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.

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.

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.

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.

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.

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.

Pepsin promotes laryngopharyngeal neoplasia by modulating signaling pathways to induce cell proliferation.

Pepsin plays an important role in laryngopharyngeal reflux (LPR), a risk factor for the development of hypopharyngeal squamous cell carcinomas (HPSCC). However, the role of pepsin in HPSCC is not clear. We show by immunohistochemistry that pepsin positivity occurs in a significant proportion of human primary HPSCC specimens, and in many cases matched adjacent uninvolved epithelia are negative for pepsin. Pepsin positivity is associated with nodal involvement, suggesting that pepsin may have a role in metastasis. Treatment of FaDu cancer cells with pepsin increased cell proliferation, possibly by inducing G1/S transition. We also observed significant changes in expression of genes involved in NF-kappaB, TRAIL and Notch signaling. Our data suggest that pepsin plays an important role in HPSCC and that targeting pepsin could have potential therapeutic benefits.

The role of pepsin in epithelia-mesenchymal transition in idiopathic subglottic stenosis.

OBJECTIVES: Idiopathic subglottic stenosis (iSGS) is commonly characterized by laryngeal fibrosis thought to arise by epithelia-mesenchymal transition (EMT) induced by chronic inflammation. Pepsin is a potent inducer of inflammation in the airways during chronic laryngopharyngeal reflux and has been observed in the subglottic mucosa of patients with iSGS, absent in normal mucosa. The aim of this study was to examine the effect of pepsin on mechanisms of EMT in laryngeal cells with implications for iSGS. STUDY DESIGN: In vitro translational research study. METHODS: Human laryngeal epithelial cell cultures were exposed to 0.1 mg/mL or 1.0 mg/mL pepsin at pH7 for 24 and 48 hours, or media pH5 ± 0.1 mg/mL pepsin for 10 minutes and harvested after 24 and 48 hours. EMT marker expression was measured by qPCR and enzyme-linked immunosorbent assays. Wound-healing scratch assay was performed on immortalized human vocal fold fibroblasts pretreated with media pH5 ± 0.1 mg/mL pepsin (10 minutes) or continuously treated with media pH7 ± 0.1 to 1 mg/mL pepsin for 24 hours. RESULTS: Pepsin yielded no effect on MMP1, MMP9, FN1, COL1A1, HAS2, or CDH1 gene expression or matrix metalloproteinase-9 or fibronectin protein expression, either alone or in the presence of weak acid. Pepsin and/or acid produced no effect on fibroblast migration. CONCLUSION: Whereas pepsin has been shown to be present in the subglottic mucosa of patients with iSGS, this in vitro acute exposure investigation does not provide evidence of a direct causal role for development of fibrosis in subglottic epithelial cell cultures. LEVELS OF EVIDENCE: NA. Laryngoscope, 130:154-158, 2020.

Preparation and Identification of Antioxidative Peptides from Pacific Herring (Clupea pallasii) Protein.

The aim of this study was to isolate and purify antioxidative peptides from Pacific herring (Clupea pallasii) protein. Five enzymes (pepsin, trypsin, papain, flavourzyme, and neutrase) were used for protein hydrolysis, and Pacific herring protein hydrolysates (PHPH) were separated by ultrafiltration. The fraction with the molecular weight below 3500 Da exhibited the highest in vitro antioxidant activities and cellular antioxidant activity. The PHPH was isolated and purified by consecutive chromatographic methods including gel filtration chromatography and reverse high-performance liquid chromatography (RP-HPLC). The purified antioxidant peptides were identified as Leu-His-Asp-Glu-Leu-Thr (MW = 726.35 Da) and Lys-Glu-Glu-Lys-Phe-Glu (MW = 808.40 Da), and the IC50 values of cellular antioxidant activity were 1.19 ± 0.05 mg/mL and 1.04 ± 0.06 mg/mL. The results demonstrate that is possible to produce natural antioxidative peptides from Pacific herring protein via enzymatic hydrolysis and purification.

Antioxidant properties and instrumental quality characteristics of spray dried Pangasius visceral protein hydrolysate prepared by chemical and enzymatic methods.

Fish protein hydrolysates are digested form of protein with various bioactive properties where, the cleavages of molecular bonds of proteins can be broken by the enzymatic and chemical process. In this study, antioxidant properties of spray dried protein hydrolysate prepared from Pangasius viscera by using enzymatic (papain and pepsin), and chemical methods (hydrochloric acid and sodium hydroxide) were evaluated. Among the different treatments, pepsin-derived visceral protein hydrolysate showed the maximum antioxidant activity when used at higher concentrations. Essential amino acids (EAA) and hydrophobic amino acids are higher in papain-derived visceral protein hydrolysate. In pepsin-derived visceral protein hydrolysate, major proportion was contributed by glycine (Gly), glutamine (Glu), proline (Pro), and asparagine (Asp). Higher amount of aromatic amino acids are found in alkali-derived FVPH. Scanning electron microscopy (SEM) images of pepsin fish visceral protein hydrolysate showed better globular structure than the other treatments. It can be concluded that among the different treatments, the visceral protein hydrolysate prepared with pepsin had better overall quality regarding antioxidant properties and papain in nutritional point of view.

Heat shock protein 70 is induced by pepsin via MAPK signaling in human nasal epithelial cells.

BACKGROUND: Recent studies have shown that laryngopharyngeal reflux is associated with chronic rhinosinusitis. Pepsin may be a key factor involved in the injury of nasal mucosal epithelial cells, but the pathogenesis remains unclear. We are to investigate whether a mitogen-activated protein kinase (MAPK) pathway regulates heat shock protein 70 (HSP70) expression in primary cultures of human nasal epithelial cells (HNEpCs) in response to pepsin stimulation. METHODS: HSP70 protein expression levels in HNEpCs were estimated by Western blot analysis after treatment with pepsin. MAPK pathway activity levels were also evaluated to elucidate the mechanism underlying the effects of pepsin on HSP70 in HNEpCs. Inhibitors of signaling pathways were used to determine the contribution of MAPKs in HSP70 response after pepsin stimulation. Cellular apoptosis and cell viability in HNEpCs after treatment with pepsin were measured. RESULTS: The expression of HSP70 increased after stimulation with pepsin and decreased after the removal of pepsin. Pepsin induced activation of p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase (JNK) 1/2. Inhibition of JNK1/2 reduced HSP70 expression in HNEpCs. The apoptosis in HNEpCs at 12 h after treatment with pepsin at pH 7.0 increased significantly when compared with the control and pH 7.0 groups. Cell viability decreased following exposure to pepsin at pH 7.0. CONCLUSION: Pepsin, even under neutral pH 7.0, increases the expression of HSP70 in HNEpCs by activating the JNK/MAPK signaling pathway. Increased HSP70 may be the protective mechanism when pepsin presents in the other parts of the body.

Molecular Cloning and Characterization of Ribosomal Protein RPS9 in Echinococcus granulosus.

Ribosomal protein S9 (RPS9) is an essential functional gene that participates in DNA repair and developmental regulations. A sequence homolog of RPS9 has been found to be upregulated in the protoscoleces (PSCs) of Echinococcus granulosus treated with artemisinin. However, E. granulosus RPS9 (EgRPS9) has not been identified before. In the present study, the 657-base pair (bp) cDNA encoding EgRPS9 was cloned. Amino acid sequence analysis showed that EgRPS9 was similar to the RSP9 proteins from Schistosoma japonicum (SjRPS9, 86%) and Schistosoma mansoni (SmRPS9, 79%). Phylogenetic tree analysis showed that EgRPS9, SmRPS9, and SjRPS9 were clustered together. We detected the EgRPS9 gene and protein expression in PSCs exposed to artesunate (AS) which displayed a dose-dependent reduction in PSC viability for 24 hr. The results showed that the EgRPS9 ratio of the 10-µM AS-treated ( P < 0.01) and 40-µM AS-treated ( P < 0.05) groups were increased from that of the control group. In addition, the level of reactive oxygen species (ROS) in the AS-treated groups increased in a dose-dependent manner compared to the level in the control group. In conclusion, the expression of EgRPS9 could be induced by ROS and might participate in the oxidative damage-based anti-parasite mechanism of AS treatment.

Interaction between Various Apple Procyanidin and Staphylococcal Enterotoxin A and Their Inhibitory Effects on Toxin Activity.

In this study, we investigated the interaction between apple polyphenols (AP; mainly consisting of procyanidin (PC) from an apple) and staphylococcal enterotoxin A (SEA), and the inhibitory effects of AP on SEA activity. According to the degree of polymerization, in particularly highly polymerized PC (more than pentamer) strongly interacted with SEA. The binding affinity of AP with SEA molecules was determined using Biacore analysis. AP reacted with SEA immobilized on a Biacore sensor chip. After treatment with pepsin and pancreatin, to examine the changes of binding affinity of AP in intragastric conditions, AP maintained interaction with SEA. We examined whether AP inhibits the proliferation and interferon-γ (IFN-γ) production induced by SEA in mouse spleen cells. AP strongly inactivated the proliferation and IFN-γ production induced by SEA. These results suggest that AP, which has a higher degree of polymerization, inactivates stronger biological activity of SEA through interaction with SEA. Our studies are the first to demonstrate the relationship between the degree of polymerization of AP and the inhibitory effects on SEA activities.

Incorporation of in vitro digestive enzymes in an intestinal epithelial cell line model for protein hazard identification.

Relatively few proteins in nature produce adverse effects following oral exposure. Of those that do, effects are often observed in the gut, particularly on intestinal epithelial cells (IEC). Previous studies reported that addition of protein toxins to IEC lines disrupted monolayer integrity but innocuous dietary proteins did not. Studies presented here investigated the effects of innocuous (bovine serum albumin, ß-lactoglobulin, RuBisCO, fibronectin) or hazardous (phytohaemagglutinin-E, concanavalin A, wheat germ agglutinin, melittin) proteins that either were untreated or exposed to digestive enzymes prior to addition to Caco-2 human IEC line monolayers. At high concentrations intact fibronectin caused an increase in monolayer permeability but other innocuous proteins did not whether exposed to digestive enzymes or not. In contrast, all untreated hazardous proteins and those that were resistant to digestion (ex. wheat germ agglutinin) disrupted monolayer integrity. However, proteins sensitive to degradation by digestive enzymes (ex. melittin) did not adversely affect monolayers when exposed to these enzymes prior to addition to IEC line monolayers. These results indicate that in vitro exposure of proteins to digestive enzymes can assist in differentiating between innocuous and hazardous proteins as another component to consider in the overall weight of evidence approach in protein hazard assessment.

Extra-Esophageal Pepsin from Stomach Refluxate Promoted Tonsil Hypertrophy.

BACKGROUND: Gastroesophageal reflux is associated with numerous pathologic conditions of the upper aerodigestive tract. Gastric pepsin within reflux contributes to immunologic reactions in the tonsil. In this study, we aimed to find the relationships between pepsin and tonsillar hypertrophy. METHODS AND FINDING: We explored the notion whether tonsillar hypertrophy was due to pepsin-mediated gastric reflux in tonsil hypertrophy. Fifty-four children with tonsil hypertrophy and 30 adults with tonsillitis were recruited before surgical treatment. Blood and tonsil tissues from each patient were harvested for analysis of changes in lymphocyte and macrophage numbers coupled with histological and biochemical analysis. Pepsin was expressed at different levels in tonsil tissues from each tonsillar hypertrophy. Pepsin-positive cells were found in the crypt epithelium, surrounding the lymphoid follicle with developing fibrosis, and also surrounding the lymphoid follicle that faced the crypt. And also, pepsin staining was well correlated with damaged tonsillar squamous epithelium and TGF-ß1 and iNOS expression in the tonsil section. In addition, pepsin and TGF-ß1-positive cells were co-localized with CD68-positive cells in the crypt and surrounding germinal centers. In comparison of macrophage responsiveness to pepsin, peripheral blood mononuclear cells (PBMNCs) were noticeably larger in the presence of activated pepsin in the child group. Furthermore, CD11c and CD163-positive cells were significantly increased by activated pepsin. However, this was not seen for the culture of PBMNCs from the adult group. CONCLUSIONS: The lymphocytes and monocytes are in a highly proliferative state in the tonsillar hypertrophy and associated with increased expression of pro-inflammatory factors as a result of exposure to stomach reflux pepsin.

Enzymatic Resistance of Corneas Crosslinked Using Riboflavin in Conjunction With Low Energy, High Energy, and Pulsed UVA Irradiation Modes.

PURPOSE: To investigate the effect of various riboflavin/ultraviolet light (UVA) crosslinking (CXL) protocols on corneal enzymatic resistance. METHODS: A total of 66 enucleated porcine eyes, with the corneal epithelium removed, were divided into 6 groups. Group 1 remained untreated. Groups 2 to 6 received riboflavin/dextran for 30 minutes. Group 3 underwent standard CXL (SCXL) with 3 mW/cm(2) UVA for 30 minutes (total energy dose 5.4 J/cm(2)). Groups 4 and 5 underwent high intensity CXL (HCXL) using 30 mW/cm(2) UVA for 3 minutes (5.4 J/cm(2)) and 30 mW/cm(2) for 4 minutes (7.2 J/cm(2)), respectively. Group 6 was exposed to 8 minutes of 30 mW/cm(2) UVA in a 10-second on/10-second off pulsed-radiation mode (p-HCXL; 7.2 J/cm(2)). A central 8-mm disk from each cornea was submerged in pepsin digest solution at 23°C and measured daily. After 13 days, the dry weight was recorded from 5 samples in each group. RESULTS: The CXL-treated corneas took longer to digest than nonirradiated corneas (P < 0.0001). Differences in digestion time also were observed between CXL groups, such that, HCXL (5.4 J/cm(2)) < SCXL (5.4 J/cm(2)) < HCXL (7.2 J/cm(2)) < p-HCXL (7.2 J/cm(2); P < 0.0001). The dry weight of the SCXL (5.4 J/cm(2)) group was higher than the HCXL (5.4 and 7.2 J/cm(2); P < 0.001) and p-HCXL 7.2 J/cm(2) (P <0.05) groups. No difference was detected between the HCXL and p-HCXL 7.2 J/cm(2) groups. CONCLUSIONS: The intensity and distribution of the crosslinks formed within the cornea vary with different UVA protocols. The precise location and amount of crosslinking needed to prevent disease progression is unknown.