of services in addition to other metrics to profile the efficiency and effectiveness of centers performing lung cancer resection across a broader spectrum of care.

Deep neural networks detect regional wall motion abnormalities and preclinical cardiovascular disease from 12-lead ECGs.

Background: Identifying regional wall motion abnormalities (RWMAs) is critical for diagnosing and risk stratifying patients with cardiovascular disease, particularly ischemic heart disease. We hypothesized that a deep neural network could accurately identify patients with regional wall motion abnormalities from a readily available standard 12-lead electrocardiogram (ECG). Methods: This observational, retrospective study included patients who were treated at Beth Israel Deaconess Medical Center and had an ECG and echocardiogram performed within 14 days of each other between 2008 and 2019. We trained a convolutional neural network to detect the presence of RWMAs, qualitative global right ventricular (RV) hypokinesis, and varying degrees of left ventricular dysfunction (left ventricular ejection fraction [LVEF] ≤50%, LVEF ≤40%, and LVEF ≤35%) identified by echocardiography, using ECG data alone. Patients were randomly split into development (80%) and test sets (20%). Model performance was assessed using area under the receiver operating characteristic curve (AUC). Cox proportional hazard models adjusted for age and sex were performed to estimate the risk of future acute coronary events. Results: The development set consisted of 19,837 patients (mean age 66.7±16.4; 46.7% female) and the test set comprised of 4,953 patients (mean age 67.5±15.8 years; 46.5% female). On the test dataset, the model accurately identified the presence of RWMA, RV hypokinesis, LVEF ≤50%, LVEF ≤40%, and LVEF ≤35% with AUCs of 0.87 (95% CI 0.858-0.882), 0.888 (95% CI 0.878-0.899), 0.923 (95% CI 0.914-0.933), 0.93 (95% CI 0.921-0.939), and 0.876 (95% CI 0.858-0.896), respectively. Among patients with normal biventricular function at the time of the index ECG, those classified as having RMWA by the model were 3 times the risk (age- and sex-adjusted hazard ratio, 2.8; 95% CI 1.9-3.9) for future acute coronary events compared to those classified as negative. Conclusions: We demonstrate that a deep neural network can help identify regional wall motion abnormalities and reduced LV function from a 12-lead ECG and could potentially be used as a screening tool for triaging patients who need either initial or repeat echocardiographic imaging.

A metabolomics pipeline highlights microbial metabolism in bloodstream infections.

The growth of antimicrobial resistance (AMR) highlights an urgent need to identify bacterial pathogenic functions that may be targets for clinical intervention. Although severe infections profoundly alter host metabolism, prior studies have largely ignored microbial metabolism in this context. Here, we describe an iterative, comparative metabolomics pipeline to uncover microbial metabolic features in the complex setting of a host and apply it to investigate gram-negative bloodstream infection (BSI) in patients. We find elevated levels of bacterially derived acetylated polyamines during BSI and discover the enzyme responsible for their production (SpeG). Blocking SpeG activity reduces bacterial proliferation and slows pathogenesis. Reduction of SpeG activity also enhances bacterial membrane permeability and increases intracellular antibiotic accumulation, allowing us to overcome AMR in culture and in vivo. This study highlights how tools to study pathogen metabolism in the natural context of infection can reveal and prioritize therapeutic strategies for addressing challenging infections.

VEXAS syndrome (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic): clinical review in a rapidly emerging field.

VEXAS syndrome is a recently described entity characterized by systemic inflammatory and hematologic manifestations. The disease was first characterized by Beck et al. in 2020 in a study characterizing 25 patients with undiagnosed adult-onset inflammatory syndromes. While the literature regarding VEXAS syndrome has grown exponentially since 2020, there is still much to be understood. This lack of information leads to challenges in both the diagnosis and treatment of patients with VEXAS syndrome. Patients will often have a variety of clinical symptoms that can lead to missed or delayed diagnoses. Additionally, awareness of VEXAS syndrome is still developing among clinicians. In this comprehensive review, we summarize the current literature regarding VEXAS syndrome, and explore clinical updates of this emerging disease state. Our aim of this review is to increase awareness regarding this new disease state and identify research areas to better understand future treatment approaches for patients with VEXAS syndrome.

Exploring the Potential of Structure-Based Deep Learning Approaches for T cell Receptor Design.

Deep learning methods, trained on the increasing set of available protein 3D structures and sequences, have substantially impacted the protein modeling and design field. These advancements have facilitated the creation of novel proteins, or the optimization of existing ones designed for specific functions, such as binding a target protein. Despite the demonstrated potential of such approaches in designing general protein binders, their application in designing immunotherapeutics remains relatively unexplored. A relevant application is the design of T cell receptors (TCRs). Given the crucial role of T cells in mediating immune responses, redirecting these cells to tumor or infected target cells through the engineering of TCRs has shown promising results in treating diseases, especially cancer. However, the computational design of TCR interactions presents challenges for current physics-based methods, particularly due to the unique natural characteristics of these interfaces, such as low affinity and cross-reactivity. For this reason, in this study, we explored the potential of two structure-based deep learning protein design methods, ProteinMPNN and ESM-IF, in designing fixed-backbone TCRs for binding target antigenic peptides presented by the MHC through different design scenarios. To evaluate TCR designs, we employed a comprehensive set of sequence- and structure-based metrics, highlighting the benefits of these methods in comparison to classical physics-based design methods and identifying deficiencies for improvement.

Ventricular Arrhythmias After Primary Percutaneous Coronary Intervention for STEMI.

Importance: Currently, mortality risk for patients with ST-segment elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (PCI) with an uncomplicated postprocedure course is low. Less is known regarding the risk of in-hospital ventricular tachycardia (VT) and ventricular fibrillation (VF). Objective: To evaluate the risk of late VT and VF after primary PCI for STEMI. Design, Setting, and Participants: This cohort study included adults aged 18 years or older with STEMI treated with primary PCI between January 1, 2015, and December 31, 2018, identified in the US National Cardiovascular Data Registry Chest Pain-MI Registry. Data were analyzed from April to December 2020. Main Outcomes and Measures: Multivariable logistic regression was used to evaluate the risk of late VT (≥7 beat run of VT during STEMI hospitalization ≥1 day after PCI) or VF (any episode of VF≥1 day after PCI) associated with cardiac arrest and associations between late VT or VF and in-hospital mortality in the overall cohort and a cohort with uncomplicated STEMI without prior myocardial infarction or heart failure, systolic blood pressure less than 90 mm Hg, cardiogenic shock, cardiac arrest, reinfarction, or left ventricular ejection fraction (LVEF) less than 40%. Results: A total of 174 126 eligible patients with STEMI were treated with primary PCI at 814 sites in the study; 15 460 (8.9%) had VT or VF after primary PCI, and 4156 (2.4%) had late VT or VF. Among the eligible patients, 99 905 (57.4%) at 807 sites had uncomplicated STEMI. The median age for patients with late VT or VF overall was 63 years (IQR, 55-73 years), and 75.5% were men; the median age for patients with late VT or VF with uncomplicated STEMI was 60 years (IQR, 53-69 years), and 77.7% were men. The median length of stay was 3 days (IQR, 2-7 days) for the overall cohort with late VT or VF and 3 days (IQR, 2-4 days) for the cohort with uncomplicated STEMI with late VT or VF. The risk of late VT or VF was 2.4% (overall) and 1.7% (uncomplicated STEMI). Late VT or VF with cardiac arrest occurred in 674 patients overall (0.4%) and in 117 with uncomplicated STEMI (0.1%). LVEF was the most significant factor associated with late VT or VF with cardiac arrest (adjusted odds ratio [AOR] for every 5-unit decrease ≤40%: 1.67; 95% CI, 1.54-1.85). Late VT or VF events were associated with increased odds of in-hospital mortality in the overall cohort (AOR, 6.40; 95% CI, 5.63-7.29) and the cohort with uncomplicated STEMI (AOR, 8.74; 95% CI, 6.53-11.70). Conclusions and Relevance: In this study, a small proportion of patients with STEMI treated with primary PCI had late VT or VF. However, late VT or VF with cardiac arrest was rare, particularly in the cohort with uncomplicated STEMI. This information may be useful when determining the optimal timing for hospital discharge after STEMI.

Transitioning from global to local computational strategies during brain-machine interface learning.

When learning to use a brain-machine interface (BMI), the brain modulates neuronal activity patterns, exploring and exploiting the state space defined by their neural manifold. Neurons directly involved in BMI control (i.e., direct neurons) can display marked changes in their firing patterns during BMI learning. However, the extent of firing pattern changes in neurons not directly involved in BMI control (i.e., indirect neurons) remains unclear. To clarify this issue, we localized direct and indirect neurons to separate hemispheres in a task designed to bilaterally engage these hemispheres while animals learned to control the position of a platform with their neural signals. Animals that learned to control the platform and improve their performance in the task shifted from a global strategy, where both direct and indirect neurons modified their firing patterns, to a local strategy, where only direct neurons modified their firing rate, as animals became expert in the task. Animals that did not learn the BMI task did not shift from utilizing a global to a local strategy. These results provide important insights into what differentiates successful and unsuccessful BMI learning and the computational mechanisms adopted by the neurons.

Bioengineered Skin for Diabetic Foot Ulcers: A Scoping Review.

Diabetic foot ulcers (DFUs) pose a significant threat to individuals with diabetes mellitus (DM), such as lower limb amputation and severe morbidity. Bioengineered skin substitutes (BSS) are alternatives to traditional interventions for treating DFUs, but their efficacy compared to standard wound care (SWC) or other treatment types, such as allografts, remains unknown. A scoping review of human studies was conducted to identify current approaches in the treatment of DFUs using BSS as compared with other treatment options. Systematic searches in PubMed, Cochrane Library, and Web of Science were conducted to identify comparative studies that enrolled 10 or more patients and evaluated wound healing outcomes (closure, time-to-healing, and area reduction). Database searches isolated articles published from 1 December 2012 to 1 December 2022 and were conducted in accordance with PRISMA-ScR guidelines. The literature search yielded 1312 articles, 24 of which were included for the qualitative analysis. Findings in these studies demonstrated that BSS outperformed SWC in all measured outcomes, suggesting that BSS may be a superior treatment for DFUs. Of the 24 articles, 8 articles compared human amniotic membrane allografts (hAMA) to BSS. Conflicting evidence was observed when comparing BSS and hAMA treatments, highlighting the need for future research.

Atomically accurate de novo design of single-domain antibodies.

Despite the central role that antibodies play in modern medicine, there is currently no way to rationally design novel antibodies to bind a specific epitope on a target. Instead, antibody discovery currently involves time-consuming immunization of an animal or library screening approaches. Here we demonstrate that a fine-tuned RFdiffusion network is capable of designing de novo antibody variable heavy chains (VHH's) that bind user-specified epitopes. We experimentally confirm binders to four disease-relevant epitopes, and the cryo-EM structure of a designed VHH bound to influenza hemagglutinin is nearly identical to the design model both in the configuration of the CDR loops and the overall binding pose.

Factors Associated With Margin Positivity After Lung Resection Surgery.

INTRODUCTION: The purpose of this study is to utilize a representative national sample to investigate the factors associated with margin positivity after attempted surgical resection. Given the changes in surgical approaches to lung cancer for the last 10 years, margin positivity and outcomes between robotic, video assisted thoracoscopic surgery (VATS) and open surgical resections may vary. METHODS: This retrospective cohort study utilized the National Cancer Database. Patients with non-small-cell lung cancer, 18 or older and who had a surgical lung resection between 2010 and 2019 were included. Demographic data, along with patient-level clinical variables were extracted. Patient-level outcome variables including 30-day, 90-day mortality and readmission rates were analyzed. Univariable and multivariable logistic regression was utilized to assess factors associated with margin positivity. RESULTS: A total of 226,884 patients were identified. Of the total cohort, 9229 had positive margins (4.2%). Patients with positive margins had statistically significant increased 30-day, 90-day mortality, as well as increased readmission rate. Older age, male sex, patients undergoing an open resection, patients who underwent a wedge resection, higher clinical stage, larger tumor size, squamous and adenosquamous histologies, and higher Charlson-Deyo Comorbidity Index were all associated with having a positive margin after resection. CONCLUSION: In conclusion, there was no difference in margin positivity when comparing robotic and VATS resection, however, open resection had increased rates of margin positivity. Increasing tumor size, clinical stage, squamous and adenosquamous histologies, male sex, and patients undergoing a wedge resection were all associated with increased rates of margin positivity.

Radioactivity from oil and gas produced water accumulated in freshwater mussels.

Legacy disposal of oil and gas produced water (OGPW) to surface water has led to radium contamination in streambed sediment creating a long-term radium source. Increased radium activities pose a potential health hazard to benthic organisms, such as freshwater mussels, as radium is capable of bioaccumulation. This project quantifies the impact of OGPW disposal on adult freshwater mussels, Eurynia dilatata, which were examined along the Allegheny River adjacent to a centralized waste treatment facility (CWT) that historically treated and then discharged OGPW. Radium isotopes (226Ra and 228Ra) were measured in streambed sediment, mussel soft tissue, and mussel hard shell collected upstream, at the outfall, 0.5 km downstream, and 5 km downstream of the CWT. Total radium activity was significantly higher (p < 0.05) in mussel tissue (mean = 3.44 ± 0.95 pCi/g), sediment (mean = 1.45 ± 0.19 pCi/g), and hard shell (mean = 0.34 ± 0.11 pCi/g) samples 0.5 km downstream than background samples collected upstream (mean = 1.27 ± 0.24; 0.91 ± 0.09; 0.10 ± 0.02 pCi/g respectively). Mussel shells displayed increased 226Ra activities up to 5 km downstream of the original discharge. Downstream soft tissue and hard shell 87Sr/86Sr ratios, as well as hard shell metal/calcium (e.g., Na/Ca; K/Ca; Mg/Ca) and 228Ra/226Ra ratios demonstrated trends towards values characteristic of Marcellus OGPW. Combined, this study demonstrates multiple lines of evidence for radium retention and bioaccumulation in freshwater mussels resulting from exposure to Marcellus OGPW.