Integrating cellular experiments, single-cell sequencing, and machine learning to identify endoplasmic reticulum stress biomarkers in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) presents a severe respiratory challenge with a poor prognosis due to the lack of reliable biomarkers. Recent evidence suggests that Endoplasmic Reticulum Stress (ERS) may be associated with IPF pathogenesis. This study focuses on uncovering ERS-associated biomarkers for IPF. METHODS: Sequencing data from diverse datasets were analyzed, utilizing differential gene expression analysis and Weighted Gene Co-expression Network Analysis (WGCNA). Endoplasmic Reticulum Stress (ERS)-related genes were extracted from the GeneCards database. Hub genes were identified through Protein-Protein Interaction (PPI) analysis. Diagnostic and prognostic models were developed using machine learning algorithms and validated across both training and validation sets. Additionally, techniques such as Cell-type Identification by Estimating Relative Subsets of RNA Transcripts and single-cell RNA sequencing were employed to identify potential IPF-related cells. These findings were further investigated to elucidate their underlying mechanisms through in vitro experiments. RESULTS: Differentially expressed genes, WGCNA-identified blue module genes, and ERS-related genes extracted from the GeneCards database were intersected, and the resulting genes were used to construct diagnostic and prognostic models. Validation using multiple datasets indicated that both the diagnostic and prognostic models possess strong predictive capabilities. PPI analysis highlighted SPP1 as a potential hub gene in IPF. Moreover, M2 macrophages were found in higher quantities in the lung tissue of IPF patients, with a significant increase in SPP1-expressing M2 macrophages compared to the control group. In vitro experiments demonstrated that exogenous SPP1 inhibited the proliferation and migration of M2 macrophages and promoted apoptosis within a certain concentration range. CONCLUSION: This study identifies ERS-related biomarkers in IPF, highlighting SPP1 and M2 macrophages. The resulting diagnostic and prognostic models offer strong predictive capabilities, unveiling new therapeutic avenues.

A pilot study of optical coherence tomography-guided transbronchial biopsy in peripheral pulmonary lesions.

BACKGROUND: The diagnosis of peripheral pulmonary lesions (PPLs) remains challenging. Despite advancements in guided transbronchial biopsy (TBB) techniques, diagnostic yields haven't reached ideal levels. Optical coherence tomography (OCT) has been developed for application in pulmonary diseases, yet no data existed evaluating effectiveness in diagnosing PPLs. RESEARCH DESIGN AND METHODS: This study included patients who underwent OCT and radial endobronchial ultrasound (R-EBUS)-guided TBB. OCT and R-EBUS imaging features were analyzed to differentiate between benign and malignant PPLs and subtypes of lung cancer. RESULTS: A total of 89 patients were included in this study. The diagnostic yield of OCT-guided TBB stood at 56.18%, R-EBUS-guided TBB was 83.15% (P<0.01). The accuracy of OCT to judge the nature of lesions was 92.59%, while R-EBUS was 77.92%. The accuracy of OCT in predicting squamous carcinoma (SCC) and adenocarcinoma were both 91.30%. CONCLUSIONS: Although the diagnostic yield of OCT-guided TBB fell short of that achieved by R-EBUS, OCT possessed the capability to judge the nature of lesions and guide the pathological classification of malignant lesions. Further extensive prospective studies are necessary to thoroughly assess the characteristics of this procedure. CLINICAL TRIAL REGISTRATION: https://register.clinicaltrials.gov/ identifier is NCT06419114.

Anoikis and SPP1 in idiopathic pulmonary fibrosis: integrating bioinformatics, cell, and animal studies to explore prognostic biomarkers and PI3K/AKT signaling regulation.

OBJECTIVE: This study aims to explore the relevance of anoikis in idiopathic pulmonary fibrosis (IPF) and identify associated biomarkers and signaling pathways. METHOD: Unsupervised consensus cluster analysis was employed to categorize IPF patients into subtypes. We utilized Weighted Gene Co-Expression Network Analysis (WGCNA) and Protein-Protein Interaction network construction to identify anoikis-related modules and key genes. A prognostic signature was developed using Lasso and multivariate Cox regression analysis. Single-cell sequencing assessed hub gene expression in various cell types, and both cell and animal experiments confirmed IPF-related pathways. RESULTS: We identified two distinct anoikis-associated subtypes with differing prognoses. WGCNA revealed essential hub genes, with SPP1 being prominent in the anoikis-related signature. The anoikis-related signature is effective in determining the prognosis of patients with IPF. Single-cell sequencing highlighted significant differences in SPP1 expression, notably elevated in fibroblasts derived from IPF patients. In vivo and in vitro experiments demonstrated that SPP1 enhances fibrosis in mouse lung fibroblasts by regulating p27 through the PI3K/Akt pathway. CONCLUSION: Our research demonstrates a robust prognostic signature associated with anoikis and highlights SPP1 as a pivotal regulator of the PI3K/AKT signaling pathway in pulmonary fibrosis.

A General and Convenient Peptide Self-Assembling Mechanism for Developing Supramolecular Versatile Nanomaterials Based on The Biosynthetic Hybrid Amyloid-Resilin Protein.

Self-assembling peptides are valuable building blocks to fabricate supramolecular biomaterials, which have broad applications from biomedicine to biotechnology. However, limited choices to induce different globular proteins into hydrogels hinder these designs. Here, an easy-to-implement and tunable self-assembling strategy, which employs Ure2 amyloidogenic peptide, are described to induce any target proteins to assemble into supramolecular hydrogels alone or in combination with notable compositional control. Furthermore, the collective effect of nanoscale interactions among amyloid nanofibrils and partially disordered elastomeric polypeptides are investigated. This led to many useful macroscopic material properties simultaneously emerging from one pure protein material, i.e. strong adhesion to any substrates under wet conditions, rapidly self--assembling into robust and porous hydrogels, adaptation to remodeling processes, strongly promoting cell adhesion, proliferation and differentiation. Moreover, he demonstrated this supramolecular material's robust performance in vitro and vivo for tissue engineering, cosmetic and hemostasis applications and exhibited superior performance compared to corresponding commercial counterparts. To the best of his knowledge, few pure protein-based materials could meet such seemingly mutually exclusive properties simultaneously. Such versatility renders this novel supramolecular nanomaterial as next-generation functional protein-based materials, and he demonstrated the sequence level modulation of structural order and disorder as an untapped principle to design new proteins.

Plant-based meat analogues enhance the gastrointestinal motility function and appetite of mice by specific volatile compounds and peptides.

Eating behavior is critical for maintaining energy homeostasis. Previous studies have found that plant-based meat analogues increased diet intake in mice compared with animal meat under a free feeding mode, however the reasons were unclear. To explore the underlying mechanisms of plant-based meat analogues increasing diet intake, mice were fed animal or plant-based pork and beef analogue diets, respectively. Biochemical and histological analyses were performed to evaluate appetite-regulating hormones and gastrointestinal motility function. Peptiomics and GC-IMS were applied to identify key substances. We found that the intake of plant-based meat analogues significantly enhanced the gastrointestinal motility function of mice. The long-term intake (68 days) of plant-based meat analogues significantly increased the muscle layer thickness of the duodenum and jejunum of mice; the activity of gastrointestinal cells of Cajal were also promoted by upregulating the expression of c-kit related signals as compared to animal meat; plant-based meat analogues intake markedly enhanced the signal intensity of the intestinal neurotransmitter 5-hydroxytryptamine (5-HT) by upregulating the expression of 5-HT synthase and receptors but downregulating its transporter and catabolic enzyme in the intestine. Moreover, plant-based meat analogues intake significantly increased levels of appetite-stimulating factors in the peripheral or hypothalamus but reduced levels of appetite-suppressing factors compared with animal meat. Specific volatile compounds were significantly associated with appetite regulating factors. Among them, 7 substances such as linalool have a potential promoting effect on food intake. Besides, different digestive peptides in gastrointestinal tract may affect eating behavior mainly through the neuroactive ligand-receptor interaction pathway, exerting hormone-like effects or influencing endocrine cell secretion. These findings preliminarily clarified the mechanism of plant-based meat analogues promoting diet intake and provided a theoretical basis for a reasonable diet.

Morphology-Based Prediction of Proliferation and Differentiation Potencies of Porcine Muscle Stem Cells for Cultured Meat Production.

Inconsistent efficiency of cell production caused by cellular quality variations has become a significant problem in the cultured meat industry. In our study, morphological information on passages 5-9 of porcine muscle stem cells (pMuSCs) from three lots was analyzed and used as input data in prediction models. Cell proliferation and differentiation potencies were measured by cell growth rate and average stained area of the myosin heavy chain. Analysis of PCA and heatmap showed that the morphological parameters could be used to discriminate the differences of passages and lots. Various morphological parameters were analyzed, which revealed that accumulating time-course information regarding morphological heterogeneity in cell populations is crucial to predicting the potencies. Based on the 36 and 60 h morphological profiles, the best proliferation potency prediction model (R2 = 0.95, RMSE = 1.1) and differentiation potency prediction model (R2 = 0.74, RMSE = 1.2) were explored. Correlation analysis demonstrated that morphological parameters selected in models are related to the quality of porcine muscle stem cells.

Tea polyphenols coated sodium alginate-gelatin 3D edible scaffold for cultured meat.

This study aimed to use edible scaffolds as a platform for animal stem cell expansion, thus constructing block-shaped cell culture meat. The tea polyphenols (TP)-coated 3D scaffolds were constructed of sodium alginate (SA) and gelatin (Gel) with good biocompatibility and mechanical support. Initially, the physicochemical properties and mechanical properties of SA-Gel-TP scaffolds were measured, and the biocompatibility of the scaffolds was evaluated by C2C12 cells. SEM results showed that the scaffold had a porous laminar structure with TP particles attached to the surface, while FT-IR results also demonstrated the encapsulation of TP coating on the scaffold. In addition, the porosity of all scaffolds was higher than 40% and the degradation rate during the incubation cycle was less than 40% and the S2-G1-TP0.1-3 h scaffold has excellent cell adhesion and extension. Subsequently, we inoculated rabbit skeletal muscle myoblasts (RbSkMC) on the scaffold and induced differentiation. The results showed good adhesion and extension behavior of RbSkMC on S2-G1-TP0.1-3 h scaffolds with high expression of myogenic differentiation proteins and genes, and SEM results confirmed the formation of myotubes. Additionally, the adhesion rate of cells on scaffolds with TP coating was 1.5 times higher than that on scaffolds without coating, which significantly improved the cell proliferation rate and the morphology of cells with extension on the scaffolds. Furthermore, rabbit-derived cultured meat had similar appearance and textural characteristics to fresh meat. These conclusions indicate the high potential of the scaffolds with TP coating as a platform for the production of cultured meat products.

SaaS sRNA promotes Salmonella intestinal invasion via modulating MAPK inflammatory pathway.

Salmonella Enteritidis is a foodborne enteric pathogen that infects humans and animals, utilizing complex survival strategies. Bacterial small RNA (sRNA) plays an important role in these strategies. However, the virulence regulatory network of S. Enteritidis remains largely incomplete and knowledge of gut virulence mechanisms of sRNAs is limited. Here, we characterized the function of a previously identified Salmonella adhesive-associated sRNA (SaaS) in the intestinal pathogenesis of S. Enteritidis. We found that SaaS promoted bacterial colonization in both cecum and colon of a BALB/c mouse model; it was preferentially expressed in colon. Moreover, our results showed that SaaS enhanced damage to mucosal barrier by affecting expressions of antimicrobial products, decreasing the number of goblet cells, suppressing mucin gene expression, and eventually reducing thickness of mucus layer; it further breached below physical barrier by strengthening invasion into epithelial cells in Caco-2 cell model as well as decreasing tight junction expressions. High throughput 16S rRNA gene sequencing revealed that SaaS also altered gut homeostasis by depleting beneficial gut microbiota while increasing harmful ones. Furthermore, by employing ELISA and western blot analysis, we demonstrated that SaaS regulated intestinal inflammation through sequential activation P38-JNK-ERK MAPK signaling pathway, which enabled immune escape at primary infection stage but strengthened pathogenesis at later stage, respectively. These findings suggest that SaaS plays an essential role in the virulence of S. Enteritidis and reveals its biological role in intestinal pathogenesis.

Effects of carboxymethyl cellulose on the emulsifying, gel and digestive properties of myofibrillar protein-soybean oil emulsion.

Improving the qualities of vegetable oil replaced animal fat meat products is particularly fascinating for the development of healthy meat products. This work was designed to investigate the effects of different carboxymethyl cellulose (CMC) concentrations (0.01 %, 0.05 %, 0.1 %, 0.2 %, and 0.5 %) on the emulsifying, gelation, and digestive properties of myofibrillar protein (MP)-soybean oil emulsions. The changes in MP emulsion characteristics, gelation properties, protein digestibility, and oil release rate were determined. Results demonstrated that CMC addition decreased the average droplet size and increased the apparent viscosity, storage modulus, and loss modulus of MP emulsions, and a 0.5 % CMC addition significantly increased the storage stability during 6 weeks. Lower CMC addition (0.01 % to 0.1 %) increased the hardness, chewiness, and gumminess of emulsion gel especially for the 0.1 % CMC addition, while higher CMC (0.5 %) content decreased the texture properties and water holding capacity of emulsion gels. The addition of CMC decreased protein digestibility during the gastric stage, and 0.01 % and 0.05 % CMC addition significantly decreased the free fatty acid release rate. In summary, the addition of CMC could improve the stability of MP emulsion and the texture properties of the emulsion gels, and decrease protein digestibility during the gastric stage.

Effects of heating rates on the self-assembly behavior and gelling properties of beef myosin.

BACKGROUND: Myosin is the most important component of myofibrillar protein, with excellent gelling properties. To date, heating treatment remains the mainstream method for forming gel in meat products, and it has the most extensive application in the field of meat industry. However, at present, there are few reports on the effects of heating rates on myosin self-assembly and aggregation behavior during heating treatment. RESULTS: The present study aimed to investigate the effects of different heating rates (1, 2, 3 and 5 °C min-1 ) on the self-assembly behavior, physicochemical, structural and gelling properties of myosin. At the lowest heating rate of 1 °C min-1 , the myosin gel had a dense microstructure, the highest elastic modulus (G') and water holding capacity compared to higher heating rates (2, 3 and 5 °C min-1 ). At higher temperatures (40, 45 °C), the surface hydrophobicity, turbidity, particle size distribution and self-assembly behavior of myosin in pre-gelling solutions showed that myosin had sufficient time to denature, underwent full structure unfolding before aggregation at the heating rate of 1°C min-1 , and formed regular and homogeneous spherical aggregates. Therefore, the myosin gel also had a better three-dimensional network. CONCLUSION: The heating rates had an important effect on the quality of myosin gels, and had theoretical implications for improving the quality of meat gel products. © 2023 Society of Chemical Industry.

Plant-Based Meat Analogues Weaken Gastrointestinal Digestive Function and Show Less Digestibility Than Real Meat in Mice.

Real meat and plant-based meat analogues have different in vitro protein digestibility properties. This study aims to further explore their in vivo digestion and absorption and their effects on the gastrointestinal digestive function of mice. Compared with the real pork and beef, plant-based meat analogues significantly reduced the number of gastric parietal cells, the levels of gastrin/CCKBR, acetylcholine/AchR, Ca2+, CAMK II, PKC, and PKA, the activity of H+, K+-ATPase, and pepsin, the duodenal villus height, and the ratio of villus height to crypt depth and downregulated the expression of most nitrogen nutrient sensors. Peptidomics revealed that plant-based meat analogues released fewer peptides during in vivo digestion and increased the host- and microbial-derived peptides. Moreover, the real beef showed better absorption properties. These results suggested that plant-based meat analogues weaken gastrointestinal digestive function of mice, and their digestion and absorption performance in vivo is not as good as the real meat.

In vitro digestion mimicking conditions in young and elderly reveals marked differences between profiles and potential bioactivity of peptides from meat and soy proteins.

We applied in vitro models of gastrointestinal (GI) digestion simulating the conditions of the GI tract of healthy adults and elderly individuals with achlorhydria (EA) to investigate differences in the digestibility of meat (chicken, beef and pork) and soy proteins. Digestibility was significantly affected by EA alterations. Peptidomics analyses revealed significant differences in peptide profiles between control and EA conditions, including number, length distribution, clustering, and differentially abundant peptides (DAPs). Our results revealed that the differences in meat peptide profiles diminished going from the gastric to intestinal phase. For soy protein, the marked differences between control and EA conditions were maintained in the gastric and intestinal phases. Higher numbers of potentially bioactive peptides were generated under the control condition compared to the EA condition. The present study provides insight into the distinct peptide profiles generated by in vitro digestion of meat and soy proteins under adult and EA GI conditions.

Phenolic modification of myofibrillar protein enhanced by ultrasound: The structure of phenol matters.

Phenolic modification of myofibrillar protein (MPN) is an essential technology in meat processing. This paper investigated the grafting reaction of three structurally relevant polyphenols (PPs), epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin (EC), and MPN, in a conventional alkaline reaction and ultrasound (UT)-assisted oxidation system. EC triggered the production of more hydroxyl radicals at an equal molar concentration, resulting in a noticeable improvement of the final grafting effect. Moreover, pronounced changes in pore area on the microscopic scale was observed in MPN-EGCG, which was ascribed to the unique chemical structure of EGCG. Additionally, the antioxidant activities of the UT-assisted EGCG group were 133.89% and 103.10% higher than those of the single MPN group (PP0) and pure EGCG group, respectively. These results emphasized the importance of the chemical structure of PPs in the process of different grafting reactions.

Interactions between the protein-epigallocatechin gallate complex and nanocrystalline cellulose: A systematic study.

In this work, the noncovalent interactions between the protein-epigallocatechin gallate (protein-EGCG) complex and nanocrystalline cellulose (NCC) were systematically investigated. Rheological properties, including large amplitude oscillation shear (LAOS), transient rheology, and conventional rheology of the mixture were also evaluated. As obtained, flexible myofibrillar protein-epigallocatechin gallate (MP-EGCG) and rigid ovalbumin-epigallocatechin gallate (Ova-EGCG) follow different rules in the stability of the regulatory system because of the difference in noncovalent interactions, triggering different rheological responses of the complexes. Additionally, MP-EGCG/NCC showed an obvious strain overshoot during LAOS flow, which could not be obtained in Ova-EGCG/NCC. Notably, the fibrous MP-EGCG network was the factor that dominated the formation of a more stable suspension system with strong hydrogen bonding, dipole-dipole interactions and/or van der Waals forces. This study can provide some ideas for the future study of the interaction between protein-polyphenol complexes and polysaccharides.

Real meat and plant-based meat analogues have different in vitro protein digestibility properties.

To explore the nutritional values of meat and meat analogues, the in vitro protein digestion of pork, beef, plant-based pork and beef were evaluated. In the gastric phase, the digestibility of pork was significantly higher than that of the plant-based pork, while the value of beef was lower than that of the plant-based beef. In the intestinal phase, both pork and beef showed higher digestibility than plant-based meat analogues. A greater number of small molecular peptides were identified from pork and beef than from plant-based meat after gastrointestinal digestion. Larger quantities of potential bioactive peptides were released from the meat than from the plant-based meat analogues during digestion. These differences were closely related to protein secondary structure, the formation of disulfide bonds and apparent viscosity of digestion solution. The findings give a new insight into the underlying mechanisms of the different phenotype responses of consumers to meat and plant-based meat.

Comparative study on the in vitro digestibility of chicken protein after different modifications.

The effects of tea polyphenols (TPPs) and ultrasound treatment (UDT) on the digestibility of chicken myofibrillar protein (MPN) in anenhanced oxidation system were investigated. As observed, the original aggregates of MPN were much lower in the UDT-assisted group than in the control protein group, and the difference widened after the incorporation of TPPs. The covalent structures of the UDT-assisted oxidation groups were verified via mass spectrometry and amino acid (AAD) measurements. The peptide abundance increased after the UDT-assisted covalent reaction and most of these peptides were derived from the structural proteins of MPNs according to the results of nano-LC-ESI-MS/MS. Digestion kinetic analysis showed that the digestion level of the EGCG-treated group was better than that of the other treated groups, regardless of the UDT-assisted covalent reaction. Overall, the combination of EGCG oxidation and UDT may be an efficient way to promote the nutritional value of the final MPN products.

Proteomic Analysis of the Protective Effect of Eriodictyol on Benzo(a)pyrene-Induced Caco-2 Cytotoxicity.

We evaluated the possible protective effects of six polyphenols on benzo(a)pyrene (BaP)-induced cytotoxicity in Caco-2 cells. We show that treatment with quinic acid, ferulic acid, homovanillic acid, trolox and BaP decreased cell viability, whereas naringenin and eriodictyol affected viability in a bi-phasic manner with low concentrations decreasing viability whereas higher concentrations increase viability. Co-treatment with 20 µM eriodictyol or naringenin reduced BaP-induced cytotoxicity, including cell apoptosis, cell cycle progression, and oxidative stress. Our results show that the protective effect of eriodictyol was superior to that of naringenin. The potential protective mechanisms of eriodictyol on BaP-induced toxicity were investigated by proteomics. We identified 80 differentially expressed proteins (DEPs) with proteins associated with genetic information processing pathway representing the highest proportion and number of proteins responding to eriodictyol treatment, including key proteins such as RPA2, SNRPA, RAD23B, NUP155 and AARS. Our results provide new knowledge on how polyphenols may prevent BaP-induced carcinogenesis.

Effects of quercetin on tenderness, apoptotic and autophagy signalling in chickens during post-mortem ageing.

The effect of quercetin on chicken breast muscle tenderness and the associated mechanism were investigated. The results indicated that quercetin significantly decreased the shear force and increased the myofibril fragmentation index (MFI). Haematoxylin-eosin-stained images showed that the internal structure of myofibril bundles in the quercetin-treated group was obviously degraded. Transmission electron microscopy showed that the myofibril structure, especially the M-line and A-band, was seriously degraded after quercetin treatment. Furthermore, quercetin treatment increased caspase-3 activity and the Bax/Bcl-2 ratio. The intensity of BiP, XBP1 and p-IRE1/IRE1 ratio increased significantly, and caspase-12 was activated. In addition, quercetin induced the transition from LC3I to LC3II and increased the expression of ATG7 and Beclin-1. The PI3K/Akt/mTOR signalling pathway was involved in the induction of autophagy and apoptosis by quercetin. These results indicated quercetin can promote meat tenderization, and activate apoptosis and autophagy pathways during post-mortem ageing.

A comprehensive review on molecular mechanism of defective dry-cured ham with excessive pastiness, adhesiveness, and bitterness by proteomics insights.

Excessive bitterness, pastiness, and adhesiveness are the main organoleptic and textural defects of dry-cured ham, which often cause a lot of financial losses to manufacturers and seriously damage the quality of the product. These sensory and textural defects are related to the protein degradation of dry-cured ham. Proteomics shows great potential to improve our understanding of the molecular mechanism of sensory and textural defects and identify biomarkers for monitoring their quality traits. This review presents some of the major achievements and considerations in organoleptic and textural defects of dry-cured ham by proteomics analysis in the recent decades and gives an overview about how to correct sensory and textural defects of dry-cured ham. Proteomics reveals that muscle proteins derived from myofibril and cytoskeleton and involved in metabolic enzymes and oxygen transport have been identified as potential biomarkers in defective dry-cured ham. Relatively high residual activities of cathepsin B and L are responsible for the excessive degradation of these protein biomarkers in defective dry-cured ham. Ultrasound-assisted mild thermal or high-pressure treatment shows a good correction for the organoleptic and textural defects of dry-cured ham by changing microstructure and conformation of muscle proteins by accelerating degradation of proteins and polypeptides into free amino acids.

Effect of incubation temperature on the binding capacity of flavor compounds to myosin.

This study was aimed to investigate the effect of incubation temperature on the binding of hexanal, octanal and 3-methylbutyraldehyde to myosin. Fluorescence quenching, Fourier transform infrared spectroscopy, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and gas chromatography-mass spectrometry (GC-MS) were employed. An increase in aldehyde concentration led to a reduction in fluorescence intensity in myosin. SPR revealed that the interactions were involved in a rapid combination and dissociation, and the dissociation constants significantly decreased from 25 to 37 °C. ITC showed that the values of entropy, enthalpy and Gibbs free energy were negative. The interactions were driven by hydrogen bonds and van der Waals forces. GC-MS further demonstrated that the highest binding capacity occurred at 37 °C between myosin and aldehydes. The findings provide a new insight into the mechanism on controlling or maintaining meat flavor.