The expression spectrum of yak epididymal epithelial cells reveals the functional diversity of caput, corpus and cauda regions.

Sperm undergo a series of changes in the epididymis region before acquiring the ability to move and fertilize, and the identification of genes expressed in a region-specific manner in the epididymis provides a valuable insight into functional differences between regions. We collected epididymal tissue from three yaks and cultured epithelial cells from the caput, corpus and cauda regions of the yak epididymis using the tissue block method. RNA sequencing analysis (RNA-seq) technology was used to detect gene expression in yak epididymal caput, corpus and cauda epithelial cells. The results showed that the DEGs were highest in the caput vs. corpus comparison, and lowest in the corpus vs. cauda comparison. Six DEGs were verified by real-time fluorescence quantitative PCR (qRT-PCR), consistent with transcriptome sequencing results. The significantly enriched DNA replication pathway in the caput vs. corpus was coordinated with cell proliferation, while upregulated DEGs such as POLD1 and MCM4 were found in the DNA replication pathway. The AMPK signaling pathway was found significantly enriched in the caput vs cauda, suggesting its involvement in sperm maturation and capacitation. The TGF beta signaling pathway was screened in the corpus vs cauda and is crucial for mammalian reproductive regulation. Upregulated DEGs (TGFB3, INHBA, INHBB) are involved in the TGF beta signaling pathway. This study provides a reference for culturing yak epididymal epithelial cells in vitro, and elucidates the transcriptional profiles of epithelial cells in different segments of the epididymis, revealing the regulatory and functional differences between different segments, providing basic data for exploring the molecular mechanism of yak sperm maturation and improving the reproductive capacity of high-altitude mammals.

The Epidemiology of Pathogens in Community-Acquired Pneumonia Among Children in Southwest China Before, During and After COVID-19 Non-pharmaceutical Interventions: A Cross-Sectional Study.

OBJECTIVE: This study aimed to investigate the pathogen epidemiology of community-acquired pneumonia (CAP) among children in Southwest China before, during and after the COVID-19 non-pharmaceutical interventions (NPIs). METHODS: Pathogen data of hospitalised children with CAP, including multiple direct immunofluorescence test for seven viruses, bacterial culture and polymerase chain reaction (PCR) for Mycoplasma pneumoniae, were analysed across three phases: Phase I (pre-NPIs: 1 January 2019 to 31 December 2019), Phase II (NPI period: 1 January 2020 to 31 December 2020) and Phase III (post-NPIs: 1 January 2023 to 31 December 2023). RESULTS: A total of 7533 cases were enrolled, including 2444, 1642 and 3447 individuals in Phases I, II and III, respectively. M. pneumoniae predominated in Phases I and III (23.4% and 35.5%, respectively). In Phase II, respiratory syncytial virus (RSV) emerged as the primary pathogen (20.3%), whereas detection rates of influenza A virus (Flu A) and M. pneumoniae were at a low level (1.8% and 9.6%, respectively). In Phase III, both Flu A (15.8%) and M. pneumoniae epidemic rebounded, whereas RSV detection rate returned to Phase I level, and detection rates of Streptococcus pneumoniae and Haemophilus influenzae decreased significantly compared to those in Phase I. Detection rates of adenovirus and parainfluenza virus type 3 decreased phase by phase. Age-stratified analysis and monthly variations supported the above findings. Seasonal patterns of multiple pathogens were disrupted during Phases II and III. CONCLUSIONS: COVID-19 NPIs exhibited a distinct impact on CAP pathogen epidemic among children, with post-NPIs increases observed in M. pneumoniae and Flu A prevalence. Continuous pathogen monitoring is crucial for effective prevention and control of paediatric CAP.

Analysis of histomorphology and SERNINA5 gene expression in different regions of epididymis of cattleyak.

The molecular mechanism of sterility in cattleyak is still unresolved. The related factors of infertility in cattleyak were studied by tissue section, SERPINA5 gene cloning and bioinformatics technology. Tissue sections of the epididymis showed poorly structured and disorganized epithelial cells in the corpus of the epididymis compared to the caput of the epididymis, while in the cauda part of the epididymis, the extra basal smooth muscle was thinner, the surface of the epithelial lumen was discontinuous and the epithelium was markedly degenerated. The results of gene cloning showed that the coding sequence (CDS) region of the SERPINA5 gene in cattleyak was 1215 bp in length, encoding a total of 404 amino acids, of which the isoleucine content was the highest, accounting for a total of 49 amino acids (12.1%). The results of real-time fluorescence quantitative PCR (qPCR) showed that the expression of the SERPINA5 gene in the epididymis caput in cattleyak was significantly higher than that in the corpus and cauda (P < 0.05), but there were no significant differences between the corpus and cauda. In the current study, histological and bioinformatics analysis, physicochemical properties, and the expression analysis of the SERPINA5 gene in different regions of the epididymis in cattleyak were carried out to explore the biological complications of cattleyak infertility.

Neuropilin-1 is a novel host factor modulating the entry of hepatitis B virus.

BACKGROUND & AIMS: Sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the cellular receptor for hepatitis B virus (HBV). However, hepatocytes expressing NTCP exhibit varying susceptibilities to HBV infection. This study aimed to investigate whether other host factors modulate the process of HBV infection. METHODS: Liver biopsy samples obtained from children with hepatitis B were used for single-cell sequencing and susceptibility analysis. Primary human hepatocytes, HepG2-NTCP cells, and human liver chimeric mice were used to analyze the effect of candidate host factors on HBV infection. RESULTS: Single-cell sequencing and susceptibility analysis revealed a positive correlation between neuropilin-1 (NRP1) expression and HBV infection. In the HBV-infected cell model, NRP1 overexpression before HBV inoculation significantly enhanced viral attachment and internalization, and promoted viral infection in the presence of NTCP. Mechanistic studies indicated that NRP1 formed a complex with LHBs and NTCP. The NRP1 b domain mediated its interaction with conserved arginine residues at positions 88 and 92 in the preS1 domain of the HBV envelope protein LHBs. This NRP1-preS1 interaction subsequently promoted the binding of preS1 to NTCP, facilitating viral infection. Moreover, disruption of the NRP1-preS1 interaction by the NRP1 antagonist EG00229 significantly attenuated the binding affinity between NTCP and preS1, thereby inhibiting HBV infection both in vitro and in vivo. CONCLUSIONS: Our findings indicate that NRP1 is a novel host factor for HBV infection, which interacts with preS1 and NTCP to modulate HBV entry into hepatocytes. IMPACT AND IMPLICATIONS: HBV infection is a global public health problem, but the understanding of the early infection process of HBV remains limited. Through single-cell sequencing, we identified a novel host factor, NRP1, which modulates HBV entry by interacting with HBV preS1 and NTCP. Moreover, antagonists targeting NRP1 can inhibit HBV infection both in vitro and in vivo. This study could further advance our comprehension of the early infection process of HBV.

Preparation and Properties of Composite Double-Network Gel for Inhibiting Coal Spontaneous Combustion.

In order to improve the inhibition effect of gel on coal spontaneous combustion, a chitosan (CS)/polyacrylamide (PAM)/metal ion (Al3+) composite double-network gel was developed in this study. The optimum formula of the composite double-network gel was determined using orthogonal experimentation. The microstructure, water retention, compressibility, and anti-destruction properties of the composite double-network gel were analyzed. The inhibition effect of the composite double-network gel on coal spontaneous combustion was studied via infrared spectroscopy and a synchronous thermal analyzer from the micro and macro perspectives. The results show that the composite double-network gel has a denser interpenetrating double-network structure and a larger void ratio than the ordinary gel. The water retention rate was 55% after standing at 150 °C for 12 h. The deformation memory ratio of the composite double-network gel was 78%, which was 26.8% higher than that of the ordinary gel, and the compressive strength also increased by 59.96%. In addition, the critical temperature point and the maximum thermal weight-loss rate temperature point decreased by 7.01 °C and 39.62 °C, respectively, and the composite double-network gel effectively reduced active functional groups in the treated coal sample, such as hydroxyl and aliphatic hydrocarbons. In this study, a CS/PAM/Al3+ composite double-network gel was produced, which exhibited good gel performance and inhibition effects, with physical effects such as the covering, wetting, and cementation of coal.

Recombinant FOXN1 fusion protein increases T cell generation in old mice.

T cell development in the thymus is dependent on the thymic microenvironment, in which thymic epithelial cells (TECs) are the major component. However, TECs undergo both a qualitative and quantitative loss during aging, which is believed to be the major factor responsible for age-dependent thymic atrophy. FOXN1 plays a critical role in TEC development and adult TECs maintenance. We have previously reported that intrathymic injection of a recombinant (r) protein containing murine FOXN1 and a protein transduction domain increases the number of TECs in mice, leading to enhanced thymopoiesis. However, intrathymic injection may not be an ideal choice for clinical applications. In this study, we produced a rFOXN1 fusion protein containing the N-terminal of CCR9, human FOXN1 and a protein transduction domain. When injected intravenously into 14-month-old mice, the rFOXN1 fusion protein enters the thymus and TECs, and enhances thymopoiesis, resulting in increased T cell generation in the thymus and increased number of T cells in peripheral lymphoid organ. Our results suggest that the rFOXN1 fusion protein has the potential to be used in preventing and treating T cell immunodeficiency in older adults.

Jellyfish-Inspired Artificial Spider Silk for Luminous Surgical Sutures.

The development of functional surgical sutures with excellent mechanical properties, good fluorescence, and high cytocompatibility is highly required in the field of medical surgeries. Achieving fibers that simultaneously exhibit high mechanical robustness, good spinnability, and durable fluorescence emission has remained challenging up to now. Taking inspiration from the spinning process of spider silk and the luminescence mechanism of jellyfish, this work reports a luminous artificial spider silk prepared with the aim of balancing the fiber spinnability and mechanical robustness. This is realized by employing highly hydrated segments with aggregation-induced luminescence for enhancing the fiber spinnability and polyhydroxyl segments for increasing the fiber mechanical robustness. Twist insertion during fiber spinning improves the fiber strength, toughness, and fluorescence emission. Furthermore, coating the fiber with an additional polymer layer results in a "sheath-core" architecture with improved mechanical properties and capacity to withstand water. This work provides a new design strategy for performing luminescent and robust surgical sutures.

Geniposide alleviates heart failure with preserved ejection fraction in mice by regulating cardiac oxidative stress via MMP2/SIRT1/GSK3ß pathway.

Heart failure with preserved ejection fraction (HFpEF) is a complex clinical syndrome with cardiac dysfunction, fluid retention and reduced exercise tolerance as the main manifestations. Current treatment of HFpEF is using combined medications of related comorbidities, there is an urgent need for a modest drug to treat HFpEF. Geniposide (GE), an iridoid glycoside extracted from Gardenia Jasminoides, has shown significant efficacy in the treatment of cardiovascular, digestive and central nervous system disorders. In this study we investigated the therapeutic effects of GE on HFpEF experimental models in vivo and in vitro. HFpEF was induced in mice by feeding with HFD and L-NAME (0.5 g/L) in drinking water for 8 weeks, meanwhile the mice were treated with GE (25, 50 mg/kg) every other day. Cardiac echocardiography and exhaustive exercise were performed, blood pressure was measured at the end of treatment, and heart tissue specimens were collected after the mice were euthanized. We showed that GE administration significantly ameliorated cardiac oxidative stress, inflammation, apoptosis, fibrosis and metabolic disturbances in the hearts of HFpEF mice. We demonstrated that GE promoted the transcriptional activation of Nrf2 by targeting MMP2 to affect upstream SIRT1 and downstream GSK3ß, which in turn alleviated the oxidative stress in the hearts of HFpEF mice. In H9c2 cells and HL-1 cells, we showed that treatment with GE (1 µM) significantly alleviated H2O2-induced oxidative stress through the MMP2/SIRT1/GSK3ß pathway. In summary, GE regulates cardiac oxidative stress via MMP2/SIRT1/GSK3ß pathway and reduces cardiac inflammation, apoptosis, fibrosis and metabolic disorders as well as cardiac dysfunction in HFpEF. GE exerts anti-oxidative stress properties by binding to MMP2, inhibiting ROS generation in HFpEF through the SIRT1/Nrf2 signaling pathway. In addition, GE can also affect the inhibition of the downstream MMP2 target GSK3ß, thereby suppressing the inflammatory and apoptotic responses in HFpEF. Taken together, GE alleviates oxidative stress/apoptosis/fibrosis and metabolic disorders as well as HFpEF through the MMP2/SIRT1/GSK3ß signaling pathway.

Plant Leaf Functional Adaptions along Urban-Rural Gradients of Jinhua City.

Environmental changes induced by urbanization may significantly alter plant survival strategies, thereby introducing uncertainties in their ability to withstand extreme heat. This study, centered on Jinhua City, distinguished urban, suburban, and rural areas to represent the various intensities of urbanization. It examined the leaf function properties of evergreen and deciduous trees common in these regions, focusing on leaf and branch characteristics. Employing an analysis of variance (ANOVA), principal component analysis (PCA), and path analysis (PA) of the plant functional traits and the climatic factors of each region, this study assessed the impact of urbanization on plant survival strategies. By tracking changes in plant functional traits from June to August, it explored the capacity of plants to acclimate to urban-warming-related heat stress across different urbanization gradients. The findings revealed that leaf thickness (LT) and stomatal size (SS) initially decreased and then increased, whereas specific leaf area (SLA) and leaf tissue density (LTD) first rose and then declined, from rural to urban regions. From June to August, branch wood density (WD), chlorophyll (Chl) content, LTD, and leaf dry matter content (LDMC) increased, whereas SLA and leaf water content (LWC) diminished, in all regions. PCA suggested that there was no significant change in the resource allocation strategy of plants (p > 0.05), with drought tolerance significantly reduced in the suburbs on the gradient of urbanization (p < 0.05). During the summer, with high temperature, plants were predominantly biased towards slow-return, conservative strategies, particularly among evergreen species. Compared to precipitation, PA revealed a significant urban warming effect. During summer, temperature was the main factor influencing resource investment strategy and drought resistance, with a notably stronger impact on the former. The high temperature in summer promoted a conservative survival strategy in plants, and the urbanization effect increased their tolerance to high temperatures.

Discovery of Highly Selective, Potent, Covalent, and Orally Bioavailable Factor XIIa Inhibitors for the Treatment of Thrombo-Inflammation.

Thrombo-inflammation is closely associated with a few severe cardiovascular and infectious diseases. Factor XIIa (FXIIa) in the intrinsic coagulation pathway plays a pivotal role in the development of thrombo-inflammation and its inhibition has emerged as a potential therapeutic approach for thrombo-inflammatory disorders. Nonetheless, as of now, few small-molecule FXIIa inhibitors have demonstrated notable effectiveness against thrombo-inflammation, with none progressing into clinical stages. Herein, we present potent, covalent, reversible, and selective small-molecule FXIIa inhibitors such as 4a and 4j obtained through structure-based drug design. Compounds 4a and 4j showed significant anticoagulation and substantial anti-inflammatory effects in vitro, coupled with exceptional plasma stability. Furthermore, in carrageenan-induced thrombosis models, 4a and 4j demonstrated remarkable dual antithrombotic and anti-inflammatory activity when administered orally. Compound 4j exhibited a favorable safety profile without obvious tissue toxicity in mice, suggesting its potential as an oral therapeutic option for thrombo-inflammation.

SepM mutation in Streptococcus mutans clinical isolates and related function analysis.

BACKGROUND: Streptococcus mutans (S. mutans) is an important pathogenic bacterium that causes dental caries, while Streptococcus gordonii (S. gordonii) is a non-cariogenic bacterium that inhibits the growth of S. mutans. The SepM protein can promote the inhibitory ability of S. mutans against S. gordonii by cleaving CSP-21 and activating the ComDE two-component system. This study was designed to explore sepM mutation in S. mutans clinical isolates and related function in the regulation of interactions with S. gordonii. METHODS: The S. mutans clinical strains that can inhibit the growth of S. gordonii constitute the inhibitory group. 286 C-serotype S. mutans strains were categorized into S. gordonii inhibitory (n = 114) and non-inhibitory bacteria (n = 172). We detected sanger sequencing of sepM gene, the expression levels of related genes and proteins in clinical isolates, obtained prokaryotic expression and purification of mutated proteins, and analyzed the effect of the target mutations on the binding between SepM and CSP-21. RESULTS: We found that C482T, G533A, and G661A missense mutations were presented at significantly higher frequency in the inhibitory group relative to the non-inhibitory group. There was no significant difference in the expression of the sepM gene between selected clinical isolates harboring the G533A mutation and the control group. The expression levels of SepM, phosphorylated ComD, and ComE in the mutation group were significantly higher than those in the control group. SepM_control and SepM_D221N (G661A at the gene level) were found to contain two residues close to the active center while SepM_G178D (G533A at the gene level) contained three residues close to the active center. At 25 °C and a pH of 5.5, SepM_D221N (G661A) exhibited higher affinity for CSP-21 (KD = 8.25 µM) than did the SepM control (KD = 33.1 µM), and at 25 °C and a pH of 7.5, SepM_G178D (G533A) exhibited higher affinity (KD = 3.02 µM) than the SepM control (KD = 15.9 µM). It means that it is pH dependent. CONCLUSIONS: Our data suggest that increased cleavage of CSP-21 by the the mutant SepM may be a reason for the higher inhibitory effect of S. mutans on S. gordonii .

Differential gene expression and gut microbiota composition in low-altitude and high-altitude goats.

Previous studies have presented evidence suggesting that altitude exerts detrimental effects on reproductive processes, yet the underlying mechanism remains elusive. Our study employed two distinct goat breeds inhabiting low and high altitudes, and conducted a comparative analysis of mRNA profiles in testis tissues and the composition of gut microbiota. The results revealed a reduced testis size in high-altitude goats. RNA-seq analysis identified the presence of 214 differentially expressed genes (DEGs) in the testis. These DEGs resulted in a weakened immunosuppressive effect, ultimately impairing spermatogenesis in high-altitude goats. Additionally, 16S rDNA amplicon sequencing recognized statistically significant variations in the abundance of the genera Treponema, unidentified_Oscillospiraceae, Desulfovibrio, Butyricicoccus, Dorea, Parabacteroides between the two groups. The collective evidence demonstrated the gut and testis played a synergistic role in causing decreased fertility at high altitudes. Our research provides a theoretical basis for future investigations into the reproductive fitness of male goats.

A structural variation in the promoter of the leucoanthocyanidin reductase gene AaLAR1 enhances freezing tolerance by modulating proanthocyanidin accumulation in kiwifruit (Actinidia arguta).

Proanthocyanidins (PAs) are important metabolites that enhance freezing tolerance of plants. Actinidia arguta, especially freezing-tolerant germplasms, accumulate abundant PAs in dormant shoots and thereby enhance freezing tolerance, but the underlying mechanism is unknown. In this study, we used two A. arguta with contrasting cold-resistant phenotypes, KL and RB, to explore the mechanisms in response to cold tolerance. We determined that a leucoanthocyanidin reductase gene (AaLAR1) was more highly expressed in freezing-tolerant KL than in freezing-sensitive RB. Moreover, overexpressing AaLAR1 in kiwifruit promoted PAs biosynthesis and enhanced cold tolerance. The AaLAR1 promoters of various A. arguta germplasms differ due to the presence of a 60-bp deletion in cold-tolerant genotypes that forms a functional binding site for MYC-type transcription factor. Yeast one-hybrid and two-hybrid, dual-luciferase reporter, bimolecular fluorescence complementation and coimmunoprecipitation assays indicated that the AaMYC2a binds to the MYC-core cis-element in the AaLAR1 promoter with the assistance of AaMYB5a, thereby promoting PAs accumulation in the shoots of cold-tolerant kiwifruit. We conclude that the variation in the AaLAR1 promoter and the AaMYC2a-AaMYB5a-AaLAR1 module shape freezing tolerance in A. arguta. The identification of a key structural variation in the AaLAR1 promoter offers a new target for resistance breeding of kiwifruit.

Plasma leucine-rich α-2 glycoprotein 1 in ST-elevation myocardial infarction: vertical variation, correlation with T helper 17/regulatory T ratio, and predictive value on major adverse cardiovascular events.

Objective: Leucine-rich α-2 glycoprotein 1 (LRG1) promotes inflammation and myocardial injury, but its clinical role in ST-elevation myocardial infarction (STEMI) is rarely disclosed. Herein, this prospective study aimed to explore the value of plasma LRG1 at different time points to predict major adverse cardiovascular event (MACE) risk in patients with STEMI. Methods: In total, 209 patients with STEMI were enrolled for determining plasma LRG1 at admission and on day (D)1/D7/D30 after admission via enzyme-linked immunosorbent assay, as well as for determination of peripheral blood T helper 17 (Th17) cells and regulatory T (Treg) cells by flow cytometry. In addition, plasma LRG1 was obtained from 30 healthy controls at enrollment. Results: LRG1 was increased in patients with STEMI at admission compared with healthy controls (P < 0.001). In patients with STEMI, LRG1 varied at different time points (P < 0.001), which elevated from admission to D1, and gradually declined thereafter. LRG1 at admission was positively associated with Th17 cells (P = 0.001) and Th17/Treg ratio (P = 0.014). LRG1 at admission (P = 0.013), D1 (P = 0.034), D7 (P = 0.001), and D30 (P = 0.010) were increased in patients with MACE compared with those without. LRG1 at D7 exhibited good ability to estimate MACE risk (area under curve = 0.750, 95% confidence interval = 0.641-0.858). LRG1 at admission > 60 µg/ml (P = 0.031) and D7 > 60 µg/ml (P = 0.018) were linked with increased accumulating MACE. Importantly, LRG1 at D7 > 60 µg/ml was independently correlated with increased MACE risk (hazard ratio = 5.216, P = 0.033). Conclusion: Plasma LRG1 increases from admission to D1 and gradually declines until D30, which positively links with Th17 cells and MACE risk in patients with STEMI.

The Role of SLIT3-ROBO4 Signaling in Endoplasmic Reticulum Stress-Induced Delayed Corneal Epithelial and Nerve Regeneration.

Purpose: In the present study, we aim to elucidate the underlying molecular mechanism of endoplasmic reticulum (ER) stress induced delayed corneal epithelial wound healing and nerve regeneration. Methods: Human limbal epithelial cells (HLECs) were treated with thapsigargin to induce excessive ER stress and then RNA sequencing was performed. Immunofluorescence, qPCR, Western blot, and ELISA were used to detect the expression changes of SLIT3 and its receptors ROBO1-4. The role of recombinant SLIT3 protein in corneal epithelial proliferation and migration were assessed by CCK8 and cell scratch assay, respectively. Thapsigargin, exogenous SLIT3 protein, SLIT3-specific siRNA, and ROBO4-specific siRNA was injected subconjunctivally to evaluate the effects of different intervention on corneal epithelial and nerve regeneration. In addition, Ki67 staining was performed to evaluate the proliferation ability of epithelial cells. Results: Thapsigargin suppressed normal corneal epithelial and nerve regeneration significantly. RNA sequencing genes related to development and regeneration revealed that thapsigargin induced ER stress significantly upregulated the expression of SLIT3 and ROBO4 in corneal epithelial cells. Exogenous SLIT3 inhibited normal corneal epithelial injury repair and nerve regeneration, and significantly suppressed the proliferation and migration ability of cultured mouse corneal epithelial cells. SLIT3 siRNA inhibited ROBO4 expression and promoted epithelial wound healing under thapsigargin treatment. ROBO4 siRNA significantly attenuated the delayed corneal epithelial injury repair and nerve regeneration induced by SLIT3 treatment or thapsigargin treatment. Conclusions: ER stress inhibits corneal epithelial injury repair and nerve regeneration may be related with the upregulation of SLIT3-ROBO4 pathway.

Microsphere-Structured Protein Hydrogel Dielectrics for Capacitive Wearable Sensors.

The desire for healthy living has created a crucial need for portable flexible health-monitoring devices based on biomaterials. Toward this end, we report a microsphere-structured hydrogel that uses bovine serum albumin (BSA) as a dielectric layer for capacitive pressure sensors. We developed a theoretical model that describes how stacking dielectric layers of spheres affects the performance of capacitive sensors. We also prepared a prototype sensor featuring the unique microsphere structure to create capacitive sensors with high sensitivity (360.91 strain sensitivity), excellent cyclical stability, and a long service life (over 5000 stretching-compression cycles). Furthermore, the design of the hydrogel sensor allows for easy integration into fabrics to create devices such as smart wristbands, which can collect a diverse range of health data. Thus, BSA-hydrogel-based sensors not only provide safe wearable devices but also advance the performance of high-sensitivity capacitive sensors.

DNA Walker-Driven Mass Nanotag Assembly System for Simultaneously Profiling Dual Markers of Oxidative Stress at Different Cellular Locations.

Simultaneous profiling of redox-regulated markers at different cellular sublocations is of great significance for unraveling the upstream and downstream molecular mechanisms of oxidative stress in living cells. Herein, by synchronizing dual target-triggered DNA machineries in one nanoentity, we engineered a DNA walker-driven mass nanotag (MNT) assembly system (w-MNT-AS) that can be sequentially activated by oxidative stress-associated mucin 1 (MUC1) and apurinic/apyrimidinic endonuclease 1 (APE1) from plasma membrane to cytoplasm and induce recycled assembly of MNTs for multiplex detection of the two markers by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). In the working cascade, the sensing process governs the separate activation of w-MNT-AS by MUC1 and APE1 in diverse locations, while the assembly process contributes to the parallel amplification of the ion signal of the characteristic mass tags. In this manner, the differences between MCF-7, HeLa, HepG2, and L02 cells in membrane MUC1 expression and cytoplasmic APE1 activation were fully characterized. Furthermore, the oxidative stress level and dynamics caused by exogenous H2O2, doxorubicin, and simvastatin were comprehensively demonstrated by tracking the fate of the two markers across different cellular locations. The proposed w-MNT-AS coupled MS method provides an effective route to probe multiple functional molecules that lie at different locations while participating in the same cellular event, facilitating the mechanistic studies on cellular response to oxidative stress and other disease-related cellular processes.