Establishment, characterization, and sensory characteristics (taste and flavor) of an immortalized muscle cell line from the seven-band grouper Epinephelus septemfasciatus: implications for cultured seafood applications.

Grouper muscle satellite cells (GMSCs) from the seven-band grouper (Epinephelus septemfasciatus) were isolated, and their growth conditions were optimized (10% fetal bovine serum, 24°C, 10 ng/mL bFGF). The cells were immortalized at passage 14 and designated as grouper immortalized muscle satellite cells (GIMSCs). DNA barcoding confirmed the grouper origin of both GMSC and GIMSC lines. GIMSCs exhibited enhanced proliferation, accelerated differentiation, and robust myotube formation compared to pre-crisis GMSCs. Western blot analysis showed upregulation of key myogenic factors (Pax7, MyoD, MyoG) and structural proteins (Desmin) in GIMSC, indicating the differentiation potential. The immortalized GIMSC line maintained consistent morphology, growth rates, and viability across multiple passages. Biocompatibility studies showed GIMSCs were compatible with bio-inks (sodium alginate, gelatin, κ-carrageenan) at 250 to 10,000 µg/mL, retaining ~ 80% viability at the highest concentration. Taste sensory analysis revealed GMSCs had the highest umami and lowest saltiness and sourness, contrasting with the muscle of the seven-band grouper, which had higher saltiness and sourness. Flavor analysis identified pronounced fishy, hot fat, and ethereal flavors in the cells at higher level than in the muscle. These findings suggest GMSCs and GIMSCs are promising for producing cultured meat with enhanced umami taste and flavors, advancing cellular agriculture and sustainable food production.

Crassostrea gigas peptide PEP-1 prevents tert-butyl hydroperoxide (t-BHP) induced oxidative stress in HepG2 cells.

Exposure to tert-butyl hydroperoxide (t-BHP) leads to cytotoxicity and oxidative stress in various organs and cell types. The bioactive peptides extracted from Oysters exhibit marked antioxidant activity. The impacts of Crassostrea gigas peptides on t-BHP-triggered oxidative stress remain largely unknown. The protective and antioxidant activity of a C.gigas peptide, PEP-1, on t-BHP-treated HepG2 cells, was investigated. PEP-1, this peptide is arginine kinase in oysters. This enzyme functions as a catalyst for the chemical reaction and serves as a phosphate transferase. Since it was the most expressed protein in the adductor muscle of oysters. Our determination showed the lowest level of a toxic concentration of t-BHP (200 µM) and the resting concentration of PEP-1 (0-1000 ng/ml). PEP-1 exerted a protective effect against t-BHP-induced apoptosis by modifying the expression of pro-and anti-apoptotic proteins. PEP-1 administration reduced nitric oxide and ROS levels while restoring levels of antioxidant proteins in t-BHP-induced cells. PEP-1 exhibited the capacity to enhance the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Therefore, the C. gigas peptide PEP-1 has demonstrated its ability to protect HepG2 cells against oxidative stress induced by t-BHP.

Establishment and Characterization of Continuous Satellite Muscle Cells from Olive Flounder (Paralichthys olivaceus): Isolation, Culture Conditions, and Myogenic Protein Expression.

Olive flounder (Paralichthys olivaceus) muscle satellite cells (OFMCs) were obtained by enzymatic primary cell isolation and the explant method. Enzymatic isolation yielded cells that reached 80% confluence within 8 days, compared to 15 days for the explant method. Optimal OFMC growth was observed in 20% fetal bovine serum at 28 °C with 0.8 mM CaCl2 and the basic fibroblast growth factor (BFGF) to enhance cell growth. OFMCs have become permanent cell lines through the spontaneous immortalization crisis at the 20th passage. Olive flounder skeletal muscle myoblasts were induced into a mitogen-poor medium containing 2% horse serum for differentiation; they fused to form multinucleate myotubes. The results indicated complete differentiation of myoblasts into myotubes; we also detected the expression of the myogenic regulatory factors myoD, myogenin, and desmin. Upregulation (Myogenin, desmin) and downregulation (MyoD) of muscle regulation factors confirmed the differentiation in OFMCs.

The Influence of κ-Carrageenan-R-Phycoerythrin Hydrogel on In Vitro Wound Healing and Biological Function.

Wound healing is widely recognized as a critical issue impacting the healthcare sector in numerous countries. The application of wound dressings multiple times in such instances can result in tissue damage, thereby increasing the complexity of wound healing. With the aim of tackling this necessity, in the present study, we have formulated a hydrogel using natural polysaccharide κ-carrageenan and phycobiliprotein R-phycoerythrin from Pyropia yezoensis. The formulated hydrogel κ-Carrageenan-R-Phycoerythrin (κ-CRG-R-PE) was analyzed for its antioxidant and antimicrobial activity. The wound healing potential of the κ-CRG-R-PE was evaluated in Hs27 cells by the wound scratch assay method. The hydrogel showed dose-dependent antioxidant activity and significant antimicrobial activity at 100 µg/mL concentration. κ-CRG-R-PE hydrogels promoted more rapid and complete wound closure than κ-Carrageenan (κ-CRG) hydrogel at 24 and 48 h. κ-CRG-R-PE hydrogels also filled the wound within 48 h of incubation, indicating that they positively affect fibroblast migration and wound healing.

L-Lysine-Modified pNIPAm-co-GMA Copolymer Hydrogel for pH- and Temperature-Responsive Drug Delivery and Fluorescence Imaging Applications.

The development of dual-stimuli-responsive hydrogels attracts much research interest owing to its unique stimuli-responsive characteristics. In this study, a poly-N-isopropyl acrylamide-co-glycidyl methacrylate-based copolymer was synthesized by incorporating N-isopropyl acrylamide (NIPAm) and a glycidyl methacrylate (GMA) monomer. The synthesized copolymer, pNIPAm-co-GMA was further modified with L-lysine (Lys) functional units and further conjugated with fluorescent isothiocyanate (FITC) to produce a fluorescent copolymer pNIPAAm-co-GMA-Lys hydrogel (HG). The in vitro drug loading and dual pH- and temperature-stimuli-responsive drug release behavior of the pNIPAAm-co-GMA-Lys HG was investigated at different pH (pH 7.4, 6.2, and 4.0) and temperature (25 °C, 37 °C, and 45 °C) conditions, respectively, using curcumin (Cur) as a model anticancer drug. The Cur drug-loaded pNIPAAm-co-GMA-Lys/Cur HG showed a relatively slow drug release behavior at a physiological pH (pH 7.4) and low temperature (25 °C) condition, whereas enhanced drug release was achieved at acidic pH (pH 6.2 and 4.0) and higher temperature (37 °C and 45 °C) conditions. Furthermore, the in vitro biocompatibility and intracellular fluorescence imaging were examined using the MDA-MB-231 cell line. Therefore, we demonstrate that the synthesized pNIPAAm-co-GMA-Lys HG system with temperature- and pH-stimuli-responsive features could be promising for various applications in biomedical fields, including drug delivery, gene delivery, tissue engineering, diagnosis, antibacterial/antifouling material, and implantable devices.

Thermosensitive Polymer-Modified Mesoporous Silica for pH and Temperature-Responsive Drug Delivery.

A mesoporous silica-based drug delivery system (MS@PNIPAm-PAAm NPs) was synthesized by conjugating the PNIPAm-PAAm copolymer onto the mesoporous silica (MS) surface as a gatekeeper that responds to temperature and pH changes. The drug delivery studies are carried out in vitro at different pH (7.4, 6.5, and 5.0) and temperatures (such as 25 °C and 42 °C, respectively). The surface conjugated copolymer (PNIPAm-PAAm) acts as a gatekeeper below the lower critical solution temperature (LCST) (<32 °C) and as a collapsed globule structure above LCST (>32 °C), resulting in controlled drug delivery from the MS@PNIPAm-PAAm system. Furthermore, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cellular internalization results support the prepared MS@PNIPAm-PAAm NPs being biocompatible and readily taken up by MDA-MB-231 cells. The prepared MS@PNIPAm-PAAm NPs, with their pH-responsive drug release behavior and good biocompatibility, could be used as a drug delivery vehicle where sustained drug release at higher temperatures is required.

Antioxidant and protective effects of a peptide (VTAL) derived from simulated gastrointestinal digestion of protein hydrolysates of Magallana gigas against acetaminophen-induced HepG2 cells.

Oxidative stress is an automatic mechanism responsible for the commencement and continuance of liver injury. In this study, an antioxidative peptide Val-Thr-Ala-Leu (VTAL) was purified from simulated gastrointestinal digestion of protein hydrolysates of the triploid oyster Magallana gigas. Significant antioxidant activity was identified, as well as a protective effect against acetaminophen (APAP)-induced human liver cancer (HepG2) cells. The results suggested that the antioxidant activity improved in a dose-dependent manner. The highest cell viability (88.105 ± 3.62%) was observed in 15 mM APAP-induced cells when treated with 25 µg/mL M. gigas peptide [M.g (pep)]. The peptide sequences include hydrophobic amino acids, which could be responsible for its chemoprotective and antioxidant activities. Treatment with M.g (pep) significantly promoted the proliferation of HepG2 cells, thus protecting them against APAP and imbuing them with significant antioxidant capacity. M.g (pep) could be beneficial for treating drug-induced oxidative stress and liver damage. Additionally, M.g (pep) could serve as an alternative to synthetic antioxidant drugs.

Antioxidant and chemoprotective peptides from simulated gastrointestinal digested (SGID) protein hydrolysate of Pyropia yezoensis against acetaminophen-induced HepG2 cells.

Excessive production of reactive oxygen and nitrogen species may result in oxidative damage to tissues and organs. Oxidative stress is a pathological mechanism that contributes to the initiation and progression of liver injury. In the present study, antioxidative peptides purified from simulated gastrointestinal-digested (SGID) protein hydrolysate of Pyropia yezoensis, showed significant antioxidant activity and also showed a protective effect against acetaminophen (N-acetyl-p-aminophenol, APAP) -induced injury in HepG2 (human liver cancer cells) cells. The antioxidant activity was increased in a dose-dependent manner. Higher cell viability (73.26 ± 0.9%) and decreasing NO levels (107.6 ± 8.9%) were observed in 15 mM APAP-induced cells when treated with the concentration of (100 µg ml-1) Pyropia peptide. Py. (pep). The sequences of the eight identified peptides present in the active fractions of the protein hydrolysate included hydrophobic and aromatic amino acids, which may have been responsible for their chemoprotective and antioxidant activities. Results indicated that the treatment with the Pyropia-peptides significantly promoted the proliferation of HepG2 cells, protecting them against APAP-mediated injury, and showed a significant antioxidant capacity. This study revealed that the Py. (pep) will be beneficial in treating drug-induced oxidative stress and liver damage conditions. Py. (pep) can also serve as a better alternative for synthetic antioxidant drugs.

A Review of Bioactive Compounds in Oyster Shell and Tissues.

Oysters are saltwater bivalves with high nutritional and medicinal value that are consumed widely around the world. As well as being highly nutritious, oysters are a low-calorie, low-cholesterol source of protein and an exceptional source of zinc, which strengthens the immune system; and a rich source of bioactive compounds, which comprise various biological activities. The present review summarizes the biological applications and bioactive compounds from oyster shells, whole tissue, gill tissue, and mantle tissue. The various biological compounds present in an oyster shell, and their chemical constituents, have applications in the food, pharmaceutical, and medical industries. Bioactive peptides and proteins obtained from the whole, mantle, and gill tissues of oysters exhibit antioxidant, antimicrobial, antihypertensive, anticancer, antifatigue, anticoagulant, and anti-wrinkle effects, as well as enhance osteoblast differentiation. This review clearly shows that oysters have great potential for functional food production and that various compounds therein can have pharmaceutical applications.

Update on Chitosan-Based Hydrogels: Preparation, Characterization, and Its Antimicrobial and Antibiofilm Applications.

Chitosan is a prominent biopolymer in research for of its physicochemical properties and uses. Each year, the number of publications based on chitosan and its derivatives increases. Because of its comprehensive biological properties, including antibacterial, antioxidant, and tissue regeneration activities, chitosan and its derivatives can be used to prevent and treat soft tissue diseases. Furthermore, chitosan can be employed as a nanocarrier for therapeutic drug delivery. In this review, we will first discuss chitosan and chitosan-based hydrogel polymers. The structure, functionality, and physicochemical characteristics of chitosan-based hydrogels are addressed. Second, a variety of characterization approaches were used to analyze and validate the physicochemical characteristics of chitosan-based hydrogel materials. Finally, we discuss the antibacterial, antibiofilm, and antifungal uses of supramolecular chitosan-based hydrogels. This review study can be used as a base for future research into the production of various types of chitosan-based hydrogels in the antibacterial and antifungal fields.

Extraction and Purification of R-Phycoerythrin Alpha Subunit from the Marine Red Algae Pyropia Yezoensis and Its Biological Activities.

Phycoerythrin is a major light-harvesting pigment of red algae and cyanobacteria that is widely used as a fluorescent probe or as a colorant in the food and cosmetic industries. In this study, phycoerythrin was extracted from the red algae Pyropia yezoensis and purified by ammonium sulfate precipitation and various chromatography methods. The purified phycoerythrin was analyzed by UV-visible and fluorescence spectroscopy. The isolated pigment had the typical spectrum of R-phycoerythrin, with a trimmer state with absorbance maxima at 497, 536, and 565 nm. It was further purified and identified by LC-MS/MS and Mascot search. It showed a 100% sequence similarity with the R-phycoerythrin alpha subunit of Pyropia yezoensis. The molecular mass was 17.97 kDa. The antioxidant activity of the purified R-phycoerythrin alpha subunit was analyzed. It showed significant antioxidant activity in ABTS and FRAP assays and had significant cytotoxicity against HepG2 cells.

Cytotoxicity against human breast carcinoma cells of silver nanoparticles biosynthesized using Capsosiphon fulvescens extract.

Targeting cancer cells with small nanoparticles is a novel and promising approach to cancer therapy. Breast cancer is the most common cancer afflicting women worldwide. In the present study, silver nanoparticles (AgNPs) were synthesized using the aqueous extract of the marine alga Capsosiphon (C.) fulvescens, and the cytotoxicity and anti-cancer activities of the nanoparticles against MCF-7 breast cancer cells were analyzed. Nanoparticle formation was confirmed by solution color change and UV-Vis spectroscopy. The size and distribution of the C. fulvescens-biosynthesized silver nanoparticles (CfAgNPs) were then examined using various analytical methods; the particle size was around 20-22 nm and spherical in shape with no agglomeration. Cytotoxicity analysis revealed that the inhibitory concentration (IC50) of CfAgNPs was 50 µg/ml. MCF-7 cell viability decreased with increasing concentrations of CfAgNPs. MCF-7 cells were evaluated for morphological changes before and after treatment with the CfAgNPs; cells treated with C. fulvescens aqueous algal extract (without CfAgNPs) showed irregular confluent aggregates with round polygonal cells, similar to the untreated control. Tamoxifen- (TMX) and CfAgNPs-treated cells show significant morphological changes. An apoptosis study was conducted using 4',6-diamidino-2-phenylindole (DAPI) staining, in which CfAgNP-treated MCF-7 cells generated bright blue fluorescence and shortened, disjointed chromatin was evident; control cells displayed less bright fluorescence. Flow cytometry analysis revealed that the percentage of cells in late apoptosis was high following treatment with TMX (77.2%) and CfAgNP (74.6%). A novel anti-cancer agent, developed by generating silver nanoparticles from C. fulvescens extract, showed strong cytotoxic activity against MCF-7 cells.

Biogenic preparation and characterization of Pyropia yezoensis silver nanoparticles (P.y AgNPs) and their antibacterial activity against Pseudomonas aeruginosa.

Marine algae play key roles in several medical, pharmaceutical, agricultural, and aquacultural applications. Furthermore, biosynthesized nanomaterials are becoming an alternative to conventional antibiotics in cost-effective, biocompatible, and non-toxic treatments for bacterial infections. This study features biogenic synthesis of silver nanoparticles using an aqueous extract of the marine red algae Pyropia yezoensis. The formation of silver nanoparticles was initially confirmed by UV-Vis spectroscopy and FTIR spectra were used to identify functional groups. The average crystalline size of the silver nanoparticles was around 20-22 nm, as determined by XRD analysis. Particle size was confirmed by SEM and TEM analyses, which also showed spherical particles without agglomeration. The antibacterial properties of the nanoparticles were assessed against both Gram-positive and Gram-negative bacterial cultures with significant activity observed against Gram negative P. aeruginosa. Our Pyropia yezoensis silver nanoparticles (P.y AgNPs) reduced the growth of P. aeruginosa at concentrations of 200 and 400 µg/ml. Our results strongly imply that P.y AgNPs may be useful in treating bacterial infections.

Characterization of recombinant protein ferritin from Pyropia yezoensis (rPyFer) and its biological activities.

Ferritins are iron-binding proteins that are basically participated in iron storage, detoxification, and immune response. In the present study, ferritin gene from the marine red algae Pyropia yezoensis was cloned into a pET21d expression vector. High-efficiency transformation was performed in Escherichia coli BL21, the recombinant protein was expressed by induction with 0.1 mM isopropyl-ß-D-thiogalactoside and purified via ammonium sulfate precipitation, anion exchange and size exclusion chromatography. The purified recombinant ferritin from P. yezoensis (rPyFer) was characterized and analyzed for its antimicrobial activity against both Gram-negative and Gram-positive bacterial cultures and exhibited significant antibacterial activity against Gram-positive cultures. The recombinant protein was also analyzed for its iron-uptake and radical-scavenging activities; rPyFer exhibited significant iron-uptake activity at low concentrations, and its radical-scavenging activity increased in a dose-dependent manner. This research will contribute to the development of new therapeutic proteins from marine algae.

Peptidyl-prolyl isomerase and the biological activities of recombinant protein cyclophilin from Pyropia yezoensis (PyCyp).

Cyclophilins are highly conserved proteins associated with peptidyl-prolyl cis-trans isomerase activity (PPIase). The present study was designed to analyze the biological activity of recombinant cyclophilin from the marine red algae Pyropia yezoensis (PyCyp). The cyclophilin gene from P. yezoensis was cloned into the pPROEX-HTA expression vector. The plasmid was transformed into BL21 Escherichia coli by high efficiency transformation. Recombinant protein was expressed using 0.1 mM IPTG and the fusion protein was purified by affinity column chromatography. The His-tag was removed by TEV protease. The recombinant protein was further purified on a HiPrep Sephacryl S-200 HR column and by reversed-phase high performance liquid chromatography with a Sep-pak plus C18 column. Purified cyclophilin was characterized by a variety of analytical methods and analyzed for its peptidyl-prolyl isomerase activity. Our recombinant PyCyp was shown to catalyze cis-trans isomerization. PyCyp was also evaluated for antimicrobial activity against both Gram-positive and Gram-negative bacteria cultures and showed significant antibacterial activity against tested pathogens. PyCyp was shown to permeabilize bacterial membranes as evidenced by increased fluorescence intensity in SYTOX Green uptake assays with Staphylococcus aureus. The radical scavenging activity of PyCyp increased in a dose-dependent manner, indicating significant antioxidant activity. This study provides information for the development of therapeutic proteins from marine algae.

Functional characterisation of bioactive peptide derived from terrestrial snail Cryptozona bistrialis and its wound-healing property in normal and diabetic-induced Wistar albino rats.

A peptide might be an exciting biomaterial or template for the development of novel wound-healing agents. In this report, it was isolated from the terrestrial snail Cryptozona bistrialis by enzymatic digestion and was evaluated for its in vitro wound-healing activity in NIH/3T3 mouse fibroblasts cell line and in vivo wound-healing activity in normal and diabetic-induced Wistar albino rats. The C. bistrialis protein was digested by the papain enzyme, and 21.79 kDa peptide (Cb-peptide) was purified by reversed-phase high-performance liquid chromatography and identified by MALDI (matrix-assisted laser desorption/ionization)-TOF analysis. The isolated Cb-peptide was characterised by various analytical methods. The peptide demonstrated a capacity to prevent the development of pathogenic bacterial and fungal cultures and proved that it promotes significant wound-healing activity in the wound scratch assay method by rapid cell migration and closure of wound. Isolated Cb-peptide was lyophilised and formulated to ointment and analysed for in vivo wound-healing activity in normal and diabetic (alloxan monohydrate)-induced Wistar albino rats. Cb-peptide ointment-treated groups showed a greater degree of wound healing and early and complete period of epithelialisation in normal and diabetic-induced Wistar albino rats. Cb-peptide ointment-treated groups showed significant excision and incision wound-healing activity. A conclusion was reached that the peptide isolated from C. bistrialis showed greater wound-healing activity compared with vehicle control and standard control.