Caspase cleavage releases a nuclear protein fragment that stimulates phospholipid scrambling at the plasma membrane.

Phospholipid scrambling in dying cells promotes phosphatidylserine exposure, a critical process for efferocytosis. We previously identified the Xkr family protein Xkr4 as a phospholipid-scrambling protein, but its activation mechanisms remain unknown. Here we show that Xkr4 is activated in two steps: dimer formation by caspase-mediated cleavage and structural change caused by activating factors. To identify the factors, we developed a new screening system, "revival screening," using a CRISPR sgRNA library. Applying this system, we identified the nuclear protein XRCC4 as the single candidate for the Xkr4 activator. Upon apoptotic stimuli, XRCC4, contained in the DNA repair complex, is cleaved by caspases, and its C-terminal fragment with an intrinsically disordered region is released into the cytoplasm. Protein interaction screening showed that the fragment interacts directly with the Xkr4 dimer to activate it. This study demonstrates that caspase-mediated cleavage releases a nuclear protein fragment for direct regulation of lipid dynamics on the plasma membrane.

Genetic and phenotypic profiling of single living circulating tumor cells from patients with microfluidics.

Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non-small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction.

A novel fully human anti-NT-ANGPTL3 antibody from phage display library exhibits potent ApoB, TG, and LDL-C lowering activities in hyperlipidemia mice.

Dyslipidemia is characterized by elevated plasma levels of low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and TG-rich lipoprotein (TGRLs) in circulation, and is closely associated with the incidence and development of cardiovascular disease. Angiopoietin-like protein 3 (ANGPTL3) deficiency has been identified as a cause of familial combined hypolipidemia in humans, which allows it to be an important therapeutic target for reducing plasma lipids. Here, we report the discovery and characterization of a novel fully human antibody F1519-D95aA against N-terminal ANGPTL3 (NT-ANGPTL3), which potently inhibits NT-ANGPTL3 with a KD as low as 9.21 nM. In hyperlipidemic mice, F1519-D95aA shows higher apolipoprotein B (ApoB) and TG-lowering, and similar LDL-C reducing activity as compared to positive control Evinacumab (56.50% vs 26.01% decrease in serum ApoB levels, 30.84% vs 25.28% decrease in serum TG levels, 23.32% vs 22.52% decrease in serum LDLC levels, relative to vehicle group). Molecular docking and binding energy calculations reveal that the F1519-D95aA-ANGPTL3 complex (10 hydrogen bonds, -65.51 kcal/mol) is more stable than the Evinacumab-ANGPTL3 complex (4 hydrogen bonds, -63.76 kcal/mol). Importantly, F1519-D95aA binds to ANGPTL3 with different residues in ANGPTL3 from Evinacumab, suggesting that F1519-D95aA may be useful for the treatment of patients resistant to Evinacumab. In conclusion, F1519-D95aA is a novel fully human anti-NT-ANGPTL3 antibody with potent plasma ApoB, TG, and LDL-C lowering activities, which can potentially serve as a therapeutic agent for hyperlipidemia and relevant cardiovascular diseases.

Ribosomal profiling of human endogenous retroviruses in healthy tissues.

Human endogenous retroviruses (HERVs) are the germline embedded proviral fragments of ancient retroviral infections that make up roughly 8% of the human genome. Our understanding of HERVs in physiology primarily surrounds their non-coding functions, while their protein coding capacity remains virtually uncharacterized. Therefore, we applied the bioinformatic pipeline "hervQuant" to high-resolution ribosomal profiling of healthy tissues to provide a comprehensive overview of translationally active HERVs. We find that HERVs account for 0.1-0.4% of all translation in distinct tissue-specific profiles. Collectively, our study further supports claims that HERVs are actively translated throughout healthy tissues to provide sequences of retroviral origin to the human proteome.

Efficacy and safety of surufatinib plus toripalimab, a chemotherapy-free regimen, in patients with advanced gastric/gastroesophageal junction adenocarcinoma, esophageal squamous cell carcinoma, or biliary tract cancer.

BACKGROUND: The programmed death 1 inhibitor toripalimab plus the angio-immuno kinase inhibitor surufatinib showed a tolerable safety profile and preliminary efficacy in patients with advanced solid tumors in a phase I study. METHODS: This open-label, multi-cohort study in China enrolled patients with advanced solid tumors who had failed or were intolerable to standard treatment into tumor-specific cohorts. Patients received surufatinib (250 mg orally, once daily) plus toripalimab (240 mg intravenously, once every three weeks). Results for three cohorts (gastric/gastroesophageal junction [GC/GEJ] adenocarcinoma, esophageal squamous cell carcinoma [ESCC], and biliary tract carcinoma [BTC]) are reported here. The primary endpoint was investigator-assessed objective response rate (ORR) per Response Evaluation criteria in Solid Tumors version 1.1. RESULTS: Between December 17, 2019, and January 29, 2021, 60 patients were enrolled (GC/GEJ, n = 20; ESCC, n = 20; BTC, n = 20). At data cutoff (February 28, 2023), ORRs were 31.6%, 30.0%, and 11.1%, respectively. Median progression-free survival was 4.1, 2.7, and 2.9 months, respectively. Median overall survival was 13.7, 10.4, and 7.0 months, respectively. Overall, grade ≥ 3 treatment-related adverse events occurred in 28 (46.7%) patients. CONCLUSIONS: Surufatinib plus toripalimab showed promising antitumor activity and a tolerable safety profile in immunotherapy-naïve patients with GC/GEJ adenocarcinoma, ESCC, or BTC. These findings warrant further study in larger randomized trials comparing surufatinib plus toripalimab with standard therapies in these tumors. CLINICALTRIALS: gov NCT04169672.

Rapid Synthesis of 3-Methyleneisoindolin-1-ones via Metal-Free Tandem Reactions of Ester-Functionalized Aziridines.

We have disclosed a novel metal-free tandem cyclization reaction for the synthesis of 3-methyleneisoindolin-1-ones starting from ester-functionalized aziridines. This strategy can be effectively promoted by DBU and carboxylic acids. Mechanistically, it involves sequential ring opening of aziridines with carboxylic acids, lactamization, and elimination of carboxylic acids.

Copper Catalyzed Three-Component Ullmann C-S Coupling in PEG for the Synthesis of 6-Aryl/alkylthio-purines.

To tackle the environmental unfriendly issue in existing synthesis strategies for 6-substitued thiopurine derivatives, such as poor step economy, frequent use of malodorous organic sulfur starting materials, toxic organic solvents, and equivalent dosage of base, we have developed a CuI-catalyzed base-free three-component Ullmann C-S coupling synthetic strategy, featured using inorganic salt Na2S as the sulfur source and nontoxic PEG-600 as the solvent. The newly developed strategy is particularly effective for the synthesis of 6-arylthiopurines. The high catalytic efficiency in PEG-600 can be rationalized by the high soluble ability of CuI catalyst, likely due to the presence of multiple oxygen coordination sites in PEG.

Dietary inflammatory index is associated with severe depression in older adults with stroke: a cross-sectional study.

Inflammation is involved in the pathogenesis of stroke and depression. We aimed to investigate the association between the dietary inflammatory index (DII) and depression in American adults with stroke. Adults with stroke were enrolled in the National Health and Nutrition Examination Survey between 2005 and 2018 in the USA. The DII was obtained from a 24-h dietary recall interview for each individual. Multivariate regression and restricted cubic spline analyses were conducted to evaluate the association between DII and depression in adults with stroke. The mean age of the 1239 participants was 63·85 years (50·20 % women), and the prevalence of depression was 18·26 %. DII showed a linear and positive association with severe depression in adults with stroke (OR 1·359; 95 % CI 1·021, 1·810; P for non-linearity = 0·493). Compared with those in the lowest tertile of the DII, adults with stroke in the third tertile of the DII had a 3·222-fold higher risk of severe depression (OR 3·222; 95 % CI 1·150, 9·026). In the stratified analyses, the association between DII score and severe depression was more significant in older adults (P for interaction = 0·010) but NS with respect to sex (P for interaction = 0·184) or smoking status (P for interaction = 0·396). No significant association was found between DII and moderate-to-moderately severe depression in adults with stroke. In conclusion, an increase in DII score was associated with a higher likelihood of severe depression in older adults with stroke.

Clinical features of anti-melanoma differentiation-associated gene 5 antibody-positive dermatomyositis with macrophage activation syndrome.

OBJECTIVES: This study aimed to describe the clinical features of patients with anti-melanoma differentiation-associated gene 5 (anti-MDA5) antibody-positive dermatomyositis (DM) who had macrophage activation syndrome (MAS). METHODS: We retrospectively examined 44 patients with anti-MDA5-positive DM and compared the clinical features between patients with MAS (n = 11) and those without (n=33). Patients without MAS were selected randomly in the same year as those with MAS at a ratio of 3:1. Among patients with MAS, we compared the features between non-survivors and survivors. We used Fisher's exact test, Student's t test, the Mann-Whitney U test and the log-rank test for statistical analysis. RESULTS: Patients complicated with MAS had a significantly higher incidence of infection, heliotrope sign, Gottron's papule, V-neck sign, and higher serum levels of ferritin, aspartate aminotransferase (AST), lactic dehydrogenase (LDH), and creatine kinase (CK) than those without MAS (p<0.05). Among the 11 patients with MAS, 4 (36.4%) died after intensive treatment. Deceased patients were older, given more combination therapy with tofacitinib (TOF) and had a higher incidence of rapid progressive interstitial lung disease, infection, heart failure and renal impairment than those who survived (p<0.05). CONCLUSIONS: Among anti-MDA5-positive DM, Infection, DM typical rashes, and higher serum levels of ferritin, AST, LDH, and CK were more common in patients complicated with MAS. The mortality of patients with MAS was high, particularly among patients who were older, given more combination therapy with TOF, and had RP-ILD, infection, heart failure and renal impairment.

Expansion and surface makers of age-associated B cells in IgG4-related disease.

OBJECTIVES: To assess the expression of age-associated B cells (ABCs), and characterise the surface markers of ABCs in patients with IgG4-related disease (IgG4-RD). METHODS: Fifty-one newly diagnosed patients with IgG4-RD, 18 IgG4-RD patients with disease remission, 34 patients with other autoimmune diseases, and 61 age- and sex-matched healthy controls (HCs) were included. Circulating ABCs, as well as surface markers were detected by flow cytometry, and tissue infiltration of ABCs were assessed by immunofluorescence (IF). The expression of ABCs in the affected organs of LatY136F knock-in (LAT) mouse models (IgG4-RD mouse model) were explored by flow cytometry and IF. RESULTS: The percentages and absolute numbers of ABCs (gated as CD21-T-bet+CD11c+) in CD19+ B cells raised remarkably in untreated IgG4-RD patients than HC, and reduced significantly after treatment. The percentage of CD27+ABCs, DN2 B cells and activated naive B cells was higher in patients with IgG4-RD than in HCs and patients with multiple autoimmune diseases, whereas the percentage was comparable with that in patients with systemic lupus erythematosus. Phenotypical analysis revealed upregulated levels of CD86, TACI, CD38, and downregulated level of CXCR3 in peripheral CD19+CD21-CD11c+ B cells of IgG4-RD patients compared with that of HC. In IgG4-RD patients, CD19+CD21- CD11c+ cells expressed higher levels of CD80, CXCR3, TACI, CD95, and BAFF-R, while lower levels of CD86, CD27, CD38, and CXCR5 compared with CD19+ CD21- CD11c- B cells. ABCs (CD11c+T-bet+ gated in B220+ cells) were increased significantly in lungs of LAT mice than that of wild type (WT) mice. CONCLUSIONS: ABCs were expanded both in the peripheral blood and affected tissues of patients with IgG4-RD as well as in the lungs of LAT mice, indicating the potential roles of ABCs in IgG4-RD pathogenesis.

UiO-66/PIM-1 Mixed-Matrix Membrane for Hexane Isomer Separation.

The separation of high-octane dibranched alkanes from naphtha is critical in the refining of gasoline. To date, research on the membrane-based separation of alkane isomers has been limited, with a particular paucity of investigations into mixed-matrix membranes. Herein, the continuous and dense UiO-66/PIM-1 mixed-matrix membrane, which was prepared through precise control of the interfacial structure, was first applied to the differentiation of C6 alkane isomers. Due to the synergistic combination of UiO-66 with differential adsorption capabilities for alkanes and PIM-1 that possesses a cross-linkable structure, the resulting UiO-66/PIM-1-(20) membrane demonstrated remarkable separation performance and high stability. Pervaporation measurements showed that the mass fraction of 2,2-dimethylbutane in the feed side was increased from 50.0 to 75.8 wt % while an excellent flux of 1700 g m-2 h-1 was maintained over a continuous 40 h period. The UiO-66/PIM-1-(20) membrane, characterized by its facile replication and processing, shows potential for large-scale fabrication. This study offers a new approach to the membrane separation of alkane isomers.

Construction and analysis of the immune effect of two different vaccine types based on Vibrio harveyi VgrG.

Vibrio harveyi poses a significant threat to fish and invertebrates in mariculture, resulting in substantial financial repercussions for the aquaculture sector. Valine-glycine repeat protein G (VgrG) is essential for the type VI secretion system's (T6SS) assembly and secretion. VgrG from V. harveyi QT520 was cloned and analyzed in this study. The localization of VgrG was determined by Western blot, which revealed that it was located in the cytoplasm, secreted extracellularly, and attached to the membrane. The effectiveness of two vaccinations against V. harveyi infection-a subunit vaccine (rVgrG) and a DNA vaccine (pCNVgrG) prepared with VgrG was evaluated. The findings indicated that both vaccines provided a degree of protection against V. harveyi challenge. At 4 weeks post-vaccination (p.v.), the rVgrG and pCNVgrG exhibited relative percent survival rates (RPS) of 71.43% and 76.19%, respectively. At 8 weeks p.v., the RPS for rVgrG and pCNVgrG were 68.21% and 72.71%, respectively. While both rVgrG and pCNVgrG elicited serum antibody production, the subunit vaccinated fish demonstrated significantly higher levels of serum anti-VgrG specific antibodies than the DNA vaccine group. The result of qRT-PCR demonstrated that the expression of major histocompatibility complex (MHC) class Iα, tumor necrosis factor-alpha (TNF-α), interferon γ (IFNγ), and cluster of differentiation 4 (CD4) were up-regulated by both rVgrG and pCNVgrG. Fish vaccinated with rVgrG and pCNVgrG exhibited increased activity of acid phosphatase, alkaline phosphatase, superoxide dismutase, and lysozyme. These findings suggest that VgrG from V. harveyi holds potential for application in vaccination.

Benzaldehyde acts as a behaviorally active component in brewer's yeast protein powder which attracts B. dorsalis through olfaction.

The Oriental fruit fly, Bactrocera dorsalis, is a significant pest that damages a variety of fruit crops. The effectiveness of chemical pesticides against such pests is limited, raising concerns about pesticide residues and resistance. Proteins naturally attract B. dorsalis and have led to the development of a management strategy known as protein bait attractant technology (BAT). Although the attraction of protein sources to B. dorsalis is well-documented, the biologically active components within these sources are not fully understood. This study employed analytical chemistry, behavioral tests, and electrophysiological techniques to investigate the behaviorally active components of beer yeast protein powder (BYPD), aiming to provide a basis for improving and developing protein baits. An olfactory trap assay confirmed the attractiveness of BYPD, and five components with high abundance were identified from its headspace volatiles using GC-MS. These components included ethanol, isoamyl alcohol, ethyl decanoate, benzaldehyde, and phenylethyl alcohol. Mixtures of these five components demonstrated significant attraction to B. dorsalis adults, with benzaldehyde identified as a potential key component. The attractiveness of benzaldehyde required a relatively large dose, and it was most attractive to adults that had been starved from dusk until the following morning. Attraction of adult flies to benzaldehyde appeared mainly mediated by inputs from olfactory receptors. While EAG data supports that ionotropic receptors could influence the detection of benzaldehyde in female adults, they did not affect female behavior towards benzaldehyde. These findings indicate that benzaldehyde is an important behaviorally active component in BYPD and offer insights for developing novel protein lures to control B. dorsalis in an environmentally friendly manner.

Structure-Guided Discovery of Diverse Cytotoxic Dimeric Xanthones/Chromanones from Penicillium chrysogenum C-7-2-1 and Their Interconversion Properties.

Xanthone-chromanone homo- or heterodimers are regarded as a novel class of topoisomerase (Topo) inhibitors; however, limited information about these compounds is currently available. Here, 14 new (1-14) and 6 known tetrahydroxanthone chromanone homo- and heterodimers (15-20) are reported as isolated from Penicillium chrysogenum C-7-2-1. Their structures and absolute configurations were unambiguously demonstrated by a combination of spectroscopic data, single-crystal X-ray diffraction, modified Mosher's method, and electronic circular dichroism analyses. Plausible biosynthetic pathways are proposed. For the first time, it was discovered that tetrahydroxanthones can convert to chromanones in water, whereas chromone dimerization does not show this property. Among them, compounds 5, 7, 8, and 16 exhibited significant cytotoxicity against H23 cell line with IC50 values of 6.9, 6.4, 3.9, and 2.6 µM, respectively.

Old trees bloom new flowers, lysosome targeted near-infrared fluorescent probe for ratiometric sensing of hypobromous acid in vitro and in vivo based on Nile red skeleton.

Hypobromous acid (HOBr), one of the significant reactive oxygen species (ROS) that acts as an important role in human immune system, however the increasing level of HOBr in human body can cause the disorder of eosinophils (EPO), leading to oxidative stress in organelles, and further causing a series of diseases. In this study, a ratiometric fluorescent probe DMBP based on Nile red skeleton was developed to detect HOBr specifically by the electrophilic substitution with HOBr. DMBP emits near-infrared (NIR) fluorescence at 653 nm, after reacting with HOBr, the emission wavelength of DMBP shifted blue and a new peak appeared at 520 nm, realizing a ratiometric examination of HOBr with a limit of detection of 89.00 nM. Based on its sensitive and specific response to HOBr, DMBP was applied in the visual imaging of HOBr in HepG2 cells and zebrafish. Foremost, probe DMBP has excellent lysosome targeting ability and NIR emission reduced the background interference of biological tissues, providing a potential analytical tool to further investigate the role of HOBr in lysosome.

Construction of cellulose-based hybrid hydrogel beads containing carbon dots and their high performance in the adsorption and detection of mercury ions in water.

Cellulose-based adsorbents have been extensively developed in heavy metal capture and wastewater treatment. However, most of the reported powder adsorbents suffer from the difficulties in recycling due to their small sizes and limitations in detecting the targets for the lack of sensitive sensor moieties in the structure. Accordingly, carbon dots (CDs) were proposed to be encapsulated in cellulosic hydrogel beads to realize the simultaneous detection and adsorption of Hg (II) in water due to their excellent fluorescence sensing performance. Besides, the molding of cellulose was beneficial to its recycling and further reduced the potential environmental risk generated by secondary pollution caused by adsorbent decomposition. In addition, the detection limit of the hydrogel beads towards Hg (II) reached as low as 8.8 × 10-8 M, which was below the mercury effluent standard declared by WHO, exhibiting excellent practicability in Hg (II) detection and water treatment. The maximum adsorption capacity of CB-50 % for Hg (II) was 290.70 mg/g. Moreover, the adsorbent materials also had preeminent stability that the hydrogel beads could maintain sensitive and selective sensing performance towards Hg (II) after 2 months of storage. Additionally, only 3.3% of the CDs leaked out after 2 weeks of immersion in water, ensuring the accuracy of Hg (II) evaluation. Notably, the adsorbent retained over 80% of its original adsorption capacity after five consecutive regeneration cycles, underscoring its robustness and potential for sustainable environmental applications.

Application Prospect of Ion-Imprinted Polymers in Harmless Treatment of Heavy Metal Wastewater.

With the rapid development of industry, the discharge of heavy metal-containing wastewater poses a significant threat to aquatic and terrestrial environments as well as human health. This paper provides a brief introduction to the basic principles of ion-imprinted polymer preparation and focuses on the interaction between template ions and functional monomers. We summarized the current research status on typical heavy metal ions, such as Cu(II), Ni(II), Cd(II), Hg(II), Pb(II), and Cr(VI), as well as metalloid metal ions of the As and Sb classes. Furthermore, it discusses recent advances in multi-ion-imprinted polymers. Finally, the paper addresses the challenges faced by ion-imprinted technology and explores its prospects for application.

A Dual-Bond Crosslinking Strategy Enabling Resilient and Recyclable Electrolyte Elastomers for Solid-State Lithium Metal Batteries.

Elastomeric solid polymer electrolytes (SPEs) are highly promising to address the solid-solid-interface issues of solid-state lithium metal batteries (LMBs), but compromises have to be made to balance the intrinsic trade-offs among their conductive, resilient and recyclable properties. Here, we propose a dual-bond crosslinking strategy for SPEs to realize simultaneously high ionic conductivity, elastic resilience and recyclability. An elastomeric SPE is therefore designed with hemiaminal dynamic covalent networks and Li+-dissociation co-polymer chains, where the -C-N- bond maintains the load-bearing covalent network under stress but is chemically reversible through a non-spontaneous reaction, the weaker intramolecular hydrogen bond is mechanically reversible, and the soft chains endow the rapid ion conduction. With this delicate structure, the optimized SPE elastomer achieves high elastic resilience without loading-unloading hysteresis, outstanding ionic conductivity of 0.2 mS cm-1 (25 °C) and chemical recyclability. Then, exceptional room-temperature performances are obtained for repeated Li plating/stripping tests, and stable cycling of LMBs with either LiFePO4 or 4.3 V-class LiFe0.2Mn0.8PO4 cathode. Furthermore, the recycled and reprocessed SPEs can be circularly reused in LMBs without significant performance degradation. Our findings provide an inspiring design principle for SPEs to address the solid-solid-interface and sustainability challenges of solid-state LMBs.

Largely Pseudocapacitive Two-Dimensional Conjugated Metal-Organic Framework Anodes with Lowest Unoccupied Molecular Orbital Localized in Nickel-bis(dithiolene) Linkages.

Although two-dimensional conjugated metal-organic frameworks (2D c-MOFs) provide an ideal platform for precise tailoring of capacitive electrode materials, high-capacitance 2D c-MOFs for non-aqueous supercapacitors remain to be further explored. Herein, we report a novel phthalocyanine-based nickel-bis(dithiolene) (NiS4)-linked 2D c-MOF (denoted as Ni2[CuPcS8]) with outstanding pseudocapacitive properties in 1 M TEABF4/acetonitrile. Each NiS4 linkage is disclosed to reversibly accommodate two electrons, conferring the Ni2[CuPcS8] electrode a two-step Faradic reaction with a record-high specific capacitance among the reported 2D c-MOFs in non-aqueous electrolytes (312 F g-1) and remarkable cycling stability (93.5% after 10,000 cycles). Multiple analyses unveil that the unique electron-storage capability of Ni2[CuPcS8] originates from its localized lowest unoccupied molecular orbital (LUMO) over the nickel-bis(dithiolene) linkage, which allows the efficient delocalization of the injected electrons throughout the conjugated linkage units without inducing apparent bonding stress. The Ni2[CuPcS8] anode is used to demonstrate an asymmetric supercapacitor device that delivers a high operating voltage of 2.3 V, a maximum energy density of 57.4 Wh kg-1, and ultralong stability over 5000 cycles.

P2X7 receptor is required for the ototoxicity caused by aminoglycoside in developing cochlear hair cells.

Aminoglycoside antibiotics (AGAs) are widely used in life-threatening infections, but they accumulate in cochlear hair cells (HCs) and result in hearing loss. Increases in adenosine triphosphate (ATP) concentrations and P2X7 receptor expression were observed after neomycin treatment. Here, we demonstrated that P2X7 receptor, which is a non-selective cation channel that is activated by high ATP concentrations, may participate in the process through which AGAs enter hair cells. Using transgenic knockout mice, we found that P2X7 receptor deficiency protects HCs against neomycin-induced injury in vitro and in vivo. Subsequently, we used fluorescent gentamicin-Fluor 594 to study the uptake of AGAs and found fluorescence labeling in wild-type mice but not in P2rx7-/- mice in vitro. In addition, knocking-out P2rx7 did not significantly alter the HC count and auditory signal transduction, but it did inhibit mitochondria-dependent oxidative stress and apoptosis in the cochlea after neomycin exposure. We thus conclude that the P2X7 receptor may be linked to the entry of AGAs into HCs and is likely to be a therapeutic target for auditory HC protection.