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.

Stomatin-like protein 3 modulates the responses of Aδ, but not C fiber bone afferent neurons to noxious mechanical stimulation in an animal model of acute experimental bone pain.

STOML3 is a membrane bound scaffolding protein that has been shown to facilitate the opening of mechanically sensitive ion channels and contribute to noxious mechanical sensation, allodynia and hyperalgesia. In this study, we aimed to determine the role of STOML3 in noxious mechanical sensitivity of bone afferent neurons and carrageenan-induced acute inflammation in the bone. An in vivo, electrophysiological bone-nerve preparation was used to make recordings of the activity and sensitivity of bone afferent neurons that innervate the tibial marrow cavity in anaesthetised rats, in response to noxious mechanical stimuli delivered to the marrow cavity, before and after injection of either the STOML3 oligomerisation inhibitor OB-1 or vehicle, in either naïve animals or animals with carrageenan-induced inflammation of the marrow cavity. A dynamic weight-bearing apparatus was used to measure weight bearing in response to inflammatory pain before and after injection of OB-1 or saline into the tibial marrow cavity in the presence of carrageenan-induced inflammation. Electrophysiological recordings revealed that Aδ, but not C bone afferent neurons have a reduced discharge frequency in response to mechanical stimulation, and that carrageenan-induced sensitisation of Aδ, but not C bone afferent neurons was attenuated by inhibition of STOML3 oligomerisation with OB-1. Animals treated with OB-1 spent a significantly greater amount of time on the limb injected with carrageenan than animals treated with saline. Our findings demonstrate that inhibition of STOML3 oligomerisation reduces inflammatory bone pain by reducing the sensitivity of Aδ bone afferent neurons to mechanical stimulation. Targeting STOML3 may be an effective approach to reduce pain from noxious pressure and/or painful inflammatory pathology in bone.

Effect of stretching on inflammation in a subcutaneous carrageenan mouse model analyzed at single-cell resolution.

Understanding the factors that influence the biological response to inflammation is crucial, due to its involvement in physiological and pathological processes, including tissue repair/healing, cancer, infections, and autoimmune diseases. We have previously demonstrated that in vivo stretching can reduce inflammation and increase local pro-resolving lipid mediators in rats, suggesting a direct mechanical effect on inflammation resolution. Here we aimed to explore further the effects of stretching at the cellular/molecular level in a mouse subcutaneous carrageenan-inflammation model. Stretching for 10 min twice a day reduced inflammation, increased the production of pro-resolving mediator pathway intermediate 17-HDHA at 48 h postcarrageenan injection, and decreased both pro-resolving and pro-inflammatory mediators (e.g., PGE2 and PGD2 ) at 96 h. Single-cell RNA sequencing analysis of inflammatory lesions at 96 h showed that stretching increased the expression of both pro-inflammatory (Nos2) and pro-resolution (Arg1) genes in M1 and M2 macrophages at 96 h. An intercellular communication analysis predicted specific ligand-receptor interactions orchestrated by neutrophils and M2a macrophages, suggesting a continuous neutrophil presence recruiting immune cells such as activated macrophages to contain the antigen while promoting resolution and preserving tissue homeostasis.

Radical Scavenging, Anti-Inflammatory, and Hepatoprotective Activities of Pentacyclic Triterpene isolated from Rosa webbiana.

INTRODUCTION: Rosa webbiana (RW) Wall Ex. Royle is used in traditional medicine in Pakistan for the treatment of several diseases including jaundice. To date, only neuroprotective potential of the plant has been evaluated. OBJECTIVE: The current study was designed to isolate bioactive compound(s) and investigate its possible radical scavenging, anti-inflammatory and hepatoprotective activities. METHODS: Column chromatography was done to isolate compounds from the chloroform fraction of RW. The compound was characterized by mass spectrometry, 1H-NMR, and 2D-NMR spectroscopy. Radical scavenging activity was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydrogen peroxide (H2O2) assays, while anti-inflammatory potential was evaluated via xylene-induced ear edema and carrageenan-induced paw edema models. For hepatoprotection, CCl4-induced model in mice was used. RESULTS: A triterpene compound (3α, 21ß-dihydroxy-olean-12-ene) was isolated from RW fruits (ARW1). The compound exhibited DPPH and H2O2 scavenging activities 61 ± 1.31% and 66 ± 0.48% respectively at 500 µg/ml. ARW1 (at 50 mg/kg) exhibited 62.9 ± 0.15% inhibition of xylene-induced ear edema and 66.6 ± 0.17% carrageenan-induced paw edema in mice. In CCl4-induced hepatotoxic mice, ARW1 significantly countered elevation in alanine transaminase (ALT), alkaline phosphatase (ALP), total bilirubin (T.B), and reduction in total protein (T.P) levels. Liver histomorphological study supported the serum biochemical profile for hepatoprotection. Moreover, ARW1 significantly attenuated the toxic changes in body and liver weight induced by CCl4. CONCLUSION: The compound ARW1 exhibited anti-radical, anti-inflammatory and hepatoprotective effects. The anti-inflammatory and hepatoprotective activities may be attributed to anti-oxidant potential of the compound.

Carrageenan impact on digestive proteolysis of meat proteins in meatballs or soluble hydrolyzed collagen.

In a health-conscious age, vivid discussion has been made on the healthfulness of processed foods and food additives. This study focuses on carrageenan (CGN), an approved but debated family of sulphated galactans from algae used as gelling, thickening and stabilizing agents but with indications of possible adverse effects, including as an inhibitor of digestive proteolysis. To challenge this inhibitory hypothesis, food-grade kappa-, iota and lambda-CGN preparations were used to produce beef meatballs whose proteolysis was studied using an in vitro digestion model coupled to various proteomic analyses. Results show that CGN anti-nutritional effects are abolished in beef meatballs. Specifically, proteomic analysis of gastric digesta of myosin light chain 1 (MYL1), alpha skeletal muscle (ACTA1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose-bisphosphate aldolase (ALDOA) reveal no appreciable differences in the profiles of bioaccessible peptides. Separate digestions of a soluble collagen hydrolysate show CGN does inhibit proteolysis of soluble collagen, therefore supporting the notion that the meat matrix confers a shielding effect that eliminates CGN ability to interfere with digestive proteolysis. Thus, this work shows that CGN ability to hinder digestive proteolysis may not apply to all foods and contributes evidence important to the discussions on CGN uses, indications and regulatory status.

Evidence and hypotheses on adverse effects of the food additives carrageenan (E 407)/processed Eucheuma seaweed (E 407a) and carboxymethylcellulose (E 466) on the intestines: a scoping review.

This scoping review provides an overview of publications reporting adverse effects on the intestines of the food additives carrageenan (CGN) (E 407)/processed Eucheuma seaweed (PES) (E 407a) and carboxymethylcellulose (CMC) (E 466). It includes evidence from human, experimental mammal and in vitro research publications, and other evidence. The databases Medline, Embase, Scopus, Web of Science Core Collection, Cochrane Database of Systematic Reviews and Epistemonikos were searched without time limits, in addition to grey literature. The publications retrieved were screened against predefined criteria. From two literature searches, 2572 records were screened, of which 224 records were included, as well as 38 records from grey literature, making a total of 262 included publications, 196 on CGN and 101 on CMC. These publications were coded and analyzed in Eppi-Reviewer and data gaps presented in interactive maps. For CGN, five, 69 and 33 research publications on humans, experimental mammals and in vitro experiments were found, further separated as degraded or native (non-degraded) CGN. For CMC, three human, 20 animal and 14 in vitro research publications were obtained. The most studied adverse effects on the intestines were for both additives inflammation, the gut microbiome, including fermentation, intestinal permeability, and cancer and metabolic effects, and immune effects for CGN. Further studies should focus on native CGN, in the form and molecular weight used as food additive. For both additives, randomized controlled trials of sufficient power and with realistic dietary exposure levels of single additives, performed in persons of all ages, including potentially vulnerable groups, are needed.

C-type lectin-like receptor-2 (CLEC-2) is a key regulator of kappa-carrageenan-induced tail thrombosis model in mice.

Kappa-carrageenan (KCG), which is used to induce thrombosis in laboratory animals for antithrombotic drug screening, can trigger platelet aggregation. However, the cell-surface receptor and related signaling pathways remain unclear. In this study, we investigated the molecular basis of KCG-induced platelet activation using light-transmittance aggregometry, flow cytometry, western blotting, and surface plasmon resonance assays using platelets from platelet receptor-deficient mice and recombinant proteins. KCG-induced tail thrombosis was also evaluated in mice lacking the platelet receptor. We found that KCG induces platelet aggregation with α-granule secretion, activated integrin αIIbß3, and phosphatidylserine exposure. As this aggregation was significantly inhibited by the Src family kinase inhibitor and spleen tyrosine kinase (Syk) inhibitor, a tyrosine kinase-dependent pathway is required. Platelets exposed to KCG exhibited intracellular tyrosine phosphorylation of Syk, linker activated T cells, and phospholipase C gamma 2. KCG-induced platelet aggregation was abolished in platelets from C-type lectin-like receptor-2 (CLEC-2)-deficient mice, but not in platelets pre-treated with glycoprotein VI-blocking antibody, JAQ1. Surface plasmon resonance assays showed a direct association between murine/human recombinant CLEC-2 and KCG. KCG-induced thrombosis and thrombocytopenia were significantly inhibited in CLEC-2-deficient mice. Our findings show that KCG induces platelet activation via CLEC-2.

Thrombosis is a serious medical condition that occurs when blood clots form in the blood vessels and can lead to heart attacks or strokes. Animal models are important for evaluating the effectiveness of drugs in thrombosis treatment. Kappa-carrageenan (KCG) is a food thickener and a substance used to induce clot formation in laboratory animals. In this study, we investigated the molecular basis of KCG-induced platelet activation, which is an important step in thrombosis development. We found that KCG activates platelets via a receptor called C-type lectin-like receptor 2 (CLEC-2), leading to a prothrombotic state in mice. We also showed that KCG-induced tail thrombosis (CTT) is significantly inhibited in CLEC-2 deficient mice. Our findings suggest that CLEC-2-mediated platelet activation plays a key role in the pathogenesis of thrombosis and CLEC-2 May participate in innate immunity as a receptor for sulfate-polysaccharide.Abbreviation; CLEC-2: C-type lectin-like receptor 2; CRP: collagen-related peptide; CTT: KCGN-induced tail thrombosis; DIC: disseminated intravascular coagulation; EDTA: ethylenediaminetetraacetic acid; GPVI: glycoprotein VI; HRP: horseradish peroxidase; KCG: Κ-Carrageenan; LAT: linker activated T cells; LDS: lithium dodecyl sulfate; LTA: light-transmittance aggregometry; MFI: mean fluorescence intensity; PFA: paraformaldehyde; PLCγ2: phospholipase C gamma 2; PS: phosphatidylserine; Syk: spleen tyrosine kinase; Co-HP: Cobalt-hematoporphyrin.

Biochemical characterization and elucidation of the action mode of a GH16 family κ-carrageenase for efficient preparation of carrageenan oligosaccharides.

κ-Carrageenan oligosaccharides have a variety of biological activities. Degradation of κ-carrageenan by κ-carrageenase leads to degradation products with different degrees of polymerization (DPs). A novel gene (CecgkA) encoding a new κ-carrageenase was cloned from Colwellia echini and heterologously expressed in Escherichia coli BL21 (DE3). The enzyme is 1104 bp in length, encodes 367 amino acid residues and has a molecular weight of 41.30 kDa. Multiple alignment analysis showed that CeCgkA belongs to the glycoside hydrolase (GH16) family and has the highest homology with the κ-carrageenase of Rhodopirellula maiorica SM1, with 58% homology. The CeCgkA showed maximum activity (453.15 U/mg) at pH 8.0 and 35 °C. Determination of biochemical properties showed that CeCgkA was a thermal recovery enzyme, and 51.6% of the initial enzyme activity was recovered by immediately placing the sample at 35 °C for 60 min after enzymatic inactivation by boiling for 10 min. K+, Na+, and EDTA had an activating effect on the enzyme activity, while Ni2+, Cu2+, and Zn2+ inhibited the activity of the enzyme. In addition, TLC and ESI-MS analysis showed that the maximum recognition unit of CecgkA was decasaccharide and that the main degradation products were disaccharides, tetrasaccharides and hexasaccharides, indicating that the enzyme is an endo-type carrageenase.

Reducing oxidative stress by κ-carrageenan and C60HyFn: The post-thaw quality and antioxidant status of Azari water buffalo bull semen.

Azeri water buffalo is a species of great interest due to the high quality of its products such as milk. Due to the decreasing trend of its number and risk of extinction in the future, our attention is directed towards ensuring the preservation of its genetic reserves by keeping its sperm. Using antioxidants in semen extender is one of the ways to reduce the detrimental effects of freezing process on post-thawed quality of spermatozoa. This study was conducted to determine the effect of κ-carrageenan (k-CRG) and C60HyFn supplemented semen extender on the quality of post-thawed Azari water buffalo spermatozoa. A total of 30 semen samples were obtained from three buffaloes using an artificial vagina (twice a week for five weeks = 10 replicates). The samples (n = 3) from each replicate were pooled and divided into equal aliquots to prepare 14 extender groups, including control (C), k-0.2, K-0.4, K-0.6, K-0.8 (containing 0.2, 0.4, 0.8 mg K-CRG/mL, respectively), C-0.1, C-0.2, C-0.4, C-0.8, C-1, C-5, C-10, C-20, and C-40 (containing 0.1, 0.2, 0.4, 0.6, 0.8, 1, 5, 10, 20, 40 µM C60HyFn, respectively), and then frozen. After thawing, motility and velocity parameters, plasma membrane integrity (PMI) and functionality (PMF), DNA damage, Hypo-osmotic swelling (HOS) test, malondialdehyde (MDA), total antioxidant capacity (TAC), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase glutathione activities and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging were evaluated. In vivo fertility was compared between k-0.6, C-1 and control groups. 60 buffalo were inseminated 24 h after the onset of estrus. The diagnosis of pregnancy was performed rectally at least 60 days after fertilization. Total and progressive motility and velocity parameters were improved by k-0.4, k-0.6, k-0.8, C-0.4, C-0.8, C-1, C-5, and C-10 groups) compared to the other groups. Plasma membranes integrity and PMF were improved by k-0.4, k-0.6, C-0.4, C-0.8, C-1, C-5, and C-10 groups compared to other groups, while in terms of sperm DNA damage K-0.4, K-0.6, K-0.8, C-0.2, C-0.4, C-0.8, C-1, C-5, and C-10 groups showed better results compared to the control group. The evidence also showed that k- 0.4, k-0.6, k-0.8, C-0.4, C-0.8, C-1, C-5, and C-10 groups could improve TAC, and decrease MDA levels. Also, k-0.4, k-0.6, k-0.8, C-0.2, C-0.4, C-0.8, C-1, C-5, and C-10 groups could improve GPx, CAT, and GSH levels, but no significant difference was found regarding SOD compared to the other groups. DPPH scavengers were tested by K-0.6, K-0.8 and C-1, C-5, C-10, C-0.8, C-0.4 and C-0.2 groups and compared to other groups improved. The fertility rate [70% (14/20)] was higher in C-1 than other groups. To conclude that k-CRG and C60HyFn supplementation can increase the quality parameters of cryopreserved buffalo semen after thawing and that 1 M C60HyFn can increase in vivo fertility of buffalo semen.

Agarolytic Pathway in the Newly Isolated Aquimarina sp. Bacterial Strain ERC-38 and Characterization of a Putative ß-agarase.

Marine microbes, particularly Bacteroidetes, are a rich source of enzymes that can degrade diverse marine polysaccharides. Aquimarina sp. ERC-38, which belongs to the Bacteroidetes phylum, was isolated from seawater in South Korea. It showed agar-degrading activity and required an additional carbon source for growth on marine broth 2216. Here, the genome of the strain was sequenced to understand its agar degradation mechanism, and 3615 protein-coding sequences were predicted, which were assigned putative functions according to their annotated functional feature categories. In silico genome analysis revealed that the ERC-38 strain has several carrageenan-degrading enzymes but could not degrade carrageenan because it lacked genes encoding κ-carrageenanase and S1_19A type sulfatase. Moreover, the strain possesses multiple genes predicted to encode enzymes involved in agarose degradation, which are located in a polysaccharide utilization locus. Among the enzymes, Aq1840, which is closest to ZgAgaC within the glycoside hydrolase 16 family, was characterized using a recombinant enzyme expressed in Escherichia coli BL21 (DE3) cells. An enzyme assay revealed that recombinant Aq1840 mainly converts agarose to NA4. Moreover, recombinant Aq1840 could weakly hydrolyze A5 into A3 and NA2. These results showed that Aq1840 is involved in at least the initial agar degradation step prior to the metabolic pathway that uses agarose as a carbon source for growth of the strain. Thus, this enzyme can be applied to development and manufacturing industry for prebiotic and antioxidant food additive. Furthermore, our genome sequence analysis revealed that the strain is a potential resource for research on marine polysaccharide degradation mechanisms and carbon cycling.

Physicochemical, structural and in vitro gastrointestinal tract release properties of ι-carrageenan/sodium caseinate synbiotic microgels produced by double-crosslinking with calcium ions and transglutaminase.

The aim of this study was to develop ι-carrageenan (ιC)/sodium caseinate (NaCas) synbiotic microgels loading Lacticasebacillus paracasei produced by double-crosslinking with calcium ions and different concentrations (0, 5, 10, and 15 U/g protein) of transglutaminase (TGase). The synbiotic microgels were coated/filled with pectic oligosaccharide (POS). Field emission scanning electron microscope (FE-SEM) and X-ray diffraction (XRD) analyses indicated that L. paracasei was successfully microencapsulated in synbiotic microgels. In Fourier transform infrared (FT-IR) analysis, the new formation of covalent and ionic crosslinking was observed in double-crosslinked synbiotic microgels. The encapsulation efficiency of L. paracasei was significantly increased from 87.82 to 97.68 % by increasing the concentration of TGase from 0 to 15 U/g protein, respectively. After exposure to simulated gastric fluid for 2 h and simulated intestinal fluid for 4 h, the survival rate of L. paracasei was significantly increased as the concentration of TGase increased.

Brain Cancer Cell-Derived Matrices and Effects on Astrocyte Migration.

Cell-derived matrices are useful tools for studying the extracellular matrix (ECM) of different cell types and testing the effects on cell migration or wound repair. These matrices typically are generated using extended culture with ascorbic acid to boost ECM production. Applying this technique to cancer cell cultures could advance the study of cancer ECM and its effects on recruitment and training of the tumor microenvironment, but ascorbic acid is potently cytotoxic to cancer cells. Macromolecular crowding (MMC) agents can also be added to increase matrix deposition based on the excluded volume principle. We report the use of MMC alone as an effective strategy to generate brain cancer cell-derived matrices for downstream analyses and cell migration studies. We cultured the mouse glioblastoma cell line GL261 for 1 week in the presence of three previously reported MMC agents (carrageenan, Ficoll 70/400, and hyaluronic acid). We measured the resulting deposition of collagens and sulfated glycosaminoglycans using quantitative assays, as well as other matrix components by immunostaining. Both carrageenan and Ficoll promoted significantly more accumulation of total collagen content, sulfated glycosaminoglycan content, and fibronectin staining. Only Ficoll, however, also demonstrated a significant increase in collagen I staining. The results were more variable in 3D spheroid culture. We focused on Ficoll MMC matrices, which were isolated using the small molecule Raptinal to induce cancer cell apoptosis and matrix decellularization. The cancer cell-derived matrix promoted significantly faster migration of human astrocytes in a scratch wound assay, which may be explained by focal adhesion morphology and an increase in cellular metabolic activity. Ultimately, these data show MMC culture is a useful technique to generate cancer cell-derived matrices and study the effects on stromal cell migration related to wound repair.

λ-Carrageenan promotes plant growth in banana via enhancement of cellular metabolism, nutrient uptake, and cellular homeostasis.

Banana (Musa acuminata) is an important fruit crop and source of income for various countries, including Malaysia. To date, current agrochemical practice has become a disputable issue due to its detrimental effect on the environment. λ-carrageenan, a natural polysaccharide extracted from edible red seaweed, has been claimed to be a potential plant growth stimulator. Hence, the present study investigates the effects of λ-carrageenan on plant growth using Musa acuminata cv. Berangan (AAA). Vegetative growth such as plant height, root length, pseudostem diameter, and fresh weight was improved significantly in λ-carrageenan-treated banana plants at an optimum concentration of 750 ppm. Enhancement of root structure was also observed in optimum λ-carrageenan treatment, facilitating nutrients uptake in banana plants. Further biochemical assays and gene expression analysis revealed that the increment in growth performance was consistent with the increase of chlorophyll content, protein content, and phenolic content, suggesting that λ-carrageenan increases photosynthesis rate, protein biosynthesis, and secondary metabolites biosynthesis which eventually stimulate growth. Besides, λ-carrageenan at optimum concentration also increased catalase and peroxidase activities, which led to a significant reduction in hydrogen peroxide and malondialdehyde, maintaining cellular homeostasis in banana plants. Altogether, λ-carrageenan at optimum concentration improves the growth of banana plants via inducing metabolic processes, enhancing nutrient uptake, and regulation of cell homeostasis. Further investigations are needed to evaluate the effectiveness of λ-carrageenan on banana plants under field conditions.

Plasma proteome responses in zebrafish following λ-carrageenan-Induced inflammation are mediated by PMN leukocytes and correlate highly with their human counterparts.

Regulation of inflammation is a critical process for maintaining physiological homeostasis. The λ-carrageenan (λ-CGN) is a mucopolysaccharide extracted from the cell wall of red algae (Chondrus crispus) capable of inducing acute intestinal inflammation, which is translated into the production of acute phase reactants secreted into the blood circulation. However, the associated mechanisms in vertebrates are not well understood. Here, we investigated the crucial factors behind the inflammatory milieu of λ-CGN-mediated inflammation administered at 0, 1.75, and 3.5% (v/w) by i.p. injection into the peritoneal cavity of adult zebrafish (ZF) (Danio rerio). We found that polymorphonuclear leukocytes (neutrophils) and lymphocytes infiltrating the ZF peritoneal cavity had short-term persistence. Nevertheless, they generate a strong pattern of inflammation that affects systemically and is enough to produce edema in the cavity. Consistent with these findings, cell infiltration, which causes notable tissue changes, resulted in the overexpression of several acute inflammatory markers at the protein level. Using reversed-phase high-performance liquid chromatography followed by a hybrid linear ion-trap mass spectrometry shotgun proteomic approach, we identified 2938 plasma proteins among the animals injected with PBS and 3.5% λ-CGN. First, the bioinformatic analysis revealed the composition of the plasma proteome. Interestingly, 72 commonly expressed proteins were recorded among the treated and control groups, but, surprisingly, 2830 novel proteins were differentially expressed exclusively in the λ-CGN-induced group. Furthermore, from the commonly expressed proteins, compared to the control group 62 proteins got a significant (p < 0.05) upregulation in the λ-CGN-treated group, while the remaining ten proteins were downregulated. Next, we obtained the major protein-protein interaction networks between hub protein clusters in the blood plasma of the λ-CGN induced group. Moreover, to understand the molecular underpinnings of these effects based on the unveiled protein sets, we performed a bioinformatic structural similarity analysis and generated overlapping 3D reconstructions between ZF and humans during acute inflammation. Biological pathway analysis pointed to the activation and abundance of diverse classical immune and acute phase reactants, several catalytic enzymes, and varied proteins supporting the immune response. Together, this information can be used for testing and finding novel pharmacological targets to treat human intestinal inflammatory diseases.

A Novel Carrageenan Metabolic Pathway in Flavobacterium algicola.

Carbohydrate-active enzymes are important components of the polysaccharide metabolism system in marine bacteria. Carrageenase is indispensable for forming carrageenan catalytic pathways. Here, two GH16_13 carrageenases showed likely hydrolysis activities toward different types of carrageenans (e.g., κ-, hybrid ß/κ, hybrid α/ι, and hybrid λ), which indicates that a novel pathway is present in the marine bacterium Flavobacterium algicola to use κ-carrageenan (KC), ι-carrageenan (IC), and λ-carrageenan (LC). A comparative study described the different features with another reported pathway based on the specific carrageenans (κ, ι, and λ) and expanded the carrageenan metabolic versatility in F. algicola. A further comparative genomic analysis of carrageenan-degrading bacteria indicated different distributions of carrageenan metabolism-related genes in marine bacteria. The crucial core genes encoding the GH127 α-3,6-anhydro-d-galactosidase (ADAG) and 3,6-anhydro-d-galactose (d-AHG)-utilized cluster have been conserved during evolution. This analysis further revealed the horizontal gene transfer (HGT) phenomenon of the carrageenan polysaccharide utilization loci (CarPUL) from Bacteroidetes to other bacterial phyla, as well as the versatility of carrageenan catalytic activities in marine bacteria through different metabolic pathways. IMPORTANCE Based on the premise that the specific carrageenan-based pathway involved in carrageenan use by Flavobacterium algicola has been identified, another pathway was further analyzed, and it involved two GH16_13 carrageenases. Among all the characterized carrageenases, the members of GH16_13 accounted for only a small portion. Here, the functional analysis of two GH16_13 carrageenases suggested their hydrolysis effects on different types of carrageenans (e.g., κ, hybrid ß/κ, hybrid α/ι-, and hybrid λ-), which led to the identification of another pathway. Further exploration enabled us to elucidate the novel pathway that metabolizes KC and IC in F. algicola successfully. The coexistence of these two pathways may provide improved survivability by F. algicola in the marine environment.

In vitro effects of λ-carrageenin in the head-kidney leucocytes of gilthead seabream (Sparus aurata).

The λ-carrageenin is a sulphated mucopolysaccharide that has been used for decades to induce experimental inflammation in mammals. However, it has been little considered in fish. We studied the in vitro effects of λ-carrageenin on gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs). For this purpose, HKLs were incubated with serial concentrations (from 0 to 1,000 µg mL-1) of λ-carrageenin for 3, 6, 12 and 24 h to assess its influence on cell viability and morphology, cell activity and modulation of several selected inflammation-related genes. The viability results demonstrated that λ-carrageenin has no negative effects on HKLs. The respiratory burst activity and phagocytic ability of HKLs after being incubated with λ-carrageenin (100 and 1,000 µg mL-1) for 24 h were increased, whereas the phagocytic capacity was inhibited by the higher dose at the same experimental time compared with control samples. However, the peroxidase activity of HKLs was not changed by incubation with λ-carrageenin. According to transmission electron microscopy results, incubation of HKLs with the higher dose of λ-carrageenin appeared to activate the cells being evident different morphological changes without sign of cell death. Furthermore, up-regulation of three proinflammatory cytokines (il1b, tnfa, and il6) and down-regulation of anti-inflammatory genes (tgfb) were denoted in HKLs incubated with carrageenin. The present results provide a detailed approach to the effects of λ-carrageenin on fish leucocytes, which could have some impact on how we understand the response of these cells when inducing an inflammatory process in fish.

Mitigation of Memory Impairment with Fermented Fucoidan and λ-Carrageenan Supplementation through Modulating the Gut Microbiota and Their Metagenome Function in Hippocampal Amyloid-ß Infused Rats.

Attenuating acetylcholinesterase and insulin/insulin-like growth factor-1 signaling in the hippocampus is associated with Alzheimer's disease (AD) development. Fucoidan and carrageenan are brown and red algae, respectively, with potent antibacterial, anti-inflammatory, antioxidant and antiviral activities. This study examined how low-molecular-weight (MW) and high-MW fucoidan and λ-carrageenan would improve memory impairment in Alzheimer's disease-induced rats caused by an infusion of toxic amyloid-ß(Aß). Fucoidan and λ-carrageenan were dissected into low-MW by Luteolibacter algae and Pseudoalteromonas carrageenovora. Rats receiving an Aß(25-35) infusion in the CA1 region of the hippocampus were fed dextrin (AD-Con), 1% high-MW fucoidan (AD-F-H), 1% low-MW fucoidan (AD-F-L), 1% high-MW λ-carrageenan (AD-C-H), and 1% low-MW λ-carrageenan (AD-C-L) for six weeks. Rats to receive saline infusion (Normal-Con) had an AD-Con diet. The AD-F-L group showed an improved memory function, which manifested as an enhanced Y-maze spontaneous alternation test, water maze, and passive avoidance tests, similar to the Normal-Con group. AD-F-L also potentiated hippocampal insulin signaling and increased the expression of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the hippocampus. AD-C-L improved the memory function mainly by increasing the BDNF content. AD-F-H and AD-C-H did not improve the memory function. Compared to AD-Con, the ascending order of AD-C-H, AD-F-H, AD-C-L, and AD-F-L increased insulin signaling by enhancing the pSTAT3®pAkt®pGSK-3ß pathway. AD-F-L improved glucose tolerance the most. Compared to AD-CON, the AD-F-L treatment increased the serum acetate concentrations and compensated for the defect of cerebral glucose metabolism. AD-Con increased Clostridium, Terrisporobacter and Sporofaciens compared to Normal-Con, and AD-F-L and AD-C-L increased Akkermentia. In conclusion, AD-F-L and AD-C-L alleviated the memory function in the rats with induced AD symptoms by modulating.

Recombinant mussel protein Pvfp5ß enhances cell adhesion of poly(vinyl alcohol)/k-carrageenan hydrogel scaffolds.

Polymeric hydrogels are increasingly considered as scaffolds for tissue engineering due to their extraordinary resemblance with the extracellular matrix (ECM) of many tissues. As cell adhesion is a key factor in regulating important cell functions, hydrogel scaffolds are often functionalized or loaded with a variety of bioactive molecules that can promote adhesion. Interesting biomimetic approaches exploit the properties of mussel-inspired recombinant adhesive proteins. In this work, we prepared hydrogel scaffolds with a 50%w mixture of k-carrageenan (kC) and polyvinyl alcohol (PVA), by a two-step physical gelation process, and we coated them with Perna viridis foot protein-5ß (Pvfp5ß). The mechanical and morphological properties of hydrogels were investigated both after conditioning with typical cell culture media and also after coating with the Pvfp5ß. The protein resulted strongly adsorbed onto the surface of the hydrogel and also able to penetrate in its interiors to a certain depth, mainly interacting with the kC component of the scaffold as resulted from the confocal analysis. Mouse embryonic fibroblasts NIH-3T3 were seeded on top of the hydrogels and cultured up to two weeks. The role of Pvfp5ß in promoting cell adhesion, spreading and colonization of the scaffold was demonstrated.

The risk of carrageenan-induced colitis is exacerbated under high-sucrose/high-salt diet.

As a common used food additive, the threat of carrageenan to colon health is controversial, and is inseparable from personal eating habits. However, no detailed descriptions are available concerning the influence of different dietary patterns on the risk of carrageenan-induced colitis. In this study, we explored the risk of κ-carrageenan-induced colitis under high-sucrose or high-salt diet in mice. Intervention with carrageenan under high-sucrose diet significantly reduced colon length and induced more serious deepening of the crypts. In addition, the intake of carrageenan under high-sucrose/high-salt diet induced more serious goblet cell reduction and increased intestinal permeability. 16S rRNA sequencing and LC-MS based metabonomic approaches were conducted to explore the changes of gut microbiota and metabolites. It was found that the intake of carrageenan under high-sucrose/high-salt diet significantly reduced the abundance of anti-inflammatory bacterium and increased the abundance of harmful bacterium, which was significantly related to the decrease of anti-inflammatory metabolites in colon, such as methyl caffeate, spermine, oleanolic acid and senecionine. Overall, high-sucrose or high-salt diet increased the risk of carrageenan-induced colitis. This reminds us to maintain good eating habits, do not prefer high-sugar or high-salt foods, and try not to consume large amounts of carrageenan continuously to maintain gut health.

Enzymatic Verification and Comparative Analysis of Carrageenan Metabolism Pathways in Marine Bacterium Flavobacterium algicola.

Marine bacteria usually contain polysaccharide utilization loci (PUL) for metabolizing red algae polysaccharides. They are of great significance in the carbon cycle of the marine ecosystem, as well as in supporting marine heterotrophic bacterial growth. Here, we described the whole κ-carrageenan (KC), ι-carrageenan (IC), and partial λ-carrageenan (LC) catabolic pathways in a marine Gram-negative bacterium, Flavobacterium algicola, which is involved carrageenan polysaccharide hydrolases, oligosaccharide sulfatases, oligosaccharide glycosidases, and the 3,6-anhydro-d-galactose (d-AHG) utilization-related enzymes harbored in the carrageenan-specific PUL. In the pathways, the KC and IC were hydrolyzed into 4-sugar-unit oligomers by specific glycoside hydrolases. Then, the multifunctional G4S sulfatases would remove their nonreducing ends' G4S sulfate groups, while the ι-neocarratetrose (Nι4) product would further lose the nonreducing end of its DA2S group. Furthermore, the neocarrageenan oligosaccharides (NCOSs) with no G4S and DA2S groups in their nonreducing ends would completely be decomposed into d-Gal and d-AHG. Finally, the released d-AHG would enter the cytoplasmic four-step enzymatic process, and an l-rhamnose-H+ transporter (RhaT) was preliminarily verified for the function for transportation of d-AHG. Moreover, comparative analysis with the reported carrageenan metabolism pathways further implied the diversity of microbial systems for utilizing the red algae carrageenan. IMPORTANCE Carrageenan is the main polysaccharide of red macroalgae and is composed of d-AHG and d-Gal. The carrageenan PUL (CarPUL)-encoded enzymes exist in many marine bacteria for decomposing carrageenan to provide self-growth. Here, the related enzymes in Flavobacterium algicola for metabolizing carrageenan were characterized for describing the catabolic pathways, notably, although the specific polysaccharide hydrolases existed that were like previous studies. A multifunctional G4S sulfatase also existed, which was devoted to the removal of G4S or G2S sulfate groups from three kinds of NCOSs. Additionally, the transformation of three types of carrageenans into two monomers, d-Gal and d-AHG, occurred outside the cell with no periplasmic reactions that existed in previously reported pathways. These results help to clarify the diversity of marine bacteria using macroalgae polysaccharides.