Pepsin immobilization on activated carbon and functionalized with glutaraldehyde and genipin for the synthesis of antioxidant peptides of goat casein.

In this article, the synthesis of antioxidant peptides in the enzymatic hydrolysis of caprine casein was analyzed at three different time points (60 min, 90 min, and 120 min) using immobilized pepsin on activated and modified carbon (AC, ACF, ACG 50, ACG 100). The immobilization assays revealed a reduction in the biocatalysts' activity compared to the free enzyme. Among the modified ones, ACG 50 exhibited greater activity and better efficiency for reuse cycles, with superior values after 60 min and 90 min. Peptide synthesis was observed under all studied conditions. Analyses (DPPH, ß-carotene/linoleic acid, FRAP) confirmed the antioxidant potential of the peptides generated by the immobilized enzyme. However, the immobilized enzyme in ACG 50 and ACG 100, combined with longer hydrolysis times, allowed the formation of peptides with an antioxidant capacity greater than or equivalent to those generated by the free enzyme, despite reduced enzymatic activity.

Oral infectivity through carnivorism in murine model of Trypanosoma cruzi infection.

Introduction: Oral transmission of T. cruzi is probably the most frequent transmission mechanism in wild animals. This observation led to the hypothesis that consuming raw or undercooked meat from animals infected with T. cruzi may be responsible for transmitting the infection. Therefore, the general objective of this study was to investigate host-pathogen interactions between the parasite and gastric mucosa and the role of meat consumption from infected animals in the oral transmission of T. cruzi. Methods: Cell infectivity assays were performed on AGS cells in the presence or absence of mucin, and the roles of pepsin and acidic pH were determined. Moreover, groups of five female Balb/c mice were fed with muscle tissue obtained from mice in the acute phase of infection by the clone H510 C8C3hvir of T. cruzi, and the infection of the fed mice was monitored by a parasitemia curve. Similarly, we assessed the infective capacity of T. cruzi trypomastigotes and amastigotes by infecting groups of five mice Balb/c females, which were infected orally using a nasogastric probe, and the infection was monitored by a parasitemia curve. Finally, different trypomastigote and amastigote inoculums were used to determine their infective capacities. Adhesion assays of T. cruzi proteins to AGS stomach cells were performed, and the adhered proteins were detected by western blotting using monoclonal or polyclonal antibodies and by LC-MS/MS and bioinformatics analysis. Results: Trypomastigote migration in the presence of mucin was reduced by approximately 30%, whereas in the presence of mucin and pepsin at pH 3.5, only a small proportion of parasites were able to migrate (∼6%). Similarly, the ability of TCTs to infect AGS cells in the presence of mucin is reduced by approximately 20%. In all cases, 60-100% of the animals were fed meat from mice infected in the acute phase or infected with trypomastigotes or amastigotes developed high parasitemia, and 80% died around day 40 post-infection. The adhesion assay showed that cruzipain is a molecule of trypomastigotes and amastigotes that binds to AGS cells. LC-MS/MS and bioinformatics analysis, also confirmed that transialidase, cysteine proteinases, and gp63 may be involved in TCTs attachment or invasion of human stomach cells because they can potentially interact with different proteins in the human stomach mucosa. In addition, several human gastric mucins have cysteine protease cleavage sites. Discussion: Then, under our experimental conditions, consuming meat from infected animals in the acute phase allows the T. cruzi infection. Similarly, trypomastigotes and amastigotes could infect mice when administered orally, whereas cysteinyl proteinases and trans-sialidase appear to be relevant molecules in this infective process.

Structural analyzes suggest that MiSSP13 and MiSSP16.5 may act as proteases inhibitors during ectomycorrhiza establishment in Laccaria bicolor.

•The signaling process during mycorrhiza establishment involves intense molecular communication between symbionts. It has been suggested that a group of protein effectors, the so-called MiSSPs, plays a broader function in the symbiosis metabolism, however, many of these remain uncharacterized structurally and functionally. •Herein we used three-dimensional protein structure modeling methods, ligand analysis, and molecular docking to structurally characterize and describe two protein effectors, MiSSP13 and MiSSP16.5, with enhanced expression during the mycorrhizal process in Laccaria bicolor. •MiSSP13 and MiSSP16.5 show structural homology with the cysteine and aspartate protease inhibitor, cocaprin (CCP1). Through structural analysis, it was observed that MiSSP13 and MiSSP16.5 have an active site similar to that observed in CCP1. The protein-protein docking data showed that MiSSP13 and MiSSP16.5 interact with the papain and pepsin proteases at sites that are near to where CCP1 interacts with these same targets, suggesting a function as inhibitor of cysteine and aspartate proteases. The interaction of MiSSP13 with papain and MiSSP16.5 with pepsin was stronger than the interaction of CCP1 with these proteases, suggesting that the MiSSPs had a greater activity in inhibiting these classes of proteases. Based on the data supplied, a model is proposed for the function of MiSSPs 13 and 16.5 during the symbiosis establishment. Our findings, while derived from in silico analyses, enable us formulate intriguing hypothesis on the function of MiSSPs in ectomycorrhization, which will require experimental validation.

[Analysis of clinical characteristics of children with adenoid hypertrophy and pharyngolaryngeal reflux].

Objectives: To explore the clinical characteristics of children with adenoid hypertrophy (AH) and laryngopharyngeal reflux (LPR) by detecting the expression of pepsin in adenoids as a standard for AH with LPR. Methods: A total of 190 children who were admitted for surgical treatment due to AH were included in the study. The main clinical symptoms of the patients were recorded, and the degree of adenoid hypertrophy was evaluated. Before the surgery, Reflux Symptom Index (RSI) and Reflux Finding Score (RFS) were used to evaluate the reflux symptoms. After the surgery, pepsin immunohistochemical staining was performed on the adenoid tissue, and according to the staining results, the patients were divided into study group (pepsin staining positive) and control group (pepsin staining negative). SPSS 19.0 software was used for statistical analysis. Quantitative data conforming to normal distribution between the two groups were tested by two-independent sample t test, and quantitative data with skewed distribution were tested by Mann-Whitney U test. Results: The positive rate of pepsin staining in the 190 AH patients was 78.4% (149/190). The study group had higher levels of preoperative symptoms such as erythema and/or congestion of the pharynx(2.1±0.7 vs. 1.8±0.6,t=2.23), vocal cord edema[1.0(0, 1.0) vs. 1.0(0, 1.0), Z=2.00], diffuse laryngeal edema[0(0, 1.0) vs. 0(0, 0), Z=2.48], posterior commissure hypertrophy[(1.4±0.6 vs. 1.1±0.5), t=2.63], and a higher total score on the RFS scale than the control group(6.2±2.7 vs. 5.0±2.6, t=2.47), with statistical differences (P<0.05). The sensitivity and specificity of RFS score in diagnosing AH with LPR were 24.8% and 80.5%, respectively. When RFS>5 was used as the positive threshold, the sensitivity and specificity of RFS score in diagnosing AH with LPR were 61.1% and 58.5%, respectively. There was a statistical difference in the number of positive cases of RFS score between the study group and the control group(91 vs. 17,χ2=5.04,P=0.032). Conclusions: LPR is common in AH children. Children with AH and LPR have specific performance in electronic laryngoscopy, such as erythema with edema in the pharynx, posterior commissure hypertrophy, and vocal cord edema.

Study of an inhibitory effect of plant polyphenolic compounds against digestive enzymes using bench-working experimental evidence predicted by molecular docking and dynamics.

The substantial nutritional content and diversified biological activity of plant-based nutraceuticals are due to polyphenolic chemicals. These chemicals are important and well-studied plant secondary metabolites. Their protein interactions are extensively studied. This relationship is crucial for the logical development of functional food and for enhancing the availability and usefulness of polyphenols. This study highlights the influence of protein types and polyphenols on the interaction, where the chemical bindings predominantly consist of hydrophobic interactions and hydrogen bonds. The interaction between polyphenolic compounds (PCs) and digestive enzymes concerning their inhibitory activity has not been fully studied. Therefore, we have examined the interaction of four digestive enzymes (α-amylase, pepsin, trypsin, and α-chymotrypsin) with four PCs (curcumin, diosmin, morin, and 2',3',4'-trihydroxychalcone) through in silico and in vitro approaches. In vitro plate assays, enzyme kinetics, spectroscopic assays, molecular docking, and simulations were performed. We observed all these PCs have significant docking scores and preferable interaction with the active site of the digestive enzymes, resulting in the reduction of enzyme activity. The enzyme-substrate binding mechanism was determined using the Lineweaver Burk plot, indicating that the inhibition occurred competitively. Among four PCs diosmin and morin has the highest interaction energy over digestive enzymes with IC50 value of 1.13 ± 0.0047 and 1.086 ± 0.0131 µM. Kinetic studies show that selected PCs inhibited pepsin, trypsin, and chymotrypsin competitively and inhibited amylase in a non-competitive manner, especially by 2',3',4'-trihydroxychalcone. This study offers insights into the mechanisms by which the selected PCs inhibit the enzymes and has the potential to enhance the application of curcumin, diosmin, morin, and 2',3',4'-trihydroxychalcone as natural inhibitors of digestive enzymes.

The molecular mechanisms of extracellular matrix-derived hydrogel therapy in idiopathic pulmonary fibrosis models.

Idiopathic Pulmonary Fibrosis (IPF) is a progressively debilitating lung condition characterized by oxidative stress, cell phenotype shifts, and excessive extracellular matrix (ECM) deposition. Recent studies have shown promising results using decellularized ECM-derived hydrogels produced through pepsin digestion in various lung injury models and even a human clinical trial for myocardial infarction. This study aimed to characterize the composition of ECM-derived hydrogels, assess their potential to prevent fibrosis in bleomycin-induced IPF models, and unravel their underlying molecular mechanisms of action. Porcine lungs were decellularized and pepsin-digested for 48 h. The hydrogel production process, including visualization of protein molecular weight distribution and hydrogel gelation, was characterized. Peptidomics analysis of ECM-derived hydrogel contained peptides from 224 proteins. Probable bioactive and cell-penetrating peptides, including collagen IV, laminin beta 2, and actin alpha 1, were identified. ECM-derived hydrogel treatment was administered as an early intervention to prevent fibrosis advancement in rat models of bleomycin-induced pulmonary fibrosis. ECM-derived hydrogel concentrations of 1 mg/mL and 2 mg/mL showed subtle but noticeable effects on reducing lung inflammation, oxidative damage, and protein markers related to fibrosis (e.g., alpha-smooth muscle actin, collagen I). Moreover, distinct changes were observed in macroscopic appearance, alveolar structure, collagen deposition, and protein expression between lungs that received ECM-derived hydrogel and control fibrotic lungs. Proteomic analyses revealed significant protein and gene expression changes related to cellular processes, pathways, and components involved in tissue remodeling, inflammation, and cytoskeleton regulation. RNA sequencing highlighted differentially expressed genes associated with various cellular processes, such as tissue remodeling, hormone secretion, cell chemotaxis, and cytoskeleton engagement. This study suggests that ECM-derived hydrogel treatment influence pathways associated with tissue repair, inflammation regulation, cytoskeleton reorganization, and cellular response to injury, potentially offering therapeutic benefits in preventing or mitigating lung fibrosis.

Lactobacillus casei-Derived Postbiotics Elevate the Bioaccessibility of Proteins via Allosteric Regulation of Pepsin and Trypsin and Introduction of Endopeptidases.

The potential of probiotics to benefit digestion has been widely reported, while its utilization in high-risk patients and potential adverse reactions have focused interest on postbiotics. A variable data-independent acquisition (vDIA)-based spatial-omics strategy integrated with unsupervised variational autoencoders was applied to profile the functional mechanism underlying the action of Lactobacillus casei-derived postbiotic supplementation in goat milk digestion in an infant digestive system, from a metabolomics-peptidomics-proteomics perspective. Amide and olefin derivatives were proved to elevate the activities of pepsin and trypsin through hydrogen bonding and hydrophobic forces based on allosteric effects, and recognition of nine endopeptidases and their cleavage to serine, proline, and aspartate were introduced by postbiotics, thereby promoting the generation of hydrophilic peptides and elevating the bioaccessibility of goat milk protein. The peptides originating from αs1-casein, ß-casein, ß-lactoglobulin, Ig-like domain-containing protein, κ-casein, and serum amyloid A protein, with multiple bioactivities including angiotensin I-converting enzyme (ACE)-inhibitory, osteoanabolic, dipeptidyl peptidase IV (DPP-IV) inhibitory, antimicrobial, bradykinin-potentiating, antioxidant, and anti-inflammatory activities, were significantly increased in the postbiotic supplementation group, which was also considered to potentially prevent necrotizing enterocolitis through inhibiting the multiplication of pathogenic bacteria and blocking signal transducer and activator of transcription 1 and nuclear factor kappa-light-chain-enhancer of activated B cells inflammatory pathways. This research deepened the understanding of the mechanism underlying the postbiotics affecting goat milk digestion, which established a critical groundwork for the clinical application of postbiotics in infant complementary foods.

Small polystyrene microplastics interfere with the breakdown of milk proteins during static in vitro simulated human gastric digestion.

Human ingestion of microplastics (MPs) is common and inevitable due to the widespread contamination of food items, but implications on the gastric digestion of food proteins are still unknown. In this study, the interactions between pepsin and polystyrene (PS) MPs were evaluated by investigating enzyme activity and conformation in a simulated human gastric environment in the presence or absence of PS MPs. The impact on food digestion was also assessed by monitoring the kinetics of protein hydrolysis through static in vitro gastric digestion of cow's milk contaminated with PS. The binding of pepsin to PS showed that the surface chemistry of MPs dictates binding affinity. The key contributor to pepsin adsorption seems to be π-π interactions between the aromatic residues and the PS phenyl rings. During quick exposure (10 min) of pepsin to increasing concentrations (222, 2219, 22188 particles/mL) of 10 µm PS (PS10) and 100 µm PS (PS100), total enzymatic activities were not affected remarkably. However, upon prolonged exposure at 1 and 2 h, preferential binding of pepsin to the small, low zeta-potential PS caused structural changes in the protein which led to a significant reduction of its activity. Digestion of cow's milk mixed with PS10 resulted in transient accumulation of larger peptides (10-35 kDa) and reduced bioavailability of short peptides (2-9 kDa) in the gastric phase. This, however, was only observed at extremely high PS10 concentration (0.3 mg/mL or 5.46E+05 particles/mL). The digestion of milk peptides, bound preferentially over pepsin within the hard corona on the PS10 surface, was delayed up to 15 min in comparison to bulk protein digestion. Intact caseins, otherwise rapidly digested, remained bound to PS10 in the hard corona for up to 15 min. This work presents valuable insights regarding the interaction of MPs, food proteins, and pepsin, and their dynamics during gastric digestion.

The relationship between keratan sulfate glycosaminoglycan density and mechanical stiffening of CXL treatment.

The corneal stroma is primarily composed of collagen fibrils, proteoglycans, and glycosaminoglycans (GAGs). It is known that corneal crosslinking (CXL) treatment improves mechanical properties of the cornea. However, the influence of stromal composition on the strengthening effect of CXL procedure has not been thoroughly investigated. The primary objective of the present research was to characterize the effect of keratan sulfate (KS) GAGs on the efficacy of CXL therapy. To this end, the CXL method was used to crosslink porcine corneal samples from which KS GAGs were enzymatically removed by keratanase II enzyme. Alcian blue staining was done to confirm the successful digestion of GAGs and uniaxial tensile experiments were performed for characterizing corneal mechanical properties. The influence of GAG removal and CXL treatment on resistance of corneal samples against enzymatic pepsin degradation was also quantified. It was found that removal of KS GAGs significantly softened corneal tensile properties (P < 0.05). Moreover, the CXL therapy significantly increased the tensile stiffness of GAG-depleted strips (P < 0.05). GAG-depleted corneal buttons were dissolved in the pepsin digestion solution significantly faster than control samples (P < 0.05). The CXL treatment significantly increased the time needed for complete pepsin digestion of GAG-depleted disks (P < 0.05). Based on these observations, we concluded that KS GAGs play a significant role in defining tensile properties and structural integrity of porcine cornea. Furthermore, the stiffening influence of the CXL treatment does not significantly depend on the density of corneal KS GAGs. The findings of the present study provided new information on the relation between corneal composition and CXL procedure mechanical effects.

Native and compactly folded in-solution conformers of pepsin are revealed and distinguished by mass spectrometric ITEM-TWO analyses of gas-phase pepstatin A - pepsin complex binding strength differences.

Pepsin, because of its optimal activity at low acidic pH, has gained importance in mass spectrometric proteome research as a readily available and easy-to-handle protease. Pepsin has also been study object of protein higher-order structure analyses, but questions about how to best investigate pepsin in-solution conformers still remain. We first determined dependencies of pepsin ion charge structures on solvent pH which indicated the in-solution existence of (a) natively folded pepsin (N) which by nanoESI-MS analysis gave rise to a narrow charge state distribution with an 11-fold protonated most intense ion signal, (b) unfolded pepsin (U) with a rather broad ion charge state distribution whose highest ion signal carried 25 protons, and (c) a compactly folded pepsin conformer (C) with a narrow charge structure and a 12-fold protonated ion signal in the center of its charge state envelope. Because pepsin is a protease, unfolded pepsin became its own substrate in solution at pH 6.6 since at this pH some portion of pepsin maintained a compact/native fold which displayed enzymatic activity. Subsequent mass spectrometric ITEM-TWO analyses of pepstatin A - pepsin complex dissociation reactions in the gas phase confirmed a very strong binding of pepstatin A by natively folded pepsin (N). ITEM-TWO further revealed the existence of two compactly folded in-solution pepsin conformers (Ca and Cb) which also were able to bind pepstatin A. Binding strengths of the respective compactly folded pepsin conformer-containing complexes could be determined and apparent gas phase complex dissociation constants and reaction enthalpies differentiated these from each other and from the pepstatin A - pepsin complex which had been formed from natively folded pepsin. Thus, ITEM-TWO turned out to be well suited to pinpoint in-solution pepsin conformers by interrogating quantitative traits of pepstatin A - pepsin complexes in the gas phase.

Nutritional properties and osteogenic activity of simulated digestion components and peptides from Larimichthys crocea.

Fish provides a range of health benefits due to its nutritional and bioactive components. However, the bioactive peptides derived from Larimichthys crocea proteins were not fully investigated, especially the beneficial effects related to bone growth in vitro. In this study, the water extract protein was subjected to the simulated in vitro digestion process, and the osteogenic effect of enzymatic hydrolysate at different digestion stages was evaluated by the proliferation of osteoblast. The protein hydrolyzates of group pepsin treatment for 1 h and pepsin treatment for 2 h showed higher osteogenic activity in vitro. Two peptides including IERGDVVVQDSPSD from pepsin treatment for 1 h and RGDLGIEIPTEK from pepsin treatment for 2 h were identified, which revealed eminent effects in terms of promoting osteoblast proliferation and enhancing ALP activity. Moreover, the available nutrients in the proteins were determined by the molecular weight distribution and free amino acid composition. Those peptides also showed stronger interaction with RGD than integrins. Therefore, the peptides from Larimichthys crocea can be used as an effective ingredient for promoting bone growth in the future.

In vitro antioxidant and anti-inflammatory properties of Artocarpus altilis (Parkinson) Fosberg (seedless breadfruit) fruit pulp protein hydrolysates.

Protein hydrolysates from dietary sources possess many physiological and biological properties. Artocarpus altilis is an evergreen multipurpose plant with many benefits. Therefore, this study evaluates in vitro antioxidant and anti-inflammatory properties of A. altilis protein hydrolysates. Protein was isolated from A. altilis and hydrolysed with pepsin and trypsin separately using different enzyme: substrate ratios (1:8, 1:16, 1:32). Antioxidant properties investigated included Fe2+-chelating, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and hydrogen peroxide radical scavenging activities. Anti-inflammatory activities were determined using effects on hypotonic solution-induced cell lysis on red blood cell membrane stabilisation and heat-induced protein denaturation. The degree of hydrolysis of trypsin hydrolysate increased with increasing enzyme-substrate ratio, while pepsin hydrolysate decreased as the enzyme-substrate ratio increased. The dominant amino acids in A. altilis protein and hydrolysates were glutamate, aspartate and leucine. Protein hydrolysates obtained from pepsin and trypsin digestion had DPPH scavenging abilities of 43.0 ± 0.01% and 22.2 ± 0.01%, respectively. However, trypsin-hydrolysed protein had a high Fe2+-chelating ability, while pepsin-hydrolysed protein had high hydrogen peroxide scavenging ability. Trypsin-hydrolysed protein showed good membrane stability and inhibition of protein denaturation. The results indicated that A. altilis protein hydrolysates possess significant antioxidant and anti-inflammatory effects and can further lend support to food industries as functional foods.

Proteolysis-driven proliferation and rigidification of pepsin-resistant amyloid fibrils.

Proteolysis of amyloids is related to prevention and treatment of amyloidosis. What if the conditions for proteolysis were the same to those for amyloid formation? For example, pepsin, a gastric protease is activated in an acidic environment, which, interestingly, is also a condition that induces the amyloid formation. Here, we investigate the competition reactions between proteolysis and synthesis of amyloid under pepsin-activated conditions. The changes in the quantities and nanomechanical properties of amyloids after pepsin treatment were examined by fluorescence assay, circular dichroism and atomic force microscopy. We found that, in the case of pepsin-resistant amyloid, a secondary reaction can be accelerated, thereby proliferating amyloids. Moreover, after this reaction, the amyloid became 32.4 % thicker and 24.2 % stiffer than the original one. Our results suggest a new insight into the proteolysis-driven proliferation and rigidification of pepsin-resistant amyloids.

Exploration of binding mechanism of apigenin to pepsin: Spectroscopic analysis, molecular docking, enzyme activity and antioxidant assays.

Pepsin plays an important role in nutrient metabolism. Apigenin (AP) is a beneficial polyphenol to human health. To enhance the bioavailability of AP and elucidate the inhibitory effect of AP on pepsin, the interaction mechanism of AP with pepsin was investigated using spectroscopic analysis and molecular docking, and the activity of pepsin and antioxidant activity of AP was also evaluated. Specifically, AP performed static quenching of pepsin and had only one binding site on pepsin. More interestingly, the interaction between AP and pepsin was spontaneous, while hydrogen bonds and van der Waals forces were the main binding forces. Generally, synchronous and three-dimensional fluorescence confirmed that AP induced the conformational changes of pepsin, and molecular docking proved the above results and illustrated the specific binding patterns. Specifically, AP inhibited the activity of pepsin, while pepsin decreased the antioxidant activity of AP. These results provided useful information for elucidating the interactions between AP and pepsin.

Purification identification and function analysis of ACE inhibitory peptide from Ulva prolifera protein.

In the present study, Ulva prolifera, an edible alga, was used to prepare angiotensin-I converting enzyme (ACE) inhibitory peptide. The algae protein was isolated and later hydrolyzed by five commercial enzymes (alcalase, papain, pepsin, trypsin, neutral protease), either individually or in combination. Hydrolysate, with the highest in vitro ACE inhibitory activity, was processed using the Sephadex-G100, ultrafiltration, HPLC-Q-TOF-MS, ADMET screening and molecular docking, respectively. The ACE inhibitory peptide DIGGL with a IC50 value of 10.32 ± 0.96 µM was then identified. The peptide against ACE by a non-competitive mode and mainly attributable to the three Conventional Hydrogen Bonds. It could activate Endothelial nitric oxide synthase activity in NO generation and reduce Endothelin-1 secretion induced by Angiotensin II in Human umbilical vein endothelial cells. Meanwhile, DIGGL could promote mice splenocytes proliferation, which was also effective when co-incubated with Con A or LPS, respectively. Besides, the anti-ACE peptide could remain active during the digestion of gastrointestinal proteases (pepsin-trypsin) in vitro.

Anti-Inflammatory, ACE Inhibitory, Antioxidative Activities and Release of Novel Antihypertensive and Antioxidative Peptides from Whey Protein Hydrolysate with Molecular Interactions.

OBJECTIVE: The aim of the study was to evaluate the whey protein hydrolysate with bio-functional attributes viz. antioxidative, anti-inflammatory and ACE inhibition efficacy and release of bioactive peptides with antioxidative and ACE-inhibitory activity by employing Pepsin. METHOD: The antioxidant, Anti-inflammatory, ACE inhibitory and proteolytic activities of the whey protein hydrolysates were studied followed by SDS-PAGE analysis and IEF. Anti-inflammatory activity of whey protein hydrolysate was also studied on RAW 264.7 cell line. The separation of the bioactive peptides from whey protein hydrolysate was achieved by RP-HPLC. The purified bioactive peptides were identified and characterized using RPLC/MS. RESULTS: WPC (Whey protein concentrate) hydrolysate with pepsin showed proteolytic activity ranging between 14.46 and 18.87 mg/ml. Using the ABTS assay, the highest antioxidative activity was observed in 10 kDa retentate (84.50%) and 3 kDa retentate (85.96%), followed by the highest proteolytic activity (13.83 mg/ml) and ACE inhibitory activity (58.37%) in a 5% WPC solution at 65 °C after 8 h of pepsin hydrolysis. When the protein hydrolysate concentration was low, the production of proinflammatory cytokines by lipopolysaccharide-treated murine macrophages (RAW 264.7) was reduced. SDS-PAGE results exhibited very little protein bands when comparing with WPC hydrolysates to insoluble WPC. There were no protein spots on 2 D gel electrophoresis and "in-solution trypsin digestion" technique have been utilized to digest protein samples directly from WPC hydrolysates. Novel antioxidative peptides and ACE inhibitory peptides were also observed by comparing two databases, i.e., BIOPEP and AHTPDB respectively. The peptide sequences used in this study were found to have excellent potential to be used as inhibitors of hACE as all of them were able to show substantial interactions against the enzyme's active site. CONCLUSIONS: The antihypertensive and antioxidative peptides from whey protein hydrolysates may be beneficial for the future development of physiologically active functional foods. Further, in vivo investigations are required to establish the health claim for each individual bioactive peptide from whey protein hydrolysate.Supplemental data for this article is available online at.

Pepsinogen/Proton Pump Co-Expression in Barrett's Esophageal Cells Induces Cancer-Associated Changes.

EDUCATIONAL OBJECTIVE: At the conclusion of this presentation, participants should better understand the carcinogenic potential of pepsin and proton pump expression in Barrett's esophagus. OBJECTIVE: Barrett's esophagus (BE) is a well-known risk factor for esophageal adenocarcinoma (EAC). Gastric H+ /K+ ATPase proton pump and pepsin expression has been demonstrated in some cases of BE; however, the contribution of local pepsin and proton pump expression to carcinogenesis is unknown. In this study, RNA sequencing was used to examine global transcriptomic changes in a BE cell line ectopically expressing pepsinogen and/or gastric H+ /K+ ATPase proton pumps. STUDY DESIGN: In vitro translational. METHODS: BAR-T, a human BE cell line devoid of expression of pepsinogen or proton pumps, was transduced by lentivirus-encoding pepsinogen (PGA5) and/or gastric proton pump subunits (ATP4A, ATP4B). Changes relative to the parental line were assessed by RNA sequencing. RESULTS: Top canonical pathways associated with protein-coding genes differentially expressed in pepsinogen and/or proton pump expressing BAR-T cells included those involved in the tumor microenvironment and epithelial-mesenchymal transition. Top upstream regulators of coding transcripts included TGFB1 and ERBB2, which are associated with the pathogenesis and prognosis of BE and EAC. Top upstream regulators of noncoding transcripts included p300-CBP, I-BET-151, and CD93, which have previously described associations with EAC or carcinogenesis. The top associated disease of both coding and noncoding transcripts was cancer. CONCLUSIONS: These data support the carcinogenic potential of pepsin and proton pump expression in BE and reveal molecular pathways affected by their expression. Further study is warranted to investigate the role of these pathways in carcinogenesis associated with BE. LEVEL OF EVIDENCE: NA Laryngoscope, 133:59-69, 2023.

Effects of autochthonous strains mixture on gut microbiota and metabolic profile in cobia (Rachycentron canadum).

The fish immune system is a topic or subject that offers a unique understanding of defensive system evolution in vertebrate heredity. While gut microbiota plays several roles in fish: well-being, promoting health and growth, resistance to bacterial invasion, regulation of energy absorption, and lipid metabolism. However, studies on fish gut microbiota face practical challenges due to the large number of fish varieties, fluctuating environmental conditions, and differences in feeding habits. This study was carried out to evaluate the impacts of supplemented three autochthonous strains, Bacillus sp. RCS1, Pantoea agglomerans RCS2, and Bacillus cereus RCS3 mixture diet on cobia fish (Rachycentron canadum). Also, chromatography, mass spectrometry and high throughput sequencing were combined to explore composition and metabolite profile of gut microbiota in juvenile cobia fed with supplemented diet. In the trial group, juvenile cobia received diets supplemented with 1 × 1012 CFU mL-1 autochthonous strains for ten weeks and a control diet without supplementation. Juvenile cobia receiving diets supplementation exhibited significantly improved growth than those without additives (control). Haematological indices, such as red blood cells, white blood cells, corpuscular haemoglobin concentration, mean corpuscular volume, haemoglobin, and mean corpuscular haemoglobin, were higher in the supplemented group. Similarly, digestive enzymes (trypsin, lipase, amylase, pepsin and cellulose, activities) activities were higher in supplemented diet with an indigenous isolates mixture. Serum biochemical parameters albumin, globulin, and total protein were significantly higher, while triglyceride, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and cholesterol showed no significant difference. On the other hand, glucose was significantly (P < 0.05) higher in the group without supplementation. On gene expression in the midgut, Immunoglobulin, Colony-stimulating factor receptor 1, major histocompatibility complex 1 were up-regulated by native isolates while T cell receptor beta, and Major histocompatibility complex 2 showed no significant difference. Gut bacterial composition was altered in fish receiving supplemented diet with autochthonous strains. Metabolomics also revealed that some metabolic pathways were considerably enriched in fish fed with supplemented diet; pathway analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment revealed that differentially expressed metabolites were involved in galactose metabolism, tryptophan metabolism, carbohydrate digestion and absorption, purine metabolism, and ABC transporters. Functional analysis of bacterial community showed that differences in enriched metabolic pathways generally comprised carbohydrate and its metabolites, nucleotide and its metabolites, amino acid and its metabolites, heterocyclic compounds, and tryptamines, cholines, pigments. The current investigation results showed that autochthonous strains mixture has significantly enhanced the growth, survival, and innate and adaptive immunities of juvenile cobia.

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.

Salivary biomarkers and esophageal disorders.

Saliva is a complex physiologic fluid that contains an abundance of biological analytes, or biomarkers. Recent research has shown that these biomarkers may be able to convey the physiologic health of a person. Work has been done linking derangements in these salivary biomarkers to a wide variety of pathologic disorders ranging from oncologic diseases to atopic conditions. The specific area of interest for this review paper is esophageal disorders. Particularly because the diagnosis and management of esophageal disorders often includes invasive testing such as esophagogastroduodenoscopy, prolonged pH monitoring, and biopsy. The aim of this review will be to explore salivary biomarkers (pepsin, bile, epidermal growth factor, and micro-RNA) that are being studied as they relate specifically to esophageal disorders. Finally, it will explore the benefits of salivary testing and identify areas of possible future research.