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Scientific Reports Sep 2022Antimicrobial peptides (AMPs) are being developed as potent alternative treatments to conventional antibiotics which are unlikely to induce bacterial resistance. They...
Antimicrobial peptides (AMPs) are being developed as potent alternative treatments to conventional antibiotics which are unlikely to induce bacterial resistance. They can be designed and modified to possess several druggable properties. We report herein a novel hybrid peptide of modified aurein (A3) and cathelicidin (P7), or A3P7, by a flipping technique. It exhibited potent antibacterial activity against both Gram-negative and -positive pathogenic bacteria but had moderate hemolytic activity. To reduce the sequence length and toxicity, C-terminal truncation was serially performed and eight truncated derivatives (AP12-AP19) were obtained. They had significantly less hemolytic activity while preserving antibacterial activity. Secondary structures of the candidate peptides in environments simulating bacterial membranes (30 mM SDS and 50% TFE), determined by CD spectroscopy, showed α-helical structures consistent with predicted in silico 3D structural models. Among the peptides, AP19 demonstrated the best combination of broad-spectrum antibacterial activity (including toward Acinetobacter baumannii) and minimal hemolytic and cytotoxic activities. A D-form peptide (D-AP19), in which all L-enantiomers were substituted with the D-enantiomers, maintained antibacterial activity in the presence of pepsin, trypsin, proteinase K and human plasma. Both isomers exhibited potent antibacterial activity against multi-drug (MDR) and extensively-drug resistant (XDR) clinical isolates of A. baumannii comparable to the traditional antibiotic, meropenem. D-AP19 displayed rapid killing via membrane disruption and leakage of intracellular contents. Additionally, it showed a low tendency to induce bacterial resistance. Our work suggested that D-AP19 could be further optimized and developed as a novel compound potentially for fighting against MDR or XDR A. baumannii.
Topics: Humans; Acinetobacter baumannii; Anti-Bacterial Agents; Bacteria; Drug Resistance, Multiple, Bacterial; Endopeptidase K; Meropenem; Microbial Sensitivity Tests; Pepsin A; Peptide Hydrolases; Trypsin
PubMed: 36151303
DOI: 10.1038/s41598-022-20236-1 -
Biomacromolecules Dec 2023Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can...
Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.
Topics: Rats; Swine; Humans; Animals; Extracellular Matrix; Papain; Decellularized Extracellular Matrix; Pepsin A; Proteomics; Hydrogels; Tissue Engineering; Tissue Scaffolds
PubMed: 38009757
DOI: 10.1021/acs.biomac.3c00602 -
Marine Drugs Jun 2022Fish collagen has been widely used in tissue engineering (TE) applications as an implant, which is generally transplanted into target tissue with stem cells for better...
Fish collagen has been widely used in tissue engineering (TE) applications as an implant, which is generally transplanted into target tissue with stem cells for better regeneration ability. In this case, the success rate of this research depends on the fundamental components of fish collagen such as amino acid composition, structural and rheological properties. Therefore, researchers have been trying to find an innovative raw material from marine origins for tissue engineering applications. Based on this concept, collagens such as acid-soluble (ASC) and pepsin-soluble (PSC) were extracted from a new type of cartilaginous fish, the blacktip reef shark, for the first time, and were further investigated for physicochemical, protein pattern, microstructural and peptide mapping. The study results confirmed that the extracted collagens resemble the protein pattern of type-I collagen comprising the α, α, β and γ chains. The hydrophobic amino acids were dominant in both collagens with glycine and hydroxyproline as major amino acids. From the FTIR spectra, α helix (27.72 and 26.32%), β-sheet (22.24 and 23.35%), β-turn (21.34 and 22.08%), triple helix (14.11 and 14.13%) and random coil (14.59 and 14.12%) structures of ASC and PSC were confirmed, respectively. Collagens retained their triple helical and secondary structure well. Both collagens had maximum solubility at 3% NaCl and pH 4, and had absorbance maxima at 234 nm, respectively. The peptide mapping was almost similar for ASC and PSC at pH 2, generating peptides ranging from 15 to 200 kDa, with 23 kDa as a major peptide fragment. The microstructural analysis confirmed the homogenous fibrillar nature of collagens with more interconnected networks. Overall, the preset study concluded that collagen can be extracted more efficiently without disturbing the secondary structure by pepsin treatment. Therefore, the blacktip reef shark skin could serve as a potential source for collagen extraction for the pharmaceutical and biomedical applications.
Topics: Acids; Amino Acids; Animals; Collagen; Collagen Type I; Fishes; Pepsin A; Sharks; Skin; Solubility
PubMed: 35736179
DOI: 10.3390/md20060376 -
Medicine Aug 2021A rapid lateral flow test (Peptest) to detect pepsin in saliva/sputum has been considered as a valuable method for diagnosing laryngopharyngeal reflux (LPR) and... (Meta-Analysis)
Meta-Analysis
BACKGROUND
A rapid lateral flow test (Peptest) to detect pepsin in saliva/sputum has been considered as a valuable method for diagnosing laryngopharyngeal reflux (LPR) and gastroesophageal reflux disease (GERD). The aim of this meta-analysis is to analyze the utility of Peptest for diagnosis of LPR and GERD.
METHODS
PubMed, EMBASE, and the Cochran Library (from January 1980 to 26 January 2020) were searched for pepsin in saliva for LPR/GERD diagnosis. Sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and area under the curve data were summarized to examine the accuracy.
RESULTS
A total of 16 articles that included 2401 patients and 897 controls were analyzed. The pooled sensitivity and specificity for the diagnosis of GERD/LPR with Peptest were 62% (95% confidence interval [CI] 49%-73%) and 74% (95% CI 50%-90%), respectively. The summarized diagnostic odds ratio and area under the curve were 5.0 (95% CI 2-19) and 0.70 (95% CI 0.66-0.74), respectively.
CONCLUSION
Peptest shows moderate diagnostic value for LPR and GERD. More studies with standard protocols should be done to verify its usefulness.
Topics: Biomarkers; Diagnosis, Differential; Gastroesophageal Reflux; Humans; Laryngopharyngeal Reflux; Pepsin A; ROC Curve; Saliva
PubMed: 34397878
DOI: 10.1097/MD.0000000000026756 -
Journal of Oleo Science 2019Abdominal fat accumulation causes metabolic syndrome, which is a cluster of metabolic abnormalities such as dyslipidemia, glucose intolerance, insulin resistance or... (Review)
Review
Abdominal fat accumulation causes metabolic syndrome, which is a cluster of metabolic abnormalities such as dyslipidemia, glucose intolerance, insulin resistance or hyperinsulinemia, and hypertension, leading to the development of diabetes and cardiovascular disease. Diets are known to contribute to the development or prevention of metabolic syndrome. Several studies have reported that the quality of dietary proteins may be an important modulator of the risk of this syndrome. We investigated the effects of consuming egg white protein (EWP) or lactic-fermented egg white (LE), an easy-to-consume form of egg white, on the development of metabolic syndrome in animal models and humans. In comparison with casein, dietary EWP decreased lymphatic lipid transport in thoracic lymph duct-cannulated rats. In an in vitro experiment, EWP pepsin hydrolysate decreased the cholesterol micellar solubility and cholesterol transfer rate from micelles to oil phase, and increased water-holding capacity, settling volume in water, and relative viscosity compared with casein pepsin hydrolysate. The daily consumption of LE for 8 weeks reduced serum total cholesterol and LDL cholesterol levels in men with mild hypercholesterolemia. Furthermore, dietary EWP reduced the body fat mass of rats by increasing the body protein mass and accelerating hepatic β-oxidation. The daily consumption of LE for 12 weeks reduced the visceral fat area and improved the ratio of the visceral to subcutaneous fat area. Taken together, these results indicated that dietary EWP and LE would be useful for preventing or alleviating metabolic syndrome.
Topics: Adipose Tissue; Animals; Cholesterol; Cholesterol, LDL; Diet; Disease Models, Animal; Egg Proteins, Dietary; Humans; Hypercholesterolemia; Intra-Abdominal Fat; Liver; Lymph; Metabolic Syndrome; Micelles; Oxidation-Reduction; Pepsin A; Proteins; Rats; Solubility; Subcutaneous Fat; Thoracic Duct
PubMed: 31168041
DOI: 10.5650/jos.ess19084 -
Food Research International (Ottawa,... Nov 2023The roles of protein composition, pH and enzymes in goat milk protein hydrolysis is still unclear and the proteolysis of low abundant goat milk proteins has received...
The roles of protein composition, pH and enzymes in goat milk protein hydrolysis is still unclear and the proteolysis of low abundant goat milk proteins has received limited attention. The aim of this study was to study the impact of protein composition and proteolytic conditions on goat milk protein hydrolysis in a simplified digestion model. Both whole milk and infant formula were hydrolyzed at pH 2 and 4, using pepsin as well as pepsin combined with pancreatin. Intact proteins were separated from digests using spin filters, followed by bottom-up proteomics of the separated proteins. Results show that under all conditions, caseins are hydrolyzed quickly. Goat casein hydrolysis in infant formula was slightly faster than in goat whole milk, possibly due to less casein coagulation during pepsin hydrolysis at both pH 2 and 4. Several low abundant immunoactive goat milk proteins, especially immunoglobulins, GLYCAM-1 and osteopontin, resisted proteolysis more than high abundant proteins, independent of the pH and enzyme used for hydrolysis. Fast hydrolysis of casein and slow hydrolysis of immunoactive proteins may indicate a good balance between protein utilization and protection of the infant by goat milk proteins.
Topics: Animals; Infant; Humans; Milk Proteins; Proteolysis; Pancreatin; Caseins; Pepsin A; Goats; Hydrogen-Ion Concentration
PubMed: 37803606
DOI: 10.1016/j.foodres.2023.113294 -
Scientific Reports Mar 2020More than 20 unique diseases such as diabetes, Alzheimer's disease, Parkinson's disease are caused by the abnormal aggregations of pathogenic proteins such as amylin,...
More than 20 unique diseases such as diabetes, Alzheimer's disease, Parkinson's disease are caused by the abnormal aggregations of pathogenic proteins such as amylin, β-amyloid (Aβ), and α-synuclein. All pathogenic proteins differ from each other in biological function, primary sequences, and morphologies; however, the proteins are toxic when aggregated. Here, we investigated the cellular toxicity of pathogenic or non-pathogenic protein aggregates. In this study, six proteins were selected and they were incubated at acid pH and high temperature. The aggregation kinetic and cellular toxicity of protein species with time were characterized. Three non-pathogenic proteins, bovine serum albumin (BSA), catalase, and pepsin at pH 2 and 65 °C were stable in protein structure and non-toxic at a lower concentration of 1 mg/mL. They formed aggregates at a higher concentration of 20 mg/mL with time and they induced the toxicity in short incubation time points, 10 min and 20 min only and they became non-toxic after 30 min. Other three pathogenic proteins, lysozyme, superoxide dismutase (SOD), and insulin, also produced the aggregates with time and they caused cytotoxicity at both 1 mg/mL and 20 mg/mL after 10 min. TEM images and DSC analysis demonstrated that fibrils or aggregates at 1 mg/mL induced cellular toxicity due to low thermal stability. In DSC data, fibrils or aggregates of pathogenic proteins had low thermal transition compared to fresh samples. The results provide useful information to understand the aggregation and cellular toxicity of pathogenic and non-pathogenic proteins.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Catalase; Cell Line; Diabetes Mellitus; Humans; Insulin; Islet Amyloid Polypeptide; Models, Molecular; Muramidase; Parkinson Disease; Pepsin A; Protein Aggregates; Protein Aggregation, Pathological; Protein Structure, Secondary; Serum Albumin, Bovine; Superoxide Dismutase; alpha-Synuclein
PubMed: 32198463
DOI: 10.1038/s41598-020-62062-3 -
Diseases of the Esophagus : Official... Mar 2023Gastroesophageal reflux disease (GERD) is primarily diagnosed based on symptoms and response to a proton-pump inhibitor (PPI) trial. Gold standard testing requires an...
Gastroesophageal reflux disease (GERD) is primarily diagnosed based on symptoms and response to a proton-pump inhibitor (PPI) trial. Gold standard testing requires an invasive endoscopic procedure, often with ambulatory pH monitoring. Salivary pepsin is a potential noninvasive modality for GERD diagnosis. This study aimed to assess diagnostic performance of salivary pepsin thresholds for GERD and determine optimal collection protocol of saliva in an external validation cohort. Over 10 months, adults with symptoms of GERD undergoing esophagogastroduodenoscopy with wireless pH-monitoring off PPI were enrolled. Saliva was self-collected by participants over 4 days across three different time points: fasting ante meridiem (AM), post-prandial, and bedtime (PM). Pepsin levels were calculated via Peptest. Pepsin variability and agreement were determined using linear mixed effects models and intraclass correlation. Validation of diagnostic threshold and performance characteristics were evaluated by receiver-operator curve analysis. Twenty participants enrolled in the study; 50% with physiologic acid exposure (acid exposure time < 4% no GERD) and 50% with elevated acid exposure (GERD). Mean pepsin concentrations were significantly lower in the AM (22.6 ± 25.2 ng/mL) compared to post-prandial (44.5 ± 36.7 ng/mL) and PM (55.4 ± 47.0 ng/mL). Agreement between pepsin concentrations across 3 days was substantial for AM samples (kappa 0.61), with lower agreement for post-prandial and PM samples. A single AM pepsin concentration of 25 ng/mL was 67% accurate for GERD with 56% sensitivity and 78% specificity. This validation study highlights fair accuracy and performance characteristics of a single fasting AM salivary pepsin concentration for the diagnosis of GERD.
Topics: Adult; Humans; Pepsin A; Sensitivity and Specificity; Gastroesophageal Reflux; Esophageal pH Monitoring; Saliva; Proton Pump Inhibitors
PubMed: 36148576
DOI: 10.1093/dote/doac063 -
The Laryngoscope Jul 2020The aim of this study was to compare the diagnostic accuracy of salivary pepsin with oropharyngeal pH monitoring using the Restech measurement system (Dx-pH) for the... (Comparative Study)
Comparative Study
OBJECTIVES
The aim of this study was to compare the diagnostic accuracy of salivary pepsin with oropharyngeal pH monitoring using the Restech measurement system (Dx-pH) for the diagnosis of laryngopharyngeal reflux (LPR).
STUDY DESIGN
Prospective cohort study.
METHODS
Seventy patients with primary symptoms related to LPR underwent gastroscopy, high-resolution manometry, pH throughout 24-hour monitoring (MII-pH), and barium esophagography between October 2015 and May 2018. In addition, an ear, nose, and throat examination was performed, including assessment of Belafsky Reflux Finding Score (RFS). Clinical symptoms were evaluated with the Belafsky Reflux Symptom Index (RSI) and the Gastrointestinal Quality of Life Index (GIQLI). Simultaneous to MII-pH, pepsin determination and Dx-pH were performed.
RESULTS
Of 70 patients, 41 (58.6%) subjects with a pathological DeMeester score showed higher mean values of pepsin (mean value: 216 ng/mL, 95% confidence interval [CI]: 172 to 260), compared to patients with a normal DeMeester score (mean value: 161 ng/mL, 95% CI: 115 to 207). Salivary pepsin showed a specificity of 86.2% and sensitivity of 41.5% for diagnosing LPR using the optimal cutoff value of 216 ng/mL. Furthermore, a significant correlation between the values of salivary pepsin and the RSI score was seen in patients with pathological results in MII-pH (r = 0.344; P = 0.046). However, elevated Dx-pH measurements showed no significant correlation with either MII-pH, RSI score, RFS score, or GIQLI score, or with the results of pepsin measurement.
CONCLUSION
Pepsin measurement in saliva could be an alternative tool to assist office-based diagnosis of LPR, whereas Dx-pH does not seem to be an adequate test.
LEVEL OF EVIDENCE
2B Laryngoscope, 130:1780-1786, 2020.
Topics: Esophageal pH Monitoring; Female; Follow-Up Studies; Humans; Hydrogen-Ion Concentration; Laryngopharyngeal Reflux; Male; Manometry; Middle Aged; Pepsin A; Pressure; Prospective Studies; Quality of Life; Reproducibility of Results; Saliva
PubMed: 31603541
DOI: 10.1002/lary.28320 -
International Journal of Biological... Apr 2022Among the matrices for enzyme immobilization, activated carbon has been standing out in immobilization processes due to its properties and to its characteristics that...
Among the matrices for enzyme immobilization, activated carbon has been standing out in immobilization processes due to its properties and to its characteristics that provide superficial modification by inserting new functional groups capable of binding the enzymes forming covalent bonds. In this study the effect of different modification methods of activated carbon (functionalization with genipin, metallization, metallization in the presence of chelating agent, and functionalization with glutaraldehyde) on efficiency of pepsin immobilization was evaluated. The effect of immobilization pH and the reaction medium on hydrolysis activity of bovine casein was also evaluated. The functionalization of activated carbon using iron ions allowed an immobilization capacity of 98.93 mg·g, with immobilization efficiency greater than 99%, and enzyme activity of 2.30 U, which was higher than the other modifications, and closer to the enzyme in the native form activity (3.32 U). In general, the carbon surface modifications were responsible for forming more stable bonds between support and enzyme, improving its proteolytic activity (from 1.84 to 2.30 U) when compared to traditional immobilization methods by adsorption and covalent binding using glutaraldehyde (from 1.04 to 1.1 U).
Topics: Adsorption; Animals; Cattle; Enzyme Stability; Enzymes, Immobilized; Glutaral; Hydrogen-Ion Concentration; Pepsin A
PubMed: 35090943
DOI: 10.1016/j.ijbiomac.2022.01.135