[Squid fat as a promising source of polyunsaturated fatty acids].

Omega-3 polyunsaturated fatty acids (ω-3 PUFA) are substances that play an important role in human metabolism. They are essential nutritional factors and can improve the functioning of individual systems and the body as a whole. The main source of ω-3 PUFA has long been fish fat, which contains PUFA in the triglyceride form. A fairly new and promising alternative to fish fat is the liver fat of the Commander squid (Berryteuthis magister), which additionally contains alkylglycerols, contains PUFA in the phospholipid form and can be obtained from squid fishery waste. The objective of the research was to carry out an analysis of scientific data, including the results of studies of the biological activity of squid fat, as well as its components that are part of other similar raw materials, and evaluate the prospects for its use in medical practice. Material and methods. During the study, various sources were analyzed, including scientific literature from electronic databases eLibrary, PubMed, Scopus, Web of Science and electronic search engines Google Academy from 2000 to 2022. Results. It is noted that squid fat has a pronounced biological activity. Its components increase innate immunity, have antitumor potential, improve the state of the body under stress, have hypolipidemiс and hypotensive effect, improve memory and attention, and also positively affect the composition and rheological parameters of blood. In addition to these effects, a positive effect of ω-3 PUFA and alkylglycerols on spermatogenesis, sperm quality and the female reproductive system has been noted. In a number of studies, alkylglycerol esters increased the permeability of the blood-brain barrier and, due to their structure, are able to form vesicles, therefore, they can be considered as raw materials for the production of new dosage forms for targeted therapy of brain tumors. In the available literature, in the case of the use of squid fat and its components, undesirable side effects have not been identified. Conclusion. Squid fat is a complete source of ω-3 PUFA and alkylglycerols, therefore it can be recommended as a dietary supplement, especially in a diet low in ω-3 PUFA.

Production of Dual Inhibitory Hydrolysate by Enzymatic Hydrolysis of Squid Processing By-product.

Squid processing by-product contains unutilized abundant proteins. In this study, 6 proteases (pepsin, protamex, trypsin, neutral protease, alkaline protease, and papain) were firstly employed to hydrolyze the squid processing by-product protein. The neutral protease-digested hydrolysate was found to have the most promising ACE (angiotensin-converting enzyme) inhibitory activity. Based on Box-Behnken design, the optimal hydrolysis process was determined to be: 52.4 ℃ of temperature, 5.7 h of time, pH 7.1, and 8151 U/g of enzyme. Under these conditions, the ACE inhibition rate and polypeptide content of the hydrolysate were 84.26% and 229.09 mg/g, respectively. Subsequently, ultrafiltration was performed, and the ACE and renin inhibitory activities of the filtrate (< 1 kDa) were the highest, reaching 87.48 ± 1.76% and 69.72 ± 1.16%, with IC50 values of 1.34 ± 0.12 mg/mL and 1.47 ± 0.06 mg/mL, respectively. However, these activities decreased to 35.15 ± 1.31% and 43.17% ± 1.42%, respectively, after digestion by simulated gastrointestinal juice. Nevertheless, this is the first report representing the neutral protease-digested hydrolysate of squid processing by-product as a potential source of both ACE and renin inhibitors.

Biochemical and subcellular characterization of a squid hnRNPA/B-like protein 2 in osmotic stress activated cells reflects molecular properties conserved in this protein family.

BACKGROUND: We have identified endogenous p65 to be an SDS-stable dimer protein composed of ~ 37 kDa hnRNPA/B-like subunits. We have investigated molecular properties involved in the stability of dimeric form, and their regulation in the transition between monomeric and dimeric forms of hnRNPA/B-like protein 2. We also investigated a cellular property conserved between squid hnRNPA/B-like protein 2 and human hnRNPA1 protein in a neuronal context. METHODS AND RESULTS: Here we show biochemical properties of a recombinant hnRNPA/B-like protein 2 (rP2) in vitro experiments, as one of p65 subunit. We found that interaction between rP2 and RNA molecules interfered with the dynamics of rP2 dimers formation, involved in disulfide bonds and/or postranslational alterations in distinct stage of SDS-stable dimers formation. In addition, we have performed immunofluorescence in SH-SY5Y cells and observed that the pEGFP-P2 fusion protein was expressed in the nucleus, similar to what is observed for human hnRNPA1 protein. CONCLUSION: Our results reinforce the idea that p65 is an SDS-stable dimer. Thus, a deeper understanding between monomeric and dimeric transition dynamic is critical into evolution of several neurodegenerative disease.

Suppressive effects of an apoptotic mimicry prepared from jumbo-flying squid-skin phospholipids on the osteoclastogenesis in receptor activator of nuclear factor kappa B ligand/macrophage colony-stimulating factor-induced RAW 264.7 cells.

BACKGROUND: Liposomes containing docosahexaenoic acid (DHA) and phosphatidylserine were claimed to inhibit osteoclast formation and bone resorption in the inflammatory status. Herein, we proposed that an apoptotic mimicry (SQ liposome) prepared from squid-skin phospholipids can explore the suppressive osteoclastogenesis. METHODS: The intermolecular fatty-acid composition in the phospholipid of squid-skin extract was analyzed by GC-FID. The SQ liposome structure was characterized by size distribution and zeta potential (ζ). RAW 264.7 cell is used to study the effect of SQ liposomes on osteoclast differentiation. Secretion of prostaglandin E2 (PGE2) and transforming growth factor-ß (TGF-ß) from RAW 264.7 cells were assayed. Antiosteoclastogenesis effects were performed via the tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) counting, bone resorption pit assay, and TRAP activity analysis. The specific gene expressions related to antiosteoclastogenesis were also detected. RESULTS: An apoptotic mimicry through the use of a single-layer liposome (SQ liposome) with phosphatidylserine exposure contains DHA (28.7%) and eicosapentaenoic acid (EPA, 11.8%). Co-treatment with receptor activator of nuclear factor kappa B ligand (RANKL)/macrophage colony-stimulating factor induced RAW 264.7-cell differentiation into mature osteoclasts, thus enhancing PGE2 and TGF-ß secretion. However, cotreatment with 1 mg/mL of SQ liposome restored (p < 0.05) the cell viabilities under the RANKL stress. Increased PGE2 levels was downregulated (p < 0.05) in cotreatments with 0.11 and 0.33 mg/mL of SQ liposome, but on the TGF-ß levels were not (p > 0.05) influenced in SQ liposome cotreatments. Cotreatments with 0.33-1 mg/mL of SQ liposome suppressed (p < 0.05) the osteoclast maturation (such as decreased MNCs and bone pit formation), inhibited TRAP activities, and downregulated the osteoclastogenesis-related gene expressions. CONCLUSION: In summary, current data support that a possible prevention of our prepared SQ liposomes which are rich in DHA and EPA on bone loss is through the suppression of osteoclastogenesis. Moreover, based on the results from this study an in vivo study warrants a further investigation.

Bioinspired and Mechanically Strong Fibers Based on Engineered Non-Spider Chimeric Proteins.

Silk-protein-based fibers have attracted considerable interest due to their low weight and extraordinary mechanical properties. Most studies on fibrous proteins focus on the recombinant spidroins, but these fibers exhibit moderate mechanical performance. Thus, the development of alternative structural proteins for the construction of robust fibers is an attractive goal. Herein, we report a class of biological fibers produced using a designed chimeric protein, which consists of the sequences of a cationic elastin-like polypeptide and a squid ring teeth protein. Remarkably, the chimeric protein fibers exhibit a breaking strength up to about 630 MPa and a corresponding toughness as high as about 130 MJ m-3 , making them superior to many recombinant spider silks and even comparable to some native counterparts. Therefore, this strategy is a novel concept for exploring bioinspired ultrastrong protein materials through the development of new types of structural chimeric proteins.

Discovery of Novel Angiotensin-Converting Enzyme Inhibitory Peptides from Todarodes pacificus and Their Inhibitory Mechanism: In Silico and In Vitro Studies.

In order to rapidly and efficiently excavate antihypertensive ingredients in Todarodes pacificus, its myosin heavy chain was hydrolyzed in silico and the angiotensin-converting enzyme (ACE) inhibitory peptides were predicted using integrated bioinformatics tools. The results showed the degree of hydrolysis (DH) theoretically achieved 56.8% when digested with papain, ficin, and prolyl endopeptidase (PREP), producing 126 ACE inhibitory peptides. By predicting the toxicity, allergenicity, gastrointestinal stability, and intestinal epithelial permeability, 30 peptides were finally screened, of which 21 had been reported and 9 were new. Moreover, the newly discovered peptides were synthesized to evaluate their in vitro ACE inhibition, showing Ile-Ile-Tyr and Asn-Pro-Pro-Lys had strong effects with a pIC50 of 4.58 and 4.41, respectively. Further, their interaction mechanisms and bonding configurations with ACE were explored by molecular simulation. The preferred conformation of Ile-Ile-Tyr and Asn-Pro-Pro-Lys located in ACE were successfully predicted using the appropriate docking parameters. The molecular dynamics (MD) result indicated that they bound tightly to the active site of ACE by means of coordination with Zn(II) and hydrogen bonding and hydrophobic interaction with the residues in the pockets of S1 and S2, resulting in stable complexes. In summary, this work proposed a strategy for screening and identifying antihypertensive peptides from Todarodes pacificus.

In situ growth of Au nanoparticles on natural melanin as biocompatible and multifunctional nanoagent for efficient tumor theranostics.

Natural melanin has been demonstrated to be a biocompatible and efficient nanoagent for the photothermal ablation of tumors, but their practical applications are limited due to their lack of typical imaging functions (CT, MRI, etc.). Thus, to achieve multifunctional melanin-based nanoagents for imaging-guided therapy, for the first time, herein, we report the in situ growth of Au nanoparticles on natural melanin as a model through a simple and safe method. The as-synthesized samples are composed of melanin nanoparticles (diameter: ∼120 nm) whose surface are decorated by small Au nanoparticles with an adjustable size ranging from ∼10 to ∼40 nm. These Au-decorated melanin (Au-M) nanocomposites exhibit satisfactory near infrared (NIR) photoabsorption and high photothermal conversion efficiency of 42.3%. Furthermore, the Au-M nanocomposites have a high X-ray attenuation coefficient and exhibit excellent biocompatibility. When the Au-M solutions were injected into the tumor of a mouse, the tumor could be detected by X-ray computed tomography (CT), photoacoustic (PA) and thermal imaging, and then be thermally ablated under the illumination of an 808 nm laser. Therefore, these Au-M nanocomposites have great potential as a novel multifunctional and biocompatible nanoagent for imaging-guided photothermal tumor ablation.

Squid type II collagen as a novel biomaterial: Isolation, characterization, immunogenicity and relieving effect on degenerative osteoarthritis via inhibiting STAT1 signaling in pro-inflammatory macrophages.

Collagen from marine organisms has a broad prospect in biomedical field, yet the knowledge on marine-derived type II collagen is rare. Herein, a novel type II collagen was successfully isolated from squid cartilage for the first time. After being characterized, the immunogenicity of squid type II collagen (SCII) was evaluated and compared with that of bovine type II collagen (BCII). Then investigations were further conducted for the impacts of SCII on pro-inflammatory macrophages and macrophage chemotaxis. The degenerative osteoarthritis (OA) -relieving effects of SCII were explored using OA rat model in vivo. Our results demonstrated that the isolated SCII maintained triple-superhelical structure of native collagen with high purity. Different from BCII, SCII presented no immunogenicity since it neither induced abnormal proliferation of lymphocytes in vitro nor changed the basic levels of IgM, IgG, anti-type II collagen IgG and CD4+/CD8+ lymphocytes ratio in vivo. Additionally, SCII also exerted prominent anti-inflammatory effects. SCII significantly reduced the production of pro-inflammatory cytokines by enhancing the activity of TCPTP and subsequently prompting the dephosphorylation of p-STAT1 in pro-inflammatory macrophages. Besides, it indirectly prevented hypertrophic changes of chondrocytes, and markedly impeded chemotaxis of macrophages. Moreover, inflammation condition in OA rats was significantly alleviated under treatment with SCII. These data suggested that the newly developed SCII could not only avoid the immunogenic risks of collagen derived from terrestrial animals, but more importantly, provide new choice for the control and treatment of OA.

Identification, Characterization, and Expression Analysis of a FMRFamide-Like Peptide Gene in the Common Chinese Cuttlefish (Sepiella japonica).

The peptide FMRFamide is one of the well-known peptides involved in multiple physiological processes in the phylum Mollusca. In this study, a FMRFamide gene (GenBank accession No. KJ933411) was identified in a cuttlefish species called Sepiella japonica and was designated as SjFMRFamide. The total length of the SjFMRFamide sequence was found to be 1880 bp while the open reading frame contained 996 bp encoding a protein of 331 amino acid residues with a predicted isoelectric point (pI) and molecular weight (MW) of 9.18 and 38.8 kDa along with a 333 bp 5'-untranslated region (UTR) and 551 bp 3'-UTR. The deduced SjFMRFamide precursor protein contains one signal peptide and expresses four kinds FMRFamide-related peptides including a single copy of FLRFamide, ALSGDAFLRFamide, and FIRFamide and multiple copies of FMRFamide. Results of phylogenetic relation analysis strongly indicated that the sequence of this gene shares high identity with the genes of known FMRFamides. Spatial expression analysis indicated the highest mRNA expression of SjFMRFamide in the brain of male and female cuttlefishes among the eight tissues analyzed. An in situ hybridization assay of the brain indicated that SjFMRFamide was transcribed in several functional lobes, which suggests that it might be related to many physiological regulatory mechanisms. This is the first study describing FMRFamide in S. japonica and the results may contribute to future studies of neuropeptide evolution or may prove useful for the development of aquaculture methods for this cuttlefish species.

Supramolecular ß-Sheets Stabilized Protein Nanocarriers for Drug Delivery and Gene Transfection.

Suckerin proteins, recently discovered in the sucker ring teeth of squids, represent a family of promising structural biomacromolecules that can form supramolecular networks stabilized by nanoconfined ß-sheets. Exploiting this feature as well as their specific amino acid composition, we demonstrate that artificial suckerin-19 (S-19) can be engineered into nanocarriers for efficient drug delivery and gene transfection in vitro and in vivo. First, we demonstrate that S-19 self-assembles into ß-sheet stabilized nanoparticles with controlled particle sizes of 100-200 nm that are able to encapsulate hydrophobic drugs for pH-dependent release in vitro, and that can effectively inhibit tumor growth in vivo. We also show that S-19 can complex and stabilize plasmid DNA, with the complexes stabilized by hydrophobic interactions of the ß-sheet domains as opposed to electrostatic interactions commonly achieved with cationic polymers, thus lowering cytotoxicity. The elevated Histidine content of S-19 appears critical to trigger endosomal escape by the proton sponge effect, thereby ensuring efficient gene transfection both in vitro and in vivo. Our study demonstrates that S-19 represents a promising functional protein nanocarrier that could be used for various drug and gene delivery applications.

ALS-linked FUS exerts a gain of toxic function involving aberrant p38 MAPK activation.

Mutations in Fused in Sarcoma/Translocated in Liposarcoma (FUS) cause familial forms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by progressive axonal degeneration mainly affecting motor neurons. Evidence from transgenic mouse models suggests mutant forms of FUS exert an unknown gain-of-toxic function in motor neurons, but mechanisms underlying this effect remain unknown. Towards this end, we studied the effect of wild type FUS (FUS WT) and three ALS-linked variants (G230C, R521G and R495X) on fast axonal transport (FAT), a cellular process critical for appropriate maintenance of axonal connectivity. All ALS-FUS variants impaired anterograde and retrograde FAT in squid axoplasm, whereas FUS WT had no effect. Misfolding of mutant FUS is implicated in this process, as the molecular chaperone Hsp110 mitigated these toxic effects. Interestingly, mutant FUS-induced impairment of FAT in squid axoplasm and of axonal outgrowth in mammalian primary motor neurons involved aberrant activation of the p38 MAPK pathway, as also reported for ALS-linked forms of Cu, Zn superoxide dismutase (SOD1). Accordingly, increased levels of active p38 MAPK were detected in post-mortem human ALS-FUS brain tissues. These data provide evidence for a novel gain-of-toxic function for ALS-linked FUS involving p38 MAPK activation.

Cloning, Characterization, and Expression Profile of Estrogen Receptor in Common Chinese Cuttlefish, Sepiella japonica.

Sex steroid hormones are widely detected in molluscs and play important roles in sex determination, gonadal tissue maturation, and gametogenesis. Nevertheless, the signaling pathways of sex steroids in cephalopod have not yet been clearly elucidated. In the present study, a full-length sequence encoding the estrogen receptor (ER) was isolated from common Chinese cuttlefish, Sepiella japonica. The sjER cDNA clone was found to contain 1,788 nucleotides including a 1,470 bp open reading frame encoding 489 amino acid (aa) residues. The deduced ER protein consisted of six nuclear receptor characteristic domains. Based on a phylogenetic analysis, the ER DNA-binding domain and ligand-binding domain are highly conserved compared to other mollusc ERs. Highest aa identities were found for sjER with common octopus (Octopus vulgaris) ER (89%) and pacific oyster (Crassostrea gigas) ER (61%). Tissue expression analysis confirmed that sjER was widely distributed among tissues and predominantly expressed in the brain, liver, gonad (testis and ovary), and other accessory sexual gland (nidamental gland). The ER expression was temporally upregulated in the brain, liver, and ovary during the early sexual maturation period in S. japonica, which is coincident with the fluctuation of ovary estradiol content. These suggest that sjER may be involved in regulating the reproductive cycle of S. japonica. A fusion protein transient transfections assay showed that sjER was mainly located in the nucleus, suggesting a possible orthodox working mechanism of S. japonica ER in the nucleus through a ligand-dependent activation of specific gene transcription.

The Amyloid Precursor Protein of Alzheimer's Disease Clusters at the Organelle/Microtubule Interface on Organelles that Bind Microtubules in an ATP Dependent Manner.

The amyloid precursor protein (APP) is a causal agent in the pathogenesis of Alzheimer's disease and is a transmembrane protein that associates with membrane-limited organelles. APP has been shown to co-purify through immunoprecipitation with a kinesin light chain suggesting that APP may act as a trailer hitch linking kinesin to its intercellular cargo, however this hypothesis has been challenged. Previously, we identified an mRNA transcript that encodes a squid homolog of human APP770. The human and squid isoforms share 60% sequence identity and 76% sequence similarity within the cytoplasmic domain and share 15 of the final 19 amino acids at the C-terminus establishing this highly conserved domain as a functionally import segment of the APP molecule. Here, we study the distribution of squid APP in extruded axoplasm as well as in a well-characterized reconstituted organelle/microtubule preparation from the squid giant axon in which organelles bind microtubules and move towards the microtubule plus-ends. We find that APP associates with microtubules by confocal microscopy and co-purifies with KI-washed axoplasmic organelles by sucrose density gradient fractionation. By electron microscopy, APP clusters at a single focal point on the surfaces of organelles and localizes to the organelle/microtubule interface. In addition, the association of APP-organelles with microtubules is an ATP dependent process suggesting that the APP-organelles contain a microtubule-based motor protein. Although a direct kinesin/APP association remains controversial, the distribution of APP at the organelle/microtubule interface strongly suggests that APP-organelles have an orientation and that APP like the Alzheimer's protein tau has a microtubule-based function.

Transcriptome analysis of the white body of the squid Euprymna tasmanica with emphasis on immune and hematopoietic gene discovery.

In the mutualistic relationship between the squid Euprymna tasmanica and the bioluminescent bacterium Vibrio fischeri, several host factors, including immune-related proteins, are known to interact and respond specifically and exclusively to the presence of the symbiont. In squid and octopus, the white body is considered to be an immune organ mainly due to the fact that blood cells, or hemocytes, are known to be present in high numbers and in different developmental stages. Hence, the white body has been described as the site of hematopoiesis in cephalopods. However, to our knowledge, there are no studies showing any molecular evidence of such functions. In this study, we performed a transcriptomic analysis of white body tissue of the Southern dumpling squid, E. tasmanica. Our primary goal was to gain insights into the functions of this tissue and to test for the presence of gene transcripts associated with hematopoietic and immune processes. Several hematopoiesis genes including CPSF1, GATA 2, TFIID, and FGFR2 were found to be expressed in the white body. In addition, transcripts associated with immune-related signal transduction pathways, such as the toll-like receptor/NF-κß, and MAPK pathways were also found, as well as other immune genes previously identified in E. tasmanica's sister species, E. scolopes. This study is the first to analyze an immune organ within cephalopods, and to provide gene expression data supporting the white body as a hematopoietic tissue.

Nine novel angiotensin I-converting enzyme (ACE) inhibitory peptides from cuttlefish (Sepia officinalis) muscle protein hydrolysates and antihypertensive effect of the potent active peptide in spontaneously hypertensive rats.

This study aimed to identify novel ACE inhibitory peptides from the muscle of cuttlefish. Proteins were hydrolyzed and the hydrolysates were then subjected to various types of chromatography to isolate the active peptides. Nine ACE inhibitory peptides were isolated and their molecular masses and amino acid sequences were determined using ESI-MS and ESI-MS/MS, respectively. The structures of the most potent peptides were identified as Val-Glu-Leu-Tyr-Pro, Ala-Phe-Val-Gly-Tyr-Val-Leu-Pro and Glu-Lys-Ser-Tyr-Glu-Leu-Pro. The first peptide displayed the highest ACE inhibitory activity with an IC50 of 5.22µM. Lineweaver-Burk plots suggest that Val-Glu-Leu-Tyr-Pro acts as a non-competitive inhibitor against ACE. Furthermore, antihypertensive effects in spontaneously hypertensive rats (SHR) also revealed that oral administration of Val-Glu-Leu-Tyr-Pro can decrease systolic blood pressure significantly (p<0.01). These results suggest that the Val-Glu-Leu-Tyr-Pro would be a beneficial ingredient for nutraceuticals and pharmaceuticals acting against hypertension and its related diseases.

Dual role of the cuttlefish salivary proteome in defense and predation.

We characterized the proteome of the posterior salivary glands of the cephalopod S. officinalis by combining de novo RNA sequencing and mass spectrometry. In silico analysis of the transcriptome revealed the occurrence of three main categories of proteins: enzymes, immune factors and toxins. Protein identification by SDS-PAGE and MALDI-TOF/TOF confirmed the occurrence of proteins essential to venom-like enzymes: peptidase S1 under four isoforms, phospholipase A2 and two toxins. The first toxin is a cystein rich secreted protein (CRISP), a common toxin found in all venomous animals. The second one is cephalotoxin, which is specific to decabrachia cephalopods. Secretions of the posterior salivary glands are transported to the cephalopodium; they are involved in prey catching but also in gamete storage, fertilization and egg-laying. The paralyzing activity and the antimicrobial effect of saliva suggest a dual role in predation and in immune defense in cuttlefish. BIOLOGICAL SIGNIFICANCE: The originality of this study lies in the use of a transcriptomic approach (de novo RNA sequencing) coupled to a proteomic approach to get an overview of posterior salivary glands in S. officinalis. In cephalopods, these glands are involved in predation, more precisely in paralyzing preys and digesting them. Our in silico analysis equally reveals a role in immune defense as observed in mammals' saliva. Our study also shows the specificity of cuttlefish venom, with the identification of cephalotoxins, proteins that are not found in octopuses. Finally, we show that cuttlefish saliva is a complex mixture that has antibacterial and crippling properties, but no lethal effect.

Shaping the microenvironment: evidence for the influence of a host galaxin on symbiont acquisition and maintenance in the squid-Vibrio symbiosis.

Most bacterial species make transitions between habitats, such as switching from free living to symbiotic niches. We provide evidence that a galaxin protein, EsGal1, of the squid Euprymna scolopes participates in both: (i) selection of the specific partner Vibrio fischeri from the bacterioplankton during symbiosis onset and, (ii) modulation of V. fischeri growth in symbiotic maintenance. We identified two galaxins in transcriptomic databases and showed by quantitative reverse-transcriptase polymerase chain reaction that one (esgal1) was dominant in the light organ. Further, esgal1 expression was upregulated by symbiosis, a response that was partially achieved with exposure to symbiont cell-envelope molecules. Confocal immunocytochemistry of juvenile animals localized EsGal1 to the apical surfaces of light-organ epithelia and surrounding mucus, the environment in which V. fischeri cells aggregate before migration into the organ. Growth assays revealed that one repeat of EsGal1 arrested growth of Gram-positive bacterial cells, which represent the cell type first 'winnowed' during initial selection of the symbiont. The EsGal1-derived peptide also significantly decreased the growth rate of V. fischeri in culture. Further, when animals were exposed to an anti-EsGal1 antibody, symbiont population growth was significantly increased. These data provide a window into how hosts select symbionts from a rich environment and govern their growth in symbiosis.

Identification of the major ACE-inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate from cuttlefish wastewater.

The aim of this work was the purification and identification of the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate recovered from a cuttlefish industrial manufacturing effluent. This process consisted on the ultrafiltration of cuttlefish softening wastewater, with a 10 kDa cut-off membrane, followed by the hydrolysis with alcalase of the retained fraction. Alcalase produced ACE inhibitors reaching the highest activity (IC50 = 76.8 ± 15.2 µg mL⁻¹) after 8 h of proteolysis. Sequential ultrafiltration of the 8 h hydrolysate with molecular weight cut-off (MWCO) membranes of 10 and 1 kDa resulted in the increased activity of each permeate, with a final IC50 value of 58.4 ± 4.6 µg mL⁻¹. Permeate containing peptides lower than 1 kDa was separated by reversed-phase high performance liquid chromatography (RP-HPLC). Four fractions (A-D) with potent ACE inhibitory activity were isolated and their main peptides identified using high performance liquid chromatography coupled to an electrospray ion trap Fourier transform ion cyclotron resonance-mass spectrometer (HPLC-ESI-IT-FTICR) followed by comparison with databases and de novo sequencing. The amino acid sequences of the identified peptides contained at least one hydrophobic and/or a proline together with positively charged residues in at least one of the three C-terminal positions. The IC50 values of the fractions ranged from 1.92 to 8.83 µg mL⁻¹, however this study fails to identify which of these peptides are ultimately responsible for the potent antihypertensive activity of these fractions.

Diamond squid (Thysanoteuthis rhombus)-derived chondroitin sulfate stimulates bone healing within a rat calvarial defect.

Chondroitin sulfate (CS) has been suggested to be involved in bone formation and mineralization processes. A previous study showed that squid-derived CS (sqCS) has osteoblastogenesis ability in cooperation with bone morphogenetic protein (BMP)-4 in vitro. However, in vivo, osteogenic potential has not been verified. In this study, we created a critical-sized bone defect in the rat calvaria and implanted sqCS-loaded gelatin hydrogel sponges (Gel) into the defect with or without BMP-4 (CS/BMP/Gel and CS/Gel, respectively). At 15 weeks, bone repair rate of CS/Gel-treated defects and CS/BMP/Gel-treated defects were 47.2% and 51.1%, respectively, whereas empty defects and defects with untreated sponges showed significantly less bone ingrowth. The intensity of von Kossa staining of the regenerated bone was less than that of the original one. Mineral apposition rates at 9 to 10 weeks were not significantly different between all treatment groups. Although bone repair was not completed, sqCS stimulated bone regeneration without BMP-4 and without external mesenchymal cells or preosteoblasts. Therefore, sqCS is a promising substance for promotion of osteogenesis.

A complex set of sex pheromones identified in the cuttlefish Sepia officinalis.

BACKGROUND: The cephalopod mollusk Sepia officinalis can be considered as a relevant model for studying reproduction strategies associated to seasonal migrations. Using transcriptomic and peptidomic approaches, we aim to identify peptide sex pheromones that are thought to induce the aggregation of mature cuttlefish in their egg-laying areas. RESULTS: To facilitate the identification of sex pheromones, 576 5'-expressed sequence tags (ESTs) were sequenced from a single cDNA library generated from accessory sex glands of female cuttlefish. Our analysis yielded 223 unique sequences composed of 186 singletons and 37 contigs. Three major redundant ESTs called SPα, SPα' and SPß were identified as good candidates for putative sex pheromone transcripts and are part of the 87 unique sequences classified as unknown. The alignment of translated SPα and SPα' revealed a high level of conservation, with 98.4% identity. Translation led to a 248-amino acid precursor containing six peptides with multiple putative disulfide bonds. The alignment of SPα-α' with SPß revealed a partial structural conservation, with 37.3% identity. Translation of SPß led to a 252-amino acid precursor containing five peptides. The occurrence of a signal peptide on SPα, SPα' and SPß showed that the peptides were secreted. RT-PCR and mass spectrometry analyses revealed a co-localization of transcripts and expression products in the oviduct gland. Preliminary in vitro experiments performed on gills and penises revealed target organs involved in mating and ventilation. CONCLUSIONS: The analysis of the accessory sex gland transcriptome of Sepia officinalis led to the identification of peptidic sex pheromones. Although preliminary functional tests suggested the involvement of the α3 and ß2 peptides in ventilation and mating stimulation, further functional investigations will make it possible to identify the complete set of biological activities expected from waterborne pheromones.