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Microbial Pathogenesis Jun 2024This study investigates the multifunctional bioactivities of pepsin-hydrolyzed jellyfish by-products (Rhopilema hispidum and Lobonema smithii), focusing on their...
This study investigates the multifunctional bioactivities of pepsin-hydrolyzed jellyfish by-products (Rhopilema hispidum and Lobonema smithii), focusing on their anti-α-glucosidase activity, anti-inflammatory effects, anti-bacterial properties, and ability to inhibit biofilm formation of Staphylococcus aureus. Our findings revealed that jellyfish protein hydrolysates, particularly from Rhopilema hispidum, exhibit significant anti-α-glucosidase activity, surpassing the well-known α-glucosidase inhibitor Acarbose. Furthermore, we demonstrated the anti-inflammatory capabilities of these hydrolysates in suppressing lipopolysaccharide (LPS)-induced nitric oxide production in murine macrophage cells. This effect was dose-dependent and non-cytotoxic, highlighting the hydrolysate potential in treating inflammation-related conditions. Regarding anti-bacterial activity, pepsin-hydrolyzed jellyfish selectively exhibited a potent effect against S. aureus, including Methicillin-susceptible and Methicillin-resistant strains. This activity was evident at minimum inhibitory concentrations (MIC) of 25 μg/mL for S. aureus ATCC10832, while a modest effect was observed against other Gram-positive strains. The hydrolysates effectively delayed bacterial growth dose-dependently, suggesting their use as alternative agents against bacterial infections. Most notably, pepsin-hydrolyzed jellyfish showed significant anti-biofilm activity against S. aureus. The umbrella section hydrolysate of Rhopilema hispidum was particularly effective, reducing biofilm formation through downregulating the icaA gene, crucial for biofilm development. Furthermore, the hydrolysates modulated the expression of the agrA gene, a key regulator in the pathogenesis of S. aureus. In conclusion, pepsin-hydrolyzed jellyfish protein hydrolysates exhibit promising multifunctional bioactivities, including anti-diabetic, anti-inflammatory, antibacterial, and anti-biofilm properties. These findings suggest their potential application in pharmaceutical and nutraceutical fields, particularly in managing diabetic risks, inflammation, bacterial infections, and combating the biofilm-associated pathogenicity of S. aureus.
Topics: Animals; Mice; Biofilms; Staphylococcus aureus; Scyphozoa; Anti-Bacterial Agents; Protein Hydrolysates; Microbial Sensitivity Tests; Anti-Inflammatory Agents; RAW 264.7 Cells; Inflammation; Macrophages; Nitric Oxide; Glycoside Hydrolase Inhibitors; Diabetes Mellitus; Pepsin A; Lipopolysaccharides
PubMed: 38641070
DOI: 10.1016/j.micpath.2024.106648 -
Food Chemistry Aug 2024Gold nanoparticles (AuNPs)-based immunochromatographic assay has gained popularity as a rapid detection method for food hazards. Synthesizing highly stable AuNPs in a...
Gold nanoparticles (AuNPs)-based immunochromatographic assay has gained popularity as a rapid detection method for food hazards. Synthesizing highly stable AuNPs in a rapid, simple and environmentally friendly manner is a key focus in this field. Here, we present a green microfluidic strategy for the rapid, automated, and size-controllable synthesis of pepsin-doped AuNPs (AuNPs@Pep) by employing glucose-pepsin as a versatile reducing agent and stabilizer. Through combining the colorimetric and photothermal (PoT) properties of AuNPs@Pep, both "signal-off" and "signal-on" formats of microfluidic paper analytical devices (PADs) were developed for detection of a small molecule antibiotic, florfenicol, and an egg allergen, ovalbumin. Compared to the colorimetric mode, a 4-fold and 3-fold improvement in limit of detection was observed in the "signal-off" detection of florfenicol and the "signal-on" detection of ovalbumin, respectively. The results demonstrated the practicality of AuNPs@Pep as a colorimetric/PoT dual-readout probe for immunochromatographic detection of food hazards at different molecular scales.
Topics: Gold; Metal Nanoparticles; Colorimetry; Green Chemistry Technology; Ovalbumin; Pepsin A; Food Contamination; Limit of Detection; Thiamphenicol
PubMed: 38636377
DOI: 10.1016/j.foodchem.2024.139311 -
Food Research International (Ottawa,... May 2024Konjac glucomannan (KGM) can significantly prolong gastrointestinal digestion. However, it is still worth investigating whether the macromolecular crowding (MMC) induced...
The effect of konjac glucomannan on enzyme kinetics and fluorescence spectrometry of digestive enzymes: An in vitro research from the perspective of macromolecule crowding.
Konjac glucomannan (KGM) can significantly prolong gastrointestinal digestion. However, it is still worth investigating whether the macromolecular crowding (MMC) induced by KGM is correlated with digestion. In this paper, the MMC effect was quantified by fluorescence resonance energy transfer and microrheology, and the digests of starch, protein, and oil were determined. The digestive enzymes were analyzed by enzyme reaction kinetic and fluorescence quenching. The results showed that higher molecular weight (604.85 ∼ 1002.21 kDa) KGM created a larger MMC (>0.8), and influenced the digestion of macronutrients; the digests of starch, protein, and oil all decreased significantly. MMC induced by KGM decreased the Michaelis-Menten constants (K and V) of pancreatic α-amylase (PPA), pepsin (PEP), and pancreatic lipase (PPL). The larger MMC (>0.8) induced by KGM resulted in the decrease of fluorescence quenching constants (K) in PPA and PPL, and the increase of K in PEP. Therefore, varying degrees of MMC induced by KGM could play a role in regulating digestion and the inhibitory effect on digestion was more significant in a relatively more crowded environment induced by KGM. This study provides theoretical support for the strategies of nutrient digestion regulation from the perspective of MMC caused by dietary fiber.
Topics: Spectrometry, Fluorescence; Mannans; Pepsin A; Macromolecular Substances; Pancreatic alpha-Amylases; Starch
PubMed: 38609226
DOI: 10.1016/j.foodres.2024.114247 -
Analytical Chemistry Apr 2024The number of therapeutic monoclonal antibodies (mAbs) is growing rapidly due to their widespread use for treating various diseases and health conditions. Assessing the...
The number of therapeutic monoclonal antibodies (mAbs) is growing rapidly due to their widespread use for treating various diseases and health conditions. Assessing the glycosylation profile of mAbs during production is essential to ensuring their safety and efficacy. This research aims to rapidly isolate and digest mAbs for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of glycans and monitoring of glycosylation patterns, potentially during manufacturing. Immobilization of an Fc region-specific ligand, oFc20, in a porous membrane enables the enrichment of mAbs from cell culture supernatant and efficient elution with an acidic solution. Subsequent digestion of the mAb eluate occurred in a pepsin-modified membrane within 5 min. The procedure does not require alkylation and desalting, greatly shortening the sample preparation time. Subsequent LC-MS/MS analysis identified 11 major mAb N-glycan proteoforms and assessed the relative peak areas of the glycosylated peptides. This approach is suitable for the glycosylation profiling of various human IgG mAbs, including biosimilars and different IgG subclasses. The total time required for this workflow is less than 2 h, whereas the conventional enzymatic release and labeling of glycans can take much longer. Thus, the integrated membranes are suitable for facilitating the analysis of mAb glycosylation patterns.
Topics: Glycosylation; Antibodies, Monoclonal; Humans; Tandem Mass Spectrometry; Polysaccharides; Chromatography, Liquid; Pepsin A; Immunoglobulin G; Animals; Membranes, Artificial
PubMed: 38607313
DOI: 10.1021/acs.analchem.4c00030 -
Ecotoxicology and Environmental Safety May 2024To study the heavy metal accumulation and its impact on insect exterior and chromosome morphology, and reveal the molecular mechanism of insects adapting to long-term...
To study the heavy metal accumulation and its impact on insect exterior and chromosome morphology, and reveal the molecular mechanism of insects adapting to long-term heavy metal compound pollution habitats, this study, in the Diaojiang river basin, which has been polluted by heavy metals(HMs) for nearly a thousand years, two Eucriotettix oculatus populations was collected from mining and non-mining areas. It was found that the contents of 7 heavy metals (As, Cd, Pb, Zn, Cu, Sn, Sb) in E. oculatus of the mining area were higher than that in the non-mining 1-11 times. The analysis of morphology shows that the external morphology, the hind wing type and the chromosomal morphology of E. oculatus are significant differences between the two populations. Based on the heavy metal accumulation,morphological change, and stable population density, it is inferred that the mining area population has been affected by heavy metals and has adapted to the environment of heavy metals pollution. Then, by analyzing the transcriptome of the two populations, it was found that the digestion, immunity, excretion, endocrine, nerve, circulation, reproductive and other systems and lysosomes, endoplasmic reticulum and other cell structure-related gene expression were suppressed. This shows that the functions of the above-mentioned related systems of E. oculatus are inhibited by heavy metal stress. However, it has also been found that through the significant up-regulation of genes related to the above system, such as ATP2B, pepsin A, ubiquitin, AQP1, ACOX, ATPeV0A, SEC61A, CANX, ALDH7A1, DLD, aceE, Hsp40, and catalase, etc., and the down-regulation of MAPK signalling pathway genes, can enhanced nutrient absorption, improve energy metabolism, repair damaged cells and degrade abnormal proteins, maintain the stability of cells and systems, and resist heavy metal damage so that E. oculatus can adapt to the environment of heavy metal pollution for a long time.
Topics: Animals; Metals, Heavy; Water Pollutants, Chemical; Grasshoppers; Environmental Monitoring; Mining; China; Adaptation, Physiological; Transcriptome; Rivers
PubMed: 38599159
DOI: 10.1016/j.ecoenv.2024.116301 -
Molecules (Basel, Switzerland) Mar 2024Pepsin, trypsin and proteinase K were used in the present study to hydrolyse the proteins from whole eggs, yolks or whites, and the resulting hydrolysates were...
Pepsin, trypsin and proteinase K were used in the present study to hydrolyse the proteins from whole eggs, yolks or whites, and the resulting hydrolysates were characterised in terms of antioxidant and IgE-binding properties, using a combination of in vitro and in silico methods. Based on the degree of hydrolysis (DH) results, the egg yolk proteins are better substrates for all the tested enzymes (DH of 6.2-20.1%) compared to those from egg whites (DH of 2.0-4.4%). The SDS-PAGE analysis indicated that pepsin and proteinase K were more efficient compared to trypsin in breaking the intramolecular peptide bonds of the high molecular weight egg proteins. For all the tested substrates, enzyme-assisted hydrolysis resulted in a significant increase in antioxidant activity, suggesting that many bioactive peptides are encrypted in inactive forms in the parent proteins. The hydrolysates obtained with proteinase K exhibited the highest DPPH radical scavenging activity (124-311 µM Trolox/g protein) and the lowest residual IgE-binding capacity. The bioinformatics tools revealed that proteinase K is able to break the integrity of the main linear IgE-binding epitopes from ovalbumin and ovomucoid. It can be concluded that proteinase K is a promising tool for modulating the intrinsic properties of egg proteins.
Topics: Antioxidants; Pepsin A; Trypsin; Endopeptidase K; Peptides; Egg Proteins; Hydrolysis; Immunoglobulin E; Protein Hydrolysates
PubMed: 38542963
DOI: 10.3390/molecules29061327 -
Biosensors Mar 2024Two fluorescent molecularly imprinted polymers (MIPs) were developed for pepsin enzyme utilising fluorescein and rhodamine b. The main difference between both dyes is...
Two fluorescent molecularly imprinted polymers (MIPs) were developed for pepsin enzyme utilising fluorescein and rhodamine b. The main difference between both dyes is the presence of two (diethylamino) groups in the structure of rhodamine b. Consequently, we wanted to investigate the effect of these functional groups on the selectivity and sensitivity of the resulting MIPs. Therefore, two silica-based MIPs for pepsin enzyme were developed using 3-aminopropyltriethoxysilane as a functional monomer and tetraethyl orthosilicate as a crosslinker to achieve a one-pot synthesis. Results of our study revealed that rhodamine b dyed MIPs (RMIPs) showed stronger binding, indicated by a higher binding capacity value of 256 mg g compared to 217 mg g for fluorescein dyed MIPs (FMIPs). Moreover, RMIPs showed superior sensitivity in the detection and quantitation of pepsin with a linear range from 0.28 to 42.85 µmol L and a limit of detection (LOD) as low as 0.11 µmol L. In contrast, FMIPs covered a narrower range from 0.71 to 35.71 µmol L, and the LOD value reached 0.34 µmol L, which is three times less sensitive than RMIPs. Finally, the developed FMIPs and RMIPs were applied to a separation-free quantification system for pepsin in saliva samples without interference from any cross-reactors.
Topics: Pepsin A; Limit of Detection; Fluorescein; Coloring Agents; Molecular Imprinting
PubMed: 38534258
DOI: 10.3390/bios14030151 -
International Journal of Biological... Apr 2024Acetaldehyde dehydrogenase 2 (ALDH2) is a crucial enzyme in alcohol metabolism, and oral administration of ALDH2 is a promising method for alcohol detoxification....
Acetaldehyde dehydrogenase 2 (ALDH2) is a crucial enzyme in alcohol metabolism, and oral administration of ALDH2 is a promising method for alcohol detoxification. However, recombinant ALDH2 is susceptible to hydrolysis by digestive enzymes in the gastrointestinal tract and is expressed as inactive inclusion bodies in E. coli. In this study, we performed three rounds of rational design to address these issues. Specifically, the surface digestive sites of pepsin and trypsin were replaced with other polar amino acids, while hydrophobic amino acids were incorporated to reshape the catalytic cavity of ALDH2. The resulting mutant DE2-852 exhibited a 45-fold increase in soluble expression levels, while its stability against trypsin and pepsin increased by eightfold and twofold, respectively. Its catalytic efficiency (k/K) at pH 7.2 and 3.2 improved by more than four and five times, respectively, with increased V and decreased K values. The enhanced properties of DE2-852 were attributed to the D457Y mutation, which created a more compact protein structure and facilitated a faster collision between the substrate and catalytic residues. These results laid the foundation for the oral administration and mass preparation of highly active ALDH2 and offered insights into the oral application of other proteins.
Topics: Humans; Aldehyde Dehydrogenase, Mitochondrial; Aldehyde Dehydrogenase; Pepsin A; Trypsin; Escherichia coli; Amino Acids
PubMed: 38521319
DOI: 10.1016/j.ijbiomac.2024.131091 -
Bio Systems Apr 2024•The signaling process during mycorrhiza establishment involves intense molecular communication between symbionts. It has been suggested that a group of protein...
•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.
Topics: Mycorrhizae; Plant Roots; Papain; Pepsin A; Aspartic Acid; Cysteine; Molecular Docking Simulation; Symbiosis; Protease Inhibitors; Laccaria
PubMed: 38513884
DOI: 10.1016/j.biosystems.2024.105194 -
Frontiers in Cellular and Infection... 2024Oral transmission of is probably the most frequent transmission mechanism in wild animals. This observation led to the hypothesis that consuming raw or undercooked meat...
INTRODUCTION
Oral transmission of 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 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
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 C8C3 of , and the infection of the fed mice was monitored by a parasitemia curve. Similarly, we assessed the infective capacity of 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 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 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.
Topics: Female; Animals; Mice; Humans; Trypanosoma cruzi; Pepsin A; Parasitemia; Disease Models, Animal; Chromatography, Liquid; Tandem Mass Spectrometry; Chagas Disease; Mucins; Communicable Diseases
PubMed: 38495650
DOI: 10.3389/fcimb.2024.1297099