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.

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.

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.

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.

Effect of Ca2+ on 3D printing accuracy and antioxidant efficacy of nano starch-lutein surimi: mechanical properties, chemical bonds and release rate.

BACKGROUND: Nano starch-lutein (NS-L) can be used in three-dimensional (3D) printed functional surimi. However, the lutein release and printing effect are not ideal. The purpose of this study was to facilitate the function and printing properties of surimi by adding the combination of calcium ion (Ca2+ ) and NS-L. RESULTS: Printing properties, lutein release and antioxidation of printed Ca2+ -NS-L-surimi were determined. The NS-L-surimi with 20 mM kg-1 Ca2+ had the best printing effects (fine accuracy, 99 ± 1%). Compared to NS-L-surimi, the structure became denser after adding Ca2+ , the gel strength, hardness, elasticity, yield stress (τ), water holding capacity of Ca2+ -NS-L-surimi increased by about 17 ± 4%, 3 ± 1%, 9 ± 2%, 20 ± 4%, 40 ± 5% respectively. These enhanced mechanical strength and self-supporting ability to resist binding deformation and improve printing accuracy. Moreover, salt dissolution and increased hydrophobic force by Ca2+ stimulated protein stretching and aggregation, leading to enhancement of gel formation. Decreased printing effects of NS-L-surimi with excessive Ca2+ (> 20 mM kg-1 ) caused by excessive gel strength and τ, leading to strong extrusion force and low extrudability. Additionally, Ca2+ -NS-L-surimi had higher digestibility and lutein release rate (increased from 55 ± 2% to 73 ± 3%), because Ca2+ made NS-L-surimi structure porous, which promoted contact of enzyme-protein. Furthermore, weakened ionic bonds reduced electron binding bondage that combined with released lutein to provide more electrons for enhancing antioxidation. CONCLUSION: Collectively, 20 mM kg-1 Ca2+ could better promote printing process and function exertion of NS-L-surimi, facilitating the application of 3D printed functional surimi. © 2023 Society of Chemical Industry.

Effects of pH and NaCl on the Spatial Structure and Conformation of Myofibrillar Proteins and the Emulsion Gel System-Insights from Computational Molecular Dynamics on Myosin of Golden Pompano.

In this study, the effects of pH and NaCl concentrations on the structure of golden pompano myosin and emulsion gel were analyzed using SEM in combination with molecular dynamics simulations (MDS). The microscopic morphology and spatial structure of myosin were investigated at different pH (3.0, 7.0, and 11.0) and NaCl concentrations (0.0, 0.2, 0.6, and 1.0 M), and their effects on the stability of emulsion gels were discussed. Our results show that pH had a greater effect on the microscopic morphology of myosin than NaCl. The MDS results show that under the condition of pH 7.0 and 0.6 M NaCl, the myosin expanded and experienced significant fluctuations in its amino acid residues. However, NaCl showed a greater effect on the number of hydrogen bonds than pH. Although changes in pH and NaCl concentrations only slightly altered the secondary structures in myosin, they, nevertheless, significantly influenced the protein spatial conformation. The stability of the emulsion gel was affected by pH changes but not NaCl concentrations, which only affect the rheology. The best elastic modulus G″ of the emulsion gel was obtained at pH 7.0 and 0.6 M NaCl. Based on the results, we conclude that pH changes have a greater influence than NaCl concentrations on the spatial structure and conformation of myosin, contributing to the instability of its emulsion gel state. The data from this study would serve as a valuable reference for emulsion gel rheology modification in future research.

Nitroarenes and nitroalkenes as potential amino sources for the synthesis of N-heterocycles.

Nitro-compounds are one of the cheapest and most readily available materials in the chemical industry and are commonly utilized as versatile building blocks. Previously, the synthesis of N-heterocycles was largely based on anilines. The utilization of nitroarenes and nitroalkenes for the synthesis of N-heterocyclic compounds can save at least one step, however, as compared to anilines. Thus, considerable attention has been paid to nitroarenes and nitroalkenes as new potential amino sources. Significant progress has been made in the reductive cyclization of nitroarenes or nitroalkenes to access various N-heterocycles in recent years. Herein, we comprehensively summarize the recent progress in the construction of N-heterocycles using nitroarenes and nitroalkenes as potential amino sources. The compatibility of the reaction substrate, its mechanism, applications, advantages, and limitations in this field are also discussed in detail.

Effects of environmental pH on protein properties and flavor factors of hairtail (Trichiurus haumela) in thermal processing.

The flavor and texture of hairtail (Trichiurus haumela) products easily change depending on the processing conditions including the programed temperature, environmental pH, and so on. In the present study, we aimed to explore the differences in the overall texture and flavor of hairtail under heat treatment with varied environmental pH. The results indicated that the secondary structure of the myofibrillar protein in thermal processed hairtail meat presented a transformation from α-helix to ß-sheet structure with the decrease of solution pH. Moreover, heat treatment in an acidic solution environment effectively improved the sensory and flavor properties of hairtail. In addition, pH-mediated changes on protein characteristics of cooked hairtail meat showed significant correlation with the texture properties, while weakly correlated with the flavor.

Improved thermal stability of roselle anthocyanin by co-pigmented with oxalic acid: Preparation, characterization and enhancement mechanism.

The enhancement effects of co-pigmentation on thermal stability of roselle anthocyanin extract (RAE) were investigated. The introduction of organic acids maintained color stability of RAE, and RAE co-pigmented with oxalic acid (OA) presented less color fading rates (19.46 ± 0.33 %) and higher redness (41.33 ± 3.51). Subsequently, suitable co-pigmentation concentration (OA:RAE = 1:2) was obtained regarding with lower ΔE (48.70 ± 2.36). Then, improvement behaviors of co-pigmentation on OA-RAE were evaluated. Results demonstrated that OA-RAE exhibited better thermal stability, as manifested by larger retention rates and more favorable thermal degradation kinetic parameters. Furthermore, both molecular docking simulation and experimental structural characterization revealed that hydrogen bonds and other non-covalent bonds made up the main parts of molecular interactions, leading to formation of stable binary complex. As a result, the aromatic ring of RAE was protected. In conclusion, the co-pigmentation of RAE via introduction of OA was effective in stability enhancement due to the generation of molecular bindings.

Novel combination strategy of high intensity focused ultrasound (HIFU) and checkpoint blockade boosted by bioinspired and oxygen-supplied nanoprobe for multimodal imaging-guided cancer therapy.

BACKGROUND: High-intensity focused ultrasound (HIFU) has shown considerable promise in treating solid tumors, but its ultrasonic energy is easily attenuated, resulting in insufficient energy accumulation in the target area. Moreover, HIFU ablation alone may inevitably lead to the presence of residual tumors, which may cause tumor recurrence and metastasis. Here, we describe a synergistic regimen combining HIFU facilitation with immunomodulation based on a novel oxygen-carrying biomimetic perfluorocarbon nanoparticle (M@P-SOP) to stimulate immunogenic cell death in tumor cells while alleviating immune suppression tumor microenvironment. METHODS: M@P-SOP was prepared by double emulsion and film extrusion method. The anticancer and antimetastatic effects of M@P-SOP were evaluated on a preclinical transplanted 4T1 tumor model by combining HIFU and immunotherapy. Flow cytometry and immunofluorescence were used to clarify the potential mechanism of HIFU+M@P-SOP and their role in anti-programmed death ligand-1 (PD-L1) therapy. RESULTS: Guided by photoacoustic/MR/ultrasound (US) multimodal imaging, M@P-SOP was abundantly enriched in tumor, which greatly enhanced HIFU's killing of tumor tissue in situ, induced stronger tumor immunogenic cell death, stimulated dendritic cell maturation and activated CD8+ T cells. At the same time, M@P-SOP released oxygen to alleviate the tumor hypoxic environment, repolarizing the protumor M2-type macrophages into antitumor M1-type. With concurrent anti-PD-L1 treatment, the antitumor immune response was further amplified to the whole body, and the growth of mimic distant tumor was effectively suppressed. CONCLUSIONS: Our findings offer a highly promising HIFU synergist for effectively ameliorating acoustic and hypoxia environment, eventually inhibiting tumor growth and metastasis by stimulating host's antitumor immunity under HIFU ablation, especially in synergizing with PD-L1 antibody immunotherapy.

Thermochromic Photonic Crystal Microspheres with Uniform Color Display and Wide Coloration Range.

Thermochromic microspheres based on poly(N-isopropylacrylamide) attract much attention in detection and sensor due to the noticeable color response and fast response rate. However, some issues such as uneven color display and narrow coloration range still limit their practical applications. Herein, novel thermochromic microspheres with homogeneous color displays and wide thermochromic range are designed by combining the microfluidic technology with the magnetically-induced self-assembly technique and copolymerizing acrylamide (AM) with N-isopropylacrylamide. The photonic crystal structure with especially even colors is fast and conveniently constructed by magnetic assembly. The addition of AM makes the microspheres more hydrophilic and thus leading to a broader coloration range. The relationship between the structural color display and both the microstructures of photonic crystals and the thermo-responsive properties of gel matrix are elucidated. The detectable temperature of microspheres rises to as high as 60°C, and displays bright iridescent color variations from orange to blue-violet in the heating process. Importantly, their shrinking or swelling equilibrium can be reached in 80 and 105 s. Such microspheres are successfully used to visually indicate the appropriate temperature of enzymatic reaction, and have great potential in practical applications such as visual temperature detection and efficiency monitoring of chemical reactions.

Emulsion gel stabilized by tilapia myofibrillar protein: Application in lipid-enhanced surimi preparation.

In this study, surimi products rich in lipids were prepared by using myofibril protein (MP) emulsion gel as carriers. The MP emulsion gel (MP concentration, c = 1.5%, oil fraction, ø = 0.68) was prepared by one-step homogenization. The emulsion gel maintained a high elastic modulus (G') after heating and freezing treatment. Confocal laser scanning microscopy revealed that the structure of the emulsion gel was a hybrid network consisting of polymers of cross-linked MP and aggregated protein-stabilized emulsion (W/O/W multiple structures) droplets. The double emulsification of the emulsion gel and MP stabilized the oil droplets in the surimi product, preventing water and oil from leaching out. The microstructure also showed smaller gaps between MPs with increased porosity, while oil droplets were stably embedded in the surimi gel matrix. Moreover, adding MP emulsion gel significantly reduced the surimi gel strength compared to adding oil directly (p < 0.05).