A microbial knowledge graph-based deep learning model for predicting candidate microbes for target hosts.

Predicting interactions between microbes and hosts plays critical roles in microbiome population genetics and microbial ecology and evolution. How to systematically characterize the sophisticated mechanisms and signal interplay between microbes and hosts is a significant challenge for global health risks. Identifying microbe-host interactions (MHIs) can not only provide helpful insights into their fundamental regulatory mechanisms, but also facilitate the development of targeted therapies for microbial infections. In recent years, computational methods have become an appealing alternative due to the high risk and cost of wet-lab experiments. Therefore, in this study, we utilized rich microbial metagenomic information to construct a novel heterogeneous microbial network (HMN)-based model named KGVHI to predict candidate microbes for target hosts. Specifically, KGVHI first built a HMN by integrating human proteins, viruses and pathogenic bacteria with their biological attributes. Then KGVHI adopted a knowledge graph embedding strategy to capture the global topological structure information of the whole network. A natural language processing algorithm is used to extract the local biological attribute information from the nodes in HMN. Finally, we combined the local and global information and fed it into a blended deep neural network (DNN) for training and prediction. Compared to state-of-the-art methods, the comprehensive experimental results show that our model can obtain excellent results on the corresponding three MHI datasets. Furthermore, we also conducted two pathogenic bacteria case studies to further indicate that KGVHI has excellent predictive capabilities for potential MHI pairs.

Gasdermin D drives focal crystalline thrombotic microangiopathy by accelerating immunothrombosis and necroinflammation.

ABSTRACT: Thrombotic microangiopathy (TMA) is characterized by immunothrombosis and life-threatening organ failure but the precise underlying mechanism driving its pathogenesis remains elusive. In this study, we hypothesized that gasdermin D (GSDMD), a pore-forming protein that serves as the final downstream effector of the pyroptosis/interleukin-1ß (IL-1ß) pathway, contributes to TMA and its consequences by amplifying neutrophil maturation and subsequent necrosis. Using a murine model of focal crystalline TMA, we found that Gsdmd deficiency ameliorated immunothrombosis, acute tissue injury, and failure. Gsdmd-/- mice exhibited a decrease in mature IL-1ß, as well as in neutrophil maturation, ß2-integrin activation, and recruitment to TMA lesions, in which they formed reduced neutrophil extracellular traps in both arteries and interstitial tissue. The GSDMD inhibitor disulfiram dose-dependently suppressed human neutrophil pyroptosis in response to cholesterol crystals. Experiments with GSDMD-deficient, human-induced, pluripotent stem cell-derived neutrophils confirmed the involvement of GSDMD in neutrophil ß2-integrin activation, maturation, and pyroptosis. Both prophylactic and therapeutic administration of disulfiram protected the mice from focal TMA, acute tissue injury, and failure. Our data identified GSDMD as a key mediator of focal crystalline TMA and its consequences, including ischemic tissue infarction and organ failure. GSDMD could potentially serve as a therapeutic target for the systemic forms of TMA.

GlcNAc-1,6-anhydro-MurNAc Moiety Affords Unusual Glycosyl Acceptor that Terminates Peptidoglycan Elongation.

Peptidoglycan (PG), an essential exoskeletal polymer in bacteria, is a well-known antibiotic target. PG polymerization requires the action of bacterial transglycosylases (TGases), which couple the incoming glycosyl acceptor to the donor. Interfering with the TGase activity can interrupt the PG assembly. Existing TGase inhibitors like moenomycin and Lipid II analogues always occupy the TGase active sites; other strategies to interfere with proper PG elongation have not been widely exploited. Inspired by the natural 1,6-anhydro-MurNAc termini that mark the ends of PG strands in bacteria, we hypothesized that the incorporation of an anhydromuramyl-containing glycosyl acceptor by TGase into the growing PG may effectively inhibit PG elongation. To explore this possibility, we synthesized 4-O-(N-acetyl-ß-d-glucosaminyl)-1,6-anhydro-N-acetyl-ß-d-muramyl-l-Ala-γ-d-Glu-l-Lys-d-Ala-d-Ala, 1, within 15 steps, and demonstrated that this anhydromuropeptide and its analogue lacking the peptide, 1-deAA, were both utilized by bacterial TGase as noncanonical anhydro glycosyl acceptors in vitro. The incorporation of an anhydromuramyl moiety into PG strands by TGases afforded efficient termination of glycan chain extension. Moreover, the preliminary in vitro studies of 1-deAA against Staphylococcus aureus showed that 1-deAA served as a reasonable antimicrobial adjunct of vancomycin. These insights imply the potential application of such anhydromuropeptides as novel classes of PG-terminating inhibitors, pointing toward novel strategies in antibacterial agent development.

Inhibition of complement factor C5a or C5aR for cholesterol crystal embolism-related vascular thrombosis with microvascular injury and its consequences.

Cholesterol crystal embolism (CCE) implies immunothrombosis, tissue necrosis, and organ failure but no specific treatments are available. As CCE involves complement activation, we speculated that inhibitors of the C5a/C5aR axis would be sufficient to attenuate the consequences of CCE like that with systemic vasculitis. Cholesterol microcrystal injection into the kidney artery of wildtype mice initiated intra-kidney immunothrombosis within a few hours followed by a sudden drop of glomerular filtration rate and ischemic kidney necrosis after 24 hours. Genetic deficiency of either C3 or C5aR prevented immunothrombosis, glomerular filtration rate drop, and ischemic necrosis at 24 hours as did preemptive treatment with inhibitors of either C5a or C5aR. Delayed C5a blockade after crystal injection still resolved crystal clots and prevented all consequences. Thus, selective blockade of C5a or C5aR is sufficient to attenuate the consequences of established CCE and prospective inhibition in high-risk patients may be clinically feasible and safe.

Large-Substrate-Terrace Confined Growth of Arrayed Ultrathin PtSe2 Ribbons on Step-Bunched Vicinal Au(001) Facets Toward Electrocatalytic Applications.

Ultrathin PtSe2 ribbons can host spin-polarized edge states and distinct edge electrocatalytic activity, emerging as a promising candidate for versatile applications in various fields. However, the direct synthesis is still challenging and the growth mechanism is still unclear. Herein, the arrayed growth of ultrathin PtSe2 ribbons on bunched vicinal Au(001) facets, via a facile chemical vapor deposition (CVD) route is reported. The ultrathin PtSe2 flakes can transform from traditional irregular shapes to desired ribbon shapes by increasing the height of bunched and unidirectionally oriented Au steps (with step height hstep) is found. This crossover, occurring at hstep ≈ 3.0 nm, defines the tailored growth from step-flow to single-terrace-confined modes, as validated by density functional theory calculations of the different system energies. On the millimeter-scale single-crystal Au(001) films with aligned steps, the arrayed ultrathin PtSe2 ribbons with tunable width of ≈20-1000 nm, which are then served as prototype electrocatalysts for hydrogen evolution reaction (HER) is achieved. This work should represent a huge leap in the direct synthesis and the mechanism exploration of arrayed ultrathin transition-metal dichalcogenides (TMDCs) ribbons, which should stimulate further explorations of the edge-related physical properties and practical applications.

Difference analysis and characteristics of incompatibility group plasmid replicons in gram-negative bacteria with different antimicrobial phenotypes in Henan, China.

BACKGROUND: Multi-drug-resistant organisms (MDROs) in gram-negative bacteria have caused a global epidemic, especially the bacterial resistance to carbapenem agents. Plasmid is the common vehicle for carrying antimicrobial resistance genes (ARGs), and the transmission of plasmids is also one of the important reasons for the emergence of MDROs. Different incompatibility group plasmid replicons are highly correlated with the acquisition, dissemination, and evolution of resistance genes. Based on this, the study aims to identify relevant characteristics of various plasmids and provide a theoretical foundation for clinical anti-infection treatment. METHODS: 330 gram-negative strains with different antimicrobial phenotypes from a tertiary hospital in Henan Province were included in this study to clarify the difference in incompatibility group plasmid replicons. Additionally, we combined the information from the PLSDB database to elaborate on the potential association between different plasmid replicons and ARGs. The VITEK mass spectrometer was used for species identification, and the VITEK-compact 2 automatic microbial system was used for the antimicrobial susceptibility test (AST). PCR-based replicon typing (PBRT) detected the plasmid profiles, and thirty-three different plasmid replicons were determined. All the carbapenem-resistant organisms (CROs) were tested for the carbapenemase genes. RESULTS: 21 plasmid replicon types were detected in this experiment, with the highest prevalence of IncFII, IncFIB, IncR, and IncFIA. Notably, the detection rate of IncX3 plasmids in CROs is higher, which is different in strains with other antimicrobial phenotypes. The number of plasmid replicons they carried increased with the strain resistance increase. Enterobacterales took a higher number of plasmid replicons than other gram-negative bacteria. The same strain tends to have more than one plasmid replicon type. IncF-type plasmids tend to be associated with MDROs. Combined with PLSDB database analysis, IncFII and IncX3 are critical platforms for taking blaKPC-2 and blaNDM. CONCLUSIONS: MDROs tend to carry more complex plasmid replicons compared with non-MDROs. The plasmid replicons that are predominantly prevalent and associated with ARGs differ in various species. The wide distribution of IncF-type plasmids and their close association with MDROs should deserve our attention. Further investigation into the critical role of plasmids in the carriage, evolution, and transmission of ARGs is needed.

Development and application of whole-chromosome painting of chromosomes 7A and 8A of Arachis duranensis based on chromosome-specific single-copy oligonucleotides.

For peanut, the lack of stable cytological markers is a barrier to tracking specific chromosomes, elucidating the genetic relationships between genomes and identifying chromosomal variations. Chromosome mapping using single-copy oligonucleotide (oligo) probe libraries has unique advantages for identifying homologous chromosomes and chromosomal rearrangements. In this study, we developed two whole-chromosome single-copy oligo probe libraries, LS-7A and LS-8A, based on the reference genome sequences of chromosomes 7A and 8A of Arachis duranensis. Fluorescence in situ hybridization (FISH) analysis confirmed that the libraries could specifically paint chromosomes 7 and 8. In addition, sequential FISH and electronic localization of LS-7A and LS-8A in A. duranensis (AA) and A. ipaensis (BB) showed that chromosomes 7A and 8A contained translocations and inversions relative to chromosomes 7B and 8B. Analysis of the chromosomes of wild Arachis species using LS-8A confirmed that this library could accurately and effectively identify A genome species. Finally, LS-7A and LS-8A were used to paint the chromosomes of interspecific hybrids and their progenies, which verified the authenticity of the interspecific hybrids and identified a disomic addition line. This study provides a model for developing specific oligo probes to identify the structural variations of other chromosomes in Arachis and demonstrates the practical utility of LS-7A and LS-8A.

Regulation mechanism and bioactivity characteristic of surfactin homologues with C14 and C15 fatty acid chains.

BACKGROUND: Surfactin, a green lipopeptide bio-surfactant, exhibits excellent surface, hemolytic, antibacterial, and emulsifying activities. However, a lack of clear understanding of the synthesis regulation mechanism of surfactin homologue components has hindered the customized production of surfactin products with different biological activities. RESULTS: In this study, exogenous valine and 2-methylbutyric acid supplementation significantly facilitated the production of C14-C15 surfactin proportions (up to 75% or more), with a positive correlation between the homologue proportion and fortified concentration. Subsequently, the branched-chain amino acid degradation pathway and the glutamate synthesis pathway are identified as critical pathways in regulating C14-C15 surfactin synthesis by transcriptome analysis. Overexpression of genes bkdAB and glnA resulted in a 1.4-fold and 1.3-fold increase in C14 surfactin, respectively. Finally, the C14-rich surfactin was observed to significantly enhance emulsification activity, achieving an EI24 exceeding 60% against hexadecane, while simultaneously reducing hemolytic activity. Conversely, the C15-rich surfactin demonstrated an increase in both hemolytic and antibacterial activities. CONCLUSION: This study presents the first evidence of a potential connection between surfactin homologue synthesis and the conversion of glutamate and glutamine, providing a theoretical basis for targeting the synthesis regulation and structure-activity relationships of surfactin and other lipopeptide compounds.

A cell-free fluorescence biosensor based on allosteric transcription factor NalC for detection of pentachlorophenol.

Pentachlorophenol (PCP) was once used as a pesticide, germicide, and preservative due to its stable properties and resistance to degradation. This study aimed to design a biosensor for the quantitative and prompt detection of capable of PCP. A cell-free fluorescence biosensor was developed while employing NalC, an allosteric Transcription Factor responsive to PCP and In Vitro Transcription. By adding a DNA template and PCP and employing Electrophoretic Mobility Shift Assay while monitoring the dynamic fluorescence changes in RNA, this study offers evidence of NalC's potential applicability in sensor systems developed for the specific detection of PCP. The biosensor showed the capability for the quantitative detection of PCP, with a Limit of Detection (LOD) of 0.21 µM. Following the addition of Nucleic Acid Sequence-Based Amplification, the fluorescence intensity of RNA revealed an excellent linear relationship with the concentration of PCP, showing a correlation coefficient (R2) of 0.9595. The final LOD was determined to be 0.002 µM. This study has successfully translated the determination of PCP into a fluorescent RNA output, thereby presenting a novel approach for detecting PCP within environmental settings.

Cylindrospermopsin enhances the conjugative transfer of plasmid-mediated multi-antibiotic resistance genes through glutathione biosynthesis inhibition.

Cylindrospermopsin (CYN), a cyanobacterial toxin, has been detected in the global water environment. However, information concerning the potential environmental risk of CYN is limited, since the majority of previous studies have mainly focused on the adverse health effects of CYN through contaminated drinking water. The present study reported that CYN at environmentally relevant levels (0.1-100 µg/L) can significantly enhance the conjugative transfer of RP4 plasmid in Escherichia coli genera, wherein application of 10 µg/L of CYN led to maximum fold change of ∼6.5- fold at 16 h of exposure. Meanwhile, evaluation of underlying mechanisms revealed that environmental concentration of CYN exposure could increase oxidative stress in the bacterial cells, resulting in ROS overproduction. In turn, this led to an upregulation of antioxidant enzyme-related genes to avoid ROS attack. Further, inhibition of the synthesis of glutathione (GSH) was also detected, which led to the rapid depletion of GSH in cells and thus triggered the SOS response and promoted the conjugative transfer process. Increase in cell membrane permeability, upregulation of expression of genes related to pilus generation, ATP synthesis, and RP4 gene expression were also observed. These results highlight the potential impact on the spread of antimicrobial resistance in water environments.

Isolation and Functional Characterization of a Constitutive Promoter in Upland Cotton (Gossypium hirsutum L.).

Multiple cis-acting elements are present in promoter sequences that play critical regulatory roles in gene transcription and expression. In this study, we isolated the cotton FDH (Fiddlehead) gene promoter (pGhFDH) using a real-time reverse transcription-PCR (qRT-PCR) expression analysis and performed a cis-acting elements prediction analysis. The plant expression vector pGhFDH::GUS was constructed using the Gateway approach and was used for the genetic transformation of Arabidopsis and upland cotton plants to obtain transgenic lines. Histochemical staining and a ß-glucuronidase (GUS) activity assay showed that the GUS protein was detected in the roots, stems, leaves, inflorescences, and pods of transgenic Arabidopsis thaliana lines. Notably, high GUS activity was observed in different tissues. In the transgenic lines, high GUS activity was detected in different tissues such as leaves, stalks, buds, petals, androecium, endosperm, and fibers, where the pGhFDH-driven GUS expression levels were 3-10-fold higher compared to those under the CaMV 35S promoter at 10-30 days post-anthesis (DPA) during fiber development. The results indicate that pGhFDH can be used as an endogenous constitutive promoter to drive the expression of target genes in various cotton tissues to facilitate functional genomic studies and accelerate cotton molecular breeding.

Metabolism, fibrosis, and apoptosis: The effect of lipids and their derivatives on keloid formation.

Keloids, pathological scars resulting from skin trauma, have traditionally posed significant clinical management challenges due to their persistence and high recurrence rates. Our research elucidates the pivotal roles of lipids and their derivatives in keloid development, driven by underlying mechanisms of abnormal cell proliferation, apoptosis, and extracellular matrix deposition. Key findings suggest that abnormalities in arachidonic acid (AA) synthesis and non-essential fatty acid synthesis are integral to keloid formation. Further, a complex interplay exists between lipid derivatives, notably butyric acid (BA), prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), and the regulation of hyperfibrosis. Additionally, combinations of docosahexaenoic acid (DHA) with BA and 15-deoxy-Δ12,14-Prostaglandin J2 have exhibited pronounced cytotoxic effects. Among sphingolipids, ceramide (Cer) displayed limited pro-apoptotic effects in keloid fibroblasts (KFBs), whereas sphingosine 1-phosphate (S1P) was found to promote keloid hyperfibrosis, with its analogue, FTY720, demonstrating contrasting benefits. Both Vitamin D and hexadecylphosphorylcholine (HePC) showed potential antifibrotic and antiproliferative properties, suggesting their utility in keloid management. While keloids remain a prevalent concern in clinical practice, this study underscores the promising potential of targeting specific lipid molecules for the advancement of keloid therapeutic strategies.

High-Performance Memristors Based on Imine-Linked Covalent Organic Frameworks Obtained by Using a Protonation Modification Strategy.

Imine-linked covalent organic frameworks (COFs) are garnering substantial interest in resistive random-access memory, attributed to their superior crystallinity, excellent chemical and thermal stability, and modifiable molecular structures. However, the development of high-performance COF-based memristors impeded by challenges such as low conjugation degree of imine bonds and poor electron delocalization ability. Herein, we report a protonation strategy to modify the imine bonds of donor-acceptor (D-A) type COFs. This modification significantly enhances the electron delocalization capability of imine bonds, lowers the energy barriers for electron injection from electrodes, and stabilizes the conductive charge transfer state, thus markedly improving device performance. The protonated COF-BTT-BPy and COF-BTT-TAPT thin films-based memristors show remarkable device performance with a high ON/OFF current ratio of 105, a low driving voltage, and outstanding endurance exceeding 600 and 1300 cycles, respectively, which is nearly twice the durability of analogous non-protonated COFs-based memristors. Notably, the protonated COF-BTT-TAPT-based memristor exhibit the highest number of cycles reported at present. This work not only unprecedentedly enhances the performance of COF-based memristors, but also provides a universal and promising approach for the molecular design and potential application of D-A type imine-linked COFs.

Removing information from working memory with a delay: Effective but not beneficial.

Ideally, removing outdated information from working memory (WM) should have two consequences: The removed content should be less accessible (removal costs), and other WM content should benefit from the freeing up of WM capacity (removal benefits). Robust removal benefits and removal costs have been demonstrated when people are told to forget items shortly after they were encoded (immediate removal). However, other studies suggest that people might be unable to selectively remove items from an already encoded set of items (delayed removal). In two experiments (n = 219; n = 241), we investigated the effectiveness and consequences of delayed removal by combining a modified version of Ecker's et al. (Journal of Memory and Language, 74, 77-90, 2014) letter updating task with a directed-forgetting in WM paradigm. We found that while delayed removal resulted in reduced memory for the to-be-forgotten item-location relations (removal costs), it failed to enhance performance for existing WM content. This contrasts sharply with immediate removal, where removal benefits can be observed. A fine-grained analysis of removal benefits shows that removal from WM proactively facilitates the subsequent encoding of new information but does not retroactively aid stored WM content.

Learning Disentangled Priors for Hyperspectral Anomaly Detection: A Coupling Model-Driven and Data-Driven Paradigm.

Accurately distinguishing between background and anomalous objects within hyperspectral images poses a significant challenge. The primary obstacle lies in the inadequate modeling of prior knowledge, leading to a performance bottleneck in hyperspectral anomaly detection (HAD). In response to this challenge, we put forth a groundbreaking coupling paradigm that combines model-driven low-rank representation (LRR) methods with data-driven deep learning techniques by learning disentangled priors (LDP). LDP seeks to capture complete priors for effectively modeling the background, thereby extracting anomalies from hyperspectral images more accurately. LDP follows a model-driven deep unfolding architecture, where the prior knowledge is separated into the explicit low-rank prior formulated by expert knowledge and implicit learnable priors by means of deep networks. The internal relationships between explicit and implicit priors within LDP are elegantly modeled through a skip residual connection. Furthermore, we provide a mathematical proof of the convergence of our proposed model. Our experiments, conducted on multiple widely recognized datasets, demonstrate that LDP surpasses most of the current advanced HAD techniques, exceling in both detection performance and generalization capability.

Ultrafast light-driven metasurfaces with an ultra-broadband frequency agile channel for sensing.

Active terahertz metasurface devices have been widely used in communication technology, optical computing and biosensing. However, numerous dynamically tunable metasurfaces are only operating at a single frequency point or in a narrow range, limiting the further possibility of the devices to meet contemporary broad-spectrum biosensing requirements. In this paper, a novel compact biosensor is proposed with an ultrawide resonance frequency agile channel shifted from 0.82 to 1.85 THz, with a tuning functionality up to 55.7%. In addition, under optical pumping irradiation, the modulator with ultra-fast response is able to complete the ultra-wideband resonant mode conversion from the Fano mode to the electromagnetically induced transparency (EIT) mode within 4 ps, and achieves a frequency shift sensitivity of 118 GHz RIU-1 and 247 GHz RIU-1 at 0.82 and 1.85 THz, respectively. This mechanism implements both refractive index and conductivity sensing functions, which provide a wealth of sensing information. Thus, this work presents the possibility of realising the detection of ultra-wide fingerprint spectra and can be extended to a wider range of optical fields.

Independent organelle and organelle-organelle interactions: essential mechanisms for malignant gynecological cancer cell survival.

Different eukaryotic cell organelles (e.g., mitochondria, endoplasmic reticulum, lysosome) are involved in various cancer processes, by dominating specific cellular activities. Organelles cooperate, such as through contact points, in complex biological activities that help the cell regulate energy metabolism, signal transduction, and membrane dynamics, which influence survival process. Herein, we review the current studies of mechanisms by which mitochondria, endoplasmic reticulum, and lysosome are related to the three major malignant gynecological cancers, and their possible therapeutic interventions and drug targets. We also discuss the similarities and differences of independent organelle and organelle-organelle interactions, and their applications to the respective gynecological cancers; mitochondrial dynamics and energy metabolism, endoplasmic reticulum dysfunction, lysosomal regulation and autophagy, organelle interactions, and organelle regulatory mechanisms of cell death play crucial roles in cancer tumorigenesis, progression, and response to therapy. Finally, we discuss the value of organelle research, its current problems, and its future directions.

Electroacupuncture reduces corpus callosum injury in rats with permanent cerebral ischemia by inhibiting the activation of high-mobility group box 1 protein and the receptor for advanced glycation end products.

Previous studies have shown that cerebral ischemia can cause white matter injury in the brain. This study aimed to investigate the potential mechanism of electroacupuncture (EA) at the Baihui (GV20) and Zusanli (ST36) acupoints in protecting white matter. Sprague-Dawley rats were used to establish permanent middle cerebral artery occlusion (pMCAO) rat models. Comprehensive motor functions were assessed using the mesh experiment. Morphological changes in the myelin sheath were assessed with Luxol fast blue staining. Morphological changes in oligodendrocytes and myelinated axons were evaluated using Nissl staining. The expressions of high-mobility group box 1 protein (HMGB1) and the receptor for advanced glycation end products (RAGE) in the corpus callosum were detected by immunohistochemical staining and Western blot analysis. pMCAO caused severe injury to the corpus callosum, evidenced by significant loss of white matter fibers and myelinated axons, and induced overexpression of HMGB1 and RAGE in the corpus callosum. EA treatment significantly improved comprehensive motor function alleviated white matter damage, and downregulated the expression of HMGB1 and RAGE. Its effects were comparable to those of FPS-ZM1, a RAGE receptor inhibitor. In conclusion, EA effectively improves comprehensive motor function in rats with cerebral infarction and alleviates corpus callosum injury. This effect may be related to the inhibition of HMGB1 and RAGE overexpression.

Intelligent double-layer film based on gellan gum/modified anthocyanin/curcumin/sodium alginate/zinc oxide for monitoring shrimp freshness.

In this study, intelligent double-layer films were prepared using modified black rice anthocyanin (MBRA)-curcumin (CUR)-gellan gum (GG) as the inner indicator layer and sodium alginate (ALG)­zinc oxide (ZnO) as the outer antimicrobial layer. The bilayer films were successfully prepared, as revealed by scanning electron microscopy, Fourier-transform infrared spectroscopy, and X-ray diffraction measurements. The mechanical characteristics, moisture content, and water vapor resistance of GG-MBRA/CUR1@ALG-ZnO, GG-MBRA/CUR2@ALG-ZnO, and GG-MBRA/CUR3@ALG-ZnO films showed significant enhancement compared to GG-MBRA/CUR3 and ALG-ZnO films. The bilayer films exhibited excellent pH responsiveness and reacted effectively to ammonia. The outer layer significantly improved the antioxidant and antibacterial properties of the inner layer. When the films were applied to shrimp, it was found that the double-layer films not only monitored the freshness of the shrimp in real-time but also were influential in extending the shelf life of the shrimp by about 1 d. Therefore, the double-layer film demonstrated potential as a smart packaging material for real-time monitoring of meat product freshness.