Alternative strategies for the recombinant synthesis, DOPA modification and analysis of mussel foot proteins - A case study for Mefp-3 from Mytilus edulis.

Mussel foot proteins (Mfps) possess unique binding properties to various surfaces due to the presence of L-3,4-dihydroxyphenylalanine (DOPA). Mytilus edulis foot protein-3 (Mefp-3) is one of several proteins in the byssal adhesive plaque. Its localization at the plaque-substrate interface approved that Mefp-3 plays a key role in adhesion. Therefore, the protein is suitable for the development of innovative bio-based binders. However, recombinant Mfp-3s are mainly purified from inclusion bodies under denaturing conditions. Here, we describe a robust and reproducible protocol for obtaining soluble and tag-free Mefp-3 using the SUMO-fusion technology. Additionally, a microbial tyrosinase from Verrucomicrobium spinosum was used for the in vitro hydroxylation of peptide-bound tyrosines in Mefp-3 for the first time. The highly hydroxylated Mefp-3, confirmed by MALDI-TOF-MS, exhibited excellent adhesive properties comparable to a commercial glue. These results demonstrate a concerted and simplified high yield production process for recombinant soluble and tag-free Mfp3-based proteins with on demand DOPA modification.

Determining the Properties that Govern Selective Ingestion and Egestion of Microplastics by the Blue Mussel (Mytilus edulis) and Eastern Oyster (Crassostrea virginica).

Suspension feeding bivalve molluscs interact with different types of microplastics (MP) suspended in the water column. Most bivalves are selective suspension feeders and, thus, do not consume all particles to which they are exposed. Selection depends upon the physicochemical properties and size of the particle. Recent work has provided evidence that blue mussels, Mytilus edulis, and eastern oysters, Crassostrea virginica, ingest and egest microspheres (polystyrene) and microfibers (nylon) differently, but whether other factors, such as polymer type and shape, mediate selection have not been explored. To investigate these factors, mussels and oysters were offered similar sized nylon (Ny) and polyester (PES) microfibers or polyethylene (PE) and polystyrene (PS) microspheres, or different sized PES microfibers during a 2 h exposure. Feces and pseudofeces were collected separately and analyzed for MPs, and the data were used to develop a linear regression model for selection. Results demonstrated clear species-specific differences in the efficiency of particle selection. Both mussels and oysters, however, exhibited size-based rejection of PES microfibers, ingesting a higher proportion of shorter fibers than longer fibers. Polymer type did not impact selection of fibers or spheres. The relative size of particles (area and perimeter) was found to be the most important factor in predicting whether a MP will be rejected or ingested.

Preparation and Hepatoprotective Activities of Peptides Derived from Mussels (Mytilus edulis) and Clams (Ruditapes philippinarum).

Low molecular weight (<5 kDa) peptides from mussels (Mytilus edulis) (MPs) and the peptides from clams (Ruditapes philippinarum) (CPs) were prepared through enzymatic hydrolysis by proteases (dispase, pepsin, trypsin, alcalase and papain). Both the MPs and the CPs showed excellent in vitro scavenging ability of free radicals including OH, DPPH and ABTS in the concentration range of 0.625−10.000 mg/mL. By contrast, the MPs hydrolyzed by alcalase (MPs-A) and the CPs hydrolyzed by dispase (CPs-D) had the highest antioxidant activities. Furthermore, MPs-A and CPs-D exhibited protective capabilities against oxidative damage induced by H2O2 in HepG2 cells in the concentration range of 25−800 µg/mL. Meanwhile, compared with the corresponding indicators of the negative control (alcohol-fed) mice, lower contents of hepatic MDA and serums ALT and AST, as well as higher activities of hepatic SOD and GSH-PX were observed in experiment mice treated with MPs-A and CPs-D. The present results clearly indicated that Mytilus edulis and Ruditapes philippinarum are good sources of hepatoprotective peptides.

"Don't Blast": blast-in-place (BiP) operations of dumped World War munitions in the oceans significantly increase hazards to the environment and the human seafood consumer.

The seas worldwide are threatened by a "new" source of pollution: millions of tons of all kind of warfare material have been dumped intentionally after World War I and II, in addition to mine barriers, failed detonations as well as shot down military planes and sunken ship wrecks carrying munitions. For example, in the German parts of the North and Baltic Sea approximately 1.6 million metric tons of toxic conventional explosives (TNT and others) and more than 5000 metric tons of chemical weapons are present. Such unexploded ordnance (UXO) constitutes a direct risk of detonation with increased human access (fisheries, water sports, cable constructions, wind farms and pipelines). Moreover, after more than 70 years of resting on the seabed, the metal shells of these munitions items corrode, such that chemicals leak out and distribute in the marine environment. Explosive chemicals such as TNT and its derivatives are known for their toxicity and carcinogenicity. In order not to endanger today's shipping traffic or the installation of pipelines and offshore plants by uncontrolled explosions, controlled blast-in-place (BiP) operations of these dangerous relics is a common practice worldwide. However, blast-in-place methods of in situ munitions disposal often result in incomplete (low-order) detonation, leaving substantial quantities of the explosive material in the environment. In the present free field investigation, we placed mussels (Mytilus spp.) as a biomonitoring system in an area of the Baltic Sea where BiP operations took place and where, by visual inspections by scientific divers, smaller and larger pieces of munitions-related materials were scattered on the seafloor. After recovery, the mussels were transferred to our laboratory and analyzed for TNT and its derivatives via gas chromatography and mass spectroscopy. Our data unequivocally demonstrate that low-order BiP operations of dumped munitions in the sea lead to multiple increases in the concentration of TNT and its metabolites in the mussels when compared to similar studies at corroding but still encased mines. For this reason, we explicitly criticize BiP operations because of the resulting environmental hazards, which can ultimately even endanger human seafood consumers.

Improved Isolation Procedures for Okadaic Acid Group Toxins from Shellfish (Mytilus edulis) and Microalgae (Prorocentrum lima).

Okadaic acid (OA) group toxins may accumulate in shellfish and can result in diarrhetic shellfish poisoning when consumed by humans, and are therefore regulated. Purified toxins are required for the production of certified reference materials used to accurately quantitate toxin levels in shellfish and water samples, and for other research purposes. An improved procedure was developed for the isolation of dinophysistoxin-2 (DTX2) from shellfish (M. edulis), reducing the number of purification steps from eight to five, thereby increasing recoveries to ~68%, compared to ~40% in a previously reported method, and a purity of >95%. Cell densities and toxin production were monitored in cultures of Prorocentrum lima, that produced OA, DTX1, and their esters, over ~1.5 years with maximum cell densities of ~70,000 cells mL-1 observed. Toxin accumulation progressively increased over the study period, to ~0.7 and 2.1 mg L-1 of OA and DTX1 (including their esters), respectively, providing information on appropriate harvesting times. A procedure for the purification of OA and DTX1 from the harvested biomass was developed employing four purification steps, with recoveries of ~76% and purities of >95% being achieved. Purities were confirmed by LC-HRMS, LC-UV, and NMR spectroscopy. Additional stability observations led to a better understanding of the chemistry of these toxins.

Blue Mussel-Derived Peptides PIISVYWK and FSVVPSPK Trigger Wnt/ß-Catenin Signaling-Mediated Osteogenesis in Human Bone Marrow Mesenchymal Stem Cells.

Marine-derived bioactive peptides have shown potential bone health promoting effects. Although various marine-derived bioactive peptides have potential nutraceutical or pharmaceutical properties, only a few of them are commercially available. This study presented an osteogenic mechanism of blue mussel-derived peptides PIISVYWK and FSVVPSPK as potential bone health promoting agents in human bone marrow-derived mesenchymal stem cells (hBMMSCs). Alkaline phosphatase (ALP) activity and mineralization were stimulated using PIISVYWK and FSVVPSPK as early and late markers of osteogenesis in a concentration-dependent manner. Western blot and RT-qPCR results revealed that PIISVYWK and FSVVPSPK increased osteoblast differentiation of hBMMSCs by activating canonical Wnt/ß-catenin signaling-related proteins and mRNAs. Immunofluorescence images confirmed nuclear translocation of ß-catenin in osteogenic differentiation. Treatment with the pharmacological inhibitor DKK-1 blocked PIISVYWK- and FSVVPSPK-induced ALP activity and mineralization, as well as mRNA expression of the canonical Wnt/ß-catenin signaling pathway in hBMMSC differentiation into osteoblasts. These findings suggested that PIISVYWK and FSVVPSPK promoted the canonical Wnt/ß-catenin signaling pathway in osteogenesis of hBMMSCs. Blue mussel-derived PIISVYWK and FSVVPSPK might help develop peptide-based therapeutic agents for bone-related diseases.

The fate of the main naturally occurring radionuclides in mussels (Mytilus edulis) and their radiological impact on human beings.

A comprehensive study was made on the activity concentrations, concentration factors, and radiation exposure impact of the main naturally occurring radionuclides in blue mussels collected in the Mediterranean Sea. The results showed that the concentrations of the measured radionuclides were site-specific and all detectable in gram-size samples of the soft tissues of the mussels, especially, some activity concentrations can reach as high as 16.8-102 Bq kg-1 for 210Po, 16.7-66.8 Bq kg-1 for 40K and 0.602-3.21 Bq kg-1 for 210Pb. The obtained mean concentration factors in the soft tissues of the mussel samples are 6.30 ± 2.40 for 238U and 234U, 4.68 ± 1.73 for 235U, (2.72 ± 1.73) × 104 for 232Th and 230Th, 480 ± 265 for 228Th, 33.3 ± 13.3 for 226Ra, 100 ± 52 for 224Ra and 29.0 ± 14.7 for 228Ra, (1.22 ± 0.72) × 104 for 210Po, 517 ± 280 for 210Pb and 2.57 ± 0.80 for 40K. The estimated mean committed effective doses of 238U, 234U, 235U, 232Th, 230Th, 228Th, 226Ra, 224Ra, 228Ra, 210Po, 210Pb and 40K to an adult due to mussel ingestion are 0.073 ± 0.027, 0.089 ± 0.035, 0.0030 ± 0.0011, 0.128 ± 0.098, 0.117 ± 0.081, 0.056 ± 0.031, 0.145 ± 0.058, 0.0487 ± 0.0250, 0.395 ± 0.200, 352 ± 209, 6.00 ± 3.25 and 1.74 ± 0.54 µSv a-1, respectively. Among the elements or nuclides, 210Po is the dominant dose contributor which contributes 96.9% of total dose fraction, and the relative dose contribution is in the order of 210Po > 210Pb > 40K > radium ≥ thorium ≥ uranium.

Proteomic investigation of the blue mussel larval shell organic matrix.

Shell matrix proteins (SMPs) are occluded within molluscan shells and are fundamental to the biological control over mineralization. While many studies have been performed on adult SMPs, those of larval stages remain largely undescribed. Therefore, this study aimed to characterize the larval shell proteome of the blue mussel for the first time and to compare it to adult mussel shell proteomes. Following development of a method for cleaning larval shells of tissue contaminants, 49 SMPs were identified using shotgun proteomics. Twenty-one proteins were independently identified in all samples indicating that they form a subset of the core larval shell proteome. These included: the blue mussel shell protein, a peroxidase domain-containing sequence, a laminin G domain-containing sequence, a ZIP domain-containing sequence and a ferric-chelate reductase 1-like sequence. Additional SMP domains identified were: fibronectin type III, BPTI/Kunitz, chitin-binding type 3, thyroglobulin and EF-hand. While key predictable molluscan shell matrix functions are identified, 67% of sequences remain unknown or uncharacterized, indicating that this shell proteome is unique to mussel larvae. Specifically, comparison with adult mytilids reveals that nine domains are exclusive to the larval shell proteome and only four domains are conserved among species and developmental stages. Thus, strong species-specific and ontogenetic variation exists in shell proteome composition.

Amino Acid Composition, Antioxidant, and Cytoprotective Effect of Blue Mussel (Mytilus edulis) Hydrolysate through the Inhibition of Caspase-3 Activation in Oxidative Stress-Mediated Endothelial Cell Injury.

Enhanced oxidative stress plays a central role in promoting endothelial dysfunction, leading to the development of atherosclerosis. In this study, we investigated the protective effects of the hydrolysates derived from blue mussel (Mytilus edulis) against H2O2-mediated oxidative injury in human umbilical vein endothelial cells (HUVECs). The blue mussel hydrolysates were prepared by enzymatic hydrolysis with eight proteases, and blue mussel-α-chymotrypsin hydrolysate (BMCH) showed the highest antioxidant activities in DPPH radical scavenging, ABTS⁺ radical scavenging, and ORAC value compared to those of the other hydrolysates. BMCH also inhibited Cu2+-mediated low density lipoprotein (LDL) oxidation. Treatment of H2O2 resulted in the decreased HUVEC viability whereas pre-treatment with BMCH increased HUVEC viability and reduced reactive oxygen species (ROS) generation. BMCH pre-treatment increased cellular antioxidant capacities, including levels of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) against H2O2-mediated oxidative stress in HUVECs. Flow cytometry and western blot analysis revealed that BMCH pre-treatment significantly reduced H2O2-mediated HUVEC apoptosis through inhibition of caspase-3 activation. Real-time-qPCR analysis showed that BMCH down-regulated expression of p53 and caspase-3 genes, as well as decreased the bax/bcl-2 ratio. Taken together, these results indicate that BMCH may be useful as functional food ingredients for protecting endothelial dysfunction or related disease.

Green sea urchins (Strongylocentrotus droebachiensis) as potential biomonitors of metal pollution near a former lead-zinc mine in West Greenland.

In this study, metal accumulation in green sea urchins (Strongylocentrotus droebachiensis) was investigated near the former Black Angel lead-zinc mine in Maarmorilik, West Greenland. Sea urchins (n = 9-11; 31-59 mm in diameter) were collected from three stations located at < 1 km, 5 km, and 12 km (reference site) away from the former mine site, respectively. After collection, tissue of the sea urchins was divided into gonads and remaining soft parts (viscera) before subjected to chemical analyses. Focus was on eight elements found in elevated concentrations in the mine waste (iron, copper, zinc, arsenic, silver, cadmium, mercury and lead). Sea urchins at the mine site contained significantly more copper, mercury and lead compared with the reference site for both the gonads and viscera, while the latter also contained significantly more iron, zinc and silver. Arsenic and cadmium were not significantly elevated in sea urchins at the mine site. Most elements were found in higher concentrations in the viscera compared with the gonads. For comprehensive monitoring of metal pollution at mine sites, a diverse selection of monitoring organisms is necessary. The study shows that green sea urchins accumulate selected metals and can be used as a monitoring organism for mining pollution, at least for iron, copper, zinc, silver, mercury and lead. However, the results also show that green sea urchins are less likely to reflect small environmental changes in loading of most metals (except iron, copper and silver) and for arsenic compared to suspension feeders such as blue mussels.

Identification of 21,22-Dehydroazaspiracids in Mussels ( Mytilus edulis) and in Vitro Toxicity of Azaspiracid-26.

Azaspiracids (AZAs) are marine biotoxins produced by the genera Azadinium and Amphidoma, pelagic marine dinoflagellates that may accumulate in shellfish resulting in human illness following consumption. The complexity of these toxins has been well documented, with more than 40 structural variants reported that are produced by dinoflagellates, result from metabolism in shellfish, or are extraction artifacts. Approximately 34 µg of a new AZA with MW 823 Da (AZA26 (3)) was isolated from blue mussels ( Mytilus edulis), and its structure determined by MS and NMR spectroscopy. AZA26, possibly a bioconversion product of AZA5, lacked the C-20-C-21 diol present in all AZAs reported thus far and had a 21,22-olefin and a keto group at C-23. Toxicological assessment of 3 using an in vitro model system based on Jurkat T lymphocyte cells showed the potency to be ∼30-fold lower than that of AZA1. The corresponding 21,22-dehydro-23-oxo-analogue of AZA10 (AZA28) and 21,22-dehydro analogues of AZA3, -4, -5, -6, -9, and -10 (AZA25, -48 (4), -60, -27, -49, and -61, respectively) were also identified by HRMS/MS, periodate cleavage reactivity, conversion from known analogues, and NMR (for 4 that was present in a partially purified sample of AZA7).

Identification and Antithrombotic Activity of Peptides from Blue Mussel (Mytilus edulis) Protein.

The blue mussel (Mytilus edulis) reportedly contains many bioactive components of nutritional value. Water-, salt- and acid-soluble M. edulis protein fractions were obtained and the proteins were trypsinized. The resultant peptides were analyzed by ultra-performance liquid chromatography quadrupole time of flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). 387 unique peptides were identified that matched 81 precursor proteins. Molecular mass distributions of the proteins and peptides were analyzed by sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE). The differences between the three protein samples were studied by Venn diagram of peptide and protein compositions. Toxicity, allergic and antithrombotic activity of peptides was predicted using database website and molecular docking respectively. The antithrombotic activity of enzymatic hydrolysate from water-, salt- and acid-soluble M. edulis protein were 40.17%, 85.74%, 82.00% at 5 mg/mL, respectively. Active mechanism of antithrombotic peptide (ELEDSLDSER) was also research about amino acid binding sites and interaction, simultaneously.

A mussel tissue certified reference material for multiple phycotoxins. Part 4: certification.

A freeze-dried mussel tissue (Mytilus edulis) reference material (CRM-FDMT1) was produced containing multiple groups of shellfish toxins. Homogeneity and stability testing showed the material to be fit for purpose. The next phase of work was to assign certified values and uncertainties to 10 analytes from six different toxin groups. Efforts involved optimizing extraction procedures for the various toxin groups and performing measurements using liquid chromatography-based analytical methods. A key aspect of the work was compensating for matrix effects associated with liquid chromatography-mass spectrometry through standard addition, dilution, or matrix-matched calibration. Certified mass fraction values are reported as mg/kg of CRM-FDMT1 powder as bottled for azaspiracid-1, -2, and -3 (4.10 ± 0.40; 1.13± 0.10; 0.96 ± 0.10, respectively), okadaic acid, dinophysistoxin-1 and -2 (1.59 ± 0.18; 0.68 ± 0.07; 3.57± 0.33, respectively), yessotoxin (2.49 ± 0.28), pectenotoxin-2 (0.66 ± 0.06), 13-desmethylspirolide-C (2.70 ± 0.26), and domoic acid (126 ± 10). Combined uncertainties for the certified values include contributions from homogeneity, stability, and characterization experiments. The commutability of CRM-FDMT1 was assessed by examining the extractability and matrix effects for the freeze-dried material in comparison with its equivalent wet tissue homogenate. CRM-FDMT1 is the first shellfish matrix CRM with certified values for yessotoxins, pectenotoxins or spirolides, and is the first CRM certified for multiple toxin groups. CRM-FDMT1 is a valuable tool for quality assurance of phycotoxin monitoring programs and for analytical method development and validation. Graphical Abstract CRM-FDMT1 is a multi-toxin mussel tissue certified reference material (CRM) to aid in development and validation of analytical methods for measuring the levels of algal toxins in seafood.

Interactive effects of seawater acidification and elevated temperature on biomineralization and amino acid metabolism in the mussel Mytilus edulis.

Seawater acidification and warming resulting from anthropogenic production of carbon dioxide are increasing threats to marine ecosystems. Previous studies have documented the effects of either seawater acidification or warming on marine calcifiers; however, the combined effects of these stressors are poorly understood. In our study, we examined the interactive effects of elevated carbon dioxide partial pressure (P(CO2)) and temperature on biomineralization and amino acid content in an ecologically and economically important mussel, Mytilus edulis. Adult M. edulis were reared at different combinations of P(CO2) (pH 8.1 and 7.8) and temperature (19, 22 and 25°C) for 2 months. The results indicated that elevated P(CO2) significantly decreased the net calcification rate, the calcium content and the Ca/Mg ratio of the shells, induced the differential expression of biomineralization-related genes, modified shell ultrastructure and altered amino acid content, implying significant effects of seawater acidification on biomineralization and amino acid metabolism. Notably, elevated temperature enhanced the effects of seawater acidification on these parameters. The shell breaking force significantly decreased under elevated P(CO2), but the effect was not exacerbated by elevated temperature. The results suggest that the interactive effects of seawater acidification and elevated temperature on mussels are likely to have ecological and functional implications. This study is therefore helpful for better understanding the underlying effects of changing marine environments on mussels and other marine calcifiers.

Multidecadal Field Data Support Intimate Links between Phytoplankton Dynamics and PCB Concentrations in Marine Sediments and Biota.

We analyzed three decades of field observations in the North Sea with additive models to infer spatiotemporal trends of chlorophyll a concentration, sediment organic carbon content, and polychlorinated biphenyls (PCBs) concentrations in mussels and sediments. By doing so, we separated long-term changes in PCB concentrations from seasonal variability. Using the inferred seasonal variability, we demonstrated that phytoplankton blooms in spring and autumn correspond to the annual maxima of the organic carbon content (r = 0.56; p = 0.004) and the PCB concentrations in sediments (r = 0.57; p = 0.004). Furthermore, we found a negative correlation between the PCB concentrations in sediments and in blue mussels (Mytilus edulis; r = -0.33, p = 0.012), which is probably related to the cleansing of the dissolved PCB phase driven by sinking organic matter during phytoplankton blooms and the filter-feeding behavior of the blue mussel. The present research demonstrates the role of seasonal phytoplankton dynamics in the environmental fate of PCBs at large spatiotemporal scales.

Improved detection of ß-N-methylamino-L-alanine using N-hydroxysuccinimide ester of N-butylnicotinic acid for the localization of BMAA in blue mussels (Mytilus edulis).

ß-N-Methylamino-L-alanine (BMAA) is an important non-protein amino acid linked to neurodegenerative diseases, specifically amyotrophic lateral sclerosis (ALS). Because it can be transferred and bioaccumulated higher up the food chain, it poses significant public health concerns; thus, improved detection methods are of prime importance for the identification and management of these toxins. Here, we report the successful use of N-hydroxysuccinimide ester of N-butylnicotinic acid (C4-NA-NHS) for the efficient separation of BMAA from its isomers and higher sensitivity in detecting BMAA compared to the current method of choice using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization. Implementation of this efficient method allowed localization of BMAA in the non-visceral tissues of blue mussels, suggesting that more efficient depuration may be required to remove this toxin prior to consumption. This is a crucial method in establishing the absence or presence of the neurotoxic amino acid BMAA in food, environmental or biomedical samples.

A mussel (Mytilus edulis) tissue certified reference material for the marine biotoxins azaspiracids.

Azaspiracids (AZAs) are lipophilic biotoxins produced by marine algae that can contaminate shellfish and cause human illness. The European Union (EU) regulates the level of AZAs in shellfish destined for the commercial market, with liquid chromatography-mass spectrometry (LC-MS) being used as the official reference method for regulatory analysis. Certified reference materials (CRMs) are essential tools for the development, validation, and quality control of LC-MS methods. This paper describes the work that went into the planning, preparation, characterization, and certification of CRM-AZA-Mus, a tissue matrix CRM, which was prepared as a wet homogenate from mussels (Mytilus edulis) naturally contaminated with AZAs. The homogeneity and stability of CRM-AZA-Mus were evaluated, and the CRM was found to be fit for purpose. Extraction and LC-MS/MS methods were developed to accurately certify the concentrations of AZA1 (1.16 mg/kg), AZA2 (0.27 mg/kg), and AZA3 (0.21 mg/kg) in the CRM. Quantitation methods based on standard addition and matrix-matched calibration were used to compensate for the matrix effects in LC-MS/MS. Other toxins present in this CRM at lower levels were also measured with information values reported for okadaic acid, dinophysistoxin-2, yessotoxin, and several spirolides.

Long-term stability and temporal trends of organic contaminants in four collections of mussel tissue frozen standard reference materials.

The stability of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and chlorinated pesticides in frozen mussel tissue Standard Reference Materials (SRMs) stored at -80 °C was assessed by analyzing samples of SRM 1974, SRM 1974a, and SRM 1974b Organics in Mussel Tissue (Mytilus edulis) periodically over 25 y, 20 y, and 12 y, respectively. The most recent analyses were performed during the certification of the fourth release of this material, SRM 1974c. Results indicate the concentrations of these persistent organic pollutants have not changed during storage at -80 °C. In addition, brominated diphenyl ethers (BDEs) were quantified in each of the materials during this study. The stability information is important for on-going monitoring studies collecting large quantities of samples for future analyses (i.e., formally established specimen banking programs). Since all four mussel tissue SRMs were prepared from mussels collected at the same site in Dorchester Bay, MA, USA, the results provide a temporal trend study for these contaminants over a 17 year period (1987 to 2004).

Ocean acidification reduces the crystallographic control in juvenile mussel shells.

Global climate change threatens the oceans as anthropogenic carbon dioxide causes ocean acidification and reduced carbonate saturation. Future projections indicate under saturation of aragonite, and potentially calcite, in the oceans by 2100. Calcifying organisms are those most at risk from such ocean acidification, as carbonate is vital in the biomineralisation of their calcium carbonate protective shells. This study highlights the importance of multi-generational studies to investigate how marine organisms can potentially adapt to future projected global climate change. Mytilus edulis is an economically important marine calcifier vulnerable to decreasing carbonate saturation as their shells comprise two calcium carbonate polymorphs: aragonite and calcite. M. edulis specimens were cultured under current and projected pCO2 (380, 550, 750 and 1000µatm), following 6months of experimental culture, adults produced second generation juvenile mussels. Juvenile mussel shells were examined for structural and crystallographic orientation of aragonite and calcite. At 1000µatm pCO2, juvenile mussels spawned and grown under this high pCO2 do not produce aragonite which is more vulnerable to carbonate under-saturation than calcite. Calcite and aragonite were produced at 380, 550 and 750µatm pCO2. Electron back scatter diffraction analyses reveal less constraint in crystallographic orientation with increased pCO2. Shell formation is maintained, although the nacre crystals appear corroded and crystals are not so closely layered together. The differences in ultrastructure and crystallography in shells formed by juveniles spawned from adults in high pCO2 conditions may prove instrumental in their ability to survive ocean acidification.

Synthesis of adhesive peptides similar to those found in blue mussel (Mytilus edulis) using papain and tyrosinase.

The blue mussel (Mytilus edulis) foot protein 5 (Mefp-5) is an adhesive protein that is mainly composed of glycine, l-lysine, and 3,4-dihydroxy-l-phenylalanine (DOPA). Thousands of adhesive pads have been analyzed in previous studies, whereby it has been found that adhesion is largely achieved by the redox-chemistry of DOPA, and that l-lysine (approximately 20 mol %) affects the formation of molecular networks. While DOPA and lysine are essential for biomimetic adhesive design, the synthesis of copolymers containing DOPA is limited, in terms of yield, by the multiple reaction steps required. Here, we synthesized adhesive peptides containing DOPA and l-lysine via two enzymatic reactions, namely, chemoenzymatic synthesis of copolypeptides of l-tyrosine and l-lysine by Papaya peptidase I (papain), as well as the enzymatic conversion from l-tyrosine to DOPA by tyrosinase. The synthesis was characterized in terms of yield, degree of polymerization, and composition of the polypeptide. In addition, the conversion of tyrosine to DOPA by tyrosinase was evaluated quantitatively by nuclear magnetic resonance and amino acid analysis. The adhesive properties of the resulting peptides, consisting of DOPA, l-lysine, and l-tyrosine, were evaluated at various pH levels with different protonation/deprotonation states. Our results show that deprotonated DOPA is required for adhesive function, and the deprotonated primary amine group of lysine induces molecular networks by varying the elastic moduli of the adhesives. In this study, we demonstrate the benefit of combining multiple enzymatic reactions, including chemoenzymatic polymerization, in obtaining new types of peptide-based materials.