Accelerating Proteomics Using Broad Specificity Proteases.

Trypsin is the gold-standard protease in bottom-up proteomics, but many sequence stretches of the proteome are inaccessible to trypsin and standard LC-MS approaches. Thus, multienzyme strategies are used to maximize sequence coverage in post-translational modification profiling. We present fast and robust SP3- and STRAP-based protocols for the broad-specificity proteases subtilisin, proteinase K, and thermolysin. All three enzymes are remarkably fast, producing near-complete digests in 1-5 min, and cost 200-1000× less than proteomics-grade trypsin. Using FragPipe resolved a major challenge by drastically reducing the duration of the required "unspecific" searches. In-depth analyses of proteinase K, subtilisin, and thermolysin Jurkat digests identified 7374, 8178, and 8753 unique proteins with average sequence coverages of 21, 29, and 37%, including 10,000s of amino acids not reported in PeptideAtlas' >2400 experiments. While we could not identify distinct cleavage patterns, machine learning could distinguish true protease products from random cleavages, potentially enabling the prediction of cleavage products. Finally, proteinase K, subtilisin, and thermolysin enabled label-free quantitation of 3111, 3659, and 4196 unique Jurkat proteins, which in our hands is comparable to trypsin. Our data demonstrate that broad-specificity proteases enable quantitative proteomics of uncharted areas of the proteome. Their fast kinetics may allow "on-the-fly" digestion of samples in the future.

In vitro model to evaluate effect of acidic pepsin on vocal fold barrier function.

The pathophysiology of laryngopharyngeal reflux (LPR) and its impact on the vocal fold is not well understood, but may involve acid damage to vocal fold barrier functions. Two different components encompass vocal fold barrier function: the mucus barrier and tight junctions. Mucus retained on epithelial microprojections protects the inside of the vocal fold by neutralizing acidic damage. Tight junctions control permeability between cells. Here we developed an in vitro experimental system to evaluate acidic injury and repair of vocal fold barrier functions. We first established an in vitro model of rat vocal fold epithelium that could survive at least one week after barrier function maturation. The model enabled repeated evaluation of the course of vocal fold repair processes. Then, an injury experiment was conducted in which vocal fold cells were exposed to a 5-min treatment with acidic pepsin that injured tight junctions and cell surface microprojections. Both of them healed within one day of injury. Comparing vocal fold cells treated with acid alone with cells treated with acidic pepsin showed that acidic pepsin had a stronger effect on intercellular permeability than acid alone, whereas pepsin had little effect on microprojections. This result suggests that the proteolytic action of pepsin has a larger effect on protein-based tight junctions than on phospholipids in microprojections. This experimental system could contribute to a better understanding of vocal fold repair processes after chemical or physical injuries, as well as voice problems due to LPR pathogenesis.

Pepsin-mediated inflammation in laryngopharyngeal reflux via the ROS/NLRP3/IL-1ß signaling pathway.

BACKGROUND: Laryngopharyngeal reflux (LPR) is one of the most common disorders in otorhinolaryngology, affecting up to 10% of outpatients visiting otolaryngology departments. In addition, 50% of hoarseness cases are related to LPR. Pepsin reflux-induced aseptic inflammation is a major trigger of LPR; however, the underlying mechanisms are unclear. The nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome has become an important bridge between stimulation and sterile inflammation and is activated by intracellular reactive oxygen species (ROS) in response to danger signals, leading to an inflammatory cascade. In this study, we aimed to determine whether pepsin causes LPR-associated inflammatory injury via mediating inflammasome activation and explore the potential mechanism. METHODS: We evaluated NLRP3 inflammasome expression and ROS in the laryngeal mucosa using immunofluorescence and immunohistochemistry. Laryngeal epithelial cells were exposed to pepsin and analyzed using flow cytometry, western blotting, and real-time quantitative PCR to determine ROS, NLRP3, and pro-inflammatorycytokine levels. RESULTS: Pepsin expression was positively correlated with ROS as well as caspase-1 and IL-1ß levels in laryngeal tissues. Intracellular ROS levels were elevated by increased pepsin concentrations, which were attenuated by apocynin (APO)-a ROS inhibitor-in vitro. Furthermore, pepsin significantly induced the mRNA and protein expression of thioredoxin-interacting protein, NLRP3, caspase-1, and IL-1ß in a dose-dependent manner. APO and the NLRP3 inhibitor, MCC950, inhibited NLRP3 inflammasome formation and suppressed laryngeal epithelial cell damage. CONCLUSION: Our findings verified that pepsin could regulate the NLRP3/IL-1ß signaling pathway through ROS activation and further induce inflammatory injury in LPR. Targeting the ROS/NLRP3 inflammasome signaling pathway may help treat patients with LPR disease.

Construction and Application of a Pepsin-Functionalized Covalent Organic Framework with Prominent Chiral Recognition Ability.

The stable Pepsin@covalent organic framework (Pepsin@COF) were constructed base on matching COF pore diameter to pepsin dimension. It exhibits excellent chiral recognition capabilities (e. e. % up to 62.63 %) and potential for enantioseparation. Furthermore, a positive correlation between the immobilized enzyme activity and chiral recognition was revealed, offering insights for the design of biocatalytic nanosystems in chiral separation.

Peptide-Based Mass Spectrometry for the Investigation of Protein Complexes.

In the last two decades, biological mass spectrometry has become the gold standard for the identification of proteins in biological samples. The technological advancement of mass spectrometers and the development of methods for ionization, gas phase transfer, peptide fragmentation as well as for acquisition of high-resolution mass spectrometric data marked the success of the technique. This chapter introduces peptide-based mass spectrometry as a tool for the investigation of protein complexes. It provides an overview of the main steps for sample preparation starting from protein fractionation, reduction, alkylation and focus on the final step of protein digestion. The basic concepts of biological mass spectrometry as well as details about instrumental analysis and data acquisition are described. Finally, the most common methods for data analysis and sequence determination are summarized with an emphasis on its application to protein-protein complexes.

Effect of Chloramphenicol as Antibiotic on the Structure and Function of Pepsin and Its Mechanism of Action.

The interaction between chloramphenicol (CHL) and pepsin (PEP), as well as the impact of CHL on PEP conformation, were investigated using spectroscopic techniques and molecular docking simulations in this study. The experimental results demonstrate that CHL exhibits a static quenching effect on PEP. The thermodynamic parameters indicate that the reaction between CHL and PEP is spontaneous, primarily driven by hydrogen bonding and van der Waals forces. Moreover, the binding distance of r<7 nm suggests the occurrence of Förster's non-radiative energy transfer between these two molecules. In the synchronous fluorescence spectrum, the maximum fluorescence intensity of PEP produced a redshift phenomenon, indicating that CHL was bound to tryptophan residues of PEP. The addition of CHL induces changes in the secondary structure of PEP, as confirmed by the observed alterations in peak values in three-dimensional fluorescence spectra. The UV spectra reveal a redshift of 3 nm in the maximum absorption peak, indicating a conformational change in the secondary structure of PEP upon addition of CHL. Circular dichroism analysis demonstrates significant alterations in the α-helix, ß-sheet, ß-turn, and random coil contents of PEP before and after CHL incorporation, further confirming its ability to modulate the secondary structure of PEP.

Usefulness of pepsin saliva measurement for the detection of primary burning mouth syndrome related to reflux.

OBJECTIVES: To study the diagnostic value of salivary pepsin tests for detecting laryngopharyngeal reflux (LPR) in patients with primary burning mouth syndrome (BMS). METHODS: Patients with BMS and asymptomatic individuals were consecutively recruited from September 2018 to June 2023. Patients underwent hypopharyngeal-esophageal impedance pH-monitoring (HEMII-pH) and saliva collections to measure pepsin. Stomatology evaluation was carried out to exclude other causes of BMS. Oral, pharyngeal and laryngeal signs and symptoms were evaluated with Reflux Sign Assessment (RSA) and Reflux Symptom Score (RSS). Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values of pepsin test were calculated considering the highest values of pepsin tests at ≥ 16, ≥ 36, and ≥ 100 ng/mL cutoffs. Receiver operating characteristic curve (ROC) was evaluated. RESULTS: Forty-nine patients with both BMS and LPR at the HEMII-pH and 21 asymptomatic individuals were recruited. Pepsin test was 83.7%, 79.6%, and 71.4% sensitive at cutoffs ≥ 16, ≥ 36, and ≥ 100 ng/mL, respectively. The ROC analysis reported that a threshold of ≥ 21.5 ng/mL was associated with sensitivity, specificity, PPV and NPV of 81.6%, 81.0%, 90.1% and 65.4%, respectively. The severity score of burning mouth symptom was significantly associated with the saliva pepsin concentration (rs = 0.263; p = 0.029) and the oral RSA (rs = 0.474; p = 0.007). CONCLUSION: Pepsin test is a valuable diagnostic approach for detecting LPR in patients with BMS. Patients with high level of saliva pepsin reported more severe burning mouth symptoms. Future studies are needed to confirm the role of LPR in the primary BMS.

Determination of pepsin in human saliva using pepsin-susceptible peptide reporter and colorimetric dipstick assay: a prospective, cross-sectional study.

A simple and effective pepsin detection assay is reported based on a pepsin-susceptible peptide (PSP) reporter degradation strategy. PSP, which can be specifically cleaved by pepsin, was modified with fluorescein isothiocyanate (FITC) and biotin at the N- and C-terminals to be used as a reporter for colorimetric detection of dipsticks. A universal lateral flow dipstick consisting of a streptavidin test line for biotin binding and a sample pad immobilized with a gold-labeled polyclonal (rabbit) anti-FITC antibody was used to verify PSP-based pepsin detection. When the PSP reporter reacts with pepsin in a tube, it cleaves into two fragments, and the cleaved fragments do not display any color on the test line. Therefore, the higher the concentration of pepsin is, the greater is the decrease in test line intensity (IT-line) and the higher is the control line intensity (IC-line). First, the PSP cleavage and dipstick assay conditions for pepsin detection was optimized. The ratio of color intensity (IT-line/IC-line) of PSP-based dipstick assay showed a linear relationship with log concentration of pepsin ranging between 4 and 500 ng/mL (R2 = 0.98, n = 6), with a limit of detection of 1.4 ng/mL. It also exhibited high specificity and good reproducibility. Finally, pepsin levels were quantified in saliva samples from healthy controls (n = 34) and patients with laryngopharyngeal reflux (LPR, n = 61). Salivary pepsin levels were higher in patients with LPR than in healthy controls. The salivary pepsin levels correlated with those measured using a conventional enzyme-linked immunosorbent assay kit. Therefore, this PSP-based dipstick assay is a convenient tool for assessing salivary pepsin levels.

Structural Catalytic Core of the Members of the Superfamily of Acid Proteases.

The superfamily of acid proteases has two catalytic aspartates for proteolysis of their peptide substrates. Here, we show a minimal structural scaffold, the structural catalytic core (SCC), which is conserved within each family of acid proteases, but varies between families, and thus can serve as a structural marker of four individual protease families. The SCC is a dimer of several structural blocks, such as the DD-link, D-loop, and G-loop, around two catalytic aspartates in each protease subunit or an individual chain. A dimer made of two (D-loop + DD-link) structural elements makes a DD-zone, and the D-loop + G-loop combination makes a psi-loop. These structural markers are useful for protein comparison, structure identification, protein family separation, and protein engineering.

Reframing prosegment-dependent folding and limits on natural protein folding landscapes from an evolutionary perspective.

In this perspective, we propose that the folding energy landscapes of model proteases including pepsin and alpha-lytic protease (αLP), which lack thermodynamic stability and fold on the order of months to millennia, respectively, should be viewed as not evolved and fundamentally distinct from their extended zymogen forms. These proteases have evolved to fold with prosegment domains and robustly self-assemble as expected. In this manner, general protein folding principles are strengthened. In support of our view, αLP and pepsin exhibit hallmarks of frustration associated with unevolved folding landscapes, such as non-cooperativity, memory effects, and substantial kinetic trapping. The evolutionary implications of this folding strategy are considered in detail. Direct applications of this folding strategy on enzyme design, finding new drug targets, and constructing tunable folding landscapes are also discussed. Together with certain proteases, growing examples of other folding "exceptions"-including protein fold switching, functional misfolding, and prevalent inability to refold-suggests a paradigm shift in which proteins may evolve to exist in a wide range of energy landscapes and structures traditionally thought to be avoided in nature.

MicroRNA-Transcription factor regulatory networks in the early strobilar development of Echinococcus granulosus protoscoleces.

BACKGROUND: Echinococcus granulosus sensu lato has a complex developmental biology with a variety of factors relating to both intermediate and final hosts. To achieve maximum parasite adaptability, the development of the cestode is dependent on essential changes in transcript regulation. Transcription factors (TFs) and miRNAs are known as master regulators that affect the expression of downstream genes through a wide range of metabolic and signaling pathways. In this study, we aimed to develop a regulatory miRNA-Transcription factor (miRNA-TF) network across early developmental stages of E. granulosus protoscoleces by performing in silico analysis, and to experimentally validate TFs expression in protoscoleces obtained from in vitro culture, and from in vivo experiments. RESULTS: We obtained list of 394 unique E. granulosus TFs and matched them with 818 differentially expressed genes which identified 41 predicted TFs with differential expression. These TFs were used to predict the potential targets of 31 differentially expressed miRNAs. As a result, eight miRNAs and eight TFs were found, and the predicted network was constructed using Cytoscape. At least four miRNAs (egr-miR-124a, egr-miR-124b-3p, egr-miR-745-3p, and egr-miR-87-3p) and their corresponding differentially expressed TFs (Zinc finger protein 45, Early growth response protein 3, Ecdysone induced protein 78c and ETS transcription factor elf 2) were highlighted in this investigation. The expression of predicted differentially expressed TFs obtained from in vitro and in vivo experiments, were experimentally validated by quantitative polymerase chain reaction. This confirmed findings of RNA-seq data. CONCLUSION: miRNA-TF networks presented in this study control some of the most important metabolic and signaling pathways in the development and life cycle of E. granulosus, providing a potential approach for disrupting the early hours of dog infection and preventing the development of the helminth in the final host.

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.

3-Hydroxyflavone - bovine serum albumin complex used as fluorescent probe for the detection of pepsin.

In this study, a method for the determination of pepsin using a 3-hydroxyflavone (3-HF)-bovine serum albumin (BSA) complex as a fluorescent probe was established. Under acidic conditions, 3-HF can react with BSA to emit strong green fluorescence, which involves an excited state intramolecular proton transfer and fluorescence resonance energy transfer. After pepsin is added, the fluorescence of the 3-HF-BSA system is quenched due to the hydrolysis of BSA. The degree of 3-HF-BSA fluorescence quenching has a good linear relationship with the concentration of pepsin in the range of 10-80 µg/mL. The correlation coefficient was R2 = 0.9932 and the detection limit was 2.41 µg/mL. This method has high selectivity and good stability and has been used to determine pepsin in real samples with satisfactory results.

Preclinical trial of the effectiveness of a safety nasogastric tube to detect the tube position based on tidal volume and pepsin assay results in the gastrointestinal tract of Macaca fascicularis.

BACKGROUND: Tube misplacement into the tracheobronchial tract is associated with pneumothorax in 0.5% of cases. NGT verification only detects the position of the tube at the end of the procedure. Therefore, a safe nasogastric tube (SNGT) was created to detect the NGT position in real time in a simple and inexpensive way. This study aimed to prove the effectiveness of the SNGT prototype in Macaca fascicularis. RESULT: An SNGT producing 50% of the TV had 100% sensitivity and specificity in detecting the position of the tube at 100% of the TV, with a sensitivity of 100% and a specificity of 87.5%. There was a significant difference between the movement of the SNGT 50% TV and SNGT 100% TV airbags (p ≤ 0.05). However, there was no significant difference between the accuracy of placement of the 50% TV SNGT, 100% TV SNGT, and conventional NGT (p > 0.05). The pepsin enzyme had better sensitivity (100%) than pH paper (91.66%) in detecting the end-of-procedure tube position. This research has the potential to advance into human clinical trials. CONCLUSION: SNGTs are highly effective in detecting the NGT position inside the respiratory and digestive tracts to prevent misplacement.

Pepsin in saliva for the diagnosis of erosive esophagitis post-sleeve gastrectomy: a prospective observational study.

BACKGROUND: Laparoscopic sleeve gastrectomy (LSG) has become the preferred bariatric procedure in many countries. However, new onset erosive esophagitis (EE) is a major shortcoming. Current recommendation is esophago-gastro-duodenoscopy (EGD) should be performed routinely at 1 year and subsequently every 2-3 years to enable the early detection of Barrett's or esophageal adenocarcinoma. This would put significant strains on resources and costs of bariatric program. Our study assesses the association between and diagnostic value of salivary pepsin concentration and endoscopically proven EE in post-LSG patients as a surrogate for EGD. METHODS: Twenty patients on routine post-LSG endoscopy between June and September 2022 were recruited for this correlational pilot study. Under supervision, fasting and post-prandial saliva sample was collected and analyzed by Peptest lateral flow device. EGD examinations were performed, and patients completed a validated 25-item QoLRAD questionnaire. RESULTS: We found a significant correlation between positive endoscopy findings of EE and salivary pepsin concentrations. The normal group had a lower mean fasting pepsin level (13.13 ng/mL ± 18.97) versus the EE-group (90.55 ng/mL ± 81.28, p = 0.009) and lower mean post-prandial pepsin level (30.50 ng/mL ± 57.72) versus the EE-group (135.09 ng/mL ± 130.17, p = 0.02). The predictive probabilities from the binary regression of fasting and post-prandial pepsin concentrations yield AUC of 0.955 ± 0.044 (95% CI 0.868 to 1.000, p < 0.001). CONCLUSION: Our study distinctively identified salivary pepsin to have excellent sensitivity and negative predictive value in EE, potentially useful to preclude the need for post-LSG EGD in asymptomatic patients with low salivary pepsin.

A prospective study of extraesophageal reflux and potential microaspiration in patients hospitalized with COVID-19 in Jordan.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lung infection has represented a global challenge. Intriguingly, it has been shown that the alveolar lung epithelium expresses little Angiotensin Converting Enzyme receptor protein (ACE2), the entry receptor for SARS-CoV-2. Upper airway establishment of infection and translocation to the lung is well documented but other anatomical niches may be relevant to potentially serious lung infection. ACE2 is heavily expressed in the gastrointestinal tract and gastrointestinal symptoms support a clinical diagnosis of Coronavirus disease 2019 (COVID-19). This suggests a research question and the need to gather patient data exploring potential aerodigestive links in SARS-CoV-2 tranlocation and infection which may be relevant in the peripheral lung. This recognizes anatomical proximity and concepts of bi-directional movement between the Gastrointestinal and lung systems in normal physiology and disease. We have therefore explored the potential for gastro oesophageal reflux disease (GORD) micro aspiration and aeorodigestive pathophysiology in a novel prospective investigation of patients hospitalized with COVID-19. METHODS: This is a prospective descriptive cohort study of 210 patients who were hospitalized with a confirmed diagnosis of COVID-19. The cohort was divided into three groups of patients based on symptom severity and radiological results. The Reflux Symptom Index (RSI) was used to evaluate the presence and severity of GOR. An RSI greater than 13 is considered to be abnormal. Patients' saliva samples were tested using enzyme-linked immunosorbent assay (ELISA) to determine the level of salivary pepsin among the cohort of patients. RESULTS: A total of 210 patients with COVID-19 were enrolled in the study with 55.2% (116/210) classified as mildly ill, 31.9% (67/210) moderately ill and 12.9% (27/210) as severely ill. 34% (72/210) of the patients had an RSI score of over 13 and a median salivary pepsin value of 54 ± 29 ng/ml which suggested an incidence of extraesophageal reflux (EOR) in around a third of patients. The presence of respiratory comorbid conditions, an RSI score of over 13 and a salivary pepsin level of > 76ng/ml increased the risk of developing a more severe COVID-19 infection. CONCLUSION: The study showed a high prevalence of EOR among the study cohort and provide the first prospective evidence suggesting the potential for aerodigestive pathophysiology including microaspiration in COVID-19 disease. We believe that the results of our study support the need for more extensive research.

Pepsin Hydrolysate from Surimi Industry-Related Olive Flounder Head Byproducts Attenuates LPS-Induced Inflammation and Oxidative Stress in RAW 264.7 Macrophages and In Vivo Zebrafish Model.

Fish head byproducts derived from surimi processing contribute about 15% of the total body weight, which are beneficial to health because they contain essential nutrients. In this study, olive flounder (OF) was the target species in order to maximize the byproduct utilization. In RAW 264.7 macrophages, the seven hydrolysates from OF head byproducts were examined for their inhibitory potential against inflammation and the oxidative stress induced by lipopolysaccharides (LPS). The pepsin hydrolysate (OFH-PH) demonstrated strong anti-inflammatory activity via the down-regulation of NO production, with an IC50 value of 299.82 ± 4.18 µg/mL. We evaluated the inhibitory potential of pro-inflammatory cytokines and PGE2 to confirm these findings. Additionally, iNOS and COX-2 protein expressions were confirmed using western blotting. Furthermore, the results from the in vivo zebrafish model demonstrated that OFH-PH decreased the LPS-elevated heart rate, NO production, cell death, and intracellular ROS level, while increasing the survival percentage. Hence, the obtained results of this study serve as a platform for future research and provide insight into the mediation of inflammatory disorders. These results suggest that OFH-PH has the potential to be utilized as a nutraceutical and functional food ingredient.

Pepsin enhances glycolysis to promote malignant transformation of vocal fold leukoplakia epithelial cells with dysplasia.

PURPOSE: The mechanism underlying malignant transformation of vocal fold leukoplakia (VFL) and the precise role of the expression of pepsin in VFL remain unclear. This study aimed to investigate the effects of acidified pepsin on VFL epithelial cell growth and migration, and also identify pertinent molecular mechanisms. METHODS: Immunochemistry and Western blotting were performed to measure glucose transporter type 1 (GLUT1), monocarboxylate transporters 4 (MCT4), and Hexokinase-II (HK-II) expressions. Cell viability, cell cycle, apoptosis, and migration were investigated by CCK-8 assay, flow cytometry and Transwell chamber assay, respectively. Glycolysis-related contents were determined using the corresponding kits. Mitochondrial HK-II was photographed under a confocal microscope using Mito-Tracker Red. RESULTS: It was found: the expression of pepsin and proportion of pepsin+ cells in VFL increased with the increased dysplasia grade; acidified pepsin enhanced cell growth and migration capabilities of VFL epithelial cells, reduced mitochondrial respiratory chain complex I activity and oxidative phosphorylation, and enhanced aerobic glycolysis and GLUT1 expression in VFL epithelial cells; along with the transfection of GLUT1 overexpression plasmid, 18FFDG uptake, lactate secretion and growth and migration capabilities of VFL epithelial cell were increased; this effect was partially blocked by the glycolysis inhibitor 2-deoxy-glucose; acidified pepsin increased the expression of HK-II and enhanced its distribution in mitochondria of VFL epithelial cells. CONCLUSION: It was concluded that acidified pepsin enhances VFL epithelial cell growth and migration abilities by reducing mitochondrial respiratory complex I activity and promoting metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis.

Saliva pepsin measurements in the detection of gastroesophageal reflux disease in laryngopharyngeal reflux patients: a cohort study.

OBJECTIVE: To study the diagnostic value of salivary pepsin measurement (Peptest) for detecting gastroesophageal reflux disease (GERD) in laryngopharyngeal reflux (LPR) patients. METHODS: Patients with reflux symptoms were consecutively recruited from January 2020 to November 2022. Patients benefited from hypopharyngeal-esophageal impedance-pH monitoring (HEMII-pH), fasting and bedtime saliva collections to measure pepsin. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values were evaluated for GERD and LPR patients considering the highest values of pepsin tests at ≥ 16, ≥ 75, and ≥ 216 ng/mL cutoffs. The relationship between HEMII-pH, endoscopic and clinical findings, and pepsin measurements was studied. RESULTS: Saliva was collected in 109 LPR patients and 30 individuals with both LPR and GERD. The total number of pharyngeal reflux events was significantly higher in GERD-LPR patients compared with LPR patients (p = 0.008). The mean fasting and bedtime pepsin saliva concentrations were similar between groups. The sensitivity of Peptest in LPR patients was 30.5%, 70.2%, and 84.0% at cutoffs ≥ 16, ≥ 75 and ≥ 216 ng/mL. In GERD-LPR group, Peptest was 80.0%, 70.0%, and 30.0% sensitive. At cutoff 16 ng/mL, Peptest reported PPV of 20.7% and 94.8% in LPR-GERD and LPR groups, respectively. NPV were 73.9% and 8.7% in GERD-LPR and LPR groups, respectively. The consistency analysis between Peptest and HEMII-pH was not significant. Peptest was significantly associated with the number of acid pharyngeal reflux events (rs = 0.182; p = 0.032). CONCLUSION: Pepsin saliva measurements appear to be not a reliable diagnostic tool for the detection of GERD in LPR patients. Future studies are needed to determine the place of Peptest in laryngopharyngeal reflux and gastroesophageal reflux diseases.

Influences of Curcuma Longa (turmeric) supplemented diet on the expression of defensive and destructive factors in indomethacin-induced ulcerated Wistar rats.

BACKGROUND: Turmeric (Curcuma longa) L. has been recognized as a plant with high medicinal value and it has been used in the prevention and treatment of many diseases. Different studies have shown that turmeric has both therapeutic and preventive effects on peptic ulcer. However, there are controversial reports about the anti-ulcerogenic potential of turmeric. Some studies also suggested that turmeric could be ulcerogenic when consumed in large quantity with no mention of the concentration at which this could take place. AIM: This study examined the influences of different concentration of turmeric rhizome powder supplemented diet on the gene expressions of both anti-ulcer and ulcer biomarkers in indomethacin-induced ulcerated rats. METHODS: The study was conducted through prophylactic treatment of test groups with turmeric at different percentage (1%, 2%, 5%, and 10%) for 28 days. Thirty-five rats were randomly divided into seven groups namely A, B, C, D (1%, 2%, 5%, and 10% groups respectively), E (standard drug group (STD)), F (ulcerogenic group) and G (normal control group (CTL)). At the end of 28 days, rats were fasted overnight and ulcer was induced in all the groups except group G by oral administration of 60mg/kg b.w (body weight) of indomethacin. The expression of defensive (Cyclo-oxygenase-1, MUCIN, and Hyme-oxygenase-1) and destructive factors (Pepsin) were then analyzed. RESULTS: Results showed that consumption of TRPSD at 1-5% increased the gene expression of protective factors when compared with animals in group F. Furthermore, Prophylactic treatment of test groups with TRPSD at 1%-5% significantly suppressed the gene expression of pepsin in comparison with animals in group F. However, TRPSD consumption at 10% down-regulated the gene expression of those protective factors. Similarly, at 10%, pepsin gene expression was not suppressed when compared with animals in group F. Conclusively, TRPSD could be gastro protective at 1%-5%. However, these potentials were abrogated in animals in group D indicating the ulcerogenic potential of turmeric at this concentration (10%) and its capability to enhance ulcerogenic action of indomethacin. CONCLUSION: Turmeric rhizome powder (TRP) have anti-ulcerogenic potential and gastro-protective effect when consumed in appropriate concentration. Consumption of TRP at 10% concentration could enhance ulcerogenic action of indomethacin (NSAIDs) thus predisposing to ulcer. Effects of turmeric rhizome powder supplemented diet (TRPSD) on the mRNA expression of protective agents (cyclo-oxygenase-1 (COX-1), mucin, and inducible heme-oxygenase (HO-1)) and destructive factor (pepsin), in indomethacin-induced ulcerated Wistar rats were explored in this paper. These were determined by prophylactic treatment of test groups with turmeric at different levels (1%, 2%, 5%, and 10%) for 28 days. Thirty-five rats were randomly divided into seven groups namely A, B, C, D (1%, 2%, 5%, and 10% groups respectively), E (standard drug group (STD)), F (ulcerogenic group) and G (normal control group (CTL)). The rats were fasted overnight and ulcer was induced in all the groups except group G by oral administration of 60mg/kg b.w (body weight) of indomethacin. The expression of defensive (Cyclo-oxygenase-1, MUCIN, and Hyme-oxygenase-1) and destructive factors (Pepsin) were then analyzed. Results showed that consumption of TRPSD at 1%-5% increased the gene expression of protective factors when compared with animals in group F. Furthermore, Prophylactic treatment of test groups with TRPSD at 1%-5% significantly suppressed the gene expression of pepsin in comparison with animals in group F. However, TRPSD consumption at 10% down-regulated the gene expression of those protective factors. Similarly, at 10%, pepsin gene expression was not suppressed when compared with animals in group F. Conclusively, TRPSD could be gastro protective at 1%-5%. However, these potentials were abrogated in animals in group D indicating the ulcerogenic potential of turmeric at this concentration (10%) and its capability to enhance ulcerogenic action of indomethacin.