Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels.

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.

Role of Ca2+ in the pepsin-induced coagulation and in the dynamic in vitro gastric digestion behavior of casein micelles.

The effect of Ca2+ on pepsin-induced hydrolysis of κ-casein and subsequent coagulation of casein micelles was studied in a micellar casein (MC) solution at pH ≈ 6.0 at 37 °C without stirring. An NaCl-supplemented MC solution was used as a positive control to assess the effect of increased ionic strength after CaCl2 addition. Quantitative determination of the released para-κ-casein during the reaction using reverse-phase high-performance liquid chromatography showed that specific hydrolysis of κ-casein by pepsin was little affected by the addition of either CaCl2 or NaCl. However, rheological properties and microstructures of curds induced by pepsin hydrolysis depended markedly on the addition of salts. Addition of CaCl2 up to 17.5 mM facilitated coagulation, with decreases in coagulation time and critical hydrolysis degree, and increases in firming rate and maximum storage modulus (G'max); further addition of CaCl2 (22.5 mM) resulted in a lower G'max. Increased ionic strength to 52.5 mM by adding NaCl retarded the coagulation and resulted in a looser curd structure. In a human gastric simulator, MC, without the addition of CaCl2, did not coagulate until the pH decreased to ≈5.0 after ≈50 min of digestion. Addition of CaCl2 facilitated coagulation of casein micelles and resulted in more cohesive curds with dense structures during digestion, which slowed the emptying rate of caseins. At the same CaCl2 concentration, a sample with higher ionic strength coagulated more slowly. This study provides further understanding on the effect of divalent (Ca2+) ions and ionic strength on the coagulation of casein micelles and the digestion behavior of milk.

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.

Evaluation of deoxythymidine-based cationic amphiphiles as antimicrobial, antibiofilm, and anti-inflammatory agents.

OBJECTIVES: We recently designed a series of cationic deoxythymidine-based amphiphiles that mimic the cationic amphipathic structure of antimicrobial peptides (AMPs). Among these amphiphiles, ADG-2e and ADL-3e displayed the highest selectivity against bacterial cells. In this study, ADG-2e and ADL-3e were evaluated for their potential as novel classes of antimicrobial, antibiofilm, and anti-inflammatory agents. METHODS: Minimum inhibitory concentrations of ADG-2e and ADL-3e against bacteria were determined using the broth microdilution method. Proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K was determined by radial diffusion and HPLC analysis. Biofilm activity was investigated using the broth microdilution and confocal microscopy. The antimicrobial mechanism was investigated by membrane depolarization, cell membrane integrity analysis, scanning electron microscopy (SEM), genomic DNA influence and genomic DNA binding assay. Synergistic activity was evaluated using checkerboard method. Anti-inflammatory activity was investigated using ELISA and RT-PCR. RESULTS: ADG-2e and ADL-3e showed good resistance to physiological salts and human serum, and a low incidence of drug resistance. Moreover, they exhibit proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K. ADG-2e and ADL-3e were found to kill bacteria by an intracellular target mechanism and bacterial cell membrane-disrupting mechanism, respectively. Furthermore, ADG-2e and ADL-3e showed effective synergistic effects when combined with several conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Importantly, ADG-2e and ADL-3e not only suppressed MDRPA biofilm formation but also effectively eradicated mature MDRPA biofilms. Furthermore, ADG-2e and ADL-3e drastically decreased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) gene expression and protein secretion in lipopolysaccharide (LPS)-stimulated macrophages, implying potent anti-inflammatory activity in LPS-induced inflammation. CONCLUSION: Our findings suggest that ADG-2e and ADL-3e could be further developed as novel antimicrobial, antibiofilm, and anti-inflammatory agents to combat bacterial infections.

Sorghum tannin extract impedes in vitro digestibility and fermentability of nutrients in the simulated porcine gastrointestinal tract.

The site and extent of digestion of sorghum nutrients affected by tannins in the intestine are not clarified. Porcine small intestine digestion and large intestine fermentation were simulated in vitro to determine the effects of sorghum tannin extract on the digestion and fermentation characteristics of nutrients in the mimicked porcine gastrointestinal tract. In experiment 1, low-tannin sorghum grain without or with 30 mg/g sorghum tannin extract were digested by porcine pepsin and pancreatin to measure in vitro digestibility of nutrients. In experiment 2, the lyophilized porcine ileal digesta from 3 barrows (Duroc × Landrace × Yorkshire, 27.75 ±â€…1.46 kg) fed the low-tannin sorghum grain without or with 30 mg/g sorghum tannin extract and the undigested residues from experiment 1 were, individually, incubated with fresh pig cecal digesta as inoculums for 48 h to simulate the porcine hindgut fermentation. The results revealed that sorghum tannin extract decreased in vitro digestibility of nutrients both by pepsin hydrolysis or pepsin-pancreatin hydrolysis (P < 0.05). Although enzymatically unhydrolyzed residues provided more energy (P = 0.09) and nitrogen (P < 0.05) as fermentation substrates, the microbial degradation of nutrients from unhydrolyzed residues and porcine ileal digesta were both decreased by sorghum tannin extract (P < 0.05). Regardless of unhydrolyzed residues or ileal digesta as fermentation substrates, microbial metabolites including the accumulative gas production excluding the first 6 h, total short-chain fatty acid and microbial protein content in the fermented solutions were decreased (P < 0.05). The relative abundances of Lachnospiraceae AC2044 and NK4A136 and Ruminococcus_1 was decreased by sorghum tannin extract (P < 0.05). In conclusion, sorghum tannin extract not only directly decreased the chemical enzymatic digestion of nutrients in the simulated anterior intestine, but also directly inhibited the microbial fermentation including microbial diversities and metabolites in the simulated posterior intestine of pigs. The experiment implies that the decreased abundances of Lachnospiraceae and Ruminococcaceae by tannins in the hindgut may weaken the fermentation capacity of microflora and thus impair the nutrient digestion in the hindgut, and ultimately reduce the total tract digestibility of nutrients in pigs fed high tannin sorghum.

Sorghum tannins may be potent negative factor to impede the nutrient utilization in sorghum grain fed to pigs. Unfortunately, the site, rate and extent of digestion of sorghum nutrients affected by tannins in the intestine remain unclear. Therefore, this experiment was conducted to determine the effects of sorghum tannin extract on the digestion and fermentation characteristics of nutrients in the in vitro simulated porcine gastrointestinal tract. The results revealed that sorghum tannin extract decreased the chemical enzymatic digestion of nutrients in the simulated anterior intestine, and inhibited the hindgut microbial fermentation including microbial diversities and metabolites (accumulative gas and short-chain fatty acid production) in the simulated posterior intestine of pigs. The experiment implies that the decreased abundances of Lachnospiraceae and Ruminococcaceae by tannins in the hindgut may weaken the fermentation capacity of microflora and thus impair the nutrient digestion in the hindgut, and ultimately may reduce the total tract nutrient digestibility in pigs fed high tannin sorghum.

The regulatory mechanism of HSP70 in endoplasmic reticulum stress in pepsin-treated laryngeal epithelium cells and laryngeal cancer cells.

BACKGROUNDS: Excessive pepsin can damage both normal laryngeal epithelial cells and laryngeal cancer (LC) cells. Heat shock protein 70 (HSP70) is closely related to pepsin. In this paper, we will explore the different significance of the regulatory role of HSP70 in endoplasmic reticulum stress (ERS) level in pepsin-treated laryngeal epithelial cells and LC cells. METHODS: In cell experiments, laryngeal epithelial cells and LC cells were selected and induced by different concentrations of pepsin. Cell activity was detected by CCK8, cell apoptosis was detected by flow cytometry, and autophagy was detected by autophagy detection kit. The expression of ER)-related proteins was detected by immunofluorescence (IF) and Western blot. Cell transfection was used to inhibit HSP70 expression in both cells, and ERS, apoptosis, and autophagy were measured using related techniques. In animal experiments, a mouse model bearing LC was established. TUNEL assay detected apoptosis, autophagy kit detected autophagy, and ER-related protein expression was detected by Western blot. RESULTS: HSP70 was increased in pepsin-stimulated laryngeal epithelial cells and LC cells, thereby inhibiting ER and ER-induced apoptosis and autophagy. Inhibition of HSP70 reduced the expression of glucose regulated protein 78 (GRP78) in pepsin-stimulated laryngeal epithelial cells and LC cells, and only inhibited downstream apoptosis-related pathways in laryngeal epithelial cells rather than in LC cells. Inhibition of HSP70 and ER could significantly promote apoptosis and inhibit tumor growth in the absence of pepsin stimulation in vivo. CONCLUSION: ER level regulated by HSP70 had different significance in laryngeal epithelial cells and LC cells treated with pepsin.

Three novel leaderless bacteriocins have antimicrobial activity against gram-positive bacteria to serve as promising food biopreservative.

BACKGROUND: Due to the detrimental effects of chemical preservatives, there has been an increasing demand for safer, healthier and natural bio-preservatives. Bacteriocins have attracted increasing interest because of their potential as natural bio-preservatives. RESULTS: We screened a large number of Bacillus thuringiensis strains and isolated one strain (B. thuringiensis P86) with antimicrobial activity against several foodborne pathogens. Three novel leaderless bacteriocins, including thucin A1, thucin A2 and thucin A3, were purified and identified from the culture supernatant of B. thuringiensis P86, whose molecular masses were 5552.02, 5578.07 and 5609.06 Da, respectively. Thucin A1 was then selected as a representative to be tested, and it exhibited potent inhibitory activity against all tested gram-positive bacteria. More importantly, thucin A1 showed stronger antimicrobial activity than nisin A against two important foodborne pathogens Bacillus cereus and Listeria monocytogenes. In addition, thucin A1 exhibited strong acid-base adaptability (pH 2-11), high endurance to heat, good stability to trypsin and pepsin, no hemolysis activity and cytotoxicity, and could effectively inhibit or eliminate Bacillus cereus and Listeria monocytogenes in skim milk. CONCLUSIONS: Our findings indicate that these novel leaderless bacteriocins are potentially promising food biopreservatives.

Novel D-form of hybrid peptide (D-AP19) rapidly kills Acinetobacter baumannii while tolerating proteolytic enzymes.

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.

Optimizing Conditions in the Acid Tolerance Test for Potential Probiotics Using Response Surface Methodology.

Acid tolerance is an important feature of probiotic development. It is one of the factors underlying the beneficial effects of probiotics in the intestine. However, the methods used by different researchers to test acid tolerance vary, causing confusion in the interpretation of the results. Therefore, in this study, we determine the optimal conditions for the acid tolerance test using response surface methodology. The factors of pH (2.5 to 3.5), exposure time (1 to 2 h), and pepsin (presence or absence) were used as independent variables, and the survival rates of seven strains (Lacticaseibacillus casei KACC 12413, Lactiplantibacillus plantarum KACC 15357, Limosilactobacillus fermentum KACC 11441, Lactiplantibacillus plantarum WCFS1, Lacticaseibacillus rhamnosus GG, Lactiplantibacillus plantarum KCTC 21024, and Lactiplantibacillus plantarum WiKim 0112) known to have probiotic properties were used as dependent variables. The results of the analysis of variance (ANOVA) indicated that the pH value and exposure time in acidic environments significantly affected the acid tolerance test model, and their interaction also had an effect (P < 0.05). Using the ANOVA results, the condition of the acid tolerance test was optimized with a target of an 85% survival rate for each strain. The optimized conditions of the acid tolerance test were as follows: pH 2.92, exposure time of 1.73 h, and presence of pepsin and pH 3, exposure time of 1.98 h, and absence of pepsin. These results can optimize strain selection with rigorous acid tolerance without confusion by unifying the conditions for the acid tolerance test. IMPORTANCE The acid tolerance test, which is the first step in selecting probiotics, is not standardized and can often cause confusion in the interpretation of results. Thus, in the present study, we optimized the conditions for the acid tolerance test using response surface methodology. These optimized conditions can be used to screen for strains with acid tolerance.

Role and mechanism of Glut-1 and H+/K+-ATPase expression in pepsin-induced development of vocal cord leukoplakia.

PURPOSE: We investigated the role of Glut-1 and H+/K+-ATPase expression in pepsin-induced development of human vocal cord leukoplakia cells (HVCLCs). Next, we analyzed the relationship between Glut-1 and H+/K+-ATPase expression with the clinicopathological features of laryngeal carcinoma. METHODS: Glut-1 and H+/K+-ATPase expression levels in HVCLCs were determined after treatment with artificial gastric juice containing pepsin and laryngeal carcinoma tissues. RESULTS: Exposure to pepsin-containing artificial gastric juice significantly enhanced the migration and proliferation of VSCLCs in a time-dependent manner. The apoptotic rate of VSCLCs decreased over time after exposure to pepsin and reached a nadir on day 7 (p < 0.01). With increasing duration of exposure to pepsin, the proportion of VSCLCs in G0/G1 phase decreased and the proportions in the S and G2/M phases significantly increased (p < 0.05). After treatment with pepsin-containing artificial gastric juice, RT-PCR and Western blotting showed that the expression of Glut-1 and H+/K+-ATPase α, ß significantly increased in HVCLCs compared to in the absence of pepsin (p < 0.05). The expression of Glut-1 and H+/K+-ATPase α, ß gradually increased from vocal cord leukoplakia (VLC) to laryngeal carcinoma (p < 0.05). Lentivirus-mediated inhibition of Glut-1 expression in VCL significantly inhibited the cells' migration and proliferation (p < 0.05) but enhanced their apoptosis (p < 0.05). Also, inhibition of Glut-1 expression resulted in an increased proportion of cells in G0/G1 phase and a significantly decreased proportion in G2/M phase (p < 0.05). CONCLUSIONS: Elevated Glut-1 expression may promote the development of VCL by upregulating laryngeal H+/K+-ATPase expression to reactivate absorbed pepsin, thus damaging the laryngeal mucosa.

The optimization of pepsin processing time to balance the mechanical and biological properties of xenogeneic bone.

Xenogeneic bone showed great prospects to treat large bone defects due to its bionic composition and structure, but the immunogenicity limited its wide applications. Previously, we developed a pepsin treating method to eliminate the immunogenicity of xenogeneic bone. In this study, we further investigated the effect of pepsin processing time on the biological and mechanical properties. The results indicated that increased pepsin treating time impaired the mechanical properties of xenogeneic bone. And MC3T3-E1 cells showed enhanced adhesion ability, as well as increased production of alkaline phosphatase and calcium nodulus production on the xenogeneic bone processed by pepsin for 24 hr (P24), as compared with xenogeneic bone processed by pepsin for 30 hr (P30) and 36 hr (P36). In addition, we found no significant inflammatory responses after implanting different xenogeneic bone into the intermuscular site of rats. These results suggested that xenogeneic bone processed by pepsin for 24 hr may be a preferable choice when using the xenogeneic bone as biomaterials for further researches.

Gastrobodies are engineered antibody mimetics resilient to pepsin and hydrochloric acid.

Protein-based targeting reagents, such as antibodies and non-antibody scaffold proteins, are rapidly inactivated in the upper gastrointestinal (GI) tract. Hydrochloric acid in gastric juice denatures proteins and activates pepsin, concentrations of which reach 1 mg/mL in the mammalian stomach. Two stable scaffold proteins (nanobody and nanofitin), previously developed to be protease-resistant, were completely digested in less than 10 min at 100-fold lower concentration of pepsin than found in the stomach. Here we present gastrobodies, a protein scaffold derived from Kunitz soybean trypsin inhibitor (SBTI). SBTI is highly resistant to the challenges of the upper GI tract, including digestive proteases, pH 2 and bile acids. Computational prediction of SBTI's evolvability identified two nearby loops for randomization, to create a potential recognition surface which was experimentally validated by alanine scanning. We established display of SBTI on full-length pIII of M13 phage. Phage selection of gastrobody libraries against the glucosyltransferase domain of Clostridium difficile toxin B (GTD) identified hits with nanomolar affinity and enzyme inhibitory activity. Anti-GTD binders retained high stability to acid, digestive proteases and heat. Gastrobodies show resilience to exceptionally harsh conditions, which should provide a foundation for targeting and modulating function within the GI tract.

Foliar Spray with Pepsin-and Papain-Whey Protein Hydrolysates Promotes the Productivity of Pea Plants Cultivated in Clay Loam Soil.

Papain and pepsin-hydrolyzed whey protein (PAH and PEH, respectively) were prepared and characterized for its degree of hydrolysis, chemical constituents (amino acid and peptides) and antioxidant activity. A field experiment was conducted at El Salheya El Gedida City, Sharqia, Egypt, during the seasons 2019 and 2020, to investigate the biological action of the foliar spray of PAH and PEH on the growth and yield of pea plants cultivated in a clay loam soil. Foliar application of the papain and pepsin-hydrolyzed whey protein (PAH and PEH, respectively) at 1000 and 2000 mg/L was applied three times after 25, 35 and 45 days from planting. All protein foliar spray treatments had significant positive effects on the uptake of N, P and K, simultaneously increasing the contents of all the photosynthetic pigments (Chlorophyll a, Chlorophyll b and Carotenoids) in a concentration-dependent manner. The most conspicuous increase was seen in Chlorophyll b (105% increase), followed by Carotenoids (91% increase). Generally, the favorable increases caused by the second level of application (2000 mg/L) were nearly 2-3 times that of the low level (1000 mg/L). Pod growth and formation indicators, e.g., no. of pod/plant, pod length and no. of seeds/pod, responded more evidently to the hydrolyzed than the intact form of whey protein treatments. Hydrolyzed whey protein foliar spray treatments achieved significantly higher increases in the global field yield components of Pisum sativum plants than the intact form, where peptic hydrolysates were significantly superior to papain hydrolysate. The treatment PEH (2000 mg/L) can be recommended as the most effective bio-stimulating foliar spray treatment for higher plant productivity when applied 25, 35 and 45 days after planting.

Pepsin Promotes Activation of Epidermal Growth Factor Receptor and Downstream Oncogenic Pathways, at Slightly Acidic and Neutral pH, in Exposed Hypopharyngeal Cells.

Pepsin refluxate is considered a risk factor for laryngopharyngeal carcinogenesis. Non-acidic pepsin was previously linked to an inflammatory and tumorigenic effect on laryngopharyngeal cells in vitro. Yet there is no clear evidence of the pepsin-effect on a specific oncogenic pathway and the importance of pH in this process. We hypothesized that less acidic pepsin triggers the activation of a specific oncogenic factor and related-signalling pathway. To explore the pepsin-effect in vitro, we performed intermittent exposure of 15 min, once per day, for a 5-day period, of human hypopharyngeal primary cells (HCs) to pepsin (1 mg/mL), at a weakly acidic pH of 5.0, a slightly acidic pH of 6.0, and a neutral pH of 7.0. We have documented that the extracellular environment at pH 6.0, and particularly pH 7.0, vs. pH 5.0, promotes the pepsin-effect on HCs, causing increased internalized pepsin and cell viability, a pronounced activation of EGFR accompanied by NF-κB and STAT3 activation, and a significant upregulation of EGFR, AKT1, mTOR, IL1ß, TNF-α, RELA(p65), BCL-2, IL6 and STAT3. We herein provide new evidence of the pepsin-effect on oncogenic EGFR activation and its related-signaling pathway at neutral and slightly acidic pH in HCs, opening a window to further explore the prevention and therapeutic approach of laryngopharyngeal reflux disease.

RNA Sequencing Reveals Cancer-Associated Changes in Laryngeal Cells Exposed to Non-Acid Pepsin.

OBJECTIVE: Laryngopharyngeal reflux (LPR) is a common affliction that contributes to laryngeal inflammation, symptoms that impact quality of life, and life-threatening illnesses such as cancer. Effective treatment strategies for LPR are lacking. Pepsin is a proinflammatory and carcinogenic element of refluxate. Investigation of molecular pathways involved in pepsin-mediated damage may lead to identification of novel biomarkers and therapeutic targets for LPR. In this study, RNA sequencing was used to examine changes in human laryngeal epithelial cells following brief pepsin insult. Cells were immortalized to generate a model to aid future study of laryngeal injury and therapeutics. STUDY DESIGN: In vitro translational. METHODS: Laryngeal epithelial cells were cultured from a patient without signs or symptoms of LPR or laryngeal cancer. Cells were treated with 0.1 mg/ml pepsin for 1 hour or normal growth media (control) prior to RNA sequencing. Cells were immortalized via HPV E6/7 and characterized by microscopy, immunohistochemistry, G-banding, and soft agar assay. RESULTS: Three hundred ninety-seven genes exhibited differences in expression with pepsin treatment (P < .05). Pathway analysis revealed association with cancer and related signaling processes including dysregulation of cancer-associated molecules, Metastasis-Associated Lung Adenocarcinoma Transcript 1 and KRT82, and the long-noncoding RNA, lipoprotein receptor-related protein 1 (LRP1)-AS, which regulates the putative pepsin receptor LRP1. CONCLUSIONS: A single, brief exposure to pepsin activated cancer-associated signaling pathways in laryngeal cells in vitro, revealing novel mechanisms by which chronic reflux may contribute to carcinogenesis. The cell line developed herein represents a novel tool in which to investigate pepsin-dysregulated pathways identified by RNA sequencing and disparities of tumor proneness of laryngeal subsites. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:121-129, 2021.

The effect of proteolytic enzymes and pH on GII.4 norovirus, during both interactions and non-interaction with Histo-Blood Group Antigens.

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis worldwide. Histo-Blood Groups Antigens (HBGAs) have been described as attachment factors, promoting HuNoV infection. However, their role has not yet been elucidated. This study aims to evaluate the ability of HBGAs to protect HuNoVs against various factors naturally found in the human digestive system. The effects of acid pH and proteolytic enzymes (pepsin, trypsin, and chymotrypsin) on GII.4 virus-like particles (VLPs) and GII.4 HuNoVs were studied, both during interactions and non-interaction with HBGAs. The results showed that GII.4 VLPs and GII.4 HuNoVs behaved differently following the treatments. GII.4 VLPs were disrupted at a pH of less than 2.0 and in the presence of proteolytic enzymes (1,500 units/mL pepsin, 100 mg/mL trypsin, and 100 mg/mL chymotrypsin). VLPs were also partially damaged by lower concentrations of trypsin and chymotrypsin (0.1 mg/mL). Conversely, the capsids of GII.4 HuNoVs were not compromised by such treatments, since their genomes were not accessible to RNase. HBGAs were found to offer GII.4 VLPs no protection against an acid pH or proteolytic enzymes.

Physicochemical characterization and self-assembly of human amniotic membrane and umbilical cord collagen: A comparative study.

The diverse application of collagen has created a need to discover renewable and economical sources with prevailing/improved physico-chemical properties. To address this scenario, the present study has extracted collagen from Human Amniotic Membrane (AM) and Umbilical cord, which are treated as medical waste and compared its physico-chemical properties. Collagen was extracted by pepsin solubilization using various salt concentrations (1 M, 2 M and 4 M). Umbilical Cord Collagen (UC) yield was 10% higher than Amniotic Membrane Collagen (AC). UC reported 58% higher sulphated glycosaminoglycan content than AC. Electrophoretic pattern of AC and UC in both disulphide bond reducing and non-reducing conditions showed bands corresponding to collagen type I, III, IV, V and XV. Collagen morphology was examined using SEM and the amino acid content was quantified by HPLC and LC-MS/MS. Triple helicity was confirmed by CD and FTIR spectra. Thermal transition temperature of AC and UC was found equivalent to animal collagen. Self-assembly, fibril morphology and spatial alignment was studied using AFM and DLS. Biocompatibility was analyzed using 3T3 fibroblast cells. In conclusion, UC with higher yield, presented with better physico-chemical, structural and biological properties than AC could serve as an efficient alternative to the existing animal collagen for diverse applications.

Measurement and visualization of cell membrane surface charge in fixed cultured cells related with cell morphology.

The diagnosis of patients with malignancies relies on the results of a clinical cytological examination. To enhance the diagnostic qualities of cytological examinations, it is important to have a detailed analysis of the cell's characteristics. There is, therefore, a need for developing a new auxiliary method for cytological diagnosis. In this study, we focused on studying the charge of the cell membrane surface of fixed cells, which is one of important cell's characteristics. Although fixed cells lose membrane potential which is observed in living cells owing to ion dynamics, we hypothesized that fixed cells still have a cell membrane surface charge due to cell membrane components and structure. We used 5 cell lines in this study (ARO, C32TG, RT4, TK, UM-UC-14). After fixation with CytoRich Red, we measured the cell membrane surface charge of fixed cells in solution using zeta potential measurements and fixed cells on glass slides, visualizing it using antibody-labeled beads and positively-charged beads. Furthermore, we measured the cell membrane surface charge of fixed cells under different conditions, such as different solution of fixative, ion concentration, pH, and pepsin treatments. The zeta potential measurements and visualization using the beads indicated that the cell membrane surface of fixed cells was negatively charged, and also that the charge varied among fixed cells. The charge state was affected by the different treatments. Moreover, the number of cell-bound beads was small in interphase, anaphase, and apoptotic cells. We concluded that the negative cell membrane surface charge was influenced by the three-dimensional structure of proteins as well as the different types of amino acids and lipids on the cell membrane. Thus, cell surface charge visualization can be applied as a new auxiliary method for clinical cytological diagnosis. This is the first systematic report of the cell membrane surface charge of fixed cells.

Effect of (-)-Epigallocatechin Gallate to Staphylococcal Enterotoxin A on Toxin Activity.

Staphylococcal enterotoxin A (SEA) functions both as superantigens that stimulate non-specific T cell proliferation as well as potent gastrointestinal toxins. We previously reported that (-)-epigallocatechin gallate (EGCG) binds to SEA. Therefore, the ability of EGCG to inhibit SEA toxin activity was examined. As a result, EGCG significantly decreased SEA-induced expression and production of interferon gamma (IFN-γ). In addition, EGCG inhibited SEA-induced spleen cell proliferation. To investigate the role of the galloyl group in EGCG on SEA cytotoxicity in more detail, the effect of the binding of a hydroxyl group at position 3 of the galloyl group in EGCG to SEA on SEA cytotoxicity was examined using two methylated EGCG. SEA cytotoxicity was significantly controlled in both (-)-3''-Me-EGCG and (-)-4''-Me-EGCG. These results suggest that EGCG inhibits toxic activity via direct interaction with SEA or without any interaction with SEA. The binding affinity between SEA and EGCG under in vivo conditions was examined using a model solution. Although after treatment under acidic and alkaline conditions, the presence of protein and the digestive tract model solution, EGCG still interacted with SEA. Our studies are the first to demonstrate the effect of the binding of EGCG to SEA on toxin activity.

Isolation of novel ACE-inhibitory peptide from naked oat globulin hydrolysates in silico approach: Molecular docking, in vivo antihypertension and effects on renin and intracellular endothelin-1.

Naked oat globulin was hydrolyzed by alcalase, flavourzyme, pepsin, and trypsin in sequence. The hydrolysates (NOGH) were purified using gel chromatography, reversed-phase high performance liquid chromatography (RP-HPLC). Finally, fraction D7d with the highest ACE-inhibitory was subjected to liquid chromatography-mass spectrometry analysis and 14 peptides were identified. Of which, peptide SSYYPFK (890.4 Da) was chose to synthesize based on in silico analysis. The SSYYPFK demonstrated high ACE-inhibitory activity (IC50 : 91.82 µM) with competitive inhibition mode, and could effectively (P < 0.05) lower the systolic blood pressure and diastolic pressure of spontaneously hypertensive rats at the concentration of 100 to 150 mg/kg body weight. Molecular docking simulation demonstrated that SSYYPFK could bind with the active site S1 of ACE via short hydrogen bonds. It could remain the ACE-inhibitory activity after simulated gastrointestinal hydrolysis. Moreover, SSYYPFK showed acceptable renin and endothelin-1 suppressing capacity (47.59% and 27.88% at 1.5 mg/mL, respectively). These results indicated that SSYYPFK may have similar antihypertensive mechanism with captopril, and could be develop to natural antihypertensive products. PRACTICAL APPLICATION: One novel ACE-inhibitory peptide SSYYPFK (890.4 Da) was identified from naked oat globulin hydrolysates. It exhibited relatively high renin and intracellular endothelin-1 suppressing capacity, and could effectively (P < 0.05) lower the systolic blood pressure and diastolic pressure of spontaneously hypertensive rats. This peptide could be used as natural and safe nutraceuticals and/or functional ingredients.