PA28γ coordinates the cross-talk between cancer-associated fibroblasts and tumor cells to promote OSCC progression via HDAC1/E2F3/IGF2 signaling.

PA28γ overexpression is aberrant and accompanied by poor patient prognosis in various cancers, the precise regulatory mechanism of this crucial gene in the tumor microenvironment remains incompletely understood. In this study, using oral squamous cell carcinoma as a model, we demonstrated that PA28γ exhibits high expression in cancer-associated fibroblasts (CAFs), and its expression significantly correlates with the severity of clinical indicators of malignancy. Remarkably, we found that elevated levels of secreted IGF2 from PA28γ+ CAFs can enhance stemness maintenance and promote tumor cell aggressiveness through the activation of the MAPK/AKT pathway in a paracrine manner. Mechanistically, PA28γ upregulates IGF2 expression by stabilizing the E2F3 protein, a transcription factor of IGF2. Further mechanistic insights reveal that HDAC1 predominantly mediates the deacetylation and subsequent ubiquitination and degradation of E2F3. Notably, PA28γ interacts with HDAC1 and accelerates its degradation via a 20S proteasome-dependent pathway. Additionally, PA28γ+ CAFs exert an impact on the tumor immune microenvironment by secreting IGF2. Excitingly, our study suggests that targeting PA28γ+ CAFs or secreted IGF2 could increase the efficacy of PD-L1 therapy. Thus, our findings reveal the pivotal role of PA28γ in cell interactions in the tumor microenvironment and propose novel strategies for augmenting the effectiveness of immune checkpoint blockade in oral squamous cell carcinoma.

Reconstruction of Extensive Maxillary Defects Using Flow-Through Fibula Free Flap With Anterolateral Thigh Free Flap.

BACKGROUND: The maxillary defects left unreconstructed or inadequately reconstructed often result in significant functional and esthetic impairments. Adequate reconstruction of extensive maxillary defects requires a sufficient volume of hard and soft tissues. METHODS: A 48-year-old male presenting bilateral extensive maxillary defects underwent secondary reconstruction with a flow-through fibula free flap in combination with an anterolateral thigh free flap. RESULTS: The use of flow-through technique allowed minimizing the problem of limited recipient vessels and the length of free flap vascular pedicle usually encountered in secondary reconstruction. The bilateral maxillary defects were successfully reconstructed, and the postoperative outcomes were uneventful. The patient was satisfied with the treatment outcomes. He is being followed up and was referred to the implantology department for the placement of osseointegrated dental implants. CONCLUSIONS: The flow-through fibula free flap, in combination with the anterolateral thigh free flap, was found reliable and feasible for this case of secondary reconstruction of bilateral maxillary defects. This technique has provided satisfactory functional and esthetic outcomes and effectively improved the patient's self-esteem.

Association between COVID 19 exposure and expression of malignant pathological features in oral squamous cell carcinoma: A retrospective cohort study.

OBJECTIVES: To analyze the relationship between the clinical and pathological characters of OSCC and COVID 19 exposure. MATERIALS AND METHODS: A retrospective cohort study in patients with OSCC with or without COVID 19 was performed. A total of 200 OSCC patients treated with surgery from 2019 to 2023 were included. Clinical and pathological features were analysed between two groups. Characters with statistical difference were further analysed by performing univariate analysis and logistic regression analysis. RESULTS: The expression of Ki67 (n = 57, 71.3 %, P < 0.001) and CyclinD1 (n = 64, 80 %, P < 0.001) in OSCC with the exposure history of COVID 19 is higher than that in patients never exposed to COVID 19. COVID 19 exposure history is an independent influencing factor for higher expression of Ki67 (OR = 4.04, 95 % CI: 1.87-8.72, P < 0.001) and CyclinD1 (OR = 5.45, 95 % CI: 2.56-11.60, P < 0.001). CONCLUSION: COVID 19 may suggest more invasive malignant biological behavior of cancer cells in OSCC.

Identification, rapid screening, docking mechanism and in vitro digestion stability of novel DPP-4 inhibitory peptides from wheat gluten with ginger protease.

To better understand the hypoglycemic potential of wheat gluten (WG), we screened dipeptidyl peptidase IV (DPP-4) inhibitory active peptides from WG hydrolysates. WG hydrolysates prepared by ginger protease were found to have the highest DPP-4 inhibitory activity among the five enzymatic hydrolysates, from which a 1-3 kDa fraction was isolated by ultrafiltration. Further characterization of the fraction with nano-HPLC-MS/MS revealed 1133 peptides. Among them, peptides with P'2 (the second position of the N-terminal) and P2 (the second position of the C-terminal) as proline residues (Pro) accounted for 12.44% and 43.69%, respectively. The peptides including Pro-Pro-Phe-Ser (PPFS), Ala-Pro-Phe-Gly-Leu (APFGL), and Pro-Pro-Phe-Trp (PPFW) exhibited the most potent DPP-4 inhibitory activity with IC50 values of 56.63, 79.45, and 199.82 µM, respectively. The high inhibitory activity of PPFS, APFGL, and PPFW could be mainly attributed to their interaction with the S2 pocket (Glu205 and Glu206) and the catalytic triad (Ser630 and His740) of DPP-4, which adopted competitive, mixed, and mixed inhibitory modes, respectively. After comparative analysis of PPFS, PPFW, and PPF, Ser was found to be more conducive to enhancing the DPP-4 inhibitory activity. Interestingly, peptides with P2 as Pro also exhibited good DPP-4 inhibitory activity. Meanwhile, DPP-4 inhibitory peptides from WG showed excellent stability, suggesting a potential application in type 2 diabetes (T2DM) therapy or in the food industry as functional components.

An epulis-like camrelizumab related reactive cutaneous capillary endothelial proliferation (RCCEP) in the oral cavity: A case report.

BACKGROUND: Immunotherapy, especially anti-PD-1 and anti-PD-L1 antibodies, have observably improved the overall survival of patients with advanced solid tumors following the unavoidable immune-related adverse events (irAEs). Camrelizumab is a novel anti-PD-1 agent with the reported most common irAEs of reactive cutaneous capillary endothelial proliferation (RCCEP). Despite it is widely occurred in the skin, oral RCCEP is rarely reported. CASE SUMMARY: A 59-year-old man complained about a painless nodule on left mandibular gingiva for two weeks. He started to inject Camrelizumab because of the recurrence of esophageal squamous cell carcinoma two month ago. An 8 mm lesion was observed on his mucosa. Several disseminated bright purple red papules were then found on his skins. The oral lesion and one lesion on his face was removed by surgery. After the final diagnosis of reactive cutaneous capillary endothelial proliferation was confirmed by histological examination. Other operable lesions on his face were removed by ligation. All the removed lesions had a good prognosis without recurrence within the follow-up visit. CONCLUSION: With the widespread use of Camrelizumab in other solid tumors, the occurrence of oral RCCEP will increase. Surgery and ligation are both effective treatment for RCCEP with a good prognosis.

Phenolic compounds in walnut pellicle improve walnut (Juglans regia L.) protein solubility under pH-shifting condition.

This study focused on the interaction of walnut protein with phenolic extracts of walnut pellicle (PEWP) under alkaline condition, leading to enhancement of protein solubility under neutral condition. First, the change of PEWP under alkaline condition was determined by RP-HPLC and mass spectrometry, and the results showed that most ellagitannins in PEWP could be retained under alkaline condition within 3 h. Interaction between PEWP and walnut protein under pH-shifting condition resulted in the remarkable increase of protein solubility (above 90%) at neutral pH. The results from SDS-PAGE and SEC showed that the improved solubility lied in the formation of large and soluble protein aggregates due to the covalent interaction among walnut protein and polyphenols. A significant change in tertiary structure of protein-phenolic complex was witnessed by fluorescence spectrum and near-UV circular dichroism. Meanwhile, walnut protein-polyphenol interaction led to a slight increase in ß-turn while a slight decrease in ß-sheet. Combined with amino acid composition, it could be illustrated that the covalent bonding for walnut protein with polyphenol mainly occurred at Lysine residues.

Growing of fungi on the stored low denatured defatted soybean meals and the hydrolysis of proteins and isoflavone glycosides by fungal enzymes.

Recently, more and more attention has been paid to the effects of fungal contamination and fungal enzymes secreted in raw grain on product quality. As the starting material of protein and active components, the quality of low denatured defatted soybean meals (LDSM) directly determines the qualities of subsequent products. In previous studies, we have revealed that infection with Aspergillus ochraceus protease causes significant hydrolysis of proteins. In this study, growing of fungi on the stored low denatured defatted soybean meals (LDSM) was analyzed by high-throughput sequencing and real-time PCR, which revealed that the abundance of Aspergillus increased significantly after storage. Twenty fungal proteases and 9 fungal glucosidases were found in stored LDSM and zymography showed that the proteases were of serine-type with some cysteine and aspartic activities. Proteolysis of the soybean storage proteins mainly occurred after the hydration of LDSM and the average molecular weight of soy proteins decreased from 57.9 kDa to 30.7 kDa after 60 min's of hydrolysis. Two-dimensional electrophoresis (2-DE) analysis found the polypeptide fragments from soybean 7S and 11S proteins with molecular weight around 10-25 kDa in the hydrated LDSM. Glycosylated isoflavones were hydrolyzed in both dry and hydrated stored LDSM which resulted in significant (p < 0.05) increase in the contents of isoflavone aglycones. This study suggested that fungi contamination be a new factor affecting the properties of LDSM derived soy protein products.

Characterization of proteases from Irpex lacteus grown on minimally denatured soybean meal.

BACKGROUND: Acid and thermal stabilities are important properties for the preparation of acidic protein beverage. It is an important method for enzymatic modification to improve the functional properties of protein. Irpex lacteus protease showed a selective hydrolysis to soy proteins. The purpose of this study was to investigate the mechanism of enzymatic hydrolysis and its effects on acid and thermal stabilities of soy proteins. RESULTS: The I. lacteus protease selectively hydrolyzed the α and α' subunits of the native soybean ß-conglycinin (7S globulin) to produce products that presented as the 55 kDa band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequences of 55 kDa polypeptides were analyzed in gel multi-enzyme digestion followed by liquid chromatography-mass spectrometry. By matching the multi-enzyme digestion peptides with the published polypeptide chain sequences of the α and α' subunits, it was confirmed that the 55 kDa polypeptides were formed by eliminating amino acid residues on both sides of the N- and C-terminals. From the published protein structure database (https://www.uniprot.org/), it is known that the cleaved peptide bonds were in extension regions. Non-selective enzyme hydrolysis of both ß-conglycinin (7S globulin) and glycinin (11S globulin), with corresponding drastic increases in the degree of hydrolysis, was observed when the substrates were preheated to the denaturation degree of 40% and above. However, 55 kDa hydrolyzed products and B polypeptides showed some extent of resistance to the proteolysis by I. lacteus protease even if denaturation degree was 100%. Both selective and non-selective hydrolysis of soy proteins by I. lacteus protease improved the acid and heat stabilities under the same hydrolysis conditions (enzyme/substrate ratio, time, and temperature). CONCLUSION: Enzymatic hydrolysis of soybean proteins by the I. lacteus protease can effectively improve the acid and thermal stabilities of proteins. This discovery is significant to avoid aggregation during processing in the beverage industry. In the near future, the protease has potential application value for modification of other proteins. © 2022 Society of Chemical Industry.

Identification of a BRAF/PA28γ/MEK1 signaling axis and its role in epithelial-mesenchymal transition in oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a chronic and insidious oral potentially malignant disorder associated with a 4-17% risk of oral squamous cell carcinoma (OSCC). Our previous study found that proteasomal activator 28 gamma (PA28γ) is frequently overexpressed in oral squamous cell carcinoma and negatively correlated with poor patient prognosis. However, the role of PA28γ in the occurrence and development of OSF remains unclear. Here, we screened PA28γ-related genes and investigated their function in OSF. We demonstrated that the expression of PA28γ was positively associated with MEK1 and gradually elevated from normal to progressive stages of OSF tissue. Arecoline, a pathogenic component of OSF, could upregulate the protein levels of PA28γ and phosphorylated MEK1 and contribute to epithelial to mesenchymal transition (EMT) in epithelial cells. Notably, PA28γ could interact with MEK1 and upregulate its phosphorylation level. Furthermore, arecoline upregulated BRAF, which can interact with PA28γ and upregulate its protein level. Additionally, BRAF, PA28γ, and MEK1 could form protein complexes and then enhance the MEK1/ERK signaling pathways. The concrete mechanism of the protein stability of PA28γ is that BRAF mediates its degradation by inhibiting its ubiquitination. These findings underscore the instrumental role of PA28γ in the BRAF/MEK1 pathway and enhanced EMT through MEK1/ERK activation in OSF.

Raw walnut kernel: A natural source for dietary proteases and bioactive proteins.

Walnut kernels are health-promoting nuts, which are mainly attributed to polyunsaturated fatty acids, phenolics, and phytosterols. However, the information concerning benefits of walnut proteins are limited. In this study, endopeptidases, aminopeptidases, carboxypeptidases, superoxide dismutases, catalases, and phospholipases with respective relative abundance of 2.730, 1.728, 0.477, 3.148, 0.743, and 0.173‰ were identified by liquid chromatography tandem mass spectrometry. These endogenous proteases exhibited activity in a broad pH range of 2-6.5, and optimal at pH 4.5 and 50 °C. Aspartic endopeptidases were predominant endopeptidases, followed by cysteine ones. There were two types of aspartic endopeptidases, one (not inhibited by pepstatin A) exerted activity at pH 2-3 and the other (inhibited by pepstatin A) optimal at pH 4.5. Carboxypeptidases were optimal at pH 4.5, and aminopeptidases exerted activity at pH near 6.5. These endogenous proteases assisted the digestion of walnut proteins, and soaking, especially peeling, greatly improved the in vitro digestibility.

Endopeptidases, exopeptidases, and glutamate decarboxylase in soybean water extract and their in vitro activity.

The proteolytic activity of some soybean endogenous proteases have been clarified in the previous studies, but the information concerning the roles of these proteases and some other unknown ones during soybean processing are scarce. Herein, 16 endopeptidases, 13 exopeptidases, 24 inhibitors (two serpin-ZX and one subtilisin inhibitor firstly identified), and one glutamate decarboxylase were identified in the soybean water extract by the liquid chromatography tandem mass spectrometry analysis. Amongst the identified endopeptidases, just the aspartic endopeptidases (optimal at pH 2.5-3 and 35-45 °C) showed the detectable proteolytic activity by the tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis and protease inhibitor assay analyses, whereas serine, cysteine, and metallo- endopeptidases (except P34 probable thiol protease) did not. Free amino acid analysis showed that the exopeptidases and glutamate decarboxylase were optimal at pH 6 and 45 °C, and by 6 h incubation, the free amino acids and γ-aminobutyric acid almost doubled.

Separation, identification and molecular binding mechanism of dipeptidyl peptidase IV inhibitory peptides derived from walnut (Juglans regia L.) protein.

Walnut protein was hydrolyzed with different proteases to evaluate the hydrolytic efficiency and dipeptidyl peptidase IV (DPP-IV) inhibitory activity in vitro. All of walnut protein hydrolysates (WPHs) exhibited DPP-IV inhibitory activity and Alcalase-derived hydrolysate (WPH-Alc) with better DPP-IV inhibitory activity of 33.90% (at 0.50 mg/mL) was subsequently separated by ultrafiltration and cation exchange chromatography on a SP Sephadex C-25 column. The results showed that fractions with lower molecular weight and higher basic amino acid residues possessed stronger DPP-IV inhibitory activity. Comparably, the obtained fraction B with the yield of 19.80% had the highest DPP-IV inhibitory activity of 76.19% at 0.25 mg/mL. Moreover, nine novel DPP-IV inhibitory peptides were identified using MALDI-TOF/TOF-MS. Molecular docking revealed the peptides could interact with DPP-IV through hydrogen bonds, salt bridges, hydrophobic interactions, π-cation bonds and π-π bonds. The walnut DPP-IV inhibitory peptides showed better stability with heating treatment, pH treatment, or in vitro gastrointestinal digestion.

Characterization of endogenous endopeptidases and exopeptidases and application for the limited hydrolysis of peanut proteins.

Research concerning the utilization of oilseed endogenous proteases is scarce. Herein, we investigated the peanut proteases and their effects on peanut proteins. Liquid chromatography tandem mass spectrometry analysis showed that peanut contained several endopeptidases and exopeptidases. Protease inhibitor assay and analysis of cleavage sites showed that the obvious proteolytic activity at pH 2-5 and 20-60 °C was from aspartic endopeptidases (optimal at pH 3) and one legumain (pH 4). The above endopeptidases destroyed five and six IgE-binding epitopes of Ara h 1 at pH 3 and 4, respectively. Ara h 1 (>95%) and arachin (50-60%) could be hydrolyzed to generate 10-20 kDa and <4 kDa peptides at pH 3, which was enhanced by the pH 3 â†’ 4 incubation. Further, the limited hydrolysis improved the gel-forming ability and in vitro digestibility (approximately 15%) of peanut proteins. Free amino acid analysis showed that the activity of exopeptidases was low at pH 2-5.

Insights into the antibacterial activity of cottonseed protein-derived peptide against Escherichia coli.

In the study, antibacterial peptides were separated and identified from cottonseed protein hydrolysates and the interactions between antibacterial peptides and Escherichia coli were further investigated. Firstly, by using a combined strategy of Amberlite CG-50 ion exchange chromatography and reversed-phase high-performance liquid chromatography, three peptides with antibacterial activity were purified and identified, including HHRRFSLY, KFMPT, and RRLFSDY. Interestingly, HHRRFSLY and RRLFSDY exhibited higher inhibition activity with the IC50 value of 0.26 mg mL-1 and 0.58 mg mL-1 (p < 0.05), respectively. Flow cytometry results showed that the incubation of antibacterial peptides with E. coli could cause damage to the integrity of the E. coli cell membrane. Transmission electron microscopy and scanning electron microscopy results revealed the damage caused to the bacterial cell surface and the leakage of cytoplasmic content by the antibacterial peptides. Molecular docking studies indicated that HHRRFSLY, KFMPT, and RRLFSDY have a good binding affinity to the active sites of the surface protein (OmpF) mainly through a hydrogen bond and salt bridge. The results here showed that the antibacterial peptides derived from cottonseed protein could be used as a good choice for functional foods or related drugs, and also shed light on further studies of antibacterial mechanism.

Proliferative ability and accumulation of cancer stem cells in oral submucous fibrosis epithelium.

OBJECTIVES: The driving force of the malignant transformation of epithelial cells during oral submucous fibrosis (OSF) is an unsettled debate. We hypothesized that the expression and accumulation of cancer stem cells (CSCs) are accompanied by epithelial atrophy in OSF. MATERIALS AND METHODS: The expression levels of Ki67 (proliferation marker), SOX2, and Bmi1 (CSC marker) in the epithelium during the early, middle, and late stages of OSF were measured by immunohistochemistry. At the same time, we focused on the expression of three proteins in OSF patients with benign hyperkeratosis and epithelial dysplasia. RESULTS: The clinical cohort study showed upregulated expression of the proliferation-associated protein Ki67 in atrophic epithelium in patients with OSF. The expression levels of SOX2 and Bmi1 showed an increasing trend in the progression of OSF. Ki67, SOX2, and Bmi1 were highly expressed in OSF tissues with dysplasia. Moreover, the three proteins were located at the epithelial and mesenchymal junctions, and their expression showed a positive correlation with each other. CONCLUSION: The results suggest that CSC accumulation could be accompanied by epithelial atrophy during OSF, which may be responsible for the driving forces for OSF carcinogenesis.

Selective Extraction and Antioxidant Properties of Thiol-Containing Peptides in Soy Glycinine Hydrolysates.

A highly selective procedure to extract thiol-containing peptides (TCPs) from complicated soy glycinin hydrolysates (SGHs) was described. This procedure included the reduction of disulfide bonds by 1,4-dithiothreitol (DTT) and enrichment of TCPs through Thiopropyl-Sephrose 6B covalent chromatography. TCPs were confirmed using a strategy based on mass shift after differential alkylation of sulfhydryl groups with iodoacetamide and N-ethylmaleimide by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS). The antioxidant activities of TCPs were evaluated using chemical assays. DTT reduction increased the concentration of sulfhydryl groups from 1.8 µmol/g to 113.8 µmol/g. The efficiency of the extraction was improved by optimizing the loading of sample, extraction and desorption time and the content of desorption reagent. Both of the adsorption and desorption process were found to fit a pseudo-second order model. MALDI-TOF-MS showed that 36 of the 45 extracted peptides were TCPs. The EC50 of TCPs for DPPH, hydroxyl radical, and superoxide anion radical was 0.1, 1.49 and 0.084 mg/mL, respectively. The reducing power of TCPs (0.2 mg/mL) was of 0.375. These results suggest that the combination of DTT reduction and Thiopropyl-Sepharose 6B covalent chromatograph was a successful pathway to extract TCPs from SGHs and the TCPs could be used as potential antioxidants.

A two-chain aspartic protease present in seeds with high affinity for peanut oil bodies.

Peanut seeds are rich in oil, which exists as oil bodies (OBs). By extraction, peanut crude OBs are obtained and can be used as a food ingredient. In a previous study, it was found that the crude OBs contained an unknown protease, which hydrolyzed the oleosins. This would disrupt the integrity of OBs, and therefore, affect their physical and oxidative stability. In this study, the protein composition of crude OBs and some properties of the unknown protease were examined. The results showed that the protease was a two-chain (32 and 9kDa) aspartic protease, which showed high affinity for OBs. The optimal pH and temperature for oleosin hydrolysis by the protease were pH 4.0 and 60°C. Interestingly, the aspartic protease not only hydrolyzed OB intrinsic proteins (oleosin, caleosin, and steroleosin), but also extrinsic proteins (especially Ara h 1 allergen and 26-30kDa arachin).

Soybean P34 Probable Thiol Protease Probably Has Proteolytic Activity on Oleosins.

P34 probable thiol protease (P34) and Gly m Bd 30K (30K) show high relationship with the protease of 24 kDa oleosin of soybean oil bodies. In this study, 9 day germinated soybean was used to separate bioprocessed P34 (P32) from bioprocessed 30K (28K). Interestingly, P32 existed as dimer, whereas 28K existed as monomer; a P32-rich sample had proteolytic activity and high cleavage site specificity (Lys-Thr of 24 kDa oleosin), whereas a 28K-rich sample showed low proteolytic activity; the P32-rich sample contained one thiol protease. After mixing with purified oil bodies, all P32 dimers were dissociated and bound to 24 kDa oleosins to form P32-24 kDa oleosin complexes. By incubation, 24 kDa oleosin was preferentially hydrolyzed, and two hydrolyzed products (HPs; 17 and 7 kDa) were confirmed. After most of 24 kDa oleosin was hydrolyzed, some P32 existed as dimer, and the other as P32-17 kDa HP. It was suggested that P32 was the protease.

Heat-induced inactivation mechanism of soybean Bowman-Birk inhibitors.

Due to the complications of the soymilk system, the heat-induced Bowman-Birk inhibitor (BBI) inactivation mechanism is not well known. In this study, two BBI samples with low and high purities were prepared from soymilk. It was confirmed that three groups (A, C, and D) of BBI, which are contained in soybean seeds, were transferred into soymilk during processing. On heating, it was found that 1) the two subdomains of BBI were not equally heat stable, 2) the conformation of BBI gradually changed, 3) some amino acid residues (namely, cystine, serine and lysine) in BBI were degraded, 4) BBI did not tend to form intermolecular cross-links with another BBI, but did slightly with non-BBI proteins. Based on some previous studies, the conformational change of BBI was attributed to ß-elimination reactions on the amino acid residues of BBI and the subsequent intramolecular reactions induced by the products yielded by the ß-elimination reactions.

An advance for removing antinutritional protease inhibitors: Soybean whey purification of Bowman-Birk chymotrypsin inhibitor by combination of two oppositely charged polysaccharides.

Two successive and selective coacervations induced by chitosan (Ch) and carrageenan (CG) were applied to remove antinutritional protease inhibitors and purify Bowman-Birk protease inhibitor (BBI) from soybean whey. At the first coacervation induced by Ch (66.7, 200, and 510kDa), only Kunitz trypsin inhibitor (KTI) and BBI complexed with Ch were extracted, while ß-amylase and soybean agglutinin remained in supernatant. The binding constants for the interaction increased on the order Ch-66.7