Elucidating regulatory processes of intense physical activity by multi-omics analysis.

BACKGROUND: Physiological and biochemical processes across tissues of the body are regulated in response to the high demands of intense physical activity in several occupations, such as firefighting, law enforcement, military, and sports. A better understanding of such processes can ultimately help improve human performance and prevent illnesses in the work environment. METHODS: To study regulatory processes in intense physical activity simulating real-life conditions, we performed a multi-omics analysis of three biofluids (blood plasma, urine, and saliva) collected from 11 wildland firefighters before and after a 45 min, intense exercise regimen. Omics profiles post- versus pre-exercise were compared by Student's t-test followed by pathway analysis and comparison between the different omics modalities. RESULTS: Our multi-omics analysis identified and quantified 3835 proteins, 730 lipids and 182 metabolites combining the 3 different types of samples. The blood plasma analysis revealed signatures of tissue damage and acute repair response accompanied by enhanced carbon metabolism to meet energy demands. The urine analysis showed a strong, concomitant regulation of 6 out of 8 identified proteins from the renin-angiotensin system supporting increased excretion of catabolites, reabsorption of nutrients and maintenance of fluid balance. In saliva, we observed a decrease in 3 pro-inflammatory cytokines and an increase in 8 antimicrobial peptides. A systematic literature review identified 6 papers that support an altered susceptibility to respiratory infection. CONCLUSION: This study shows simultaneous regulatory signatures in biofluids indicative of homeostatic maintenance during intense physical activity with possible effects on increased infection susceptibility, suggesting that caution against respiratory diseases could benefit workers on highly physical demanding jobs.

Comprehensive evaluation and efficient classification of BRCA1 RING domain missense substitutions.

BRCA1 is a high-risk susceptibility gene for breast and ovarian cancer. Pathogenic protein-truncating variants are scattered across the open reading frame, but all known missense substitutions that are pathogenic because of missense dysfunction are located in either the amino-terminal RING domain or the carboxy-terminal BRCT domain. Heterodimerization of the BRCA1 and BARD1 RING domains is a molecularly defined obligate activity. Hence, we tested every BRCA1 RING domain missense substitution that can be created by a single nucleotide change for heterodimerization with BARD1 in a mammalian two-hybrid assay. Downstream of the laboratory assay, we addressed three additional challenges: assay calibration, validation thereof, and integration of the calibrated results with other available data, such as computational evidence and patient/population observational data to achieve clinically applicable classification. Overall, we found that 15%-20% of BRCA1 RING domain missense substitutions are pathogenic. Using a Bayesian point system for data integration and variant classification, we achieved clinical classification of 89% of observed missense substitutions. Moreover, among missense substitutions not present in the human observational data used here, we find an additional 45 with concordant computational and functional assay evidence in favor of pathogenicity plus 223 with concordant evidence in favor of benignity; these are particularly likely to be classified as likely pathogenic and likely benign, respectively, once human observational data become available.

The Wildland Firefighter Exposure and Health Effect (WFFEHE) Study: Rationale, Design, and Methods of a Repeated-Measures Study.

The wildland firefighter exposure and health effect (WFFEHE) study was a 2-year repeated-measures study to investigate occupational exposures and acute and subacute health effects among wildland firefighters. This manuscript describes the study rationale, design, methods, limitations, challenges, and lessons learned. The WFFEHE cohort included fire personnel ages 18-57 from six federal wildland firefighting crews in Colorado and Idaho during the 2018 and 2019 fire seasons. All wildland firefighters employed by the recruited crews were invited to participate in the study at preseason and postseason study intervals. In 2019, one of the crews also participated in a 3-day midseason study interval where workplace exposures and pre/postshift measurements were collected while at a wildland fire incident. Study components assessed cardiovascular health, pulmonary function and inflammation, kidney function, workplace exposures, and noise-induced hearing loss. Measurements included self-reported risk factors and symptoms collected through questionnaires; serum and urine biomarkers of exposure, effect, and inflammation; pulmonary function; platelet function and arterial stiffness; and audiometric testing. Throughout the study, 154 wildland firefighters participated in at least one study interval, while 144 participated in two or more study interval. This study was completed by the Centers for Disease Control and Prevention's National Institute for Occupational Safety and Health through a collaborative effort with the U.S. Department of Agriculture Forest Service, Department of the Interior National Park Service, and Skidmore College. Conducting research in the wildfire environment came with many challenges including collecting study data with study participants with changing work schedules and conducting study protocols safely and operating laboratory equipment in remote field locations. Forthcoming WFFEHE study results will contribute to the scientific evidence regarding occupational risk factors and exposures that can impact wildland firefighter health over a season and across two wildland fire seasons. This research is anticipated to lead to the development of preventive measures and policies aimed at reducing risk for wildland firefighters and aid in identifying future research needs for the wildland fire community.

Wildland firefighter exposure to smoke and COVID-19: A new risk on the fire line.

Throughout the United States, wildland firefighters respond to wildfires, performing arduous work in remote locations. Wildfire incidents can be an ideal environment for the transmission of infectious diseases, particularly for wildland firefighters who congregate in work and living settings. In this review, we examine how exposure to wildfire smoke can contribute to an increased likelihood of SARS-CoV-2 infection and severity of coronavirus disease (COVID-19). Human exposure to particulate matter (PM), a component of wildfire smoke, has been associated with oxidative stress and inflammatory responses; increasing the likelihood for adverse respiratory symptomology and pathology. In multiple epidemiological studies, wildfire smoke exposure has been associated with acute lower respiratory infections, such as bronchitis and pneumonia. Co-occurrence of SARS-CoV-2 infection and wildfire smoke inhalation may present an increased risk for COVID-19 illness in wildland firefighters due to PM based transport of SARS CoV-2 virus and up-regulation of angiotensin-converting enzyme II (ACE-2) (i.e. ACE-2 functions as a trans-membrane receptor, allowing the SARS-CoV-2 virus to gain entry into the epithelial cell). Wildfire smoke exposure may also increase risk for more severe COVID-19 illness such as cytokine release syndrome, hypotension, and acute respiratory distress syndrome (ARDS). Current infection control measures, including social distancing, wearing cloth masks, frequent cleaning and disinfecting of surfaces, frequent hand washing, and daily screening for COVID-19 symptoms are very important measures to reduce infections and severe health outcomes. Exposure to wildfire smoke may introduce additive or even multiplicative risk for SARS-CoV-2 infection and severity of disease in wildland firefighters. Thus, additional mitigative measures may be needed to prevent the co-occurrence of wildfire smoke exposure and SARS-CoV-2 infection.

ETV4 Is Necessary for Estrogen Signaling and Growth in Endometrial Cancer Cells.

Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression, however, the molecular mechanisms underlying ER's regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells, and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways. ETV4 loss affects chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture, where hollow organoids were formed and in vivo in the context of estrogen-dependent growth. These results show that ETV4 plays an important role in estrogen signaling in endometrial cancer cells. SIGNIFICANCE: Estrogen receptor alpha (ER) is a key oncogene in endometrial cancer. This study uncovers ETV4 as an important factor in controlling the activity of ER and the growth of endometrial cancer cells. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/6/1234/F1.large.jpg.

Electrostatic repulsion causes anticooperative DNA binding between tumor suppressor ETS transcription factors and JUN-FOS at composite DNA sites.

Many different transcription factors (TFs) regulate gene expression in a combinatorial fashion, often by binding in close proximity to each other on composite cis-regulatory DNA elements. Here, we investigated how ETS TFs bind with the AP1 TFs JUN-FOS at composite DNA-binding sites. DNA-binding ability with JUN-FOS correlated with the phenotype of ETS proteins in prostate cancer. We found that the oncogenic ETS-related gene (ERG) and ETS variant (ETV) 1/4/5 subfamilies co-occupy ETS-AP1 sites with JUN-FOS in vitro, whereas JUN-FOS robustly inhibited DNA binding by the tumor suppressors ETS homologous factor (EHF) and SAM pointed domain-containing ETS TF (SPDEF). EHF bound ETS-AP1 DNA with tighter affinity than ERG in the absence of JUN-FOS, possibly enabling EHF to compete with ERG and JUN-FOS for binding to ETS-AP1 sites. Genome-wide mapping of EHF- and ERG-binding sites in prostate epithelial cells revealed that EHF is preferentially excluded from closely spaced ETS-AP1 DNA sequences. Structural modeling and mutational analyses indicated that adjacent positively charged surfaces from EHF and JUN-FOS use electrostatic repulsion to disfavor simultaneous DNA binding. Conservation of positive residues on the JUN-FOS interface identified E74-like ETS TF 1 (ELF1) as an additional ETS TF exhibiting anticooperative DNA binding with JUN-FOS, and we found that ELF1 is frequently down-regulated in prostate cancer. In summary, divergent electrostatic features of ETS TFs at their JUN-FOS interface enable distinct binding events at ETS-AP1 DNA sites, which may drive specific targeting of ETS TFs to facilitate distinct transcriptional programs.

Work Practices and Respiratory Health Status of Appalachian Coal Miners With Progressive Massive Fibrosis.

OBJECTIVE: The aim of this study was to characterize workplace practices and respiratory health among coal miners with large opacities consistent with progressive massive fibrosis (PMF) who received care at a federally funded black lung clinic network in Virginia. METHODS: Participants were interviewed about their workplace practices and respiratory health. Medical records were reviewed. RESULTS: Nineteen former coal miners were included. Miners reported cutting rock, working downwind of dust-generating equipment, nonadherence to mine ventilation plans (including dust controls), improper sampling of respirable coal mine dust exposures, working after developing respiratory illness, and suffering from debilitating respiratory symptoms. CONCLUSION: Consistent themes of suboptimal workplace practices contributing to development of PMF emerged during the interviews. Some of the practices reported were unsafe and unacceptable. Further research is needed to determine the prevalence of these factors and how best to address them.

ETV4 and AP1 Transcription Factors Form Multivalent Interactions with three Sites on the MED25 Activator-Interacting Domain.

The recruitment of transcriptional cofactors by sequence-specific transcription factors challenges the basis of high affinity and selective interactions. Extending previous studies that the N-terminal activation domain (AD) of ETV5 interacts with Mediator subunit 25 (MED25), we establish that similar, aromatic-rich motifs located both in the AD and in the DNA-binding domain (DBD) of the related ETS factor ETV4 interact with MED25. These ETV4 regions bind MED25 independently, display distinct kinetics, and combine to contribute to a high-affinity interaction of full-length ETV4 with MED25. High-affinity interactions with MED25 are specific for the ETV1/4/5 subfamily as other ETS factors display weaker binding. The AD binds to a single site on MED25 and the DBD interacts with three MED25 sites, allowing for simultaneous binding of both domains in full-length ETV4. MED25 also stimulates the in vitro DNA binding activity of ETV4 by relieving autoinhibition. ETV1/4/5 factors are often overexpressed in prostate cancer and genome-wide studies in a prostate cancer cell line indicate that ETV4 and MED25 occupy enhancers that are enriched for ETS-binding sequences and are both functionally important for the transcription of genes regulated by these enhancers. AP1-motifs, which bind JUN and FOS transcription factor families, were observed in MED25-occupied regions and JUN/FOS also contact MED25; FOS strongly binds to the same MED25 site as ETV4 AD and JUN interacts with the other two MED25 sites. In summary, we describe features of the multivalent ETV4- and AP1-MED25 interactions, thereby implicating these factors in the recruitment of MED25 to transcriptional control elements.

The Effects of Pleural Plaques on Longitudinal Lung Function in Vermiculite Miners of Libby, Montana.

BACKGROUND: This study was conducted to assess associations of pleural plaques (PP) and longitudinal lung function in vermiculite miners of Libby, Montana who are occupationally exposed to asbestos. High-resolution computed tomography (HRCT) was used to identify asbestos-related findings in former Libby vermiculite miners. We investigated annual lung function decline in miners with PP only and compared them to miners with normal HRCT findings. MATERIALS AND METHODS: HRCTs from 128 miners were categorized into the following 4 diagnostic groups: (1) normal computed tomography scan (n = 9); (2) PP only (n = 72); (3) PP and interstitial fibrosis (n = 26) and (4) additional HRCT abnormalities (n = 21) such as rounded atelectasis, diffuse pleural thickening, pleural effusions or pulmonary nodules or tumor >1cm in diameter. Random intercept and slope linear mixed-effect regression models identified differences in lung function decline between miners with asbestos-associated outcomes and those with normal HRCT. Models were adjusted for follow-up time, body mass index, smoking status, latent exposure period and employment years. Interactions for smoking status with age and smoking status with pleural plaque severity were examined. RESULTS: Miners with PP only did not have an accelerated decline in lung function between 40 and 80 years. Miners with PP and additional HRCT abnormalities displayed significantly accelerated declines in forced expiratory volume in 1 second and diffusing capacity of the lungs for carbon monoxide (P = 0.05 and P < 0.01, respectively). Plaque severity did not affect lung function decline. However, smokers with extensive plaques displayed accelerated loss in diffusing capacity of the lungs for carbon monoxide and forced expiratory volume in 1 second when compared to nonsmoking miners with mild plaque formation. CONCLUSIONS: PP alone did not significantly affect lung function decline in vermiculite miners of Libby, Montana.

Identification and Quantification of Intravenous Therapy Drugs Using Normal Raman Spectroscopy and Electrochemical Surface-Enhanced Raman Spectroscopy.

Errors in intravenous (IV) drug therapies can cause human harm and even death. There are limited label-free methods that can sensitively monitor the identity and quantity of the drug being administered. Normal Raman spectroscopy (NRS) provides a modestly sensitive, label-free, and completely noninvasive means of IV drug sensing. In the case that the analyte cannot be detected within its clinical range with Raman, a label-free surface-enhanced Raman spectroscopy (SERS) approach can be implemented to detect the analyte of interest. In this work, we demonstrate two individual cases where we use NRS and electrochemical SERS (EC-SERS) to detect IV therapy analytes within their clinically relevant ranges. We implement NRS to detect gentamicin, a commonly IV-administered antibiotic and EC-SERS to detect dobutamine, a drug commonly administered after heart surgery. In particular, dobutamine detection with EC-SERS was found to have a limit of detection 4 orders of magnitude below its clinical range, highlighting the excellent sensitivity of SERS. We also demonstrate the use of hand-held Raman instrumentation for NRS and EC-SERS, showing that Raman is a highly sensitive technique that is readily applicable in a clinical setting.

Lung Function before and after a Large Chlorine Gas Release in Graniteville, South Carolina.

RATIONALE: On January 6, 2005 a train derailment led to an estimated 54,915-kg release of chlorine at a local textile mill in Graniteville, South Carolina. OBJECTIVES: We used the employee health spirometry records of the textile to identify enduring effects of chlorine gas exposure resulting from the incident on the lung function of workers employed at the textile mill. METHODS: Spirometry records from 1,807 mill workers (7,332 observations) were used from 4 years before and 18 months after the disaster. Longitudinal analysis using marginal regression models produced annual population mean estimates for FEV1, FVC, and FEV1/FVC ratio. Covariate adjustment was made for sex, age, smoking, height, season tested, technician, obesity, season × year interactions, and smoker × year interactions. The increased prevalence of mill workers having accelerated FEV1 decline was also evaluated after the chlorine spill. MEASUREMENTS AND MAIN RESULTS: In the year of the accident, we observed a significant reduction in mean FEV1 (-4.2% predicted; P = 0.019) when compared with the year before the incident. In the second year, partial recovery in the mean FVC % predicted level was seen, but the cohort's average FEV1/FVC ratio continued to decrease over time. Severe annual FEV1 decline was most prevalent in the year of the accident, and independent of mill worker smoking status. CONCLUSIONS: The Graniteville mill worker cohort revealed significant reductions in lung function immediately after the chlorine incident. Improvement was seen in the second year; but the proportion of mill workers experiencing accelerated FEV1 annual decline significantly increased in the 18 months after the chlorine incident.

Pleural plaques and their effect on lung function in Libby vermiculite miners.

BACKGROUND: Multiple studies have investigated the relationship between asbestos-related pleural plaques (PPs) and lung function, with disparate and inconsistent results. Most use chest radiographs to identify PPs and simple spirometry to measure lung function. High-resolution CT (HRCT) scanning improves the accuracy of PP identification. Complete pulmonary function tests (PFTs), including spirometry, lung volumes, and diffusing capacity of the lung for carbon monoxide, provide a more definitive assessment of lung function. The goal of this study was to determine, using HRCT scanning and complete PFTs, the effect of PPs on lung function in Libby vermiculite miners. METHODS: The results of HRCT scanning and complete PFTs performed between January 2000 and August 2012 were obtained from the medical records of 166 Libby vermiculite miners. Multivariate regression analyses with Tukey multivariate adjustment were used to assess statistical associations between the presence of PPs and lung function. Adjustments were made for age, BMI, smoking history, duration of employment, and years since last occupational asbestos exposure. RESULTS: Nearly 90% of miners (n = 149) had evidence of PPs on HRCT scan. No significant differences in spirometry results, lung volumes, or diffusing capacity of the lung for carbon monoxide were found between miners with PPs alone and miners with normal HRCT scans. Miners with both interstitial fibrosis and the presence of PPs had a significantly decreased total lung capacity in comparison with miners with normal HRCT scans (P = .02). Age, cumulative smoking history, and BMI were significant covariates that contributed to abnormal lung function. CONCLUSIONS: Asbestos-related PPs alone have no significant effect on lung function in Libby vermiculite miners.

Dual views of SRF: a genomic exposure.

Esnault and colleagues (pp. 943-958) take a genomics approach to investigate the role of SRF (serum response factor) in the serum response of fibroblasts. The well-established dual role of SRF with alternative cofactors and responsiveness to two signaling pathways is illustrated at the genome-wide level, yet new insight comes from this global picture.

Respiratory symptoms and lung function 8-10 months after community exposure to chlorine gas: a public health intervention and cross-sectional analysis.

BACKGROUND: We implemented a community based interventional health screening for individuals located within one mile of a 54 metric tons release of liquid chlorine following a 16 tanker car train derailment on 6 January, 2005 in Graniteville, South Carolina, USA. Public health intervention occurred 8-10 months after the event, and provided pulmonary function and mental health assessment by primary care providers. Its purpose was to evaluate those exposed to chlorine for evidence of ongoing impairment for medical referral and treatment. We report comparative analysis between self-report of respiratory symptoms via questionnaire and quantitative spirometry results. METHODS: Health assessments were obtained through respiratory symptom and exposure questionnaires, simple spirometry, and physical exam. Simple spirometry was used as the standard to identify continued breathing problems. Sensitivity, specificity, positive and negative predictive values were applied to evaluate the validity of the respiratory questionnaire. We also identified the direction of discrepancy between self-reported respiratory symptoms and spirometry measures. Generalized estimation equations determined prevalence ratios for abnormal spirometry based on the presence of participant persistent respiratory symptoms. Covariate adjustment was made for participant age, sex, race, smoking and educational status. RESULTS: Two hundred fifty-nine people participated in the Graniteville health screening; 53 children (mean age = 11 years, range: <1-16), and 206 adults (mean age = 50 years, range: 18-89). Of these, 220 (85%) performed spirometry maneuvers of acceptable quality. Almost 67% (n = 147) displayed abnormal spirometry, while 50% (n = 110) reported persistent new-onset respiratory symptoms. Moreover, abnormal spirometry was seen in 65 participants (29%) who did not report any discernible breathing problems. This represented a net 16.8% underreporting of symptoms. Sensitivity and specificity of questionnaire self-report of symptoms were low at 55.8% and 61.6%, respectively. Persistent cough (41%) and shortness of breath (39%) were the most frequently reported respiratory symptoms. CONCLUSION: Eight to ten months after acute chlorine exposure, the Graniteville health screening participants under-reported respiratory symptoms when compared to abnormal spirometry results. Sensitivity and specificity were low, and we determined that relying upon the self-report questionnaire was not adequate to objectively assess the lung health of our population following irritant gas exposure.

Drosophila melanogaster muscle LIM protein and alpha-actinin function together to stabilize muscle cytoarchitecture: a potential role for Mlp84B in actin-crosslinking.

Stabilization of tissue architecture during development and growth is essential to maintain structural integrity. Because of its contractile nature, muscle is especially susceptible to physiological stresses, and has multiple mechanisms to maintain structural integrity. The Drosophila melanogaster Muscle LIM Protein (MLP), Mlp84B, participates in muscle maintenance, yet its precise mechanism of action is still controversial. Through a candidate approach, we identified α-actinin as a protein that functions with Mlp84B to ensure muscle integrity. α-actinin RNAi animals die primarily as pupae, and Mlp84B RNAi animals are adult viable. RNAi knockdown of Mlp84B and α-actinin together produces synergistic early larval lethality and destabilization of Z-line structures. We recapitulated these phenotypes using combinations of traditional loss-of-function alleles and single-gene RNAi. We observe that Mlp84B induces the formation of actin loops in muscle cell nuclei in the absence of nuclear α-actinin, suggesting Mlp84B has intrinsic actin cross-linking activity, which may complement α-actinin cross-linking activity at sites of actin filament anchorage. These results reveal a molecular mechanism for MLP stabilization of muscle and implicate reduced actin crosslinking as the primary destabilizing defect in MLP-associated cardiomyopathies. Our data support a model in which α-actinin and Mlp84B have important and overlapping functions at sites of actin filament anchorage to preserve muscle structure and function.

The Immediate Pulmonary Disease Pattern following Exposure to High Concentrations of Chlorine Gas.

Background. Classification of pulmonary disease into obstructive, restrictive, and mixed patterns is based on 2005 ATS/ERS guidelines and modified GOLD criteria by Mannino et al. (2003), but these guidelines are of limited use for simple spirometry in situations involving mass casualties. Aim. The purpose of this study was to apply these guidelines to patients who underwent simple spirometry following high concentration of chlorine gas inhalation after a train derailment in Graniteville, South Carolina. Methods. We retrospectively investigated lung functions in ten patients. In order to classify pulmonary disease pattern, we used 2005 ATS/ERS guidelines and modified GOLD criteria along with our own criteria developed using available simple spirometry data. Results. We found predominant restrictive pattern in our patients with both modified GOLD and our criteria, which is in contrast to other chlorine exposure studies where obstructive pattern was more common. When compared to modified GOLD and our criteria, 2005 ATS/ERS guidelines underestimated the frequency of restrictive disease. Conclusion. Diagnosis of pulmonary disease patterns is of importance after irritant gas inhalation. Acceptable criteria need to be developed to evaluate pulmonary disease through simple spirometry in events leading to mass casualty and patient surge in hospitals.