Sequence grammar underlying the unfolding and phase separation of globular proteins.

Aberrant phase separation of globular proteins is associated with many diseases. Here, we use a model protein system to understand how the unfolded states of globular proteins drive phase separation and the formation of unfolded protein deposits (UPODs). We find that for UPODs to form, the concentrations of unfolded molecules must be above a threshold value. Additionally, unfolded molecules must possess appropriate sequence grammars to drive phase separation. While UPODs recruit molecular chaperones, their compositional profiles are also influenced by synergistic physicochemical interactions governed by the sequence grammars of unfolded proteins and cellular proteins. Overall, the driving forces for phase separation and the compositional profiles of UPODs are governed by the sequence grammars of unfolded proteins. Our studies highlight the need for uncovering the sequence grammars of unfolded proteins that drive UPOD formation and cause gain-of-function interactions whereby proteins are aberrantly recruited into UPODs.

ARAF mutations confer resistance to the RAF inhibitor belvarafenib in melanoma.

Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAFV600-mutant melanoma, they are ineffective in non-BRAFV600 mutant cells1-3. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAFV600E- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAFV600E- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors.

Pach-ing It in: The Peculiar Organization of Mammalian Pachytene Chromosomes.

In this issue of Molecular Cell, Wang et al. (2019) use Hi-C to visualize at high resolution the complex reprogramming of chromatin architecture during spermatogenesis in rhesus monkeys and mice. They find that pachytene spermatocytes have a unique chromosome organization that may result from the presence of the synaptonemal complex and transcription-associated proteins.

Maintenance of CTCF- and Transcription Factor-Mediated Interactions from the Gametes to the Early Mouse Embryo.

The epigenetic information present in mammalian gametes and whether it is transmitted to the progeny are relatively unknown. We find that many promoters in mouse sperm are occupied by RNA polymerase II (Pol II) and Mediator. The same promoters are accessible in GV and MII oocytes and preimplantation embryos. Sperm distal ATAC-seq sites containing motifs for various transcription factors are conserved in monkeys and humans. ChIP-seq analyses confirm that Foxa1, ERα, and AR occupy distal enhancers in sperm. Accessible sperm enhancers containing H3.3 and H2A.Z are also accessible in oocytes and preimplantation embryos. Furthermore, their interactions with promoters in the gametes persist during early development. Sperm- or oocyte-specific interactions mediated by CTCF and cohesin are only present in the paternal or maternal chromosomes, respectively, in the zygote and 2-cell stages. These interactions converge in both chromosomes by the 8-cell stage. Thus, mammalian gametes contain complex patterns of 3D interactions that can be transmitted to the zygote after fertilization.

A safety mechanism enables tissue-specific resistance to protein aggregation during aging in C. elegans.

During aging, proteostasis capacity declines and distinct proteins become unstable and can accumulate as protein aggregates inside and outside of cells. Both in disease and during aging, proteins selectively aggregate in certain tissues and not others. Yet, tissue-specific regulation of cytoplasmic protein aggregation remains poorly understood. Surprisingly, we found that the inhibition of 3 core protein quality control systems, namely chaperones, the proteasome, and macroautophagy, leads to lower levels of age-dependent protein aggregation in Caenorhabditis elegans pharyngeal muscles, but higher levels in body-wall muscles. We describe a novel safety mechanism that selectively targets newly synthesized proteins to suppress their aggregation and associated proteotoxicity. The safety mechanism relies on macroautophagy-independent lysosomal degradation and involves several previously uncharacterized components of the intracellular pathogen response (IPR). We propose that this protective mechanism engages an anti-aggregation machinery targeting aggregating proteins for lysosomal degradation.

Structural insights into the multifunctionality of rabies virus P3 protein.

Viruses form extensive interfaces with host proteins to modulate the biology of the infected cell, frequently via multifunctional viral proteins. These proteins are conventionally considered as assemblies of independent functional modules, where the presence or absence of modules determines the overall composite phenotype. However, this model cannot account for functions observed in specific viral proteins. For example, rabies virus (RABV) P3 protein is a truncated form of the pathogenicity factor P protein, but displays a unique phenotype with functions not seen in longer isoforms, indicating that changes beyond the simple complement of functional modules define the functions of P3. Here, we report structural and cellular analyses of P3 derived from the pathogenic RABV strain Nishigahara (Nish) and an attenuated derivative strain (Ni-CE). We identify a network of intraprotomer interactions involving the globular C-terminal domain and intrinsically disordered regions (IDRs) of the N-terminal region that characterize the fully functional Nish P3 to fluctuate between open and closed states, whereas the defective Ni-CE P3 is predominantly open. This conformational difference appears to be due to the single mutation N226H in Ni-CE P3. We find that Nish P3, but not Ni-CE or N226H P3, undergoes liquid-liquid phase separation and this property correlates with the capacity of P3 to interact with different cellular membrane-less organelles, including those associated with immune evasion and pathogenesis. Our analyses propose that discrete functions of a critical multifunctional viral protein depend on the conformational arrangements of distant individual domains and IDRs, in addition to their independent functions.

An epidermal growth factor receptor-targeting immunotoxin based on IgG shows potent antitumor activity against head and neck cancer.

The epidermal growth factor receptor (EGFR) is an important target for cancer therapies. Many head and neck cancer (HNC) cells have been reported to overexpress EGFR; therefore, anti-EGFR therapies have been attempted in patients with HNC. However, its clinical efficacy is limited owing to the development of drug resistance. In this study, we developed an EGFR-targeting immunotoxin consisting of a clinically proven anti-EGFR IgG (cetuximab; CTX) and a toxin fragment (LR-LO10) derived from Pseudomonas exotoxin A (PE) using a novel site-specific conjugation technology (peptide-directed photo-crosslinking reaction), as an alternative option. The immunotoxin (CTX-LR-LO10) showed specific binding to EGFR and properties of a typical IgG, such as stability, interactions with receptors of immune cells, and pharmacokinetics, and inhibited protein synthesis via modification of elongation factor-2. Treatment of EGFR-positive HNC cells with the immunotoxin resulted in apoptotic cell death and the inhibition of cell migration and invasion. The efficacy of CTX-LR-LO10 was evaluated in xenograft mouse models, and the immunotoxin exhibited much stronger tumor suppression than CTX or LR-LO10. Transcriptome analyses revealed that the immunotoxins elicited immune responses and altered the expression of genes related to its mechanisms of action. These results support the notion that CTX-LR-LO10 may serve as a new therapeutic agent targeting EGFR-positive cancers.

Discovery of a dual-action small molecule that improves neuropathological features of Alzheimer's disease mice.

Alzheimer's disease (AD) is characterized by complex, multifactorial neuropathology, suggesting that small molecules targeting multiple neuropathological factors are likely required to successfully impact clinical progression. Acid sphingomyelinase (ASM) activation has been recognized as an important contributor to these neuropathological features in AD, leading to the concept of using ASM inhibitors for the treatment of this disorder. Here we report the identification of KARI 201, a direct ASM inhibitor evaluated for AD treatment. KARI 201 exhibits highly selective inhibition effects on ASM, with excellent pharmacokinetic properties, especially with regard to brain distribution. Unexpectedly, we found another role of KARI 201 as a ghrelin receptor agonist, which also has therapeutic potential for AD treatment. This dual role of KARI 201 in neurons efficiently rescued neuropathological features in AD mice, including amyloid beta deposition, autophagy dysfunction, neuroinflammation, synaptic loss, and decreased hippocampal neurogenesis and synaptic plasticity, leading to an improvement in memory function. Our data highlight the possibility of potential clinical application of KARI 201 as an innovative and multifaceted drug for AD treatment.

Recruitment of CTCF to an Fto enhancer is responsible for transgenerational inheritance of BPA-induced obesity.

The mechanisms by which environmentally-induced epiphenotypes are transmitted transgenerationally in mammals are poorly understood. Here we show that exposure of pregnant mouse females to bisphenol A (BPA) results in obesity in the F2 progeny due to increased food intake. This epiphenotype can be transmitted up to the F6 generation. Analysis of chromatin accessibility in sperm of the F1-F6 generations reveals alterations at sites containing binding motifs for CCCTC-binding factor (CTCF) at two cis-regulatory elements (CREs) of the Fto gene that correlate with transmission of obesity. These CREs show increased interactions in sperm of obese mice with the Irx3 and Irx5 genes, which are involved in the differentiation of appetite-controlling neurons. Deletion of the CTCF site in Fto results in mice that have normal food intake and fail to become obese when ancestrally exposed to BPA. The results suggest that epigenetic alterations of Fto can lead to the same phenotypes as genetic variants.

Inhibition of receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation and bone resorption by Gryllus bimaculatus extract: An in vitro study.

Excessive bone-resorbing osteoclast activity during bone remodeling is a major feature of bone diseases, such as osteoporosis. Therefore, the inhibition of osteoclast formation and bone resorption can be an effective therapeutic target for various bone diseases. Gryllus biomaculatus (GB) has recently been approved as an alternative food source because of its high nutritional value and environmental sustainability. Traditionally, GB has been known to have various pharmacological properties, including antipyretic and blood pressure-lowering activity, and it has recently been reported to have various biological activities, including protective effects against inflammation, oxidative stress, insulin resistance, and alcohol-induced liver injury. However, the effect of GB on osteoclast differentiation and bone metabolism has not yet been demonstrated. In this study, we confirmed the inhibitory effect of GB extract (GBE) on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. To determine the effect of GBE on RANKL-induced osteoclast differentiation and function, we performed TRAP and F-actin staining, as well as a bone-resorbing assay. The intracellular mechanisms of GBE responsible for the regulation of osteoclastogenesis were revealed by Western blot analysis and quantitative real-time polymerase chain reaction. We investigated the relationship between GBE and expression of osteoclast-specific molecules to further elucidate the underlying mechanisms. It was found that GBE significantly suppressed osteoclastogenesis by decreasing the phosphorylation of Akt, p38, JNK, and ERK, as well as Btk-PLCγ2 signaling, in pathways involved in early osteoclastogenesis as well as through the subsequent suppression of c-Fos, NFATc1, and osteoclastogenesis-specific marker genes. Additionally, GBE inhibited the formation of F-actin ring-positive osteoclasts and bone resorption activity of mature osteoclasts. Our findings suggest that GBE is a potential functional food and therapeutic candidate for bone diseases involving osteoclasts.

Rare Case of Echinostoma cinetorchis Infection, South Korea.

A woman in South Korea who underwent a colonoscopy for occasional gastrointestinal discomfort had 4 adult flukes of Echinostoma cinetorchis showing 37 collar spines around the oral sucker recovered from the terminal ileum through the ascending colon. Partial gene sequencing showed high identity with E. cinetorchis.

Exploring Molecular Genetic Alterations and RAF Fusions in Melanoma: A Belvarafenib Expanded Access Program in Patients with RAS/RAF-Mutant Melanoma.

BACKGROUND: Melanoma incidence is on the rise in East Asia, yet studies of the molecular landscape are lacking in this population. We examined patients with melanoma who underwent next-generation sequencing (NGS) at a single tertiary center in South Korea, focusing on patients harboring NRAS or RAF alterations who received belvarafenib, a pan-RAF dimer inhibitor, through the Expanded Access Program (EAP). PATIENTS AND METHODS: Data were collected from 192 patients with melanoma who underwent NGS between November 2017 and May 2023. Variant call format data were obtained and annotated. Patients in the EAP received 450 mg twice daily doses of belvarafenib. RESULTS: Alterations in the RAS/RTK pathway were the most prevalent, with BRAF and NRAS alteration rates of 22.4% and 17.7%, respectively. NGS enabled additional detection of fusion mutations, including 6 BRAF and 1 RAF1 fusion. Sixteen patients with NRAS or RAF alterations received belvarafenib through the EAP, and disease control was observed in 50%, with 2 patients demonstrating remarkable responses. CONCLUSIONS: Our study highlights the value of NGS in detecting BRAF, NRAS mutations and RAF fusions, expanding possibilities for targeted therapies in malignant melanoma. Belvarafenib showed clinical benefit in patients harboring these alterations. Ongoing trials will provide further insights into the safety and efficacy of belvarafenib.

Uvaol ameliorates lipid deposition in hyperlipidemic hepatocytes by suppressing protein-tyrosine phosphatase 1B/ER stress signaling.

Uvaol (UV), a pentacyclic triterpene found in olives and virgin olive oil, is known for its anti-inflammatory and antioxidant effects in various disease models. While olive oil is reported to reduce obesity and insulin resistance, the specific impact of UV on liver lipid metabolism and its molecular mechanisms are not fully understood. In this study, hepatic lipid accumulation was measured using oil red O staining, and protein expression levels in liver cells were assessed via Western blot analysis. Apoptosis was evaluated through cell viability and caspase 3 activity assays. UV treatment reduced lipid accumulation, fatty acid uptake, apoptosis, and ER stress in palmitate-treated liver cells. Additionally, UV enhanced fatty acid oxidation. Mechanistically, increased SIRT6 expression and autophagy were observed in UV-treated cells. SIRT6-targeted siRNA or 3-methyladenine blocked the effects of UV in hyperlipidemic cells. In conclusion, UV improves SIRT6/autophagy signaling, reducing lipid deposition and apoptosis in liver cells under high lipid conditions. This in vitro study provides strong evidence for potential therapeutic strategies for hepatic steatosis.

Safety of Gadoxetate Disodium for Hepatobiliary MRI in Children and Adolescents.

Background Despite a proven role in the characterization of liver lesions, use of the gadolinium-based contrast agent (GBCA) gadoxetate disodium at MRI is limited in children due to a lack of comparative safety data. Purpose To evaluate the safety of the GBCA gadoxetate disodium (a linear ionic hepatobiliary contrast agent [HBA]) in children and adolescents, compared with extracellular contrast agents (ECA). Materials and Methods A retrospective analysis was conducted in children and adolescents aged 18 years or younger who underwent HBA-enhanced MRI at one of three tertiary hospitals from January 2010 to December 2022. The incidence of GBCA-associated acute adverse events was compared between MRI examinations with a HBA and those with ECA. Severity was categorized according to American College of Radiology guidelines (mild, moderate, or severe). (a) Propensity score matching using multivariable logistic regression models and (b) inverse probability of treatment weighting analysis based on nine covariates (age, sex, asthma, allergic rhinitis, chronic urticaria or atopy, food allergy, drug allergy, premedication, and history of GBCA-associated adverse events) were used for confounder adjustment. Results A total of 1629 MRI examinations (ECA, n = 1256; HBA, n = 373) in 1079 patients were included (mean age, 8.6 years ± 6.5; 566 girls). The per-examination incidence of GBCA-associated acute adverse events showed no evidence of a difference, with rates of 0.9% (11 of 1256 examinations) for ECA and 1.3% (five of 373 examinations) for HBA (odds ratio [OR], 1.55 [95% CI: 0.54, 4.46]; P = .42). Acute adverse events were all mild with ECA, whereas with HBA, they were mild for four patients and moderate for one patient. There was no evidence of a difference in the incidence of acute adverse events, even in propensity score matching (OR, 1.33 [95% CI: 0.30, 5.96]; P = .71) and inverse probability of treatment weighting analysis (OR, 0.84 [95% CI: 0.25, 2.86]; P = .78). Conclusion Gadoxetate disodium showed no difference in acute adverse events compared with ECA in children and adolescents, with further large-scale pediatric studies required to confirm its safety. © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Otero in this issue.

Association between high levels of nitrogen dioxide and increased cumulative incidence of lung cancer in patients with idiopathic pulmonary fibrosis.

BACKGROUND: Lung cancer is a fatal complication of idiopathic pulmonary fibrosis (IPF) with a poor prognosis. However, the association between individual exposure to air pollutants and lung cancer development in patients with IPF is unknown. This study aimed to assess the effect of individual exposure to nitrogen dioxide (NO2) on lung cancer development in patients with IPF. METHODS: We enrolled 1085 patients from an IPF cohort in the Republic of Korea (mean age 65.6 years, males 80.6%). We estimated individual-level long-term exposures to NO2 at the patients' residential addresses using a national-scale exposure prediction model based on data from air quality regulatory monitoring stations. To evaluate the association between NO2 levels and lung cancer development in IPF, we used an individual- and area-level covariates adjusted model as our primary model. RESULTS: The estimated average annual NO2 concentration was 23.1 ppb. During a median follow-up of 4.3 years, 86 patients (7.9%) developed lung cancer. NO2 concentration was associated with lung cancer development in an unadjusted model (HR 1.219; p=0.042), while a marginal association was found in the primary model (HR 1.280; p=0.084). When NO2 concentration was stratified by the median value (21.0 ppb), exposure to high NO2 levels (≥21.0 ppb) was associated with a 2.0-fold increase in the risk of lung cancer development (HR 2.023; p=0.047) in the primary model. CONCLUSION: Individual exposure to high NO2 levels may increase the risk of lung cancer development in patients with IPF.

Sensitivity, Specificity, and Cutoff Identifying Optic Atrophy by Macular Ganglion Cell Layer Volume in Syndromic Craniosynostosis.

PURPOSE: To determine the sensitivity, specificity, and cutoff of macular ganglion cell layer (GCL) volume consistent with optic atrophy in children with syndromic craniosynostosis and to investigate factors independently associated with reduction in GCL volume. DESIGN: Retrospective cross-sectional study. PARTICIPANTS: Patients with syndromic craniosynostosis evaluated at Boston Children's Hospital (2010-2022) with reliable macular OCT scans. METHODS: The latest ophthalmic examination that included OCT macula scans was identified. Age at examination, sex, ethnicity, best-corrected logarithm of the minimum angle of resolution (logMAR) visual acuity, cycloplegic refraction, and funduscopic optic nerve appearance were recorded in addition to history of primary or recurrent elevation in intracranial pressure (ICP), Chiari malformation, and obstructive sleep apnea (OSA). Spectral-domain OCT software quantified segmentation of macula retinal layers and was checked manually. MAIN OUTCOME MEASURES: The primary outcome was determining sensitivity, specificity, and optimal cutoff of GCL volume consistent with optic atrophy. The secondary outcome was determining whether previously elevated ICP, OSA, Chiari malformation, craniosynostosis diagnosis, logMAR visual acuity, age, or sex were independently associated with lower GCL volume. RESULTS: Median age at examination was 11.9 years (interquartile range, 8.5-14.8 years). Fifty-eight of 61 patients (112 eyes) had reliable macula scans, 74% were female, and syndromes represented were Apert (n = 14), Crouzon (n = 17), Muenke (n = 6), Pfeiffer (n = 6), and Saethre-Chotzen (n = 15). Optimal cutoff identifying optic atrophy was a GCL volume < 1.02 mm3 with a sensitivity of 83% and specificity of 77%. Univariate analysis demonstrated that significantly lower macular GCL volume was associated with optic atrophy on fundus examination (P < 0.001), Apert syndrome (P < 0.001), history of elevated ICP (P = 0.015), Chiari malformation (P = 0.001), OSA (P < 0.001), male sex (P = 0.027), and worse logMAR visual acuity (P < 0.001). Multivariable median regression analysis confirmed that only OSA (P = 0.005), optic atrophy on fundus examination (P = 0.003), and worse logMAR visual acuity (P = 0.042) were independently associated with lower GCL volume. CONCLUSIONS: Surveillance for optic atrophy by GCL volume may be useful in a population where cognitive skills can limit acquisition of other key ophthalmic measures. It is noteworthy that OSA is also associated with lower GLC volume in this population. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Impact of ambient temperature on respiratory disease: a case-crossover study in Seoul.

BACKGROUND: Respiratory diseases contribute to global morbidity and mortality, and temperature is a significant factor. We investigated the association between ambient temperature and emergency department (ED) visits for various respiratory diseases in Seoul, South Korea. METHODS: Using data from the National Emergency Department Information System (2008-2017), we analysed 1,616,644 ED visits for respiratory diseases, categorised according to the Korean Standard Classification of Diseases 7th revision codes (J00-J99). Using a time-stratified case-crossover design and a distributed lag nonlinear model, we investigated the effect of temperature exposure on ED visits for respiratory diseases, calculating the relative risk (RR) for the maximum risk temperature (MaxRT) of both cold and hot extremes compared to the minimum risk temperature (MinRT). RESULTS: Cold temperatures (MaxRT: -9.0 °C) resulted in a 2.68-fold increase (RR = 2.68, 95% CI = 2.26-3.14) in ED visits for total respiratory diseases, while hot temperatures (MaxRT: 29.4 °C) led to a 1.26-fold increase (RR = 1.26, 95% CI = 1.11-1.42) compared to the MinRT (24.8 °C). Cold temperatures increased the risk of most respiratory diseases, except interstitial lung disease, whereas hot temperatures increased ED visits for acute upper respiratory infections and influenza. Cold temperatures increased ED visits for all age groups, especially those aged 18-64 (RR = 3.54, 95% CI = 2.90-4.33), while hot temperatures significantly affected those < 18 (RR = 1.45, 95% CI = 1.27-1.66). The risk levels were similar in both males and females, regardless of hot and cold temperatures. CONCLUSION: Our findings underscore the significant impact of both cold and heat exposure on ED visits for respiratory diseases, with varying intensities and risk profiles across different population groups.

Smoking status and clinical outcome in idiopathic pulmonary fibrosis: a nationwide study.

BACKGROUND: Smoking status has been linked to the development of idiopathic pulmonary fibrosis (IPF). However, the effect of smoking on the prognosis of patients with IPF is unclear. We aimed to investigate the association between smoking status and all-cause mortality or hospitalisation by using national health claims data. METHODS: IPF cases were defined as people who visited medical institutions between January 2002 and December 2018 with IPF and rare incurable disease exempted calculation codes from the National Health Insurance Database. Total 10,182 patients with available data on smoking status were included in this study. Ever-smoking status was assigned to individuals with a history of smoking ≥ 6 pack-years. The multivariable Cox proportional hazard model was used to evaluate the association between smoking status and prognosis. RESULTS: In the entire cohort, the mean age was 69.4 years, 73.9% were males, and 45.2% were ever smokers (current smokers: 14.2%; former smokers: 31.0%). Current smokers (hazard ratio [HR]: 0.709; 95% confidence interval [CI]: 0.643-0.782) and former smokers (HR: 0.926; 95% CI: 0.862-0.996) were independently associated with all-cause mortality compared with non-smokers. Current smokers (HR: 0.884; 95% CI: 0.827-0.945) and former smokers (HR: 0.909; 95% CI: 0.862-0.959) were also associated with a reduced risk of all-cause hospitalisation compared with non-smokers. A non-linear association between smoking amount and prognosis was found in a spline HR curve and showed increasing risk below 6 pack-years. CONCLUSION: Ever-smoking status may be associated with favourable clinical outcomes in patients with IPF.

Non-thermal atmospheric pressure plasma induces selective cancer cell apoptosis by modulating redox homeostasis.

BACKGROUND: Anticancer treatments aim to selectively target cancer cells without harming normal cells. While non-thermal atmospheric pressure plasma (NTAPP) has shown anticancer potential across various studies, the mechanisms behind its selective action on cancer cells remain inadequately understood. This study explores the mechanism of NTAPP-induced selective cell death and assesses its application in cancer therapy. METHODS: We treated HT1080 fibrosarcoma cells with NTAPP and assessed the intracellular levels of mitochondria-derived reactive oxygen species (ROS), mitochondrial function, and cell death mechanisms. We employed N-acetylcysteine to investigate ROS's role in NTAPP-induced cell death. Additionally, single-cell RNA sequencing was used to compare gene expression in NTAPP-treated HT1080 cells and human normal fibroblasts (NF). Western blotting and immunofluorescence staining examined the expression and nuclear translocation of nuclear factor erythroid 2-related factor 2 (NRF2), a key antioxidant gene transcription factor. We also evaluated autophagy activity through fluorescence staining and transmission electron microscopy. RESULTS: NTAPP treatment increased ROS levels and induced mitochondrial dysfunction, leading to apoptosis in HT1080 cells. The involvement of ROS in selective cancer cell death was confirmed by N-acetylcysteine treatment. Distinct gene expression patterns were observed between NTAPP-treated NF and HT1080 cells, with NF showing upregulated antioxidant gene expression. Notably, NRF2 expression and nuclear translocation increased in NF but not in HT1080 cells. Furthermore, autophagy activity was significantly higher in normal cells compared to cancer cells. CONCLUSIONS: Our study demonstrates that NTAPP induces selective cell death in fibrosarcoma cells through the downregulation of the NRF2-induced ROS scavenger system and inhibition of autophagy. These findings suggest NTAPP's potential as a cancer therapy that minimizes damage to normal cells while effectively targeting cancer cells.

Predictors for emergency readmission in patients with ureteral calculi: a focus on pain management and stone location.

BACKGROUND: The management of patients with ureteral calculi in the emergency department (ED) remains challenging due to high revisit rates. PURPOSE: To identify predictors of revisits among patients with ureteral calculi in the ED. DESIGN, SETTING, AND PARTICIPANTS: Data from patients who presented at a tertiary academic hospital in Seoul, Republic of Korea, between February 2018 and December 2019, were analyzed retrospectively. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Variables, including the respiratory rate (RR), estimated glomerular filtration rate (eGFR), duration of pain, number of analgesic doses, location of ureteral calculi, and ED length of stay (LOS) were examined using logistic regression. We also examined some additional variables included in the STONE and CHOKAI scoring systems to examine their association with revisit. RESULTS: Significant predictors of revisits included the number of analgesic doses and the location of ureteral calculi. Patients who required multiple analgesic doses or those with proximal or mid-ureteral calculi were more likely to revisit the ED. Although the STONE and CHOKAI scores could predict uncomplicated ureteral calculi, we found that the CHOKAI score is a valuable tool for predicting the likelihood of patient revisits (p = 0.021). CONCLUSIONS: Effective pain management and consideration of calculi location are important for predicting patient revisits. More research is required to validate findings, develop precise predictive models, and empower tailored care for high-risk patients. In patients with ureteral calculi in the ED, the number of analgesics given and stone location predict return visits. Proximal ureteral calculi on CT may require early urologic intervention to prevent pain-related revisits.