Targeted sphingolipidomics indicates increased C22-C24:16 ratios of virtually all assayed classes in liver, kidney, and plasma of fumonisin-fed chickens.

The biological properties of sphinganine-(d18:0)-, sphingosine-(d18:1)-, deoxysphinganine-(m18: 0)-, deoxysphingosine-(m18:1)-, deoxymethylsphinganine-(m17:0)-, deoxymethylsphingosine-(m17:1)-, sphingadienine-(d18:2)-, and phytosphingosine-(t18:0)-sphingolipids have been reported to vary, but little is known about the effects of fumonisins, which are mycotoxins that inhibit ceramide synthase, on sphingolipids other than those containing d18:0 and d18:1. Thirty chickens divided into three groups received a control diet or a diet containing 14.6 mg FB1 + FB2/kg for 14 and 21 days. No effects on health or performance were observed, while the effects on sphingoid bases, ceramides, sphingomyelins, and glycosylceramides in liver, kidney, and plasma varied. The t18:0 forms were generally unaffected by fumonisins, while numerous effects were found for m18:0, m18:1, d18:2, and the corresponding ceramides, and these effects appeared to be similar to those observed for d18:0-, and d18:1-ceramides. Partial least square discriminant analysis showed that d18:1- and d18:0-sphingolipids are important variables for explaining the partitioning of chickens into different groups according to fumonisins feeding, while m17:1-, m18:0-, m18:1-, d18:2-, and t18:0-sphingolipids are not. Interestingly, the C22-C24:C16 ratios measured for each class of sphingolipid increased in fumonisin-fed chickens in the three assayed matrices, whereas the total amounts of the sphingolipid classes varied. The potential use of C22-C24:C16 ratios as biomarkers requires further study.

Marine-Sulfated Polysaccharides Extracts Exhibit Contrasted Time-Dependent Immunomodulatory and Antiviral Properties on Porcine Monocytes and Alveolar Macrophages.

Porcine respiratory complex syndrome has a strong economic impact on the swine breeding sector, as well as a clear repercussion on the wellbeing of the animals, leading to overuse of antimicrobial molecules. Algal extracts used in short-term treatments are empirically recognized by farmers as having a positive effect on pigs' health, however, their mechanisms of action are not well known and more research is needed. Herein we studied the short and median term impact of three algal extracts, in vitro, on the pro-inflammatory and antiviral responses of porcine primary blood monocytes and alveolar macrophages, as well as the susceptibility of the treated cells to infection by Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) and the Aujeszky's Disease Virus (ADV). All extracts presented a pro-inflammatory short-term effect, associated for two of them, with an inhibition of the PRRSV replication. Conversely, the three extracts presented an anti-inflammatory median term effect, with no impact on PRRSV replication. The observed immune modulation prompts us to test, in vivo, the anti-PRRSV action of algal extracts and strengthen the interest for this natural resource.

The sulphated polysaccharides extract ulvans from Ulva armoricana limits Marek's disease virus dissemination in vitro and promotes viral reactivation in lymphoid cells.

BACKGROUND: Marek's disease (MD) is a highly contagious lymphoproliferative disease of chickens caused by an alphaherpesvirus, Marek's disease virus (MDV). MD is presently controlled by systematic vaccination of animals, which protects efficiently against the development of clinical disease. However, MDV vaccines do not prevent the multiplication and spread of MDV field strains and may favor the emergence of strains with increased virulence. Therefore, MDV persists to be a major problem for the poultry industry and the development of new alternative strategies to control MDV is needed. Seaweed extracts have previously been shown to exert immunomodulatory and antiviral activities, especially against herpesviruses. The objective of the present study was to explore the effect of Ulva armoricana extracts on MDV infection in vitro. RESULTS: We could demonstrate that the ulvan extract as well as its vitamin-enriched formulation reduce the viral load by about 80% at 24 h post-infection in infected chicken fibroblasts at concentrations that are innocuous for the cells. We also observed a substantial decrease in MDV plaque size suggesting that ulvans impede MDV cell-to-cell spread in vitro. Moreover, we showed that ulvan extract could promote MDV reactivation in lymphoid cells. CONCLUSIONS: Our data provide the first evidence that the use of the ulvan extract could be a good alternative to limit MDV infection in poultry.

Strong Alterations in the Sphingolipid Profile of Chickens Fed a Dose of Fumonisins Considered Safe.

Fumonisins (FB) are mycotoxins known to exert most of their toxicity by blocking ceramide synthase, resulting in disruption of sphingolipid metabolism. Although the effects of FB on sphinganine (Sa) and sphingosine (So) are well documented in poultry, little information is available on their other effects on sphingolipids. The objective of this study was to analyze the effects of FB on the hepatic and plasma sphingolipidome in chickens. The first concern of this analysis was to clarify the effects of FB on hepatic sphingolipid levels, whose variations can lead to numerous toxic manifestations. The second was to specify the possible use of an alteration of the sphingolipidome as a biomarker of exposure to FB, in addition to the measurement of the Sa:So ratio already widely used. For this purpose, we developed an UHPLC MS/MS method that enabled the determination of 82 SL, including 10 internal standards, in chicken liver and plasma. The validated method was used to measure the effects of FB administered to chickens at a dose close to 20 mg FB1 + FB2/kg feed for 9 days. Significant alterations of sphingoid bases, ceramides, dihydroceramides, glycosylceramides, sphingomyelins and dihydrosphingomyelins were observed in the liver. In addition, significant increases in plasma sphinganine 1-phosphate, sphingosine 1-phosphate and sphingomyelins were observed in plasma. Interestingly, partial least-squares discriminant analysis of 11 SL in plasma made it possible to discriminate exposed chickens from control chickens, whereas analysis of Sa and So alone revealed no difference. In conclusion, our results show that the effects of FB in chickens are complex, and that SL profiling enables the detection of exposure to FB when Sa and So fail.

Activation of chicken gamma-delta T lymphocytes by a purified ulvan extract.

Chicken γδ T lymphocytes are present in a variety of tissues such as blood, spleen and intestine. They constitute a major cytotoxic population. In chicken, Salmonella immunization as well as vaccination against Newcastle disease virus are accompanied by an increase of γδ T lymphocytes in peripheral blood, which may be activated, and thus represent a protective immune response. It has been published that activation of avian γδ T cells can occur in a MHC non-restricted manner. Ulvans are complex sulfated polysaccharides composed of disaccharide repetitions found in the cell walls of green algae belonging to the genus Ulva. We recently demonstrated that a purified ulvan extract activates chicken heterophils and monocytes in vivo through TLR2 and TLR4 receptors when given in drinking water. We demonstrate here, that the same extract given once in drinking water at 25 and 50 mg/l, results in increased membrane expression of Major Histocompatibility Complex class 2 as soon as day 2, as detected using flow cytometry. We conclude chicken γδ T lymphocytes to be activated, or at least primed, in vivo, with the extract. Further experiments are required to fully understand whether their activation or priming is the result of direct and/or indirect mechanisms.

Polar Lipids of Commercial Ulva spp. of Different Origins: Profiling and Relevance for Seaweed Valorization.

Macroalgae of the genus Ulva have long been used as human food. Local environmental conditions, among other factors, can have an impact on their nutrient and phytochemical composition, as well as on the value of the seaweed for food and non-food applications. This study is the first to initiate a comparison between commercial Ulva spp. from different European origins, France (FR, wild-harvested Ulva spp.), and Portugal (PT, farm-raised Ulva rigida), in terms of proximate composition, esterified fatty acids (FA), and polar lipids. The ash content was higher in PT samples, while FR samples had higher levels of proteins, lipids, and carbohydrates and other compounds. The profile of esterified FA, as well as FA-containing polar lipids at the class and species levels were also significantly different. The FR samples showed about three-fold higher amount of n-3 polyunsaturated FA, while PT samples showed two-fold higher content of monounsaturated FA. Quantification of glycolipids and phospholipids revealed, respectively, two-fold and three-fold higher levels in PT samples. Despite the differences found, the polar lipids identified in both batches included some lipid species with recognized bioactivity, valuing Ulva biomass with functional properties, increasing their added value, and promoting new applications, namely in nutraceutical and food markets.

Ulvan Activates Chicken Heterophils and Monocytes Through Toll-Like Receptor 2 and Toll-Like Receptor 4.

Responsiveness to invasive pathogens, clearance via the inflammatory response, and activation of appropriate acquired responses are all coordinated by innate host defenses. Toll-like receptor (TLR) ligands are potent immune-modulators with profound effects on the generation of adaptive immune responses. This property is being exploited in TLR-based vaccines and therapeutic agents in chickens. However, for administering the TLR agonist, all previous studies used in ovo, intra-muscular or intra-venous routes that cannot be performed in usual farming conditions, thus highlighting the need for TLR ligands that display systemic immune effects when given orally (per os). Here we have demonstrated that an ulvan extract of Ulva armoricana is able to activate avian heterophils and monocytes in vitro. Using specific inhibitors, we have evidenced that ulvan may be a new ligand for TLR2 and TLR4; and that they regulate heterophil activation in slightly different manner. Moreover, activation of heterophils as well as of monocytes leads to release pro-inflammatory cytokines, including interleukin1-ß, interferon α and interferon γ, through pathways that we partly identified. Finally, when given per os to animals ulvan induces heterophils and monocytes to be activated in vivo thus leading to a transient release of pro-inflammatory cytokines with plasma concentrations returning toward baseline levels at day 3.

A randomized controlled double-blind clinical trial comparing versus placebo the effect of an edible algal extract (Ulva Lactuca) on the component of depression in healthy volunteers with anhedonia.

BACKGROUND: The effects of the seaweed extract were evaluated on the animal model equivalent of depression compared with a control group treated with the carrier (spring water) and a reference group treated with Imipramine and showed significative effect. This clinical trial was intended to confirm in humans the potential efficacy identified in animals. The primary objective was to compare against a placebo the effect of Ulva L.L extract in healthy volunteers whose anhedonia was characterized by a component of depression. METHODS: Single-centre double-blind randomized placebo-controlled clinical trial on parallel arms of two groups of 45 subjects. The study could include men or women aged 18 to 65 years with anhedonia characterized by a Snaith Hamilton Pleasure Scale score (SHAPS) of ≥5 and feeling low morale for at least four weeks characterized by a component of depression evaluated on the Quick Inventory of Depressive Symptomatology - Self Report (QIDS-SR). Evaluation criteria: QIDS-SR; Patient Global Improvement Impression (PGII) and Clinical Global Improvement Impression (CGII). RESULTS: 86 subjects were included in the trial: 42 in the placebo group and 44 Ulva group. At D84, QIDS-SR significantly decreased more in the Ulva.L.L. group than in the placebo group (p: 0.0389). This difference is essentially linked to an improvement of the sleep disorders (p: 0.0219), of the psychomotor consequences (p: 0.002) and of the nutrition behaviour (p: 0.0694). 90.1% have the feeling of being improved in the Ulva group vs 72.5% in the placebo group (p: 0.0114) and in parallel 90.9% of the practitioners have the feeling that the subject has improved vs 70.8% (p: 0.0214). CONCLUSION: This double-blind randomized placebo-controlled trial shows that daily intake for three months of a water-soluble extract of Ulva L.L. continues to significantly improve the component of depression of subjects presenting anhedonia compared with a placebo. TRIAL REGISTRATION: Trial retrospectively registred on ClinicalTrial.gov under ID: NCT03545399 Date: 05/22/2018.

A novel unsaturated ß-glucuronyl hydrolase involved in ulvan degradation unveils the versatility of stereochemistry requirements in family GH105.

Ulvans are cell wall matrix polysaccharides in green algae belonging to the genus Ulva. Enzymatic degradation of the polysaccharide by ulvan lyases leads to the production of oligosaccharides with an unsaturated ß-glucuronyl residue located at the non-reducing end. Exploration of the genomic environment around the Nonlabens ulvanivorans (previously Percicivirga ulvanivorans) ulvan lyase revealed a gene highly similar to known unsaturated uronyl hydrolases classified in the CAZy glycoside hydrolase family 105. The gene was cloned, the protein was overexpressed in Escherichia coli, and enzymology experiments demonstrated its unsaturated ß-glucuronyl activity. Kinetic analysis of purified oligo-ulvans incubated with the new enzyme showed that the full substrate specificity is attained by three subsites that preferentially bind anionic residues (sulfated rhamnose, glucuronic/iduronic acid). The three-dimensional crystal structure of the native enzyme reveals that a trimeric organization is required for substrate binding and recognition at the +2 binding subsite. This novel unsaturated ß-glucuronyl hydrolase is part of a previously uncharacterized subgroup of GH105 members and exhibits only a very limited sequence similarity to known unsaturated ß-glucuronyl sequences previously found only in family GH88. Clan-O formed by families GH88 and GH105 was singular in the fact that it covered families acting on both axial and equatorial glycosidic linkages, respectively. The overall comparison of active site structures between enzymes from these two families highlights how that within family GH105, and unlike for classical glycoside hydrolysis, the hydrolysis of vinyl ether groups from unsaturated saccharides occurs independently of the α or ß configuration of the cleaved linkage.

Persicivirga ulvanivorans sp. nov., a marine member of the family Flavobacteriaceae that degrades ulvan from green algae.

A rod shaped, Gram-stain-negative, chemo-organotrophic, heterotrophic, strictly aerobic, non-gliding bacterium, designated strain PLR(T), was isolated from faeces of the mollusc Aplysia punctata (Mollusca, Gastropoda) that had been fed with green algae belonging to the genus Ulva. The novel strain was able to degrade ulvan, a polysaccharide extracted from green algae (Chlorophyta, Ulvophyceae). The taxonomic position of strain PLR(T) was investigated by using a polyphasic approach. Strain PLR(T) was dark orange, oxidase-positive, catalase-positive and grew optimally at 25 °C, at pH 7.5 and in the presence of 2.5 % (w/v) NaCl with an oxidative metabolism using oxygen as the electron acceptor. Nitrate could not be used as the electron acceptor. Strain PLR(T) had a Chargaff's coefficient (DNA G+C content) of 35.3 mol%. Phylogenetic analysis based on the sequence of the 16S rRNA gene placed the novel strain in the family Flavobacteriaceae (phylum 'Bacteroidetes'), within a clade comprising Stenothermobacter spongiae, Nonlabens tegetincola, Sandarakinotalea sediminis, Persicivirga xylanidelens and Persicivirga dokdonensis. The closest neighbours of strain PLR(T) were P. xylanidelens and P. dokdonensis, sharing 95.2 and 95.5 % 16S rRNA gene sequence similarity, respectively. Phylogenetic inference and differential phenotypic characteristics demonstrated that strain PLR(T) represents a novel species of the genus Persicivirga, for which the name Persicivirga ulvanivorans sp. nov. is proposed. The type strain is PLR(T) ( = CIP 110082(T) = DSM 22727(T)).

Enzymatic degradation of κ-carrageenan in aqueous solution.

Enzymatic degradation of standard κ-carrageenan and the low-gelling hybrid κ-/µ-carrageenan were conducted using recombinant Pseudoalteromonas carrageenovora κ-carrageenase. The initial velocity of the enzyme was determined as a function of varying Tris or NaI concentrations and at constant 200 mM cosolutes concentration, adjusting NaI and Tris concentrations accordingly. In both cases, we observed strong inhibition of the enzyme with increasing amounts of iodide. The characterization of the κ- and κ-/µ-carrageenan ordering by optical rotation and the visualization of iodide binding on carrageenan by (127)I NMR revealed that inhibition was not caused by the disordered-ordered transition of carrageenan in NaI, but by iodide binding. These results were confirmed by analysis of the degradation products by gel permeation chromatography. Degradation of carrageenan in the disordered state led to a rapid decrease in molecular mass and the production of all possible neo-κ-carrabiose oligomers. In the ordered conformation, the degradation kinetics, the decrease of average molecular weight, and the chain population distribution of degradation products varied with iodide concentration. These observations were interpreted to be the result of increasing amounts of bound iodide on carrageenan helices that, in turn, impede enzyme catalysis. Based on these results, we propose a single-helix ordered conformation state for κ-carrageenan and reject the previously advocated double-helix model.