Insights into the fabrication and antibacterial effect of fibrinogen hydrolysate-carrageenan loading apigenin and quercetin composite hydrogels.

Escherichia coli and Staphylococcus aureus are the most prevalent pathogenic bacteria, often resulting in the foodborne disease outbreaks through food spoilage and foodborne infections. To prevent and control food spoilage and foodborne infections induced by Escherichia coli and Staphylococcus aureus, the antibacterial hydrogels were fabricated using fibrinogen hydrolysate-carrageenan (AHs-C) and flavonoids (apigenin and quercetin), and the antibacterial effect of the composite hydrogels against Escherichia coli and Staphylococcus aureus was further investigated. The results of mechanical property exhibited that the composite hydrogels with 0.2 % of apigenin and quercetin (AHs-C-Ap/Que) showed the highest hardness and swelling property compared with the separate addition of apigenin or quercetin. Scanning electron microscopy and atomic force microscopy showed that the dense networks were formed in the hydrogels of AHs-C-Ap/Que., and the average roughness of AHs-C-Ap/Que. significantly increased to 30.70 nm compared with AHs-C. 1H NMR and FTIR spectra demonstrated that apigenin and quercetin were bound to AHs-C by hydrogen bond, hydrophobic interaction and Schiff base, where the interactions between Ap/Que. and AHs-C was stronger compared with the separate addition of apigenin or quercetin. The hydrogels of AHs-C-Ap/Que. showed the highest antibacterial capacity and antibacterial adhesion against Escherichia coli and Staphylococcus aureus. The antibacterial adhesion assay showed that 99 % removal ratios for E. coli and S. aureus were observed in AHs-C-Ap/Que. hydrogels, which showed a great potential to prevent food spoilage and foodborne infections.

Ultrasound Depolymerization and Characterization of Poly- and Oligosaccharides from the Red Alga Solieria chordalis (C. Agardh) J. Agardh 1842.

Red seaweed carrageenans are frequently used in industry for its texturizing properties and have demonstrated antiviral activities that can be used in human medicine. However, their high viscosity, high molecular weight, and low skin penetration limit their use. Low-weight carrageenans have a reduced viscosity and molecular weight, enhancing their biological properties. In this study, ι-carrageenan from Solieria chordalis, extracted using hot water and dialyzed, was depolymerized using hydrogen peroxide and ultrasound. Ultrasonic depolymerization yielded fractions of average molecular weight (50 kDa) that were rich in sulfate groups (16% and 33%) compared to those from the hydrogen peroxide treatment (7 kDa, 6% and 9%). The potential bioactivity of the polysaccharides and low-molecular-weight (LMW) fractions were assessed using WST-1 and LDH assays for human fibroblast viability, proliferation, and cytotoxicity. The depolymerized fractions did not affect cell proliferation and were not cytotoxic. This research highlights the diversity in the biochemical composition and lack of cytotoxicity of Solieria chordalis polysaccharides and LMW fractions produced by a green (ultrasound) depolymerization method.

An Antibacterial, Highly Sensitive Strain Sensor Based on an Anionic Copolymer Interpenetrating with κ-Carrageenan.

Polysaccharide-based hydrogels are suitable for use in the field of flexible bioelectronics due to their benign mechanical properties and biocompatibility. However, the preparation of hydrogel sensors with high performance without affecting their physicochemical properties (e.g., flexibility, toughness, self-healing, and antibacterial activity) remains a challenge and needs to be solved. Herein, a metal ion cross-linking reinforced, double network hydrogel was formed from a 2-acrylamide-2-methylpropanesulfonic acid (AMPS) copolymer interpenetrating κ-carrageenan (CAR), followed by immersing the gel in a Cu2+ ion solution to obtain an antibacterial CAR/P(AM-co-AMPS)-Cu2+ conductive hydrogel. LiCl was added as the electrolyte. The presence of the LiCl electrolyte and sulfonated molecular chain units not only gives the hydrogel good electrical conductivity (conductivity up to 2.68 S/m) but also improves the sensitivity of the hydrogel as a stress-strain sensor, with a hydrogel sensitivity GF of up to 3.76 in the 20%-100% strain range and response time of up to 280 ms. The CAR double-helical structure and sol-gel properties and the interaction of multiple noncovalent bonds between polymers provide the hydrogel with excellent self-healing, with a self-healing efficiency of 68%. In addition, the electrostatic interaction of Cu2+ with Escherichia coli cells can inhibit their growth, exhibiting good antibacterial properties with an inhibition circle diameter of 20.5 mm. This work could provide an effective strategy for antibacterial multifunctional CAR-based bionic sensors.

Fabrication of smart nanogel based on carrageenan and green coffee extract as a long-term antifouling agent to improve biofilm prevention in food production.

This study investigates the extract of the bioactive compounds from green coffee extract (GCE) and the loading of two different concentrations of GCE (1% and 2%) onto carrageenan nanogels (CAR NGs) to compare their antibacterial and antibiofilm effects with unloaded nanogels (NGs). The bioactive compounds of GCE were characterized using GC-MS analysis. The GCE1 and GCE2 were successfully deposited onto the surface of CAR NGs. The antibacterial and antibiofilm potential of prepared NGs were conducted against some foodborne pathogens (E. coli O157, Salmonella enterica, Staphylococcus aureus, and Listeria monocytogenes). The results of GC-MS analysis indicated that there were identified 16 bioactive compounds in GCE, including caffeine (36.27%), Dodemorph (9.04%), and D-Glycero-d-ido-heptose (2.44%), contributing to its antimicrobial properties. The antibacterial coatings demonstrated a notable antimicrobial effect, showing zone of inhibition (ZOI) diameters of up to 37 mm for GCE2 loaded CAR NGs. The minimum inhibitory concentration (MIC) values for GCE2 loaded CAR NGs were 80 ppm for E. coli O157, and 120 ppm for S. enterica, S. aureus, and L. monocytogenes, achieving complete bacterial inactivation within 10-15 min of exposure. Both GCE1 and GCE2 loaded CAR NGs significantly reduced biofilm cell densities on stainless steel (SS) materials for E. coli O157, S. enterica, S. aureus, and L. monocytogenes, with reductions ranging from 60% to 95%. Specifically, biofilm densities were reduced by up to 95% for E. coli O157, 89% for S. enterica, 85% for S. aureus, and 80% for L. monocytogenes. Results of the toxicity evaluation indicated that the NGs were non-toxic and biocompatible, with predicted EC50 values proved their biocompatibility and safety. These results recommended that GCE loaded CAR NGs are promising as natural antimicrobial agents for enhancing food safety and extending shelf life. Further, the study concluded that incorporating GCE into CAR NGs is an effective strategy for developing sustainable antimicrobial coatings for the food industry and manufacturing.

Exploring the amyloid degradation potential of nanoformulated carrageenan-bridging in vitro and in vivo perspectives.

Amyloids, with their ß-sheet-rich structure, contribute to diabetes, neurodegenerative diseases, and amyloidosis by aggregating within diverse anatomical compartments. Insulin amyloid (IA), sharing structural resemblances with amyloids linked to neurological disorders, acts as a prototype, while compounds capable of degrading these fibrils hold promise as therapeutic agents for amyloidosis intervention. In this research, liposomal nanoformulated iota carrageenan (nCG) was formulated to disrupt insulin amyloids, demonstrating about a 17-20 % higher degradation efficacy compared to conventional carrageenan through thioflavin T fluorescence, dynamic light scattering analysis, and turbidity quantification. The biocompatibility of the nCG and nCG-treated insulin amyloids was evaluated through MTT assay, live-dead cell assay on V79 cells, and hemolysis testing on human blood samples to establish their safety for use in vitro. Zebrafish embryos were utilized to assess in vivo biocompatibility, while adult zebrafish were employed to monitor the degradation capacity of IA post subcutaneous injection, with fluorescence emitted by the fish captured via IVIS. This demonstrated that the formulated nCG exhibited superior anti-amyloid efficacy compared to carrageenan alone, while both materials demonstrated biocompatibility. Furthermore, through docking simulations, an exploration was conducted into the molecular mechanisms governing the inhibition of the target protein pancreatic insulin by carrageenan.

Integrated self-assembly and cross-linking technology engineered photodynamic antimicrobial film for efficient preservation of perishable foods.

A new antibacterial film was constructed to combat the severe spoilage of fruits and vegetables caused by microorganisms. Specifically, photoresponsive cinnamaldehyde-tannic­iron acetate nanospheres (CTF NPs) were prepared using ultrasonic-triggered irreversible equilibrium self-assembly and ionic cross-linking co-driven processes and were integrated into the matrix of κ-carrageenan (KC) (CTF-KC films) as functional fillers. The CTF0.4-KC film (KC film doped with 0.4 mg/mL CTF NPs) showed a 99.99% bactericidal rate against both E. coli and S. aureus, extended the storage period of cherry tomatoes from 20 to 32 days. The introduction of CTF enhanced the barrier, thermal stability, and mechanical strength properties, albeit with a slight compromise on transparency. Furthermore, the biosafety of the CTF0.4-KC film was confirmed through hemolysis and cytotoxicity tests. Together, the aforementioned results demonstrated the outstanding antibacterial and fresh-keeping properties of CTF0.4-KC. These desirable properties highlight the potential use of CTF0.4-KC films in food preservation applications.

Facile preparation of a pH-sensitive biocompatible nanocarrier based on magnetic layered double hydroxides/Cu MOFs-chitosan crosslinked к-carrageenan for controlled doxorubicin delivery to breast cancer cells.

Recently, the biocompatibility of hydrogel nanoparticles has gained considerable research attention in the field of drug delivery. In this regard, we design a pH-controlled nanocarrier based on magnetic layered double hydroxides/copper metal-organic framework-chitosan crosslinked к-carrageenan hydrogel nanoparticles (LDH-Fe3O4/Cu MOF-DOX-CS@CAR) for targeted release from DOX to breast cancer cells. FT-IR, EDX, XRD, FE-SEM, VSM, and Zeta potential investigated the chemical structure of hydrogel nanoparticles. The encapsulation efficiency and drug loading capacity of the DOX were obtained to be 96.1 % and 9.6 %, respectively. The cumulative release of DOX from LDH-Fe3O4/Cu MOF-DOX-CS@CAR at pH 5.5 and 7.4 after 72 h was 60.3 % and 22.6 %, respectively. These in vitro release results confirmed the controlled release and pH-response behavior of hydrogel nanoparticles. Also, the mechanism of DOX release from LDH-Fe3O4/Cu MOF-DOX-CS@CAR hydrogel nanoparticles showed that the Korsmeyer-Peppas model with Fickian diffusion is the best-fitting model for describing the release behavior of DOX from hydrogel nanoparticles. The cellular cytotoxicity and DAPI tests of the prepared LDH and LDH-Fe3O4/Cu MOF toward L929 non-cancerous cells and MCF-7 breast cancer cells confirm its relative biocompatibility and safety. Whereas, LDH-Fe3O4/Cu MOF-DOX-CS@CAR hydrogel nanoparticles toward MCF-7 breast cancer cells had higher cytotoxicity effects due to the targeted and controlled release of DOX to MCF-7 cells. The in vitro DPPH, hemolysis assay, colloidal stability, and enzymatic degradation proved the excellent antioxidant activity (71.81 %), blood compatibility (less than 5 %), better stability, and biodegradation behavior of hydrogel nanoparticles. On these findings, the present study suggests the potential of the prepared LDH-Fe3O4/Cu MOF-DOX-CS@CAR hydrogel nanoparticles as a pH-controlled drug delivery system for cancer treatment and various biomedical uses.

An antibacterial film using κ-carrageenan loaded with benzyl isothiocyanate nanoemulsion: Characterization and application in beef preservation.

Benzyl isothiocyanate (BITC) is a naturally active bacteriostatic substance and κ-carrageenan (KC) is a good film-forming substrate. In the present study, a nanoemulsion incorporating BITC was fabricated with a particle size of 224.1 nm and an encapsulation efficiency of 69.2 %. Subsequently, the acquired BITC nanoemulsion (BITC-NE) was incorporated into the KC-based film, and the light transmittance of the prepared composite films was lower than that of the pure KC film. Fourier transform infrared spectroscopy and scanning electron microscopy revealed that BITC-NE was compatible with the KC matrix. BITC-NE incorporation enhanced the tensile strength of the KC-based films by 33.7 %, decreased the elongation at break by 33.8 %, decreased the water vapor permeability by 60.1 %, increased the maximum thermal degradation temperature by 48.8 %, and decreased the oxygen permeability by 42 % (p < 0.05). Furthermore, the composite films showed enhanced antimicrobial activity against Staphylococcus aureus, Salmonella typhimurium, and Pseudomonas fluorescens. The developed KC-based composite films were applied to wrap raw beef, which significantly delayed the increase in total viable count, total volatile base nitrogen content, and thiobarbituric acid reactive substances, and prolonged the shelf-life of the raw beef by up to 10 days. These results indicated that the composite films prepared by incorporating BITC nanoemulsions into KC matrices have great antimicrobial application potential.

Biomimetic kartogenin containing κ-carrageenan hydrogel for nucleus pulposus regeneration.

Intervertebral disc degeneration is a clinical disease that reduces the quality of patient's life. The degeneration usually initiates in the nucleus pulposus (NP), hence the use of hydrogels represents a promising therapeutic approach. However, the viscoelastic nature of hydrogel and its ability to provide biomimetic architecture and biochemical cues influence the regeneration capability. This study focused on tuning the physical nature of a glycosaminoglycan hydrogel (κ-carrageenan) as well as the release kinetics of a chondrogenic factor (kartogenin - KGN) through physical cross-linking. For this, κ-carrageenan was cross linked with 2.5 % and 5 % potassium chloride (KCl) for 15 and 30 min and loaded with KGN molecule at 50 µM and 100 µM. The tight network structure with low water retention and degradation property was seen in hydrogel cross-linked with increased KCl concentration and time. However, optimal degradation along with NP mimicking viscoelastic nature was exhibited by 5 wt% KCl treated hydrogel (H3 hydrogel). All hydrogel groups exhibited burst KGN release at 24 h followed by a sustained release for 5 days. However, hydrogel cross-linked with 5 wt% KCl enhanced chondrogenic differentiation, mainly at lower KGN dose. In summary, this study shows the potential application of biomimetic KGN laden carrageenan hydrogel in NP regeneration.

Carrageenan-ferrocene-eicosapentaenoic acid composite hydrogel induce ferroptosis and apoptosis for anti-tumor recurrence and metastasis.

The immune-suppressive microenvironment of solid tumors is a key factor limiting the effectiveness of immunotherapy, which seriously threatens human life and health. Ferroptosis and apoptosis are key cell-death pathways implicated in cancers, which can synergistically activate tumor immune responses. Here, we developed a multifunctional composite hydrogel (CE-Fc-Gel) based on the self-assembly of poloxamer 407, cystamine-linked ιota-carrageenan (CA)-eicosapentaenoic acid (EPA), and ferrocene (Fc). CE-Fc-Gel improved targeting in tumor microenvironment due to its disulfide bonds. Moreover, CE-Fc-Gel promoted lipid peroxidation, enhanced reactive oxygen species (ROS) production, and decreased glutathione peroxidase 4 (GPX4), inducing ferroptosis by the synergistic effect of Fc and EPA. CE-Fc-Gel induced apoptosis and immunogenic cell death (ICD), thereby promoting dendritic cells (DCs) maturation and T cell infiltration. As a result, CE-Fc-Gel significantly inhibited primary and metastatic tumors in vivo. Our findings provide a novel strategy for enhancing tumor immunotherapy by combining apoptosis, ferroptosis, and ICD.

Xenogeneic versus allogeneic serum and macromolecular crowding in human tenocyte cultures.

Allogeneic serum and tissue-specific extracellular matrix have been shown to maintain permanently differentiated cell phenotype in culture. This is of particular importance for human tenocytes, a cell population that readily loses its function during ex vivo culture. With these in mind, herein we extracted human tenocytes using either foetal bovine serum or human serum, cultured them in the absence and presence of carrageenan and Ficoll®, the most widely used macromolecular crowding agents (to induce tissue-specific extracellular matrix deposition), and assessed cellular function, via metabolic activity, viability, proliferation and immunofluorescence for collagen related molecules, non-collagenous molecules and transmembrane molecules. At day 7, longest time point assessed, neither carrageenan nor Ficoll® significantly affected metabolic activity, viability and proliferation in either serum and human serum significantly increased metabolic activity and proliferation. At day 7, in the absence of macromolecular crowding, cells in human serum deposited significantly lower collagen type VI, biglycan, versican and tenomodulin than cells in foetal bovine serum. Interestingly, at day 7, in comparison to the no macromolecular crowding group, carrageenan in foetal bovine serum induced the highest effect, as judged by the highest number of significantly increased molecules (collagen type I, collagen type IV, collagen type V, collagen type VI, transforming growth factor ß1, matrix metalloproteinase 14, lumican, versican, scleraxis and integrin α2ß1). These data, although contradict previous observations where human serum outperformed foetal bovine serum, at the same time, support the use of foetal bovine serum in the development of cell-based medicines.

Fabrication and characterization of carrageenan-based multifunctional films integrated with gallic acid@ZIF-8 for beef preservation.

The development of environmentally friendly biodegradable films is urgently required for reducing the plastic pollution crisis and ensuring food safety. Thus, here we aimed to prepare ZIF-8 that has delivery ability for gallic acid (GA) and further incorporated this material (GA@ZIF-8) into carrageenan (CA) matrix to obtain a series of CA-GA@ZIF-8 films. This design significantly improved the mechanical strength and UV barrier and reduced water vapor permeability, moisture content, and swelling rate of the CA films. CA-GA@ZIF-8 films exhibited sustainable release of GA and controlled migration of Zn2+ up to 144 h in a high-fat food simulator. Also, the composite films performed high-efficiency antioxidant activities (83.29 % for DPPH and 62.11 % for ABTS radical scavenging activity) and 99.51 % antimicrobial effects against Escherichia coli O157:H7 after 24 h. The great biocompatibility of GA@ZIF-8 and CA-GA@ZIF-8-10 % was confirmed by hemolysis, cell cytotoxicity, and mice model. Finally, the preservation experiments showed that CA-GA@ZIF-8 films could effectively maintain freshness and reduce the growth of microorganisms and oxidation of lipids during the preservation of beef. These results suggest that CA-GA@ZIF-8 films hold promising potential for improving the quality preservation of beef.

Tropane Alkaloid Isolated from Erythroxylum bezerrae Exhibits Neuropharmacological Potential in an Adult Zebrafish (Danio rerio) Model.

This study carried out to investigate the anti-inflammatory and antinociceptive effect of tropane alkaloid (EB7) isolated from E. bezerrae. It evaluated the toxicity and possible involvement of ion channels in the antinociceptive effect of EB7, as well as its anti-inflammatory effect in adult zebrafish (Zfa). Docking studies with EB7 and COX-1 and 2 were also performed. The tested doses of EB7 (4, 20 and 40 mg/kg) did not show any toxic effect on Zfa during the 96h of analysis (LD50>40 mg/kg). They did not produce any alteration in the locomotor behavior of the animals. Furthermore, EB7 showed promising pharmacological effects as it prevented the nociceptive behavior induced by hypertonic saline, capsaicin, formalin and acid saline. EB7 had its analgesic effect blocked by amiloride involving the neuromodulation of ASICs in Zfa. In evaluating the anti-inflammatory activity, the edema induced by κ-carrageenan 3.5 % was reduced by the dose of 40 mg/kg of EB7 observed after the fourth hour of analysis, indicating an effect similar to that of ibuprofen. Molecular docking results indicated that EB7 exhibited better affinity energy when compared to ibuprofen control against the two evaluated targets binding at different sites in the cocrystallized COX-1 and 2 inhibitors.

Common food additive carrageenan inhibits proglucagon expression and GLP-1 secretion by human enteroendocrine L-cells.

Proglucagon mRNA expression and GLP-1 secretion by cultured human L-cells (NCI-H716) were inhibited following exposure to λ-carrageenan, a commonly used additive in processed foods. Carrageenan is composed of sulfated or unsulfated galactose residues linked in alternating alpha-1,3 and beta-1,4 bonds and resembles the endogenous sulfated glycosaminoglycans. However, carrageenan has unusual alpha-1,3-galactosidic bonds, which are not innate to human cells and are implicated in immune responses. Exposure to carrageenan predictably causes inflammation, and carrageenan impairs glucose tolerance and contributes to insulin resistance. When cultured human L-cells were deprived overnight of glucose and serum and then exposed to high glucose, 10% FBS, and λ-carrageenan (1 µg/ml) for 10 minutes, 1 h, and 24 h, mRNA expression of proglucagon and secretion of GLP-1 were significantly reduced, compared to control cells not exposed to carrageenan. mRNA expression of proglucagon by mouse L-cells (STC-1) was also significantly reduced and supports the findings in the human cells. Exposure of co-cultured human intestinal epithelial cells (LS174T) to the spent media of the carrageenan-treated L-cells led to a decline in mRNA expression of GLUT-2 at 24 h. These findings suggest that ingestion of carrageenan-containing processed foods may impair the production of GLP-1, counteract the effect of GLP-1 receptor agonists and induce secondary effects on intestinal epithelial cells.

Enhanced probiotic viability in innovative double-network emulsion gels: Synergistic effects of the whey protein concentrate-xanthan gum complex and κ-carrageenan.

In this study, the whey protein concentrate and xanthan gum complex obtained by specific pH treatment, along with κ-carrageenan (KC), were used to encapsulate Lactobacillus acidophilus JYLA-191 in an emulsion gel system. The effects of crosslinking and KC concentration on the visual characteristics, stability, mechanical properties, and formation mechanism of emulsion gels were investigated. The results of optical imaging, particle size distribution, and rheology exhibited that with the addition of crosslinking agents, denser and more homogeneous emulsion gels were formed, along with a relative decrease in the droplet size and a gradual increase in viscosity. Especially when the concentration of citric acid (CA) was 0.09 wt%, KC was 0.8 wt%, and K+ was present in the system, the double-network emulsion gel was stable at high temperatures and in freezing environments, and the swelling ratio was the lowest (9.41%). Gastrointestinal tract digestive treatments and pasteurization revealed that the probiotics encapsulated in the double-network emulsion gel had a higher survival rate, which was attributed to the synergistic cross-linking of CA and K+ biopolymers to construct the emulsion gels. Overall, this study highlights the potential of emulsion gels to maintain probiotic vitality and provides valuable insights for developing inventive functional foods.

Electrospun nanofibrous scaffolds as a platform to reduce melanoma tumour growth, recurrence, and promote post-resection wound repair.

Wound healing following skin tumour surgery still remains a major challenge. To address this issue, polysaccharide-loaded nanofibrous mats have been engineered as skin patches on the wound site to improve wound healing while simultaneously eliminating residual cancer cells which may cause cancer relapse. The marine derived polysaccharides kappa-carrageenan (KCG) and fucoidan (FUC) were blended with polydioxanone (PDX) nanofibers due to their inherent anti-cancer activity conferred by the sulphate groups as well as their immunomodulatory properties which can reduce inflammation resulting in accelerated wound healing. KCG and FUC were released sustainably from the blend nanofibers via the Korsmeyer-Peppas kinetics. MTT assays, live/dead staining and SEM images demonstrated the toxicity of KCG and FUC towards skin cancer MP 41 cells. In addition, MP 41 cells showed reduced metastatic potential when grown on KCG or FUC containing mats. Both KCG and FUC were non- cytotoxic to healthy L 929 fibroblast cells. In vivo studies on healthy Wistar rats confirmed the non-toxicity of the nanofibrous patches as well as their improved and scarless wound healing potential. In vivo studies on tumour xenograft model further showed a reduction of 7.15 % in tumour volume in only 4 days following application of the transdermal patch.

Carrageenan/polyvinyl alcohol composite film reinforced with spermidine carbon dots: An active packaging material with dual-mode antibacterial activity.

Exploring biopolymer-based antibacterial packaging materials is promising to tackle the issues caused by petroleum plastic pollution and microbial contamination. Herein, a novel packaging material with two antibacterial modes, continuous and efficient, is constructed by dispersing positively charged spermidine carbon dots (Spd-CDs) in a carrageenan/polyvinyl alcohol (CP) composite biopolymer. The obtained nanocomposite film (CP/CDs film) not only gradually releases the ultra-small Spd-CDs but also rapidly generates reactive oxygen species to inhibit the reproduction of E. coli and S. aureus. Benefiting from the complementary advantages of carrageenan and polyvinyl alcohol, as well as the addition of Spd-CDs, the CP/CDs films exhibit high transparency, good mechanical performance, water vapor barrier ability, low migration, etc. The CP/CDs film as a packaging material is validated to be effective in preventing microbial contamination of pork samples. Our prepared nanocomposite film with sustainability and efficient antibacterial properties is expected as food active packaging.

Carrageenan maintains the contractile phenotype of vascular smooth muscle cells by increasing macromolecular crowding in vitro.

BACKGROUND: The contractile phenotype of vascular smooth muscle cells (VSMCs) results in good diastolic and contractile capacities, and its altered function is the main pathophysiological basis for diseases such as hypertension. VSMCs exist as a synthetic phenotype in vitro, making it challenging to maintain a contractile phenotype for research. It is widely recognized that the common medium in vitro is significantly less crowded than in the in vivo environment. Additionally, VSMCs have a heightened sense for detecting changes in medium crowding. However, it is unclear whether macromolecular crowding (MMC) helps maintain the VSMCs contractile phenotype. PURPOSE: This study aimed to explore the phenotypic, behavioral and gene expression changes of VSMCs after increasing the crowding degree by adding carrageenan (CR). METHODS: The degree of medium crowding was examined by a dynamic light scattering assay; VSMCs survival and activity were examined by calcein/PI cell activity and toxicity and CCK-8 assays; VSMCs phenotypes and migration were examined by WB and wound healing assays; and gene expression was examined by transcriptomic analysis and RT-qPCR. RESULTS: Notably, 225 µg/mL CR significantly increased the crowding degree of the medium and did not affect cell survival. Simultaneously, CR significantly promoted the contraction phenotypic marker expression in VSMCs, shortened cell length, decreased cell proliferation, and inhibited cell migration. CR significantly altered gene expression in VSMCs. Specifically, 856 genes were upregulated and 1207 genes were downregulated. These alterations primarily affect the cellular ion channel transport, microtubule movement, respiratory metabolism, amino acid transport, and extracellular matrix synthesis. The upregulated genes were primarily involved in the cytoskeleton and contraction processes of VSMCs, whereas the downregulated genes were mainly involved in extracellular matrix synthesis. CONCLUSIONS: The in vitro study showed that VSMCs can maintain the contractile phenotype by sensing changes in the crowding of the culture environment, which can be maintained by adding CR.

Evaluation of Kappa-carrageenan supplementation in extender for post-thaw Kajli ram sperm quality.

Cryopreservation is one of the reliable techniques for long-term storage of sperm. The success of this technique depends on the choice of cryoprotectant; therefore, a plethora of literature has reported the effects of different cryoprotective agents so far. Kappa-carrageenan (κ-carrageenan) is a hydrocolloid polysaccharide extracted from red marine seaweed. Its unique property makes it a promising option as a non-colligative cryoprotectant. The current study aims to evaluate the cryoprotective effect of k-carrageenan along with glycerol on ram sperm quality both after equilibration and freezing. Nine Kajli rams were utilized in this experiment for semen collection through an artificial vagina maintained at 42°C. Qualified samples were diluted in tris egg yolk glycerol (TEYG) extender containing different concentrations of k-carrageenan as 0 mg/mL (control), 0.2, 0.5, 0.8 and 1 mg/mL. Post-thaw assessment was done at 37°C after 24 h of storage, which showed a significant improvement (p < .05) in sperm viability, motility, membrane and acrosome integrity in an extender containing k-carrageenan at a concentration of 0.5 mg/mL compared to control. It is concluded from the current study that the combination of glycerol and 0.5 mg/mL concentration of k-carrageenan improved the sperm post-thaw quality.

In vitro immune-depression and anti-inflammatory activities of cantharidin on gilthead seabream (Sparus aurata) leucocytes activated by λ-carrageenan.

Cantharidin is a natural compound with known therapeutic applications in humans. The aim of this study was to investigate the in vitro effects of cantharidin on gilthead seabream (Sparus aurata) head kidney leucocytes (HKL) stimulated with λ-carrageenan. HKLs were incubated for 24 h with cantharidin (0, 2.5 and 5 µg mL-1) and λ-carrageenan (0 and 1000 µg mL-1). The results showed that HKL viability only decreased by 15.2% after incubated with 5 µg mL-1 of cantharidin and λ-carrageenan. Cantharidin increased the peroxidase activity of HKLs only when incubated in combination with λ-carrageenan. Besides this, cantharidin inhibited the respiratory burst and phagocytic activities. Furthermore, cantharidin induced morphological changes in HKLs (apoptotic and vacuolization signs) that were enhanced when incubated with λ-carrageenan. Considering the analysis of the selected gene expression studied in HKLs [NF-κB subunits (rela, relb, crel, nfkb1, nfkb2), proinflammatory cytokines (il1b, tnfa), anti-inflammatory cytokines (il10, tgfb) and caspases (casp1, casp3, casp8, casp9)], although λ-carrageenan up-regulated the expression of the proinflammatory gene il1b, λ-carrageenan and cantharidin down-regulated its expression in HKLs. In addition, cantharidin up-regulated casp3 and casp9 expression. The casp3 and casp9 gene expression was down-regulated while casp1 gene expression was up-regulated in HKLs incubated with both cantharidin and λ-carrageenan. All the effects of cantharidin are related to its inhibitory effect on protein phosphatases, which induce apoptosis at long exposure times, and minimize the effects of λ-carrageenan. The present results provide detailed insight into the immune-depressive and anti-inflammatory properties of cantharidin on immune cells, which could be of interest to the aquaculture sector.