Single cell RNA sequencing provides novel cellular transcriptional profiles and underlying pathogenesis of presbycusis.

Age-related hearing loss (ARHL) or presbycusis is associated with irreversible progressive damage in the inner ear, where the sound is transduced into electrical signal; but the detailed mechanism remains unclear. Here, we sought to determine the potential molecular mechanism involved in the pathogeneses of ARHL with bioinformatics methods. A single-cell transcriptome sequencing study was performed on the cochlear samples from young and aged mice. Detection of identified cell type marker allowed us to screen 18 transcriptional clusters, including myeloid cells, epithelial cells, B cells, endothelial cells, fibroblasts, T cells, inner pillar cells, neurons, inner phalangeal cells, and red blood cells. Cell-cell communications were analyzed between young and aged cochlear tissue samples by using the latest integration algorithms Cellchat. A total of 56 differentially expressed genes were screened between the two groups. Functional enrichment analysis showed these genes were mainly involved in immune, oxidative stress, apoptosis, and metabolic processes. The expression levels of crucial genes in cochlear tissues were further verified by immunohistochemistry. Overall, this study provides new theoretical support for the development of clinical therapeutic drugs.

Targeted sonogenetic modulation of GABAergic interneurons in the hippocampal CA1 region in status epilepticus.

Rationale: Sonogenetics is an advanced ultrasound-based neurostimulation approach for targeting neurons in specific brain regions. However, the role of sonogenetics in treating status epilepticus (SE) remains unclear. Here, we aimed to investigate the effects of ultrasound neurostimulation and MscL-G22S (a mechanosensitive ion channel that mediates Ca2+ influx)-mediated sonogenetics (MG-SOG) in a mouse model of kainic acid (KA)-induced SE. Methods: For MG-SOG, a Cre-dependent AAV expressing MscL-G22S was injected into parvalbumin (PV)-cre and somatostatin (SST)-cre mice to induce the expression of MscL-G22S-EGFP in PV interneurons (PV-INs) and SST interneurons (SST-INs), respectively; mice were stimulated with continuous pulses of ultrasound stimulation during the latency of generalized seizures (GSs), the latency to SE, in SE model mice. We performed calcium fiber photometry, patch-clamp recording, local field potential recording, and SE monitoring to investigate the role of MG-SOG in treating SE. Results: First, we observed obvious neuronal activation in the hippocampal CA1 region in SE model mice. Both excitatory neurons (ENs) and GABAergic interneurons (GABA-INs) in the CA1 region were activated in SE model mice; however, the inhibitory effect of GABA-INs on ENs seemed to be insufficient to reduce EN excitability despite the increased activation of GABA-INs in SE model mice. Thus, we speculated that MG-SOG-induced activation of GABA-INs, mainly SST-INs and PV-INs, in the CA1 region may protect against SE. We found that MG-SOG-mediated PV-IN activation in the CA1 region ameliorated SE and changed SE-related electrophysiological abnormalities in the CA1 region; however, MG-SOG-induced SST-IN activation in the CA1 region did not ameliorate SE. Conclusions: MG-SOG-mediated activation of PV-INs had a positive effect on relieving SE. Our work may promote the development of sonogenetic neurostimulation techniques for treating SE.

An Oxidative Stress Nano-Amplifier for Improved Tumor Elimination and Combined Immunotherapy.

Amplifying oxidative stress to disrupt intracellular redox homeostasis can accelerate tumor cell death. In this work, an oxidative stress amplifier (PP@T) is prepared for enhanced tumor oxidation therapy to reduce tumor growth and metastases. The nano-amplifier has been successfully constructed by embedding MTH1 inhibitor (TH588) in the PDA-coated porphyrin metal-organic framework PCN-224. The controllable-released TH588 is demonstrated from pores can hinder MTH1-mediated damage-repairing process by preventing the hydrolysis of 8-oxo-dG, thereby amplifying oxidative stress and exacerbating the oxidative DNA damage induced by the sonodynamic therapy of PP@T under ultrasound irradiation. Furthermore, PP@T can effectively induce immunogenic cell death to trigger systemic anti-tumor immune response. When administered in combination with immune checkpoint blockade, PP@T not only impedes the progression of the primary tumor but also achieves obvious antimetastasis in breast cancer murine models, including orthotopic and artificial whole-body metastasis models. Furthermore, the nanoplatform also provides photoacoustic imaging for in vivo treatment guidance. In conclusion, by amplifying oxidative stress and reactive oxygen species sensitized immunotherapy, this image-guided nanosystem shows potential for highly specific, effective combined therapy against tumor cells with negligible side-effects to normal cells which will provide a new insight for precise tumor treatment.

Fabrication and characterization of oleic acid/sesame protein isolate/ poly (vinyl) alcohol core-shell nanofibers: Mitigating lipid oxidation by emulsion electrospinning.

Formulated oil-in-water (O/W) emulsions of oleic acid (OA) using sesame protein isolate (SPI) were processed via emulsion electrospinning with poly (vinyl) alcohol (PVA) to fabricate core-shell nanofibers for lipid oxidation prevention. The emulsion droplet size and viscosity increased as the oil volume fraction rose from 5 % to 30 %. The morphology tests and Fourier transform infrared spectroscopy (FTIR) confirmed the uniformity of nanofibers and OA encapsulation with hydrogen bonding. The thermal stability, mechanical properties, and water contact angle (WCA) of the nanofiber films improved with increased OA content. Encapsulation efficiency was 94.76 % and storage stability was maintained for 7 days in 5 % oil fraction nanofibers. The nanofibers showed lower oxidation and superior oxidative resistance to free OA, with the lowest peroxide value (POV, 2.14 mmol/L) and thiobarbituric acid-reactive substances (TBARS, 36.75 µmol/L). In conclusion, the OA/SPI/PVA (PE) core-shell nanofibers via emulsion electrospinning are efficient for fatty acid encapsulation in functional foods.

Single-cell dissection of the human blood-brain barrier and glioma blood-tumor barrier.

The blood-brain barrier (BBB) serves as a crucial vascular specialization, shielding and nourishing brain neurons and glia while impeding drug delivery. Here, we conducted single-cell mRNA sequencing of human cerebrovascular cells from 13 surgically resected glioma samples and adjacent normal brain tissue. The transcriptomes of 103,230 cells were mapped, including 57,324 endothelial cells (ECs) and 27,703 mural cells (MCs). Both EC and MC transcriptomes originating from lower-grade glioma were indistinguishable from those of normal brain tissue, whereas transcriptomes from glioblastoma (GBM) displayed a range of abnormalities. Among these, we identified LOXL2-dependent collagen modification as a common GBM-dependent trait and demonstrated that inhibiting LOXL2 enhanced chemotherapy efficacy in both murine and human patient-derived xenograft (PDX) GBM models. Our comprehensive single-cell RNA sequencing-based molecular atlas of the human BBB, coupled with insights into its perturbations in GBM, holds promise for guiding future investigations into brain health, pathology, and therapeutic strategies.

Chitosan/polyvinyl alcohol food packaging incorporated with purple potato anthocyanins and nano-ZnO: Application on the preservation of hairtail (Trichiurus haumela) during chilled storage.

The objective of this work was to evaluate the potential application of chitosan/PVA food packaging films incorporating nano-ZnO and purple potato anthocyanins for preserving chilled hairtail pieces. The hairtail pieces were packaged with chitosan/PVA (CP) and chitosan/PVA/nano-ZnO/purple potato anthocyanins (CPZP), respectively, and Control named without any packaging. The changes in pH, total volatile basic nitrogen (TVB-N), total bacterial colony (TVC), thiobarbituric acid (TBA), color value, and sensory evaluation scores of hairtail pieces were periodically determined. Notably, pH, TVC, TVB-N and TBA values of CPZP group on day 15 were 11.67 %, 23.71 %, 80.73 %, and 35.07 %, respectively, lower than Control group. In addition, CPZP group also performed the best in color and sensory evaluation. These results indicated that CPZP, an active food packaging, could extend the shelf-life of hairtail at least 6 days. Overall, chitosan/PVA food films incorporated with nano-ZnO and purple potato anthocyanins (180 mg/100 mL) provides a potential application in food preservation.

A review: Interactions between protein from blue foods and functional components in delivery systems: Function exertion and transmembrane transport by in vitro digestion/cells model.

Functional compounds (FCs) had some functions, which are affected easily by digestion and transmembrane transport leading to low absorption rates, such as lutein, quercetin, xylo-oligosaccharide. Protein from blue foods is a potential bioactive compound, which had higher bioavailability, especially for bioactive peptides (BBPs). The BBPs has great limitations, especially the variability under pepsin digestion. However, the limitation of single FCs and BBPs in bioavailability might can be complemented by mixture of different bioactive compounds. Therefore, this review provides an in-depth study on the function and mechanism of different FCs/BBPs and their mixtures. Specifically, digestion effect of mixtures on function and transmembrane transport mechanisms of different bioactive compounds were exhibited to elaborate interactions between BBPs and FCs in delivery systems (function and bioavailability). Combination of FCs/BBPs could enhance bioactive compounds function by mutual complement of function mechanisms, as well as improving the function after digestion by regulating digestion process. Moreover, transmembrane absorption and transport of FCs/BBPs also could be facilitated by mixtures due to complement of transmembrane mechanism (endocytosis, protein channels, cell bypass way). This manuscript lays a foundation for the development of active ingredient bioavailability in functional food processing.

Comparison of lipid profiles of male and female silkworm (Bombyx mori) pupae through high-resolution mass spectrometry-based lipidomics and chemometrics.

Silkworm (Bombyx mori) pupae lipid profiles were analyzed using high-resolution mass spectrometry-based lipidomics. A total of 241 lipid molecular species were annotated with high confidence in male and female silkworm pupae. Triacylglycerol (TG), phosphoethanolamine (PE) and phosphocholine (PC) were the main lipid subclasses of silkworm pupae, accounting for 63, 41 and 38 lipid molecular species, respectively. No unique lipid molecular species were identified, but there were differences in the abundance of lipid molecular species between male and female silkworm pupae. Therefore, the differences in the lipid abundance of male and female silkworm pupae were analyzed by chemometrics. As a result, 8 lipid molecular species were screened for differential lipids. Hierarchical clustering analysis (HCA) showed that male and female silkworm pupae samples formed two distinct branches, indicating that these differential lipids could potentially be used as biomarkers to distinguish between male and female silkworm pupae.

Ca2+-nano starch-lutein endowed 3D printed surimi with antioxidation and mutual reinforcing transmembrane transport mechanisms via hepg2 and caco-2 cells model.

To better enhance printing effects meanwhile casting functionality, antioxidation and absorption of bioactive component in printed Ca2+-nano starch (NS)-lutein (L)-surimi were investigated. Results shown that Ca2+-NS-L promoted surimi printability due to enhanced gel strength and denser structure. Mixing Ca2+-NS-L endowed printed surimi with antioxidation (DPPH, ABTS, hydroxyl radical, Fe2+ reduction were 42 %, 79 %, 65 %, 0.104 mg·mL-1, respectively) due to the ability of lutein with more -OH groups and conjugate bonds to capture free radicals. It also manifested in cellular antioxidation that Ca2+-NS-L-surimi regulated the level of Nrf2 to protect gene expression of antioxidases (SOD, CAT, GSH-Px increased by 30-180 %, compared to damaged cells) through keap1-Nrf2-ARE pathway. Additionally, lutein absorption and transportation of Ca2+-NS-L-surimi increased by 20 %, compared to NS-L. Possibly, combination of samples and membrane was facilitated by surface hydrophobic, promoting endocytosis. Meanwhile, digestive surimi (peptides) with acidic-alkaline amino acids and negative charges made samples be attracted and moved in bypass parts under electrostatic traction and repulsion (electrostatic domain) to promote transport process. Also, Ca2+ facilitated CaM expression in membrane and formed Ca2+ channel by combining with CaM to accelerate entry of samples into cells. Conclusively, Ca2+-NS-L both strengthened printability of surimi and antioxidation, promoting application of printed functional surimi.

Combination of MHI148 Targeted Photodynamic Therapy and STING Activation Inhibits Tumor Metastasis and Recurrence.

Metastasis and recurrence are notable contributors to mortality associated with breast cancer. Although immunotherapy has shown promise in mitigating these risks after conventional treatments, its effectiveness remains constrained by significant challenges, such as impaired antigen presentation by dendritic cells (DCs) and inadequate T cell infiltration into tumor tissues. To address these limitations, we developed a multifunctional nanoparticle platform, termed GM@P, which consisted of a hydrophobic shell encapsulating the photosensitizer MHI148 and a hydrophilic core containing the STING agonist 2'3'-cGAMP. This design elicited robust type I interferon responses to activate antitumor immunity. The GM@P nanoparticles loaded with MHI148 specifically targeted breast cancer cells. Upon exposure to 808 nm laser irradiation, the MHI148-loaded nanoparticles produced toxic reactive oxygen species (ROS) to eradicate tumor cells through photodynamic therapy (PDT). Notably, PDT stimulated immunogenic cell death (ICD) to foster the potency of antitumor immune responses. Furthermore, the superior photoacoustic imaging (PAI) capabilities of MHI148 enabled the simultaneous visualization of diagnostic and therapeutic procedures. Collectively, our findings uncovered that the combination of PDT and STING activation facilitated a more conducive immune microenvironment, characterized by enhanced DC maturation, infiltration of CD8+ T cells, and proinflammatory cytokine release. This strategy stimulated local immune responses to augment systemic antitumor effects, offering a promising approach to suppress tumor growth, inhibit metastasis, and prevent recurrence.

Differentiation of three commercial tuna species through GC-Q-TOF and UPLC-Q/Orbitrap mass spectrometry-based metabolomics and chemometrics.

Food fraud is common in the tuna industry because of the economic benefits involved. Ensuring the authenticity of tuna species is crucial for protecting both consumers and tuna stocks. In this study, GC-Q-TOF and UPLC-Q/Orbitrap mass spectrometry-based metabolomics were used to investigate the metabolite profiles of three commercial tuna species (skipjack tuna, bigeye tuna and yellowfin tuna). A total of 22 and 77 metabolites were identified with high confidence using GC-Q-TOF and UPLC-Q/Orbitrap mass spectrometry, respectively. Further screening via chemometrics revealed that 38 metabolites could potentially serve as potential biomarkers. Hierarchical cluster analysis showed that the screened metabolite biomarkers successfully distinguished the three tested tuna species. Furthermore, a total of 27 metabolic pathways were identified through enrichment analysis based on the Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways.

Discrimination of three commercial tuna species through species-specific peptides: From high-resolution mass spectrometry discovery to MRM validation.

The risk of tuna adulteration is high driven by economic benefits. The authenticity of tuna is required to protect both consumers and tuna stocks. Given this, the study is designed to identify species-specific peptides for distinguishing three commercial tropical tuna species. The peptides derived from trypsin digestion were separated and detected using ultrahigh-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF/MS) in data-dependent acquisition (DDA) mode. Venn analysis showed that there were differences in peptide composition among the three tested tuna species. The biological specificity screening through the National Center for Biotechnology Information's Basic Local Alignment Search Tool (NCBI BLAST) revealed that 93 peptides could serve as potential species-specific peptides. Finally, the detection specificity of species-specific peptides of raw meats and processed products was carried out by multiple reaction monitoring (MRM) mode based on a Q-Trap mass spectrometer. The results showed that three, one and two peptides of Katsuwonus pelamis, Thunnus obesus and Thunnus albacores, respectively could serve as species-specific peptides.

The luxS deletion reduces the spoilage ability of Shewanella putrefaciens: An analysis focusing on quorum sensing and activated methyl cycle.

The luxS mutant strains of Shewanella putrefaciens (SHP) were constructed to investigate the regulations of gene luxS in spoilage ability. The potential regulations of AI-2 quorum sensing (QS) system and activated methyl cycle (AMC) were studied by analyzing the supplementation roles of key circulating substances mediated via luxS, including S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), homocysteine (Hcy) and 4,5-dihydroxy-2,3-pentanedione (DPD). Growth experiments revealed that the luxS deletion led to certain growth limitations of SHP, which were associated with culture medium and exogenous additives. Meanwhile, the decreased biofilm formation and diminished hydrogen sulfide (H2S) production capacity of SHP were observed after luxS deletion. The relatively lower total volatile base nitrogen (TVB-N) contents and higher sensory scores of fish homogenate with luxS mutant strain inoculation also indicated the weaker spoilage-inducing effects after luxS deletion. However, these deficiencies could be offset with the exogenous supply of circulating substances mentioned above. Our findings suggested that the luxS deletion would reduce the spoilage ability of SHP, which was potentially attributed to the disorder of AMC and AI-2 QS system.

Integrative analysis of transcriptome and proteome profiles in primary and recurrent glioblastoma.

PURPOSE: Glioblastoma (GBM) is the most common and aggressive primary brain tumor characterized by poor prognosis and high recurrence. The underlying molecular mechanism that drives tumor progression and recurrence is unclear. This study is intended to look for molecular and biological changes that play a key role in GBM recurrence. EXPERIMENTAL DESIGN: An integrative transcriptomic and proteomic analysis was performed on three primary GBM and three recurrent GBM tissues. Omics analyses were conducted using label-free quantitative proteomics and whole transcriptome sequencing. RESULTS: A significant difference was found between primary GBM and recurrent GBM at the transcriptional level. Similar to other omics studies of cancer, a weak overlap was observed between transcriptome and proteome, and Procollagen C-Endopeptidase Enhancer 2 (PCOLCE2) was observed to be upregulated at mRNA and protein levels. Analysis of public cancer database revealed that high expression of PCOLCE2 is associated with poor prognosis of patients with GBM and that it may be a potential prognostic indicator. Functional and environmental enrichment analyses revealed significantly altered signaling pathways related to energy metabolism, including mitochondrial ATP synthesis-coupled electron transport and oxidative phosphorylation. CONCLUSIONS AND CLINICAL RELEVANCE: This study provides new insights into the recurrence process of GBM through combined transcriptomic and proteomic analyses, complementing the existing GBM transcriptomic and proteomic data and suggesting that integrated multi-omics analyses may reveal new disease features of GBM.

The highly stable indicator film incorporating roselle anthocyanin co-pigmented with oxalic acid: Preparation, characterization and freshness monitoring application.

A novel stable PVA/HPMC/roselle anthocyanin (RAE) indicator film co-pigmented with oxalic acid (OA) was prepared, its properties, application effects and stability enhancement mechanism were investigated correspondingly. The structural characterization revealed that more stable network was formed due to the co-pigmentation facilitated generation of molecular interactions. Meanwhile, the co-pigmentation improved film mechanical and hydrophobic properties compared to both PVA/HPMC/RAE newly prepared (PHRN) or stored (PHRS) film, expressing as higher tensile strength values (12.25% and 14.44% higher than PHRN and PHRS), lower water solubility (7.22% and 10.09% lower than PHRN and PHRS) and water vapor permeability values (33.20% and 21.05% lower than PHRN and PHRS) of PVA/HPMC/RAE/OA newly prepared (PHON) or stored (PHOS) film. Compared with the PHRS film, the PHOS film still presented more distinguishable color variations when being applied to monitor shrimp freshness, owing to the stabilization behaviors of co-pigmentation in anthocyanin conformation. Hence, the co-pigmentation was an effective strategy to enhance film stability, physical and pH-responsive properties after long term storage, leading to better film monitoring effects when applied in real-time freshness monitoring.

Comprehensive Transcriptomic Profiling of m6A Modification in Age-Related Hearing Loss.

Age-related hearing loss (ARHL), also known as presbycusis, is one of the most common neurodegenerative disorders in elderly individuals and has a prevalence of approximately 70-80% among individuals aged 65 and older. As ARHL is an intricate and multifactorial disease, the exact pathogenesis of ARHL is not fully understood. There is evidence that transcriptional dysregulation mediated by epigenetic modifications is widespread in ARHL. However, the potential role of N6-methyladenosine (m6A) modification, as a crucial component of epigenetics, in ARHL progression remains unclear. In this study, we confirmed that the downregulation of m6A modification in cochlear tissues is related to ARHL and found that the expression of the m6A methylation regulators Wilms tumour suppressor-1-associated protein (WTAP), methyltransferase-like 3 (METTL3), ALKB homologous protein 5 (ALKBH5) and fat mass and obesity-associated protein (FTO) is decreased significantly at the mRNA and protein levels in ARHL mice. Then, we used methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and RNA sequencing (RNA-Seq) to identify the differentially m6A-methylated genes in the cochlear tissues of ARHL mice. A total of 3438 genes with differential m6A methylation were identified, of which 1332 genes were m6A-hypermethylated and 2106 genes were m6A-hypomethylated in the ARHL group compared to the control group according to MeRIP-seq. Further joint analysis of RNA-Seq and MeRIP-Seq data showed that 262 genes had significant differences in both mRNA expression and m6A methylation. GO and KEGG analyses indicated that 262 unique genes were enriched mainly in the PI3K-AKT signalling pathway. In conclusion, the results of this study reveal differential m6A methylation patterns in the cochlear tissues of ARHL mice, providing a theoretical basis for further study of the pathogenesis of ARHL and potential therapeutic strategies.

Ionic strength-mediated protein and flavor studies on thermally processed hairtail pieces.

This study aimed to investigate the impact of different ionic strengths on the texture, protein, and flavor of thermally processed hairtail pieces. Incorporating salt ions into the heat treatment process had a positive impact on the quality of the cooked hairtail pieces. The pieces treated with 2 M NaCl showed superior texture and sensory scores. The ionic strength had a significant positive correlation with the chewiness and cohesion of cooked hairtail (p < 0.01). Furthermore, the myofibrillar protein content and total sulfhydryl content increased significantly. Circular dichroism spectra analysis revealed a transition in the protein structure from a ß-sheet structure to an α-helical structure as the ionic strength decreased. The ionic strength had a significant impact on the interaction between protein and flavor compounds. Specifically, it impacted the expression of certain volatile components (p < 0.05). Our study suggests that selecting the appropriate cooking method is crucial for both healthiness and sensory quality of processed hairtail products, and ionic strength mediation is superior in both aspects.

Potential roles for lncRNA Mirg/Foxp1 in an ARHL model created using C57BL/6J mice.

Age-related hearing loss (ARHL) is associated with hair cell apoptosis, but the underlying mechanism of hair cell apoptosis remains unclear. Here, we investigated the expression profiles of long noncoding RNAs (lncRNAs) and mRNAs in an ARHL model created with C57BL/6 J mice using RNA sequencing and found that the expression of several lncRNAs was significantly correlated with apoptosis-associated mRNAs in the cochlear tissues of old mice compared to young mice. We found that lncRNA Mirg was upregulated in the cochlear tissues of old mice compared to young mice and its overexpression promoted apoptosis in House Ear Institute-Organ of Corti 1 (HEI-OC1). H2O2-induced oxidative stress increased HEI-OC1 cell apoptosis by upregulating lncRNA Mirg. Furthermore, the expression of lncRNA Mirg and Foxp1 showed the highest correlation coefficient in the cochlear tissues of old mice, and lncRNA Mirg promoted HEI-OC1 cell apoptosis by increasing Foxp1 expression. In conclusion, our findings suggest that lncRNA Mirg expression correlates with cell apoptosis-associated mRNAs in the ARHL model created using C57BL/6 J mice and that oxidative stress-induced lncRNA Mirg promotes HEI-OC1 cell apoptosis by increasing Foxp1 expression. These data suggest the potential therapeutic significance of targeting lncRNA Mirg/Foxp1 signaling in ARHL.

Enhanced fresh-keeping capacity of printed surimi by Ca2+-nano starch-lutein and its nondestructive freshness monitoring based on 4D printed anthocyanin.

To solve undiscernible freshness changes of printed functional surimi while maintaining printed shape, 4D printable color-changing material were prepared. Firstly, based on results of printing properties and fresh-keeping of Ca2+-NS-L-surimi, it showed better printing effects (enhanced mechanical strength) and good preservation (inhibition of amino acids decomposition, bacterial growth). However, freshness changes of printed Ca2+-NS-L-surimi were not distinguished directly. To avoid that, 4D printable color-changing material-anthocyanin-hydroxypropyl methyl cellulose-xanthan gum-carrageenan (AHXK) was prepared for indicating freshness through discoloration. Printing results showed AHX with 5 % K had the most suitable mechanical strength (appropriate gel strength, texture, rheology) for printing. Based on that, AHXK had stable color (ΔE fluctuation <5) and was sensitive to pH and ammonia (obvious discoloration; ΔE > 10). Actual freshness monitoring results (co-printing of AHXK-surimi) exhibited significant discolorations, especially for HXK with 0.75 % A. It became green during refrigeration of 3-5 d (keeping fresh, ΔE < 4), brighter green at 7 d (decreased freshness, ΔE > 6), turned yellow at 9 d (spoilage, ΔE > 16), which were distinguished significantly with naked eyes rather than traditional freshness determining. In conclusion, printed AHXK-functional surimi exhibited good printing, preservation and nondestructive freshness monitoring, facilitating application of 3D printed functional surimi.