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The mitochondrial genome of plants contains an open reading frame, orfx, which encodes a protein classified as very rare and which has so far escaped mass spectrometric detection. The protein resembles the c-subunit of bacterial twin-arginine-motif-dependent protein translocases (TatC). Using native prefractionation of mitochondrial protein complexes from Arabidopsis and trapped-ion mobility spectrometry (TIMS) time-of-flight mass spectrometry, we report the identification of three peptides of the orfx protein. Our experimental approach was used to trace the native forms of the protein and show that it is present in protein complexes in the size range of 450-530 kDa, which also contain TatB. We suggest that mitochondrial TatBC complexes in plants may bind to late assembly intermediates of respiratory chain complex III.
The 3-min all-out test is considered a time-efficient alternative for estimating critical power (CP) and curvature constant (W'), by means of endtest power (EP) and work done above endtest power (WEP), respectively. This review aims to assess its reliability and validity with respect to methodology. Following the PRISMA 2020 guidelines, a systematic literature search was conducted on June 20th 2025, using a specified search term in PubMed and Web of Science. Included studies were English-language, peer-reviewed original articles, that provided empirical data on the 3-min all-out test in cycling and used appropriate protocols for assessing reliability/validity. Quality of studies was assessed using a modified critical appraisal tool. Meta-analysis for validity was done using pooled Hedge's gz with random effects synthesis and 95%prediction intervals. Twenty studies comprising N = 284 participants met all inclusion criteria. Thereof, 15 studies examined validity, 8 reliability and 3 both. Included studies demonstrated methodological heterogeneity and small sample sizes (n = 6-21). Test-retest reliability demonstrated 'good' to 'excellent' agreement for EP (ICC = 0.89-0.99) and WEP (ICC = 0.76-0.98). EP systematically overestimates CP (gz= 0.408 [0.024, 0.792]) while WEP underestimates W' (gz= -0.389 [-0.954, 0.176]). Whereas the 3-min all-out test provides reliable results for EP and WEP if tests are conducted within three weeks, its validity seems to largely depend on the methodological approach. Certain power profiles and/or isokinetic 3-min tests lead to better estimates of CP, while the variability of WEP does not allow to accurately estimate W'. Familiarisation trials and proper verbal encouragement during the 3-min test are mandatory.
An agent-based discrete computational model biologically calibrated to Pseudomonas aeruginosa migration is used to explore the impacts of bacterial reversals and wrap mode on the efficiency of motion in different environments, both with and without chemotaxis. It is first shown that wrap mode increases the exploration of continuous multimodal chemotactic profiles such as those produced by biologically relevant fungal networks. For cells undergoing a run-reverse pattern, it is shown that the bacteria are likely to remain at the first local chemoattractant maximal production site on a hypha they find. However, with wrap mode, the bacteria can more easily escape these local sites to further explore their neighboring environment along the fungi, suggesting that wrap mode may be beneficial for migration along the fungi in liquid. In a different set of simulations of bacterial motion close to an isolated chemotactic source, wrap mode is shown to increase the ability of a bacterium to reorient toward the source while reducing the overall motion required for similar chemotactic efficiency as a run-reverse strategy, suggesting a potential metabolic benefit. In contrast, model simulations show that wrap mode can increase the rate of dispersal of P. aeruginosa in a nonchemotactic environment.
Inappropriate activation of the mitogen-activated protein kinase (MAPK) pathway, often stemming from activating mutations in RAS or RAF, represents one of the most common oncogenic events in human cancer. However, currently approved RAS/RAF inhibitors target specific mutants that are only present in a small proportion of RAS- or RAF-activated tumors. Current MEK inhibitors (MEKi) are associated with class-effect toxicities and tumor escape via CRAF bypass of MEK blockade. To address these issues, we designed atebimetinib to resist CRAF-mediated bypass and enable a pharmacokinetic/pharmacodynamic (PK/PD) profile termed deep cyclic inhibition (DCI), an approach that transiently but deeply suppresses oncogenic signaling while allowing daily physiologic reset. Unlike chronic or intermittent approaches, DCI optimized depth and duration of MEK suppression on a daily cycle, blunting rebound signaling while preserving recovery windows for normal tissues. Functioning as an allosteric, selective MEKi, atebimetinib potently inhibited ERK phosphorylation in vitro and in vivo, resisted CRAF bypass, and was well tolerated and more efficacious in xenograft models of cancer compared with current MEK inhibitors. Overall, these findings establish atebimetinib as a dual-MEK inhibitor with the potential to provide mutation-agnostic inhibition of the MAPK pathway while leveraging DCI, designed to avoid toxicities associated with existing MEK inhibitors. Atebimetinib offers MEK inhibition that is durable and tolerable, supporting the potential of this therapeutic paradigm.
This study aimed to explore latent categories of nursing and medical students' knowledge, attitude and practice intentions regarding advance care planning (ACP), and to identify the factors influencing these categories. It sought to provide an evidence base for developing personalized, precision-targeted advance care planning educational interventions. From October to December 2023, a stratified cluster random sampling method was employed to select a sample of 1006 undergraduate students in clinical medicine and nursing (Years 1-4) from a medical university in eastern China. A total of 935 valid questionnaires were recovered. Research instruments included a self-developed demographic questionnaire, the advance care planning knowledge, attitude and practice intentions Questionnaire, the Death Attitude Profile-Revised, and the Meaning in Life Questionnaire. Latent profile analysis determined the optimal number of categories; logistic regression identified factors influencing different advance care planning knowledge, attitude and practice intentions categories; a nomogram was constructed using R software, and predictive performance was evaluated using area under the curve (AUC), Hosmer-Lemeshow test, and 1000-bootstrap calibration curves. Latent profile analysis identified two latent categories: a "collaborative" group (33.6% of the sample) and a "lagging" group (66.4% of the sample). The collaborative group scored higher than the lagging group across all dimensions of advance care planning knowledge, attitude and practice intentions questionnaire. Logistic regression analysis indicated: palliative care training (OR = 1.5, 1.028-2.190), resuscitation experience (OR = 0.64, 0.424-0.966), clinical medicine student (OR = 0.537, 0.376-0.766), urban student (OR = 0.626, 0.419-0.935), high death escape scores, low natural acceptance scores, and low meaning in life questionnaire scores were more likely to be classified as "lagging type". Senior students (OR = 7.217, 4.089-12.737) and those with positive major attitude (OR = 3.572, 1.228-10.386) were more likely to be classified as "collaborative type". The nomogram demonstrated good discriminatory ability (AUC = 0.864; 95% CI 0.841-0.887; Brier score = 0.14) and high calibration (χ2 = 13.845, p = 0.086; calibration slope = 0.97). The established nomogram model identified the influencing factors of the knowledge, attitude and practice Intentions level of advance care planning among medical undergraduates, providing empirical evidence for the educational intervention of advance care planning for nursing and medical undergraduates. Universities needed to carry out refined advance care planning education in a targeted manner based on the different characteristics of students. To strengthen the validity of the research, it was planned, conducted and reported in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist. All participants contributed to the conducting of this study by completing self-reported questionnaires.
m5C modification plays a vital role in the progression of human cancers, including breast cancer (BC), but the function of NOP2/Sun RNA methyltransferase 2 (NSUN2), an RNA m5C modification enzyme, remains largely unclear. qRT-PCR, western blot and IHC assays were carried out to determine the expression of NSUN2, poly (ADP-ribose) polymerase 1 (PARP9) and Y box binding protein 1 (YBX1). EdU, flow cytometry, transwell, tube formation and sphere formation assays were conducted to evaluate cell proliferation, apoptosis, invasion, angiogenesis and stemness, respectively. ELISA was performed to examine the concentrations of inflammatory factors. RIP and pull-down assays were used to verify the interaction between NSUN2/YBX1 and PARP9. Murine xenograft model was constructed to explore the functions of NSUN2 and PARP9 in tumor growth in vivo. PARP9 silencing suppressed the proliferation, invasion, angiogenesis, stemness and immune escape, and promoted apoptosis in BC cells in vitro. Mechanically, NSUN2 functioned as an m5C writer that catalyzed m5C modification of PARP9 mRNA, while YBX1 acted as an m5C reader that recognized the modified transcript and enhanced its stability, thereby elevating PARP9 expression in BC cells. YBX1 could elevate PARP9 expression in BC cells. Moreover, NSUN2 knockdown restrained the malignant behaviors of BC cells, with PARP9 overexpression restored the effects. In addition, NSUN2 knockdown blocked tumorigenesis in vivo by regulating PARP9 expression. NSUN2-mediated m5C modification stabilizes PARP9 mRNA and is associated with enhanced BC progression and immune escape. The results broadened our understanding of the pathogenic mechanism of BC.
Universal behavior in far-from-equilibrium systems is driven by interactions between transport processes and noise structure. The Kardar-Parisi-Zhang (KPZ) framework predicts that extensions incorporating conserved currents or temporally correlated noise give rise to distinct growth morphologies and universality classes, yet direct experimental realization has remained elusive. Here, we report atomically resolved Sn thin-film growth on Sb-doped MnBi2Te4, revealing a sharp dynamical crossover between two fundamentally different regimes. Early-stage growth follows Villain-Lai-Das Sarma (VLDS) scaling, forming two-dimensional islands and stanene layers. Beyond a critical deposition time, temporally correlated noise becomes the dominant factor, driving the nucleation of α-Sn clusters, their development into faceted grains, and the coexistence of faceted β-Sn. Molecular dynamics simulations and Auger electron spectroscopy reveal that adatom escape serves as the microscopic origin of this temporally correlated noise, offering a mechanism for the observed universality crossover. These findings establish that temporal noise correlations can fundamentally alter the universality class of a growing interface, linking atomistic kinetics to emergent universal behavior.
Coronary Artery Disease (CAD) is a leading cause of mortality worldwide and is primarily associated with atherosclerotic plaque formation, resulting in coronary artery stenosis. The accurate prediction of CAD using deep learning models is often constrained by the limitations of conventional optimization techniques, including premature convergence and limited adaptability of the models. To address these challenges, this study proposes a Dynamic Optuna Hybrid Harris Hawks Optimization (Dynamic Optuna H-HHO) framework to enhance the performance of deep learning-based CAD prediction models. The proposed approach integrates dynamic parameter adjustment, adaptive escape energy mechanisms, and Optuna-based hyperparameter tuning. The framework was applied to optimize several deep-learning classifiers, including ResNet-50, VGG-16, InceptionV3, and MobileNet, using a coronary artery stenosis dataset. The performance was evaluated through a comparative analysis with models optimized using the conventional Hybrid Harris Hawks Optimization (H-HHO) algorithm. The experimental results indicate that the proposed Dynamic Optuna H-HHO framework consistently improves the predictive accuracy across all evaluated models. InceptionV3 achieved the highest accuracy of 97.9%, followed by MobileNet with 97.6%, compared with the maximum accuracy of 82.46% obtained using traditional HHO-based optimization. By combining adaptive optimization strategies with automated hyperparameter tuning, the proposed framework provides a robust and scalable solution for improving the accuracy of coronary artery disease prediction.
Neonatal seizures are high-risk clinical events that require rapid recognition but present subtle symptoms. Although simulation-based education supports clinical competence, the use of escape-room-based approaches in neonatal nursing education remains underexplored. The aim of the study was to explore the experiences of nursing students regarding escape room-based simulation for managing neonatal seizure scenarios. This descriptive qualitative study involved 8 undergraduate nursing students engaged in a 5-stage escape-room simulation using a high-fidelity neonatal simulator (Super Tory S2220, Gaumard Scientific), which included seizure assessment, cause identification, intervention selection, information interpretation, and communication. Data were collected through semistructured focus group interviews and researcher observations. Qualitative data were analyzed using Giorgi's descriptive phenomenological approach to identify essential themes. Five themes and 12 subthemes were organized across 5 experiential dimensions- environmental, cognitive, behavioral, relational, and value. These included authentic immersion within a high-stakes environment (environmental), practical expansion of theoretical knowledge (cognitive), strengthening clinical competence and enhancing self-efficacy (behavioral), collaborative team dynamics and multifaceted communication competence (relational), and professional self-reflection and formation of responsibility (value). As a promising gamified educational modality, escape-room-based simulations provide valuable insights into how students experience the enhancement of technical proficiency, clinical judgment, and teamwork, offering a valuable framework that prepares nursing students for complex, real-world neonatal emergency challenges.
In most animals, a small number of descending neurons (DNs) connect the brain to circuits and motor neurons (MNs) in the nerve cord. To understand how brain signals generate behavior, it is critical to understand the organization of the neural pathways linking DNs to MNs. In companion papers, we introduced a densely reconstructed connectome of the Drosophila Male Adult Nerve Cord (MANC; Takemura et al., 2024), including cell types and developmental lineages (Marin et al., 2024), which provides complete connectivity of the ventral nerve cord (VNC) at synaptic resolution. Here, we present a first look at the organization of the networks connecting DNs to MNs. We first proofread and curated all DNs and MNs, then systematically matched their morphology to light microscopy data. We report both broad organizational patterns of the entire network and fine-scale analysis of selected circuits of interest. We discover that direct DN-MN connections are infrequent and identify neuron communities putatively linked to control of different motor systems, including walking, flight steering and power generation, and coordinated action of wings and legs. Our analyses generate hypotheses for future functional experiments and empowers others to investigate these and other circuits of the VNC in richer mechanistic detail.
Piezocatalytic water splitting offers a sustainable route for hydrogen evolution, yet is challenged by weak polarity and slow charge separation kinetics under stress. Herein, we report strong polar hydroxylated Bi2O2CO3 (BOC) synthesized scalably, characterized by noncentrosymmetric (NCS) planar triangular [CO3] groups and surface hydroxyls, which applies as a robust piezocatalyst for hydrogen evolution. The aligned NCS planar [CO3] triangulars in BOC enable oriented accumulation of dipole moments to produce strong spontaneous polarization, and the intrinsic delocalized π-electrons within these structural units construct a conjugation freeway that minimizes charge migration resistance. Further external mechanical stress triggers a highly anisotropic lattice response; specifically, compression along the b-axis induces extreme geometric and electronic asymmetries that amplify the interlayer internal electric field (IEF) for charge separation. When synergistically coupled with surface hydroxylation, this stress-induced structural distortion significantly lowers the work function and interfacial kinetic barrier for electron escape. Consequently, the hydroxylated BOC catalyst achieves an ultrahigh piezocatalytic H2 evolution rate of 3055 µmol·g-1·h-1 and a record mechanical-to-hydrogen (MTH) energy conversion efficiency of 0.31% in pure water. It also maintains robust H2 evolution from real-world aquatic matrices, including rainwater, seawater, and antibiotic wastewater. This work establishes a polar group design-oriented paradigm for exploiting advanced piezocatalysts.
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Diabetic cardiomyopathy (DbCM), a severe complication of type 2 diabetes mellitus (T2DM), is driven by glucolipid metabolism dysregulation-induced oxidative stress and ferroptosis. Current clinical management, primarily relying on glucose-lowering agents, shows limited efficacy due to poor myocardial targeting efficiency and inability to intervene in these interconnected pro-death pathways. Here we develop a cardiac-targeting multifunctional nanozyme (Ru/CoMn-LDH@CTP) by loading Ru single atoms onto layered double hydroxides (LDHs) and conjugating with cardiac-targeting peptide (CTP) for DbCM treatment. This nanozyme exhibits synergistic superoxide dismutase/catalase-mimetic activities and acid stability for broad-spectrum reactive oxygen species (ROS) scavenging. CTP modification enables efficient myocardial targeting and lysosomal escape for mitochondrial delivery. In vitro, Ru/CoMn-LDH@CTP effectively alleviates glucolipotoxicity-induced oxidative damage, reducing mitochondrial ROS to 54.47% and cardiomyocyte apoptosis to 18.76% of those in the injury group. In a DbCM mouse model, the nanozyme selectively accumulates in cardiac lesions, attenuates oxidative damage, reduces fibrosis to 38.68% of the original level, thereby suppressing ventricular remodeling and promoting cardiac functional recovery. Mechanistically, Ru/CoMn-LDH@CTP inhibits ferroptosis by upregulating protective proteins (GPX4, xCT, and CAT), while downregulating ACSL1 and 4-HNE. This work presents a smart targeted nanozyme with multi-enzyme synergy and acid stability, offering a novel therapeutic strategy for DbCM and related cardiovascular diseases.
Programmed death ligand 1 (PD-L1)-driven immune escape is a crucial mechanism in the progression of hepatocellular carcinoma (HCC). Furthermore, Krüppel-like factor 16 (KLF16) may be a key molecule that regulates this process. Therefore, the role of KLF16 in PD-L1-driven immune escape in HCC was investigated. Hepa1-6 cells were subcutaneously injected into the right dorsum of BALB/c mice and nude mice to establish tumor-bearing models. The abundance of CD8+ T cells in tumor tissues was detected by flow cytometry. RT‒qPCR, Western blotting, immunofluorescence, and immunohistochemistry were used to analyze the expression of key genes and proteins. KLF16 is significantly highly expressed in HCC tissues and is associated with enrichment of the T-cell receptor signaling pathway. Knockdown of KLF16 inhibited PD-L1 expression in Hepa1-6 cells, whereas overexpression of KLF16 promoted PD-L1 expression. KLF16 knockdown significantly suppressed tumor growth, and the inhibitory effect was markedly stronger in immunocompetent mice than in nude mice. Additionally, KLF16 knockdown increased CD8+ T-cell infiltration in tumor-bearing tissues, as well as the number of granzyme B (GzmB)- and interferon-γ (IFN-γ)-positive CD8+ T cells, and enhanced the antitumor efficacy of combined anti-PD-1 and anti-CTLA-4 therapy. Mechanistically, KLF16 did not directly regulate PD-L1 transcription. Instead, KLF16 bound to the promoter of PRKDC (the gene encoding the catalytic subunit of DNA-dependent protein kinase, DNA-PK) and activated PRKDC transcription. The resulting elevation of DNA-PK then may phosphorylate PD-L1, thereby enhancing PD-L1 protein stability. KLF16 promotes PD-L1 expression by transcriptionally activating PRKDC, promoting immune escape in HCC. KLF16 knockdown enhances the efficacy of immune checkpoint inhibitors, suggesting that KLF16 is a potential target for HCC immunotherapy.
A mathematical model is developed to investigate the relative contribution of macromolecule transport across the glomerular filtration surface and that across mesangial area to glomerular size-selectivity. Endothelial fenestrae are assumed to be filled with glycosaminoglycans. Glomerular basement membrane (GBM) is a hydrogel containing two types of fibers. Slit diaphragm is a row of parallel cylinders with inter-fiber spacing following a lognormal distribution. Glomerular mesangium is viewed as a Brinkman medium with solute diffusivity and convection rate calculated from hydrodynamic forces exerted on confined spheres. Comparison between calculated sieving coefficients and those of Ficolls from in vivo studies demonstrates that inclusion of fluxes across the filtration surface and mesangial area, although capable of explaining small and medium-sized solute sieving, underestimates filtration of macromolecules with radii larger than 5 nm. Based on electron micrographs displaying red blood cells escaping through openings at the junction between the filtration surface and mesangium, the location with maximum shear stress, the present study examines effects of these openings using low-Reynolds-number hydrodynamics. Even though such effects on filtration of small and moderate-sized solutes are negligible, the presence of possibly shear-induced openings amplifies sieving of large macromolecules, yielding calculated sieving coefficients that agree well with those obtained from urinalysis in healthy humans and patients with diabetic nephropathy for the entire range of solute radii. While the glomerular filtration surface is the main pathway for small and moderate-sized solutes, main passages of large macromolecules are likely to be through the shear-induced openings, explaining the "upper limit" of glomerular size-selectivity.
Multiple myeloma (MM), a hematological malignancy, remains an incurable disease due to the development of resistance to the treatment; thus, there is an urgent need for new and effective therapeutic strategies, particularly for patients who do not respond to standard therapies. High levels of Cluster of Differentiation 47 (CD47) expression have been reported in MM and are associated with disease progression. CD47 acts as a cancer immune escape mechanism by binding to SIRPα protein, resulting in inhibiting phagocytosis of macrophages and NK cell activity. Therefore, blocking the CD47 signaling pathway has emerged as a promising strategy for cancer immunotherapy. In this study, we confirmed that MM cells have high CD47 expression. We generated and characterized a tri-specific killer engager targeting CD47, namely TriKE-CD47, that targets both CD47 on MM cells and CD16 on NK cells. Additionally, it incorporates an IL-15 moiety to enhance NK cell proliferation. TriKE-CD47 treatment promoted a remarkable proliferation of NK cells overexpressing CD16 (N6 cells). Co-culturing MM cells with N6 cells, primary NK cells, and monocyte-derived macrophages in the presence of 200 ng of TriKE-CD47 significantly improved NK cytotoxicity and macrophage phagocyte activities against MM cells. Notably, the efficacy of TriKE-CD47 was directly correlated with CD47 expression levels on the target cells reflecting the specificity of TriKE-CD47 to target antigen. Furthermore, TriKE-CD47 effectively suppressed tumor growth in MM xenograft mice models. Taken together, these findings strongly supported that TriKE-CD47 could be a potential therapeutic for MM patients.
Unpartnered heterosexual women constitute a significant demographic within nursing home populations, yet their sexuality remains largely understudied. Although recent healthcare policies and research focus on improving residents' sexual wellbeing, they often reflect heteronormative assumptions, for instance by prioritizing life-long sex for heterosexual monogamous couples. While the heteronormativity of nursing home spaces has been highlighted in critical gerontological approaches that draw on LGBTQ+ lived experiences, its structuring of heterosexual women's lived experiences remains under-researched. This paper explores this gap through the stories of two heterosexual women living in dementia or psychiatric care units in a Belgian nursing home, both with a history of sex work. Embedded in a wider study using in-depth interviews, talking group sessions, and participant observation, it examines their experiences, desires, and imagination of sex and how these are entangled within the nursing home space. It reveals notions of later life sex and desire that extend beyond frameworks focused on activity or performance, to include imagination as a meaningful dimension. Moreover, the women's imageries of alternative relationships, spaces, and strategies for evading the discomforts of institutional life emerge as disruptors of heteronormative assumptions. Through this analysis, we expand understandings of women's heterosexuality in later life beyond heteronormativity and beyond binaries of desexualization and successful ageing. We underscore the value of intersectional and Critical New Materialist lenses that center on embodiment and practices within (imagined) space, rather than focusing primarily on heterosexual identity. These perspectives illuminate textured lived experiences that contribute to diversified understandings of sexual wellbeing in later life.
Although opioids are effective analgesics, they can lead to problematic drug use behaviors that underlie opioid use disorder (OUD). Opioids also cause gut microbiota dysbiosis, which is linked to altered opioid responses. We used a longitudinal paradigm of voluntary oral morphine self-administration to capture multiple facets of drug seeking and preserve both individual behavioral responses and individual gut microbiota variation to investigate the role of the gut microbiota in a mouse model of OUD. Although all the mice consumed morphine, only a subset of the mice that transitioned to a state we defined statistically as compulsive. In compulsive mice, morphine constricted natural variability and fragmented the microbiota community networks, which convergently reorganized to form robust novel connections post-morphine. In contrast, the more variable communities of non-compulsive mice were highly interconnected during morphine disturbance and displayed more continuity post-morphine, suggesting greater flexibility and adaptability. Compulsive mice displayed a greater loss of functional diversity and a shift in favor of potential pathobionts, whereas non-compulsive mice better preserved genera associated with gut health and broader functional diversity. These findings highlight the potential role of persistent and stable opioid-induced microbiota dysbiosis in long-term behavioral changes underlying OUD and contributing to vulnerability to relapse.
RNA viruses form membraneless condensates in host cells to drive replication, but whether these compartments also regulate host RNAs remains unclear. Using MERFISH-based subcellular transcriptomics, we quantified cellular mRNA recruitment into Ebola virus condensates under basal and IFN-stimulated states. We find that in the basal state, cellular RNAs with minimally folded coding regions are selectively recruited. Under IFN-stimulation, however, interferon-stimulated genes (ISGs) with structured 3'UTRs concentrate in viral condensates. We find that both features, minimally folded coding regions and structured 3'UTRs, are conserved in the viral RNA genome, supporting viral genome retention in condensates. In parallel, for cellular mRNAs, we find that partitioning into condensates escapes decay, prolonging RNA-half-life, and amplifying rather than dampening ISG expression. Fruit bats, which do not experience severe disease for RNA viruses, instead have ISGs with reduced 3'UTR folding, and may evade condensate-sequestration, enabling balanced antiviral responses. This selective stabilization links condensate function to RNA regulation as a molecular determinant of viral and host co-evolution and disease pathogenesis.
Programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) constitute a core immune checkpoint axis that restrains T cell activation. Under physiological conditions, the interaction between PD-1 and PD-L1 maintains peripheral tolerance and prevents tissue damage from excessive immune responses. In tumor microenvironment, this axis is frequently activated through upregulation of PD-L1 on cancer cells, causing T cell dysfunction and tumor immune escape. PD-1/PD-L1 blockades reactivate exhausted T cells to kill cancer cells. However, some cancer patients fail to respond to PD-1/PD-L1 inhibitors, and the underlying mechanisms remain incompletely revealed. Thus, it is important to unveil the regulatory mechanisms that govern PD-1 and PD-L1 expression in tumor microenvironment. This review systematically summarizes the regulation of PD-1/PD-L1 expression at the genetic, epigenetic, transcriptional, and posttranscriptional levels. Beyond expression levels, posttranslational modifications and membrane trafficking determine PD‑L1 surface availability and sensitivity to PD-1/PD-L1 inhibitors. In this review, the rapidly expanding clinical trials of PD-1/PD-L1 blockades and future research directions are also discussed.