Understanding how chemical stress perturbs human lung physiology requires models that capture dynamic molecular responses in real time. Here, we established a CRISPR/Cas9-engineered human induced pluripotent stem cell (hiPSC)-derived lung organoid expressing endogenous G3BP1–mCherry, enabling live, non-destructive visualization of stress granule (SG) formation under toxicant exposure. The organoids recapitulated airway and alveolar epithelial diversity and displayed lamellar body-like ultrastructures, indicating advanced maturation. Time-lapse imaging revealed rapid and reversible SG dynamics across chemically distinct stressors, while cytotoxicity assays showed that these organoids are significantly more sensitive than conventional 2D or cancer-derived lung models. Importantly, SG dynamics were linked to exposure duration–dependent changes in epithelial barrier integrity, indicating that SG formation precedes overt epithelial injury and serves as an early indicator of toxicant-induced cellular stress. Integration with high-content screening enabled quantitative, image-based analysis of cellular stress phenotypes, greatly enhancing throughput and mechanistic insight, thereby provided next-generation New Approach Methodologies for lung toxicity assessment. Together, this hiPSC-derived lung organoid SG reporter platform links early molecular stress adaptation to tissue-level responses, offering a predictive and mechanistically informative framework for human-relevant lung toxicity evaluation. ● CRISPR-engineered human lung organoids express endogenous G3BP1–mCherry reporter ● Live imaging enables rapid and reversible visualization of stress granules ● Stress granule dynamics act as an early and sensitive biomarkers of chemical stress ● Lung organoids show higher chemical sensitivity than immortalized and cancer cell models ● Reporter enables non-destructive, real-time monitoring of lung toxicant responses

Cells constantly encounter environmental and physiological fluctuations that challenge homeostasis and threaten viability. In response to these cues, specific proteins and nucleic acids engage in multivalent interactions and undergo phase separation to form membraneless assemblies known as biomolecular condensates. Nuclear condensates include paraspeckles, nuclear speckles, and Cajal bodies, while cytoplasmic condensates include stress granules, processing bodies, RNA transport granules, U-bodies, and Balbiani bodies. These assemblies regulate transcription, splicing fidelity, RNA stability, translational reprogramming, and integration of signaling pathways, thereby serving as dynamic platforms for metabolic regulation and physiological adaptation. However, dysregulation of these condensates has been increasingly recognized as a central pathogenic mechanism in neurodegenerative diseases, cancers, and viral infections, contributing to toxic protein aggregation, nucleic acid dysregulation, and aberrant cell survival signaling. This review provides a comprehensive synthesis of the molecular mechanisms governing condensation, delineates the diverse types and functions of major biomolecular condensates, and examines therapeutic approaches based on their pathophysiological relevance to disease development and progression. Furthermore, we highlight the cutting-edge technologies, including CRISPR/Cas-based imaging, optogenetic manipulation, and AI-driven phase separation prediction tools, which enable the real-time monitoring and precision targeting of cytoplasmic biomolecular condensates. These insights underscore the emerging potential of biomolecular condensates as both biomarkers and therapeutic targets, paving the way for precision medicine approaches in condensate-associated diseases.

INTRODUCTION: Natural Killer (NK) cell therapy has shown strong potential for cancer treatment; however, NK cell efficacy is limited by their short lifespan and suppression within the tumor microenvironment. One factor contributing to this suppression may be the activation of the cyclic GMP-AMP synthase (cGAS) pathway. Since the discovery of cGAS as a DNA sensor, there has been renewed interest in DNA sensing mechanisms, although the role of DNA sensing-mediated innate immune responses on NK cells remains unclear. Poxin, a protein derived from DNA viruses, is known to degrade the cGAS signaling product cyclic GMP-AMP (cGAMP) and could potentially alleviate DNA sensing-related stress in NK cells. OBJECTIVES: This study aims to investigate whether Poxin expression can inhibit DNA sensing pathways in NK cells, thereby enhancing their cytotoxic function and anti-tumor activity. METHODS: We generated NK-92 cells expressing a Poxin transgene and evaluated their response to DNA sensing activation. The cGAS-STING pathway was stimulated through either exogenous plasmid transfection or irradiation to induce endogenous double-stranded DNA (dsDNA). We assessed NK-92 and umbilical cord blood derived primary NK (pNK) cells activity through cytotoxicity assay and analysis of perforin and granzyme B expression levels, performed RNA-seq analysis to explore the mechanism and conducted functional assays to evaluate anti-tumor efficacy of NK-92 and chimeric antigen receptors (CAR)-NK-92 cells. RESULTS: Poxin expression significantly inhibited the cGAS-STING pathway, reducing activation in response to both plasmid transfection and irradiation-induced dsDNA. RNA-seq analysis indicated increased levels of cytotoxic mediators, including perforin and granzyme B, in Poxin-expressing NK-92 cells. Furthermore, these transgenic NK-92/CAR-NK-92 cells exhibited enhanced anti-tumor activity compared to controls. CONCLUSION: Poxin effectively suppresses DNA sensing-mediated innate immunity in NK cells, enhancing their cytotoxicity and anti-tumor effectiveness.

Cells produce multiple mRNAs through alternative splicing, which ensures proteome diversity. Because most human genes undergo alternative splicing, key components of signal transduction pathways are no exception. Cells regulate various signal transduction pathways, including those associated with cell proliferation, development, differentiation, migration, and apoptosis. Since proteins produced through alternative splicing can exhibit diverse biological functions, splicing regulatory mechanisms affect all signal transduction pathways. Studies have demonstrated that proteins generated by the selective combination of exons encoding important domains can enhance or attenuate signal transduction and can stably and precisely regulate various signal transduction pathways. However, aberrant splicing regulation via genetic mutation or abnormal expression of splicing factors negatively affects signal transduction pathways and is associated with the onset and progression of various diseases, including cancer. In this review, we describe the effects of alternative splicing regulation on major signal transduction pathways and highlight the significance of alternative splicing.

Understanding how chemical stress perturbs human lung physiology requires models that capture dynamic molecular responses in real time. Here, we established a CRISPR/Cas9-engineered human induced pluripotent stem cell (hiPSC)-derived lung organoid expressing endogenous G3BP1–mCherry, enabling live, non-destructive visualization of stress granule (SG) formation under toxicant exposure. The organoids recapitulated airway and alveolar epithelial diversity and displayed lamellar body-like ultrastructures, indicating advanced maturation. Time-lapse imaging revealed rapid and reversible SG dynamics across chemically distinct stressors, while cytotoxicity assays showed that these organoids are significantly more sensitive than conventional 2D or cancer-derived lung models. Importantly, SG dynamics were linked to exposure duration–dependent changes in epithelial barrier integrity, indicating that SG formation precedes overt epithelial injury and serves as an early indicator of toxicant-induced cellular stress. Integration with high-content screening enabled quantitative, image-based analysis of cellular stress phenotypes, greatly enhancing throughput and mechanistic insight, thereby provided next-generation New Approach Methodologies for lung toxicity assessment. Together, this hiPSC-derived lung organoid SG reporter platform links early molecular stress adaptation to tissue-level responses, offering a predictive and mechanistically informative framework for human-relevant lung toxicity evaluation. ● CRISPR-engineered human lung organoids express endogenous G3BP1–mCherry reporter ● Live imaging enables rapid and reversible visualization of stress granules ● Stress granule dynamics act as an early and sensitive biomarkers of chemical stress ● Lung organoids show higher chemical sensitivity than immortalized and cancer cell models ● Reporter enables non-destructive, real-time monitoring of lung toxicant responses

Cells constantly encounter environmental and physiological fluctuations that challenge homeostasis and threaten viability. In response to these cues, specific proteins and nucleic acids engage in multivalent interactions and undergo phase separation to form membraneless assemblies known as biomolecular condensates. Nuclear condensates include paraspeckles, nuclear speckles, and Cajal bodies, while cytoplasmic condensates include stress granules, processing bodies, RNA transport granules, U-bodies, and Balbiani bodies. These assemblies regulate transcription, splicing fidelity, RNA stability, translational reprogramming, and integration of signaling pathways, thereby serving as dynamic platforms for metabolic regulation and physiological adaptation. However, dysregulation of these condensates has been increasingly recognized as a central pathogenic mechanism in neurodegenerative diseases, cancers, and viral infections, contributing to toxic protein aggregation, nucleic acid dysregulation, and aberrant cell survival signaling. This review provides a comprehensive synthesis of the molecular mechanisms governing condensation, delineates the diverse types and functions of major biomolecular condensates, and examines therapeutic approaches based on their pathophysiological relevance to disease development and progression. Furthermore, we highlight the cutting-edge technologies, including CRISPR/Cas-based imaging, optogenetic manipulation, and AI-driven phase separation prediction tools, which enable the real-time monitoring and precision targeting of cytoplasmic biomolecular condensates. These insights underscore the emerging potential of biomolecular condensates as both biomarkers and therapeutic targets, paving the way for precision medicine approaches in condensate-associated diseases.

INTRODUCTION: Natural Killer (NK) cell therapy has shown strong potential for cancer treatment; however, NK cell efficacy is limited by their short lifespan and suppression within the tumor microenvironment. One factor contributing to this suppression may be the activation of the cyclic GMP-AMP synthase (cGAS) pathway. Since the discovery of cGAS as a DNA sensor, there has been renewed interest in DNA sensing mechanisms, although the role of DNA sensing-mediated innate immune responses on NK cells remains unclear. Poxin, a protein derived from DNA viruses, is known to degrade the cGAS signaling product cyclic GMP-AMP (cGAMP) and could potentially alleviate DNA sensing-related stress in NK cells. OBJECTIVES: This study aims to investigate whether Poxin expression can inhibit DNA sensing pathways in NK cells, thereby enhancing their cytotoxic function and anti-tumor activity. METHODS: We generated NK-92 cells expressing a Poxin transgene and evaluated their response to DNA sensing activation. The cGAS-STING pathway was stimulated through either exogenous plasmid transfection or irradiation to induce endogenous double-stranded DNA (dsDNA). We assessed NK-92 and umbilical cord blood derived primary NK (pNK) cells activity through cytotoxicity assay and analysis of perforin and granzyme B expression levels, performed RNA-seq analysis to explore the mechanism and conducted functional assays to evaluate anti-tumor efficacy of NK-92 and chimeric antigen receptors (CAR)-NK-92 cells. RESULTS: Poxin expression significantly inhibited the cGAS-STING pathway, reducing activation in response to both plasmid transfection and irradiation-induced dsDNA. RNA-seq analysis indicated increased levels of cytotoxic mediators, including perforin and granzyme B, in Poxin-expressing NK-92 cells. Furthermore, these transgenic NK-92/CAR-NK-92 cells exhibited enhanced anti-tumor activity compared to controls. CONCLUSION: Poxin effectively suppresses DNA sensing-mediated innate immunity in NK cells, enhancing their cytotoxicity and anti-tumor effectiveness.

Cells produce multiple mRNAs through alternative splicing, which ensures proteome diversity. Because most human genes undergo alternative splicing, key components of signal transduction pathways are no exception. Cells regulate various signal transduction pathways, including those associated with cell proliferation, development, differentiation, migration, and apoptosis. Since proteins produced through alternative splicing can exhibit diverse biological functions, splicing regulatory mechanisms affect all signal transduction pathways. Studies have demonstrated that proteins generated by the selective combination of exons encoding important domains can enhance or attenuate signal transduction and can stably and precisely regulate various signal transduction pathways. However, aberrant splicing regulation via genetic mutation or abnormal expression of splicing factors negatively affects signal transduction pathways and is associated with the onset and progression of various diseases, including cancer. In this review, we describe the effects of alternative splicing regulation on major signal transduction pathways and highlight the significance of alternative splicing.

Alternative pre-mRNA splicing is key to proteome diversity; however, the biological roles of alternative splicing (AS) in signaling pathways remain elusive. Here, we focus on TEA domain transcription factor 1 (TEAD1), a YAP binding factor in the Hippo signaling pathway. Public database analyses showed that expression of YAP-TEAD target genes negatively correlated with the expression of a TEAD1 isoform lacking exon 6 (TEAD1ΔE6) but did not correlate with overall TEAD1 expression. We confirmed that the transcriptional activity and oncogenic properties of the full-length TEAD1 isoform were greater than those of TEAD1ΔE6, with the difference in transcription related to YAP interaction. Furthermore, we showed that RNA-binding Fox-1 homolog 2 (RBFOX2) promoted the inclusion of TEAD1 exon 6 via binding to the conserved GCAUG element in the downstream intron. These results suggest a regulatory mechanism of RBFOX2-mediated TEAD1 AS and provide insight into AS-specific modulation of signaling pathways.

Deoxyribonucleic acid (DNA) data storage has attracted significant attention as an alternative to conventional digital storage media due to its high information density and long-term durability. However, biochemical processes involved in DNA synthesis, polymerase chain reaction (PCR) amplification, and sequencing inevitably introduce errors, which lead to an important challenge to reliable data recovery. In this study, an error-resilient DNA encoding scheme based on the classical Hamming coding system is described. Specifically, the 8-bit American standard code for information interchange data representing a single character is expanded into a 12-bit codeword by inserting four parity bits using a Hamming code, which enables the detection and correction of single-bit errors. Subsequently, the resulting 12-bit binary codeword is converted into a DNA sequence using a base-4 encoding scheme, in which the quaternary symbols 0, 1, 2, and 3 are mapped to the nucleotides adenine, cytosine, guanine, and thymine, respectively. During decoding, parity checks inherent to the Hamming code are employed to identify and correct erroneous bits, thereby allowing accurate reconstruction of the original data. Furthermore, PCR-induced DNA sequence errors were experimentally introduced and analyzed, demonstrating that the embedded Hamming code effectively detects and corrects errors under practical biochemical conditions. This work shows that fundamental digital error-correcting codes can be effectively integrated into DNA-based storage systems, providing a modular encoding framework that can be extended to other coding schemes for reliable DNA data storage.

Exhaled breath analysis of volatile organic compounds (VOCs) has emerged as a promising non-invasive approach for clinical diagnostics, as breath VOCs can reflect disease-associated metabolic, inflammatory, and microbiological changes. For the reason, VOC-based diagnostics have been explored in a wide range of clinical settings, with vigorous research in cancer, infectious diseases, and respiratory diseases. However, differences in breath sampling, analytical platforms, and data processing strategies have limited comparability across studies and hindered robust clinical translation. In this review, we summarize clinical studies applying exhaled VOC analysis for disease diagnostics, organized by major disease groups, and compare analytical techniques, diagnostic performance, and key limitations. In cancer research, distinct VOC patterns have been reported across various cancer types, with lung cancer being the most extensively investigated. In infectious diseases, studies have mainly focused on viral respiratory infections and pneumonia, reporting VOC profiles associated with specific pathogens and host immune responses. In respiratory diseases such as chronic obstructive pulmonary disease (COPD) and asthma, VOC signatures have been linked to inflammatory status and disease activity. This paper highlights current achievements and shared methodological limitations in breath VOC research and emphasizes the need for standardized protocols and large-scale validation to facilitate routine clinical application.

Tetramethylthiuram disulfide (TMTD), widely used in rubber manufacturing and agriculture, presents occupational inhalation hazards, yet its effects on human lung epithelial cells remain poorly characterized. Here, we investigated TMTD-induced cellular stress responses in A549 lung epithelial cells, focusing on stress granule formation, oxidative stress, and DNA damage. TMTD induced concentration-dependent cytotoxicity, with brief exposure producing effects comparable to continuous exposure, indicating persistent cellular damage. Using live-cell imaging with A549 G3BP1-GFP knock-in cells, we demonstrated that TMTD rapidly triggered SG formation within minutes, accompanied by marked eIF2α phosphorylation. TMTD exposure caused dramatic intracellular ROS accumulation and robust γ-H2AX phosphorylation. Antioxidant rescue experiments using N-acetylcysteine confirmed that oxidative stress directly drives SG formation and DNA damage. Repeated TMTD exposure significantly increased apoptotic cell populations, demonstrating that cells cannot recover from recurrent exposure. Our findings reveal a mechanistic cascade whereby TMTD induces oxidative stress, triggers SG formation as an adaptive response, causes DNA damage, and ultimately leads to apoptosis when cellular stress overwhelms protective mechanisms. This study establishes stress granule formation as a sensitive early biomarker for TMTD exposure and highlights significant respiratory health risks for workers in rubber and agricultural industries, supporting the need for re-evaluation of occupational exposure limits and implementation of stringent protective measures.

Background/Aim: Plasma-activated medium (PAM), which generates reactive oxygen and nitrogen species (RONS), has emerged as a promising anti-cancer approach. However, although PAM effectively eliminates bulk tumor cells, surviving populations often acquire enhanced stem cell-like properties, suggesting selective resistance in cancer stem cells (CSCs). This study aimed to elucidate the molecular mechanisms underlying CSC resistance to PAM-induced oxidative stress in non-small cell lung cancer (NSCLC). Materials and Methods: CD133+ and CD133− subpopulations were isolated from A549 lung cancer cells to evaluate their sensitivity to PAM-induced cytotoxicity. Intracellular reactive oxygen species (ROS) levels, apoptosis, and antioxidant defense mechanisms were assessed in vitro, while therapeutic efficacy was examined using xenograft mouse models. Results: CD133+ A549 cells exhibited marked resistance to PAM-induced apoptosis compared with CD133− cells, accompanied by significantly reduced intracellular ROS accumulation. Peroxiredoxin 5 (PRDX5) was identified as a key antioxidant enzyme that was highly up-regulated in CD133+ cells and essential for maintaining redox homeostasis. Genetic silencing of PRDX5 in CD133+ cells significantly enhanced PAM-mediated cytotoxicity both in vitro and in vivo, restoring sensitivity to oxidative damage. Furthermore, combining PRDX5 knockdown with PAM treatment or co-administering PAM with paclitaxel substantially suppresses tumor growth in xenograft models, reducing tumor burden and inflammatory responses. These findings reveal PRDX5 as a key survival factor enabling CSCs to evade plasma therapy and suggest that targeting PRDX5 alongside PAM or conventional chemotherapy could improve treatment outcomes in NSCLC. Conclusion: PRDX5 plays a central role in mediating oxidative stress resistance in CD133+ lung cancer stem-like cells, enabling their survival following PAM treatment. Targeting PRDX5, either alone or in combination with PAM or conventional chemotherapy, represents a promising strategy to overcome cancer stem cell–mediated resistance and improve therapeutic outcomes in non-small cell lung cancer.