Energy Transfer Between i-Motif DNA Encapsulated Silver Nanoclusters and Fluorescein Amidite Efficiently Visualizes the Redox State of Live Cells.

The redox regulation, maintaining a balance between oxidation and reduction in living cells, is vital for cellular homeostasis, intricate signaling networks, and appropriate responses to physiological and environmental cues. Here, a novel redox sensor, based on DNA-encapsulated silver nanoclusters (DNA/AgNCs) and well-defined chemical fluorophores, effectively illustrating cellular redox states in live cells is introduced. Among various i-motif DNAs, the photophysical property of poly-cytosines (C20)-encapsulated AgNCs that sense reactive oxygen species (ROS) is adopted. However, the sensitivity of C20/AgNCs is insufficient for evaluating ROS levels in live cells. To overcome this drawback, the ROS sensing mechanism of C20/AgNCs through gel electrophoresis, mass spectrometry, and small-angle X-ray scattering is primarily defined. Then, by tethering fluorescein amidite (FAM) and Cyanine 5 (Cy5) dyes to each end of the C20/AgNCs sensor, an Energy Transfer (ET) between AgNCs and FAM is achieved, resulting in intensified green fluorescence upon ROS detection. Taken together, the FAM-C20/AgNCs-Cy5 redox sensor enables dynamic visualization of intracellular redox states, yielding insights into oxidative stress-related processes in live cells.

O-GlcNAcylation of RBM14 contributes to elevated cellular O-GlcNAc through regulation of OGA protein stability.

Dysregulation of O-GlcNAcylation has emerged as a potential biomarker for several diseases, particularly cancer. The role of OGT (O-GlcNAc transferase) in maintaining O-GlcNAc homeostasis has been extensively studied; nevertheless, the regulation of OGA (O-GlcNAcase) in cancer remains elusive. Here, we demonstrated that the multifunctional protein RBM14 is a regulator of cellular O-GlcNAcylation. By investigating the correlation between elevated O-GlcNAcylation and increased RBM14 expression in lung cancer cells, we discovered that RBM14 promotes ubiquitin-dependent proteasomal degradation of OGA, ultimately mediating cellular O-GlcNAcylation levels. In addition, RBM14 itself is O-GlcNAcylated at serine 521, regulating its interaction with the E3 ligase TRIM33, consequently affecting OGA protein stability. Moreover, we demonstrated that mutation of serine 521 to alanine abrogated the oncogenic properties of RBM14. Collectively, our findings reveal a previously unknown mechanism for the regulation of OGA and suggest a potential therapeutic target for the treatment of cancers with dysregulated O-GlcNAcylation.

Tailed-Hoogsteen Triplex DNA Silver Nanoclusters Emit Red Fluorescence upon Target miRNA Sensing.

MicroRNAs (miRNAs) are small RNA molecules, typically 21‒22 nucleotides in size, which play a crucial role in regulating gene expression in most eukaryotes. Their significance in various biological processes and disease pathogenesis has led to considerable interest in their potential as biomarkers for diagnosis and therapeutic applications. In this study, a novel method for sensing target miRNAs using Tailed-Hoogsteen triplex DNA-encapsulated Silver Nanoclusters (DNA/AgNCs) is introduced. Upon hybridization of a miRNA with the tail, the Tailed-Hoogsteen triplex DNA/AgNCs exhibit a pronounced red fluorescence, effectively turning on the signal. It is successfully demonstrated that this miRNA sensor not only recognized target miRNAs in total RNA extracted from cells but also visualized target miRNAs when introduced into live cells, highlighting the advantages of the turn-on mechanism. Furthermore, through gel-fluorescence assays and small-angle X-ray scattering (SAXS) analysis, the turn-on mechanism is elucidated, revealing that the Tailed-Hoogsteen triplex DNA/AgNCs undergo a structural transition from a monomer to a dimer upon sensing the target miRNA. Overall, the findings suggest that Tailed-Hoogsteen triplex DNA/AgNCs hold great promise as practical sensors for small RNAs in both in vitro and cell imaging applications.

Innate mechanism of mucosal barrier erosion in the pathogenesis of acquired colitis.

The colonic mucosal barrier protects against infection, inflammation, and tissue ulceration. Composed primarily of Mucin-2, proteolytic erosion of this barrier is an invariant feature of colitis; however, the molecular mechanisms are not well understood. We have applied a recurrent food poisoning model of acquired inflammatory bowel disease using Salmonella enterica Typhimurium to investigate mucosal barrier erosion. Our findings reveal an innate Toll-like receptor 4-dependent mechanism activated by previous infection that induces Neu3 neuraminidase among colonic epithelial cells concurrent with increased Cathepsin-G protease secretion by Paneth cells. These anatomically separated host responses merge with the desialylation of nascent colonic Mucin-2 by Neu3 rendering the mucosal barrier susceptible to increased proteolytic breakdown by Cathepsin-G. Depletion of Cathepsin-G or Neu3 function using pharmacological inhibitors or genetic-null alleles protected against Mucin-2 proteolysis and barrier erosion and reduced the frequency and severity of colitis, revealing approaches to preserve and potentially restore the mucosal barrier.

Proposal of a Methodology for Prediction of Indoor PM2.5 Concentration Using Sensor-Based Residential Environments Monitoring Data and Time-Divided Multiple Linear Regression Model.

This study aims to propose an indoor air quality prediction method that can be easily utilized and reflects temporal characteristics using indoor and outdoor input data measured near the indoor target point as input to calculate indoor PM2.5 concentration through a multiple linear regression model. The atmospheric conditions and air pollution detected in one-minute intervals using sensor-based monitoring equipment (Dust Mon, Sentry Co Ltd., Seoul, Korea) inside and outside houses from May 2019 to April 2021 were used to develop the prediction model. By dividing the multiple linear regression model into one-hour increments, we attempted to overcome the limitation of not representing the multiple linear regression model's characteristics over time and limited input variables. The multiple linear regression (MLR) model classified by time unit showed an improvement in explanatory power by up to 9% compared to the existing model, and some hourly models had an explanatory power of 0.30. These results indicated that the model needs to be subdivided by time period to more accurately predict indoor PM2.5 concentrations.

O-GlcNAcylation of RIPK1 rescues red blood cells from necroptosis.

Necroptosis is a type of cell death with excessive inflammation and organ damage in various human diseases. Although abnormal necroptosis is common in patients with neurodegenerative, cardiovascular, and infectious diseases, the mechanisms by which O-GlcNAcylation contributes to the regulation of necroptotic cell death are poorly understood. In this study, we reveal that O-GlcNAcylation of RIPK1 (receptor-interacting protein kinase1) was decreased in erythrocytes of the mouse injected with lipopolysaccharide, resulting in the acceleration of erythrocyte necroptosis through increased formation of RIPK1-RIPK3 complex. Mechanistically, we discovered that O-GlcNAcylation of RIPK1 at serine 331 in human (corresponding to serine 332 in mouse) inhibits phosphorylation of RIPK1 at serine 166, which is necessary for the necroptotic activity of RIPK1 and suppresses the formation of the RIPK1-RIPK3 complex in Ripk1 -/- MEFs. Thus, our study demonstrates that RIPK1 O-GlcNAcylation serves as a checkpoint to suppress necroptotic signaling in erythrocytes.

Poly-ADP ribosylation of p21 by tankyrases promotes p21 degradation and regulates cell cycle progression.

p21WAF1/Cip1 acts as a key negative regulator of cell cycle progression, which can form complexes with cyclin-dependent kinases together with specific cyclins to induce cell cycle arrest at specific stages. p21 protein levels have been shown to be regulated primarily through phosphorylation and ubiquitination during various stages of the cell cycle. Although phosphorylation and ubiquitin-dependent proteasomal degradation of p21 have been well established, other post-translational modifications that contribute to regulation of p21 stability and function remain to be further elucidated. Here, we show that p21 degradation and its function are controlled by tankyrases, which are members of the poly(ADP-ribose) polymerase (PARP) protein family. p21 interacts with tankyrases via newly defined tankyrase-binding motifs and is PARylated by tankyrases in vitro and in vivo, suggesting that PARylation is a new post-translational modification of p21. Up-regulation of tankyrases induces ubiquitin-dependent proteasomal degradation of p21 through an E3 ligase RNF146, thus promoting cell cycle progression in the G1/S phase transition. On the contrary, inhibition of tankyrases by knockdown or inhibitor treatment stabilizes p21 protein and leads to cell cycle arrest in the G1 phase. Together, our data demonstrate that tankyrase may function as a new molecular regulator that controls the protein levels of p21 through PARylation-dependent proteasomal degradation. Hence, a novel function of the tankyrase-p21 axis may represent a new avenue for regulating cell cycle progression.

O-GlcNAcylation: An Emerging Protein Modification Regulating the Hippo Pathway.

The balance between cellular proliferation and apoptosis and the regulation of cell differentiation must be established to maintain tissue homeostasis. These cellular responses involve the kinase cascade-mediated Hippo pathway as a crucial regulator. Hence, Hippo pathway dysregulation is implicated in diverse diseases, including cancer. O-GlcNAcylation is a non-canonical glycosylation that affects multiple signaling pathways through its interplay with phosphorylation in the nucleus and cytoplasm. An abnormal increase in the O-GlcNAcylation levels in various cancer cells is a potent factor in Hippo pathway dysregulation. Intriguingly, Hippo pathway dysregulation also disrupts O-GlcNAc homeostasis, leading to a persistent elevation of O-GlcNAcylation levels, which is potentially pathogenic in several diseases. Therefore, O-GlcNAcylation is gaining attention as a protein modification that regulates the Hippo pathway. This review presents a framework on how O-GlcNAcylation regulates the Hippo pathway and forms a self-perpetuating cycle with it. The pathological significance of this self-perpetuating cycle and clinical strategies for targeting O-GlcNAcylation that causes Hippo pathway dysregulation are also discussed.

Coagulation factor protein abundance in the pre-septic state predicts coagulopathic activities that arise during late-stage murine sepsis.

BACKGROUND: Although sepsis accounts for 1 in 5 deaths globally, few molecular therapies exist for this condition. The development of effective biomarkers and treatments for sepsis requires a more complete understanding of host responses and pathogenic mechanisms at early stages of disease to minimize host-driven pathology. METHODS: An alternative to the current symptom-based approach used to diagnose sepsis is a precise assessment of blood proteomic changes during the onset and progression of Salmonella Typhimurium (ST) murine sepsis. FINDINGS: A distinct pattern of coagulation factor protein abundance was identified in the pre-septic state- prior to overt disease symptoms or bacteremia- that was predictive of the dysregulation of fibrinolytic and anti-coagulant activities and resultant consumptive coagulopathy during ST murine sepsis. Moreover, the changes in protein abundance observed generally have the same directionality (increased or decreased abundance) reported for human sepsis. Significant overlap of ST coagulopathic activities was observed in Gram-negative Escherichia coli- but not in Gram-positive staphylococcal or pneumococcal murine sepsis models. Treatment with matrix metalloprotease inhibitors prevented aberrant inflammatory and coagulopathic activities post-ST infection and increased survival. Antibiotic treatment regimens initiated after specific changes arise in the plasma proteome post-ST infection were predictive of an increase in disease relapse and death after cessation of antibiotic treatment. INTERPRETATION: Altered blood proteomics provides a platform to develop rapid and easy-to-perform tests to predict sepsis for early intervention via biomarker incorporation into existing blood tests prompted by patient presentation with general malaise, and to stratify Gram-negative and Gram-positive infections for appropriate treatment. Antibiotics are less effective in microbial clearance when initiated after the onset of altered blood proteomics as evidenced by increased disease relapse and death after termination of antibiotic therapy. Treatment failure is potentially due to altered bacterial / host-responses and associated increased host-driven pathology, providing insight into why delays in antibiotic administration in human sepsis are associated with increased risk for death. Delayed treatment may thus require prolonged therapy for microbial clearance despite the prevailing notion of antibiotic de-escalation and shortened courses of antibiotics to improve drug stewardship. FUNDING: National Institutes of Health, U.S. Army.

Remodeling of host glycoproteins during bacterial infection.

Protein glycosylation is a common post-translational modification found in all living organisms. This modification in bacterial pathogens plays a pivotal role in their infectious processes including pathogenicity, immune evasion, and host-pathogen interactions. Importantly, many key proteins of host immune systems are also glycosylated and bacterial pathogens can notably modulate glycosylation of these host proteins to facilitate pathogenesis through the induction of abnormal host protein activity and abundance. In recent years, interest in studying the regulation of host protein glycosylation caused by bacterial pathogens is increasing to fully understand bacterial pathogenesis. In this review, we focus on how bacterial pathogens regulate remodeling of host glycoproteins during infections to promote the pathogenesis. [BMB Reports 2021; 54(11): 541-544].

O-GlcNAcylated p53 in the liver modulates hepatic glucose production.

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.

Neu3 neuraminidase induction triggers intestinal inflammation and colitis in a model of recurrent human food-poisoning.

Intestinal inflammation is the underlying basis of colitis and the inflammatory bowel diseases. These syndromes originate from genetic and environmental factors that remain to be fully identified. Infections are possible disease triggers, including recurrent human food-poisoning by the common foodborne pathogen Salmonella enterica Typhimurium (ST), which in laboratory mice causes progressive intestinal inflammation leading to an enduring colitis. In this colitis model, disease onset has been linked to Toll-like receptor-4-dependent induction of intestinal neuraminidase activity, leading to the desialylation, reduced half-life, and acquired deficiency of anti-inflammatory intestinal alkaline phosphatase (IAP). Neuraminidase (Neu) inhibition protected against disease onset; however, the source and identity of the Neu enzyme(s) responsible remained unknown. Herein, we report that the mammalian Neu3 neuraminidase is responsible for intestinal IAP desialylation and deficiency. Absence of Neu3 thereby prevented the accumulation of lipopolysaccharide-phosphate and inflammatory cytokine expression in providing protection against the development of severe colitis.

O-GlcNAc stabilizes SMAD4 by inhibiting GSK-3ß-mediated proteasomal degradation.

O-linked ß-N-acetylglucosamine (O-GlcNAc) is a post-translational modification which occurs on the hydroxyl group of serine or threonine residues of nucleocytoplasmic proteins. It has been reported that the presence of this single sugar motif regulates various biological events by altering the fate of target proteins, such as their function, localization, and degradation. This study identified SMAD4 as a novel O-GlcNAc-modified protein. SMAD4 is a component of the SMAD transcriptional complex, a major regulator of the signaling pathway for the transforming growth factor-ß (TGF-ß). TGF-ß is a powerful promoter of cancer EMT and metastasis. This study showed that the amount of SMAD4 proteins changes according to cellular O-GlcNAc levels in human lung cancer cells. This observation was made based on the prolonged half-life of SMAD4 proteins. The mechanism behind this interaction was that O-GlcNAc impeded interactions between SMAD4 and GSK-3ß which promote proteasomal degradation of SMAD4. In addition, O-GlcNAc modification on SMAD4 Thr63 was responsible for stabilization. As a result, defects in O-GlcNAcylation on SMAD4 Thr63 attenuated the reporter activity of luciferase, the TGF-ß-responsive SMAD binding element (SBE). This study's findings imply that cellular O-GlcNAc may regulate the TGF-ß/SMAD signaling pathway by stabilizing SMAD4.

Mutual regulation between OGT and XIAP to control colon cancer cell growth and invasion.

O-GlcNAc transferase (OGT) is an enzyme that catalyzes the O-GlcNAc modification of nucleocytoplasmic proteins and is highly expressed in many types of cancer. However, the mechanism regulating its expression in cancer cells is not well understood. This study shows that OGT is a substrate of the E3 ubiquitin ligase X-linked inhibitor of apoptosis (XIAP) which plays an important role in cancer pathogenesis. Although LSD2 histone demethylase has already been reported as an E3 ubiquitin ligase in lung cancer cells, we identified XIAP as the main E3 ubiquitin ligase in colon cancer cells. Interestingly, OGT catalyzes the O-GlcNAc modification of XIAP at serine 406 and this modification is required for the E3 ubiquitin ligase activity of XIAP toward specifically OGT. Moreover, O-GlcNAcylation of XIAP suppresses colon cancer cell growth and invasion by promoting the proteasomal degradation of OGT. Therefore, our findings regarding the reciprocal regulation of OGT and XIAP provide a novel molecular mechanism for controlling cancer growth and invasion regulated by OGT and O-GlcNAc modification.

O-GlcNAcylation of Mef2c regulates myoblast differentiation.

Unlike other types of glycosylation, O-GlcNAcylation is a single glycosylation which occurs exclusively in the nucleus and cytosol. O-GlcNAcylation underlie metabolic diseases, including diabetes and obesity. Furthermore, O-GlcNAcylation affects different oncogenic processes such as osteoblast differentiation, adipogenesis and hematopoiesis. Emerging evidence suggests that skeletal muscle differentiation is also regulated by O-GlcNAcylation, but the detailed molecular mechanism has not been fully elucidated. In this study, we showed that hyper-O-GlcNAcylation reduced the expression of myogenin, a transcription factor critical for terminal muscle development, in C2C12 myoblasts differentiation by O-GlcNAcylation on Thr9 of myocyte-specific enhancer factor 2c. Furthermore, we showed that O-GlcNAcylation on Mef2c inhibited its DNA binding affinity to myogenin promoter. Taken together, we demonstrated that hyper-O-GlcNAcylation attenuates skeletal muscle differentiation by increased O-GlcNAcylation on Mef2c, which downregulates its DNA binding affinity.

O-GlcNAcylation on LATS2 disrupts the Hippo pathway by inhibiting its activity.

The Hippo pathway controls organ size and tissue homeostasis by regulating cell proliferation and apoptosis. The LATS-mediated negative feedback loop prevents excessive activation of the effectors YAP/TAZ, maintaining homeostasis of the Hippo pathway. YAP and TAZ are hyperactivated in various cancer cells which lead to tumor growth. Aberrantly increased O-GlcNAcylation has recently emerged as a cause of hyperactivation of YAP in cancer cells. However, the mechanism, which induces hyperactivation of TAZ and blocks LATS-mediated negative feedback, remains to be elucidated in cancer cells. This study found that in breast cancer cells, abnormally increased O-GlcNAcylation hyperactivates YAP/TAZ and inhibits LATS2, a direct negative regulator of YAP/TAZ. LATS2 is one of the newly identified O-GlcNAcylated components in the MST-LATS kinase cascade. Here, we found that O-GlcNAcylation at LATS2 Thr436 interrupted its interaction with the MOB1 adaptor protein, which connects MST to LATS2, leading to activation of YAP/TAZ by suppressing LATS2 kinase activity. LATS2 is a core component in the LATS-mediated negative feedback loop. Thus, this study suggests that LATS2 O-GlcNAcylation is deeply involved in tumor growth by playing a critical role in dysregulation of the Hippo pathway in cancer cells.

O-Linked N-Acetylglucosamine Modification of Mitochondrial Antiviral Signaling Protein Regulates Antiviral Signaling by Modulating Its Activity.

Post-translational modifications, including O-GlcNAcylation, play fundamental roles in modulating cellular events, including transcription, signal transduction, and immune signaling. Several molecular targets of O-GlcNAcylation associated with pathogen-induced innate immune responses have been identified; however, the direct regulatory mechanisms linking O-GlcNAcylation with antiviral RIG-I-like receptor signaling are not fully understood. In this study, we found that cellular levels of O-GlcNAcylation decline in response to infection with Sendai virus. We identified a heavily O-GlcNAcylated serine-rich region between amino acids 249-257 of the mitochondrial antiviral signaling protein (MAVS); modification at this site disrupts MAVS aggregation and prevents MAVS-mediated activation and signaling. O-GlcNAcylation of the serine-rich region of MAVS also suppresses its interaction with TRAF3; this prevents IRF3 activation and production of interferon-ß. Taken together, these results suggest that O-GlcNAcylation of MAVS may be a master regulatory event that promotes host defense against RNA viruses.

Accelerated Aging and Clearance of Host Anti-inflammatory Enzymes by Discrete Pathogens Fuels Sepsis.

Sepsis is a life-threatening inflammatory syndrome accompanying a bloodstream infection. Frequently secondary to pathogenic bacterial infections, sepsis remains difficult to treat as a singular disease mechanism. We compared the pathogenesis of murine sepsis experimentally elicited by five bacterial pathogens and report similarities among host responses to Gram-negative Salmonella and E. coli. We observed that a host protective mechanism involving de-toxification of lipopolysaccharide by circulating alkaline phosphatase (AP) isozymes was incapacitated during sepsis caused by Salmonella or E. coli through activation of host Toll-like receptor 4, which triggered Neu1 and Neu3 neuraminidase induction. Elevated neuraminidase activity accelerated the molecular aging and clearance of AP isozymes, thereby intensifying disease. Mice deficient in the sialyltransferase ST3Gal6 displayed increased disease severity, while deficiency of the endocytic lectin hepatic Ashwell-Morell receptor was protective. AP augmentation or neuraminidase inhibition diminished inflammation and promoted host survival. This study illuminates distinct routes of sepsis pathogenesis, which may inform therapeutic development.

Recurrent infection progressively disables host protection against intestinal inflammation.

Intestinal inflammation is the central pathological feature of colitis and the inflammatory bowel diseases. These syndromes arise from unidentified environmental factors. We found that recurrent nonlethal gastric infections of Gram-negative Salmonella enterica Typhimurium (ST), a major source of human food poisoning, caused inflammation of murine intestinal tissue, predominantly the colon, which persisted after pathogen clearance and irreversibly escalated in severity with repeated infections. ST progressively disabled a host mechanism of protection by inducing endogenous neuraminidase activity, which accelerated the molecular aging and clearance of intestinal alkaline phosphatase (IAP). Disease was linked to a Toll-like receptor 4 (TLR4)-dependent mechanism of IAP desialylation with accumulation of the IAP substrate and TLR4 ligand, lipopolysaccharide-phosphate. The administration of IAP or the antiviral neuraminidase inhibitor zanamivir was therapeutic by maintaining IAP abundance and function.

Psychological effects of a disastrous hydrogen fluoride spillage on the local community.

BACKGROUND: On September 27, 2012, at 3:43 pm, a hydrogen fluoride spill occurred in a manufacturing plant located at the 4th complex of the Gumi National Industrial Complex in Gumi City, South Korea. The present study aimed to evaluate the psychological effects of the hydrogen fluoride spill on the members of the community and to investigate their relationships with physical symptoms and changes in psychological effects occurring as time passed after the accident. METHODS: The 1st phase involved a survey of 1359 individuals that was conducted 1 month after the spill, and the 2nd phase involved a survey of 711 individuals that was conducted 7 months after the accident. The questionnaires included items for assessing demographic characteristics, hydrogen fluoride exposure level, physical symptoms, and psychological status. Physical symptoms were assessed to determine the persistence of irritations. Psychological status was assessed to investigate the impact of event level using the Impact of Event Scale - Revised Korean version (IES-R-K), and the anxiety level was assessed using the Beck Anxiety Inventory (BAI). RESULTS: As the hydrogen fluoride exposure level increased, the impact of event and anxiety levels increased significantly both 1 and 7 months after the accident (p < 0.05). The mean score of the impact of event levels decreased significantly from 33.33 ± 14.64 at 1 month after the accident to 28.68 ± 11.80 at 7 months after the accident (p < 0.05). The mean score of the anxiety levels increased significantly from 5.16 ± 6.59 at 1 month after the accident to 6.79 ± 8.41 at 7 months after the accident (p < 0.05). The risk of persistent physical symptoms at 7 months after the accident was significantly higher in females. The risk of persistent physical symptoms also increased significantly, with increasing age, hydrogen fluoride exposure, and impact of event levels (p < 0.05). CONCLUSIONS: The present study found that the impact of event level and anxiety level increased with increasing hydrogen fluoride exposure. Anxiety levels persisted even after time passed. The risk of persistent physical symptoms at 7 months after the accident was higher in females, and it increased with increasing age, hydrogen fluoride exposure level, and impact of event levels.