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Engineered interleukin-18 (IL-18) has attracted interest as a cytokine-based treatment. However, knowledge-based mutagenesis of IL-18 has been reported for only a few regions of the protein structures, including binding sites I and II. When coupled with the binding region mutant (E6K), the non-binding residue of IL-18, Thr63 (T63), has been shown to increase the flexibility of the binding loop. Nevertheless, the function of Thr63 in conformational regulation is still unknown. Using homology modeling, molecular dynamics simulation, and structural analysis, we investigated the effects of Thr63 alteration coupling with E6K on conformational change pattern, binding loop flexibility, and the hydrogen bond network. The results indicate that the 63rd residue was significantly associated with hydrogen-bond relaxation at the core β-barrel binding sites I and II Glu85-Ile100 loop. This result provided conformational and flexible effects to binding sites I and III by switching their binding loops and stabilizing the 63rd residue cavity. These findings may pave the way for the conceptualization of a new design for IL-18 proteins by modifying non-binding residues for structure-based drug development. [ABSTRACT FROM AUTHOR]

Trypsin from the digestive tract of harpiosquillid mantis shrimp (HMS) was purified using ammonium sulfate precipitation and a soybean trypsin inhibitor‐CNBr‐activated Sepharose 4B affinity column. The purified trypsin (PTRP‐HMS) had a purity of 30.4‐fold, and a yield of 14.5% was obtained. PTRP‐HMS had the molecular weight of 23.0 kDa as examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and only one isoform was detected by native‐PAGE. Its optimal temperature and pH were 55°C and 8.5, respectively. TLCK almost completely inhibited the activity of trypsin. The PTRP‐HMS had a Michaelis–Menten constant (Km) and catalytic constant (Kcat) of 0.87 mM and 13.04 s−1, respectively, toward Nα‐benzoyl‐l‐arginine 4‐nitroanilide hydrochloride. When chitooligosaccharide (COS) and COS‐catechin (COS‐CAT) conjugates were examined for inhibition toward the PTRP, the latter exhibited higher efficacy in inhibiting the trypsin. Both COS and COS‐CAT conjugate showed mixed‐type inhibition kinetics. As a consequence, COS and COS‐CAT conjugate could be used as natural additives for inhibiting trypsin in whole HMS, thus retarding the softening and lengthening the shelf‐life of HMS during the iced storage. Practical Application: Harpiosquillid mantis shrimp (HMS) is of high demand due to its delicacy. However, its meat undergoes rapid softening within 2–3 days when stored in ice. Understanding causative proteolytic enzymes, especially trypsin from digestive tract, paves a way for preventing their negative impact on HMS eating quality. Employment of safe inhibitors, for example, chitooligosaccharide (COS) or COS conjugated with catechin, could inhibit HMS trypsin. Overall, softening of whole HMS containing trypsin in its digestive tract can be impeded, especially when treated with COS‐CAT. This finding is beneficial for the HMS local vendor or exporter, in which HMS quality can be maintained. [ABSTRACT FROM AUTHOR]

Our study aimed to evaluate tumour retardation effects of low-piperine fractional Piper nigrum extract (PFPE or LP-PE) related to enhancement of immune response. Results showed that PFPE at doses of 50 (low), 100 (intermediated) and 150 (high) mg/kg BW had a tumour incidence of 20, 20 and 0%, respectively. PFPE at low dose inhibited cytokines/chemokines in the early stages of cancer however it could not suppress longer than 3 months. Intermediated dose of PFPE inhibited cancer cytokines/chemokines including sICAM-1, CX3CL1, CXCL7, VEGF, TIMP-1, CCL5, CCL20, CXCL1, CXCL5, CXCL9 and IL-1β. Interestingly, PFPE stimulated IFN- γ at 3 months after treatment in all doses. Intermediated and high doses of PFPE increased Th1 and reduced Th2. Moreover, only a high dose of PFPE reduced Treg. Additionally, PFPE reduced ER-α and NF-κB and increased IL-6R and STAT3 proteins in tumour tissues. Our findings indicate that PFPE suppressed breast cancer via immune modulation. [ABSTRACT FROM AUTHOR]

Bambara groundnut (BG)-based films containing seed coat extract at different concentrations were prepared and characterized. BG seed coat extract (BGSCE) had a total phenolic content of 708.38 mg GAE/g dry extract. BGSCE majorly consisted of quercetin 3-galactoside, rutin, and azaleatin 3-arabinoside. BGSCE exhibited ABTS and DPPH radical scavenging activities (ABTS-RSAs and DPPH-RSAs), a ferric reducing antioxidant power (FRAP), and an oxygen radical absorbance capacity (ORAC) of 66.44, 4.98, 4.42, and 0.91 mmol Trolox equivalent/g dry extract, respectively. When BGSCE at various concentrations (0–8%, w/w, protein content) was incorporated into the BG protein isolate (BG-PI)-based films, film containing 4% BGSCE exhibited higher thickness, tensile strength, elongation at break, water vapor and UV-light barrier properties, and a*-value (redness) than the control film (p < 0.05). Films containing BGSCE had greater ABTS-RSA, FRAP, and ORAC than the control film (p < 0.05). An FTIR analysis elucidated that the proteins interacted with phenolic compounds in BGSCE. Nonetheless, less thermal stability was attained in films added with BGSCE. Hence, the addition of BGSCE possessing antioxidant activity exhibited an important role in properties and characteristics of BG-PI-based film. The developed active film could be applied as packaging material possessing antioxidant property for food applications. [ABSTRACT FROM AUTHOR]

Summary: Encapsulation efficiency (EE), structure, antioxidant and antimicrobial activities of liposomes loaded with chitooligosaccharide–gallic acid conjugate (COS–GAL) as affected by cholesterol (CHO) and ascorbyl palmitate (AP) as stabilisers were investigated. In addition, release kinetics and in vitro digestion of selected liposomes were also studied. The higher EE was obtained for liposome (AP/COS–GAL) stabilised by 0.50% AP, when used to encapsulate 0.10% COS–GAL conjugate. The highest EE was noticed for 0.25% CHO when used to encapsulate 1.00% conjugate (CHO/COS–GAL) as compared to the counterparts (P < 0.05). Almost similar antioxidant and antimicrobial activities were noticed for all COS–GAL liposomes and COS–GAL powder. Microstructure study suggested the formation of bilayer in liposomes. CHO/COS–GAL showed a lower release efficiency than the AP/COS–GAL. CHO/COS–GAL was also able to protect conjugate during the in vitro digestion. Therefore, liposomes could be used as a promising carrier of COS–GAL conjugate in both foods as well as medical systems. [ABSTRACT FROM AUTHOR]

Bio-calcium derived from fish frames may offer several advantages for osteoporosis prevention. This study aimed to evaluate the effects of bio-calcium derived from skipjack tuna frames on bone loss in ovariectomized rats. Tuna bio-calcium was prepared through enzymatic hydrolysis, defatting, bleaching, and grinding processes. The bioavailability of calcium was tested using the Caco-2 cell monolayer model, showing that 13% of tuna bio-calcium was absorbed, compared to 10% for calcium carbonate. Rats were divided into the five following groups: (1) OVX, (2) sham-operated, (3), OVX + estrogen-treated (4) OVX + calcium carbonate-treated, and (5) OVX + tuna bio-calcium-treated. All groups were raised for eight weeks. Tuna bio-calcium was able to increase BV/TV by 26% in the femur and 29% in the tibia, compared to 13% and 17% in the OVX group, respectively. Trabecular thickness in the femur upsurged to 360 µm in the tuna group, while a thickness of 290 µm was observed in the control. Additionally, osteoclast numbers were reduced to 5 N.Oc/mm in the femur and 6 N.Oc/mm in the tibia in the tuna group, compared to 35 and 45 N.Oc/mm in the control. Overall, tuna bio-calcium effectively prevented bone loss and can serve as a promising natural alternative for managing osteoporosis. [ABSTRACT FROM AUTHOR]

KRAS G12D mutation is prevalent in various cancers and is associated with poor prognosis. This study aimed to identify potential drug candidates targeting KRAS G12D using combined machine learning, virtual screening, molecular docking, and molecular dynamics (MD) simulations. The training and test sets are constructed based on a selection of inhibitors targeting the KRAS G12D mutant from the ChEMBL library. A random forest machine learning algorithm is developed to predict potential KRAS G12D binders. Molecular docking and the MM/PBSA binding energy are used to identify the lead compounds. The compound NPC489264 is identified as the top candidate, exhibiting favorable docking energy for the KRAS G12D mutant (−13.16 kcal mol−1). A hydrogen bond between the mutated Asp12 residue in the KRAS G12D mutant and NPC489264 is found to be a key interaction between these 2 molecules. MD simulations and MM/PBSA analysis revealed the strong binding affinity of NPC489264 to the G12D mutant (−5.49 kcal mol−1) compared to the wild type (10.17 kcal mol−1). These findings suggest that NPC489264 is a promising lead compound for further development of KRAS G12D‐targeted cancer therapies. [ABSTRACT FROM AUTHOR]

Summary: Freshness and physicochemical properties of myofibrillar protein (MP) from decapitated Harpiosquillid mantis shrimp (HMS) treated with pulsed electric field (PEF), followed by soaking in 2% chitooligosaccharide (D‐P‐COS) and whole HMS (U‐W) during storage in iced water of 3 days were investigated. Lower rate of increase in K‐value, freshness index, in D‐P‐COS was observed in comparison with U‐W. For physicochemical properties of MP, MP from U‐W had gradual decrease in ATPase activity throughout the storage of 3 days, whereas no drastic alteration was attained in MP from D‐P‐COS. Surface hydrophobicity and carbonyl contents of MP from both samples upsurged up to 3 days, nevertheless the increases were more pronounced in D‐P‐COS. The secondary conformation changes were affected by decapitation, treatment and storage time. Decapitation and treatments maintained freshness, however, slightly enhanced physicochemical changes of MP from HMS. [ABSTRACT FROM AUTHOR]

Lipid oxidation is among vital deteriorative reactions associated with quality loss of foods, and some oxidation products have toxicity when consumed. Maillard Reaction Products (MRPs) obtained from salmon frame protein hydrolysate (PH) were determined for antioxidant activities. MRPs exhibited superior antioxidant efficacy in both ß-carotene linoleate and lecithin liposome systems to PH (p<0.05). MRPs also showed preventive effect toward oxidation in comminuted pork system (p<0.05). The antioxidant potential of MRPs was dose-dependent in all systems tested. Based on cytotoxicity toward Caco-2 cells, MRPs up to 10 mg/mL had no toxicity toward cells when the exposure time was up to 48 h (p>0.05). In addition, the less absorption of MRPs across the Caco-2 cell monolayer was observed as evidenced by the decreased absorbance at 420 nm in the basolateral side (p<0.05). However, small free peptides in MRPs could pass through monolayer effectively. Furthermore, following translocation across the Caco-2 cell monolayer, MRPs exhibited marginally reduced DPPH and ABTS radical scavenging activities, ferric reducing antioxidant capacity and metal chelating activity. Overall, MRPs were therefore a promising safe antioxidant in various food systems, thus improving oxidative stability of treated foods. [ABSTRACT FROM AUTHOR]

Pre-cooked bone is a waste product generated during tuna processing and can serve as a potential source of biocalcium (BC). Generally, non-collagenous protein and fat must be removed properly from bone. A NaCl solution can be used to remove such proteins, while fish lipase can be used in a green process, instead of solvent, for fat removal. Thus, this study aimed to investigate the impact of NaCl pretreatment at different concentrations in combination with heat to eliminate non-collagenous proteins, and to implement fish lipase treatments at varying levels for fat removal, for BC production from pre-cooked tuna bone. Optimal NaCl pretreatment of bone was achieved when a 5% NaCl solution at 80 °C was used for 150 min. The lowest lipid content was obtained for bone defatted with crude lipase extract (CLE) at 0.30 Unit/g of bone powder for 2 h. BC powder from bone defatted with CLE (DF-BC) possessed greater contents of ash, calcium, and phosphorus and smaller particle sizes than the control BC powder. X-ray diffractograms suggested that both BC powders consisted of hydroxyapatite as a major compound, which had a crystallinity of 62.92–63.07%. An elemental profile confirmed the presence of organic and inorganic matter. Thus, BC powder could be produced from pre-cooked tuna bone using this 'green process'. [ABSTRACT FROM AUTHOR]

Summary: The effects of chitooligosaccharide–gallic acid conjugate (CGC) and its liposome (CGLP) at two levels (1.25 and 2.50%, w/v) on quality and shelf‐life of milk kept at 4 °C were studied. Microbial counts including total viable, pathogenic and spoilage bacteria were the lowest for CGC‐fortified milk, especially when 2.50% (w/v) was used (CGC‐2.50) than remaining samples (P < 0.05). Lower thiobarbituric acid reactive substances and peroxide values were obtained for CGC‐2.50 than other samples (P < 0.05). However, higher acidity values and decreased pH and lightness were noticed in the aforementioned sample (P < 0.05). Moreover, all fortified milk samples had higher bioactivities during in vitro digestion, in which CGC‐2.50 had the highest bioactivities (P < 0.05). Higher retention of unsaturated fatty acids content in CGC‐2.50 was obtained than other counterparts (P < 0.05). Therefore, CGC efficiently extended milk shelf‐life with high acceptability at 4 °C for a minimum of 6 days, whereas control had a shelf‐life of less than 4 days. [ABSTRACT FROM AUTHOR]

Chitooligosaccharide (COS) and gallic acid (GA) are natural compounds with anti-cancer properties, and their conjugate (COS–GA) has several biological activities. Herein, the anti-cancer activity of COS–GA in SW620 colon cancer cells was investigated. MTT assay was used to evaluate cell viability after treatment with 62.5, 122, and 250 µg/mL of COS, GA, and COS–GA for 24 and 48 h. The number of apoptotic cells was determined using flow cytometry. Proteomic analysis was used to explore the mechanisms of action of different compounds. COS–GA and GA showed a stronger anti-cancer effect than COS by reducing SW620 cell proliferation at 125 and 250 µg/mL within 24 h. Flow cytometry revealed 20% apoptosis after COS–GA treatment for 24 h. Thus, GA majorly contributed to the enhanced anti-cancer activity of COS via conjugation. Proteomic analysis revealed alterations in protein translation and DNA duplication in the COS group and the structural constituents of the cytoskeleton, intermediate filament organization, the mitochondrial nucleoid, and glycolytic processes in the COS–GA group. Anti-cancer-activity-related proteins were altered, including CLTA, HSPA9, HIST2H2BF, KRT18, HINT1, DSP, and VIM. Overall, the COS–GA conjugate can serve as a potential anti-cancer agent for the safe and effective treatment of colon cancer. [ABSTRACT FROM AUTHOR]

Freeze-dried tuna pepsin powder (TPP) was prepared using maltodextrin (10%) and trehalose (5%), while trypsin-loaded beads (TLB) with 5% glycerol were obtained via chitosan/alginate ionotropic gelation. The storage stability of TPP and TLB and their proteolytic activity toward red kidney bean protein (RKB), threadfin bream surimi (TBS) and egg white protein (EWP) in varying simulated in vitro gastrointestinal (GI) tract conditions were studied. The intestinal transepithelial transportation of generated peptides was also carried out through Caco-2 cell monolayers after the cytotoxicity test. Enzyme activity was dropped when TPP and TLB in blister packs were kept for 10 weeks of storage at room (28 °C) and refrigerated (4 °C) temperatures. TPP and TLB at a level of 50% (w/w of proteins) effectively hydrolyzed RKB, TBS and EWP in a simulated in vitro GI tract, as indicated by marked protein degradation and increased degree of hydrolysis. Some peptides generated after GI digestion could transport through Caco-2 cell monolayers. Those peptides had different molecular size distribution and antioxidant activities. The highest antioxidant activity was observed for RKB hydrolysate after passing through the Caco-2 cell monolayer. Therefore, TPP and TLB from skipjack tuna viscera could potentially be used for enzyme supplementation to help digest food proteins. Food-derived bioactive peptides generated after GI digestion could assist in improving human health due to their antioxidant activity. [ABSTRACT FROM AUTHOR]

Molecular classifications for urothelial bladder cancer appear to be promising in disease prognostication and prediction. This study investigated the novel molecular subtypes of muscle invasive bladder cancer (MIBC). Tumor samples and normal tissues of MIBC patients were submitted for transcriptome sequencing. Expression profiles were clustered using K-means clustering and principal component analysis. The molecular subtypes were also applied to The Cancer Genome Atlas (TCGA) dataset and analyzed for clinical outcome correlation. Three molecular subtypes of MIBC were discovered, clusters A, B, and C. The most differentially upregulated genes in cluster A were BDKRB1, EDNRA, AVPR1A, PDGFRB, and TNC, while the most upregulated genes in cluster C were collagen-related genes, PDGFRB, and PRKG1. For cluster B, COL6A3, COL1A2, COL6A2, tenascin C, and fibroblast growth factor 2 were statistically suppressed. When the centroids of clustering on PCA were applied to TCGA data, the clustering significantly predicted survival outcomes. Cluster B had the best overall survival (OS), and cluster C was associated with poor OS but exhibited the best response to perioperative chemotherapy. Among all groups, cluster B had a better pathologic response to neoadjuvant chemotherapy (40%). Based on the results of the present study, the novel clusters of subtype MIBC appear potentially suitable for integration into clinical practice. [ABSTRACT FROM AUTHOR]

Context: Many natural extracts have been shown to minimize the toxicity of doxorubicin (Dox). Low piperine Piper nigrum L. (Piperaceae) extract (PFPE) is a natural extract containing many types of antioxidants that may reduce Dox toxicities. Objective: To evaluate the effect of PFPE in attenuating the side effects of Dox. Materials and methods: Tumour-bearing Sprague Dawley rats were divided into five groups including normal, vehicle, 100mg/kg BW of PFPE plus 2mg/kg BW of Dox (P100þDox), 100 mg/kg BW of PFPE plus 2mg/kg BW of Dox (P200þDox) and Dox. Rats were treated with Dox and/or PFPE three times/week for 4 weeks. Tumour burden, blood parameters, weight of internal organs and immunological data were investigated. Results: The addition of 200 mg/kg PFPE significantly restored the levels of AST from 174.60 ± 45.67 U/L in the Dox group near to normal levels at 109.80 ± 4.99 U/L. The combination of PFPE and Dox also decreased the levels of CXCL7, TIMP-1, sICAM-1 and L-selectin about 1.4-1.6-fold compared to Dox group. Feeding rats with 200mg/kg BW of PFPE combination with Dox slightly increased Th1 from 161.67 ± 14.28 cells in Dox group to 200.75 ± 5.8 cells meanwhile suppressed Treg from 3088 ± 78 cells in Dox to 2561 ± 71 cells. Discussion and conclusions: This study showed that PFPE ameliorated Dox toxicity in many aspects indicating the role of antioxidant and other substances in the extract on toxicity attenuation. This suggested the using of PFPE may be valuable for Dox treated patients. [ABSTRACT FROM AUTHOR]