E3 Ubiquitin Ligase CHIP Inhibits Haemocyte Proliferation and Differentiation via the Ubiquitination of Runx in the Pacific Oyster.

Mollusca first evolve primitive immune cells (namely, haemocytes), which assemble a notable complex innate immune system, which are continuously produced through proliferation and differentiation and infused in the haemolymph. As a typical E3 ligase, CHIP is critical for immune cell turnover and homeostasis in vertebrates. In this study, a CHIP homolog (CgCHIP) with a high expression in haemocytes was identified in oysters to investigate its role in the proliferation and differentiation of ancient innate immune cells. CgCHIP exhibited a widespread distribution across all haemocyte subpopulations, and the knockdown of CgCHIP altered the composition of haemocytes as examined by flow cytometry. Mechanistically screened with bioinformatics and immunoprecipitation, a key haematopoietic transcription factor CgRunx was identified as a substrate of CgCHIP. Moreover, amino acids in the interacted intervals of CgCHIP and CgRunx were determined by molecular docking. Experimental evidence from an in vitro culture model of an agranulocyte subpopulation and an in vivo oyster model revealed that the knockdown of CgCHIP and CgRunx had opposing effects on agranulocyte (precursor cells) differentiation and granulocyte (effector cells) proliferation. In summary, CgCHIP negatively regulated agranulocyte differentiation and granulocyte proliferation by mediating the ubiquitination and degradation of CgRunx in oysters. These results offer insight into the involvement of ubiquitylation in controlling haemocyte turnover in primitive invertebrates.

Testing combined effects of environmental trace metals/arsenic and marine trophic status on the bioaccumulation in Pacific oysters: Insights from 22-site field samplings.

Pacific oysters were sampled from 22 human-impacted sites in northeastern Japan to measure Cr, Cu, Zn, Pb, Cd, and As. The hazard quotient was slightly >1 for Cu and/or As at two sites, but <1 for all metal species and As at the other sites, indicating low human health risks. Oysters' Cu, Zn, and Pb contents were positively related to their concentrations in the sediment, while Cr and As were not. Oysters' Cu and Zn were negatively related to the inorganic nitrogen in seawater, while oysters' Pb and As showed positive relationships with the particulate organic carbon. These findings suggest that marine trophic status affects oysters' metal uptake differently among the metal species. Furthermore, oysters' Cr, Cu, Zn, and Pb contents were negatively related to their eicosapentaenoic acid content and condition index. Therefore, the nutritional conditions of oysters may influence their elimination or accumulation of these metals.

Effects of deep, air and vacuum frying on oyster quality and protein-mediated mechanism analysis via TMT quantitative proteomics.

Fried oyster is a popular aquatic food product in East Asia, but nutrient loss during thermal processing become a significant concern. The goal of this research was to examine the impact of distinct frying techniques, including deep frying (DF), air frying (AF), and vacuum frying (VF), on the nutritional, textural and flavor characteristics of oysters. The VF method demonstrated superior retention of beneficial properties and flavor, and reduced protein and lipid oxidation compared to the DF and AF methods. Furthermore, proteomic analysis of oysters was attempted to explain the molecular mechanisms governing the influence of key differential proteins. 20 major differential proteins, including actin-2 protein, tryptophan 2,3-dioxygenase and 1-alph, involved in oyster protein oxidation were identified, annotated and analyzed to elucidate their influence mechanisms. This research provides a deeper understanding of intricate interactions between frying techniques and oyster biochemistry, which offers valuable implications for enhancing food quality in seafood industry.

Metabolic dysfunctions in pearl oysters following recurrent marine heatwaves.

Marine heatwaves (MHWs) have become more frequent, intense and extreme in oceanic systems in the past decade, resulting in mass mortality events of marine invertebrates and devastating coastal marine ecosystems. While metabolic homeostasis is a fundamental requirement in stress tolerance, little is known about its role under intensifying MHWs conditions. Here, we investigated impacts of MHWs on the metabolism in pearl oysters (Pinctada maxima) - an ecologically and economically significant bivalve species in tropical ecosystems. Activities of digestive enzymes (gastric proteases, lipases, and amylases) did not significantly respond to various scenario of recurrent MHWs varying from 24 °C to 28 °C (moderate) and 32 °C (severe). The metabolomics analysis revealed nine and five key metabolism pathways under both MHWs scenarios. Specifically, pathways associated with energy metabolism were impaired by moderate MHWs, manifesting in downregulation of differential metabolite (The nicotinic acid and N-acetyl-glutamic acid). The content of CDP-ethanolamine was significantly decrease, and the perturbations of oxidative stress caused by the decreased of content of D-glutamine. Metabolites related to a suite of body functions (e.g., the lipid metabolism, biomineralization, and antioxidant defenses) showed significantly negative responses by severe MHWs. These findings reveal the metabolic impairments of marine bivalves when subjected to MHWs varying in intensity and frequency, implying cascading consequences which deserve further investigation.

Tamoxifen induces biochemical responses in Pacific oysters Magallana gigas (Thunberg, 1793) at environmentally relevant concentrations.

The activities of catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), and glutathione-S-transferase (GST) were evaluated in the gills (GI) and digestive gland (DG) of Magallana gigas oysters exposed to tamoxifen (TAM) at environmental concentrations of 10 and 100 ng L-1 for 1 and 4 days. A higher CAT activity in the GI and DG and higher GPx activity only in the DG was observed of oysters exposed to both concentrations after 1 day. Furthermore, a significant increase in GR and G6PDH, was detected in the DG after 1 day of exposure to 10 ng L-1 and only G6PDH activity increase after 1 day of exposure to 10 ng L-1 in the GI. This suggests that the DG is a tissue more sensitive to TAM exposure and was confirmed with the individual Integrated Biomarker Response version 2 index (IBRv2i), highlighting the acute stress caused by TAM and a cellular adaptation.

Exploring the co-exposure effects of environmentally relevant microplastics and an estrogenic mixture on the metabolome of the Sydney rock oyster.

In aquatic environments the concurrent exposure of molluscs to microplastics (MPs) and estrogens is common, as these pollutants are frequently released by wastewater treatment plants into estuaries. Therefore, this study aimed to evaluate the independent and co-exposure impacts of polyethylene microplastics (PE-MPs) and estrogenic endocrine-disrupting chemicals (EEDCs) at environmentally relevant concentrations on polar metabolites and morphological parameters of the Sydney rock oyster. A seven-day acute exposure revealed no discernible differences in morphology; however, significant variations in polar metabolites were observed across oyster tissues. The altered metabolites were mostly amino acids, carbohydrates and intermediates of the Kreb's cycle. The perturbation of metabolites were tissue and sex-specific. All treatments generally showed an increase of metabolites relative to controls - a possible stimulatory and/or a potential hormetic response. The presence of MPs impeded the exposure of adsorbed and free EEDCs potentially due to the selective feeding behaviour of oysters to microplastics, favouring algae over similar-sized PE-MPs, and the formation of an eco/bio-corona involving faeces, pseudo-faeces, natural organic matter, and algae.

Genome-wide identification of the HSP70 genes in Pacific oyster Magallana gigas and their response to heat stress.

Heat shock protein 70 (HSP70), the most prominent and well-characterized stress protein in animals, plays an important role in assisting animals in responding to various adverse conditions. In the present study, a total of 113 HSP70 gene family members were identified in the updated genome of Magallana gigas (designated MgHSP70) (previously known as Crassostrea gigas). There were 75, 12, 11, and 8 HSP70s located in the cytoplasm, nucleus, mitochondria, and endoplasmic reticulum, respectively, and 7 HSP70s were located in both the nucleus and cytoplasm. Among 113 MgHSP70 genes, 107 were unevenly distributed in 8 chromosomes of M. gigas with the greatest number in chromosome 07 (61 genes, 57.01%). The MgHSP70 gene family members were mainly assigned into five clusters, among which the HSPa12 subfamily underwent lineage-specific expansion, consisting of 89 members. A total of 68 MgHSP70 genes (60.18%) were tandemly duplicated and formed 30 gene pairs, among which 14 gene pairs were under strong positive selection. In general, the expression of MgHSP70s was tissue-specific, with the highest expression in labial palp and gill and the lowest expression in adductor muscle and hemocytes. There were 35, 31, and 47 significantly upregulated genes at 6, 12, and 24 h after heat shock treatment (28 °C), respectively. The expression patterns of different tandemly duplicated genes exhibited distinct characteristics after shock treatment, indicating that these genes may have different functions. Nevertheless, genes within the same tandemly duplicated group exhibit similar expression patterns. Most of the tandemly duplicated HSP70 gene pairs showed the highest expression levels at 24 h. This study provides a comprehensive description of the MgHSP70 gene family in M. gigas and offers valuable insights into the functions of HSP70 in the mollusc adaptation of oysters to environmental stress.

Temporal distribution of microplastics and other anthropogenic particles in four marine species from the Atlantic coast (France).

The characterization of microplastic (MP) contamination in marine species is increasing as concerns about environmental and food safety are more and more discussed. Here, we reported a quantitative and qualitative assessment of the contamination by anthropogenic particles (from visual sorting; AP) and MP (plastic-made) in the whole soft body or digestive tract of marine species. Four commercial species were studied, namely the Pacific oyster (Magallana gigas), the spiny spider crab (Maja sp.), the common sole (Solea solea) and seabass (Dicentrarchus labrax or punctatus). AP and MP uptake were studied over three to four seasons depending on the species. After tissues digestion, particles were extracted under a stereomicroscope and morphometric characteristics were reported. Then, polymers were identified by ATR-FTIR spectroscopy. Seasonal variations were mainly described in the Pacific oyster as AP uptake was lower in autumn and MP uptake was higher in spring. These variations may be linked to the reproduction and growth cycles of this species. Moreover, seabass ingestion was lower in autumn compared to winter. Contamination in spider crabs and soles showed either weak or no seasonal trends, both quantitatively and qualitatively. Overall, AP contamination in all studied species ranged from 1.17 ± 1.89 AP.ind-1 (in sole) to 4.07 ± 6.69 AP.ind-1 (in seabass) while MP contamination ranged from 0.10 ± 0.37 MP.ind-1 (in sole) to 1.09 ± 3.06 MP.ind-1 (in spider crab). Fibers were mostly reported in all species (at least 77.7%), along with cellulosic polymers (at least 43.7%). AP and MP uptake were detected in all species and at almost all seasons, with the only exception of the common sole during autumn. Therefore, this study emphasizes the ubiquity of AP and MP contamination in marine species and provides new knowledges about seasonal uptake by commercial species.

A novel route of intercellular copper transport and detoxification in oyster hemocytes.

Bivalve hemocytes are oyster immune cells composed of several cellular subtypes with different functions. Hemocytes accumulate high concentrations of copper (Cu) and exert critical roles in metal sequestration and detoxification in oysters, however the specific biochemical mechanisms that govern this have yet to be fully uncovered. Herein, we demonstrate that Cu(I) is predominately sequestered in lysosomes via the Cu transporter ATP7A in hemocytes to reduce the toxic effects of intracellular Cu(I). We also found that Cu(I) is translocated along tunneling nanotubes (TNTs) relocating from high Cu(I) cells to low Cu(I) cells, effectively reducing the burden caused by overloaded Cu(I), and that ATP7A facilitates the efflux of intracellular Cu(I) in both TNTs and hemocyte subtypes. We identify that elevated glutathione (GSH) contents and heat-shock protein (Hsp) levels, as well as the activation of the cell cycle were critical in maintaining the cellular homeostasis and function of hemocytes exposed to Cu. Cu exposure also increased the expression of membrane proteins (MYOF, RalA, RalBP1, and cadherins) and lipid transporter activity which can induce TNT formation, and activated the lysosomal signaling pathway, promoting intercellular lysosomal trafficking dependent on increased hydrolase activity and ATP-dependent activity. This study explores the intracellular and intercellular transport and detoxification of Cu in oyster hemocytes, which may help in understanding the potential toxicity and fate of metals in marine animals.

Reactivity and bioconcentration of stable cesium in a hyperturbid fluvial-estuarine continuum: A combination of field observations and geochemical modeling.

Effective, post-accidental management needs an accurate understanding of the biogeochemical behavior of radionuclides in surface environments at a regional scale. Studies on stable isotopes (element homologs) can improve this knowledge. This work focuses on the biogeochemical behavior of stable cesium (Cs) along a major European fluvial-estuarine system, the Gironde Estuary (SW France). We present results obtained from (i) a long-term monitoring (2014-2017) of dissolved (Csd) and particulate (Csp) Cs concentrations at five sites along the freshwater continuum of the Garonne watershed, (ii) Csd and Csp concentrations during four oceanographic campaigns at contrasting hydrological conditions along longitudinal profiles of the estuarine system, (iii) a 24 h cycle of Csp at the estuary mouth, and (iv) a historical trend of Cs bioconcentration in wild oysters at the estuary mouth (RNO/ROCCH, 1984-2017). In addition, we model the partitioning of Cs within the estuarine environment for clay mineral interactions via PhreeqC. At fluvial sites, we observe a geogenic dependence of the Csp and a seasonal variability of Csd, with a downstream increase of the solid-liquid partitioning (log10 Kd values from 3.64 to 6.75 L kg-1) for suspended particulate matter (SPM) < 200 mg L-1. Along the estuarine salinity gradients, Cs shows a non-conservative behavior where fresh SPM (defined as Cs-depleted particles recently put in contact with Csd) act as a Cs sink during both flood and low discharge (drought) conditions. This sorption behavior was explained by the geochemical model, highlighting the relevance of ionic strength, water and SPM residence times. However, at high salinities, the overall log10 Kd value decreases from 6.02 to 5.20 for SPM ∼300-350 mg L-1 due to the Csd oceanic endmember. Despite wild oysters showing low bioconcentration factors (∼1220 L kg-1) at the estuary mouth, they are sensitive organisms to Cs fluxes.

Effect of baking on the structure and bioavailability of protein-binding zinc from oyster (Crassoetrea hongkongensis).

To compare the bioavailability of protein-binding zinc, we investigated the impact of baking on the structure of zinc-binding proteins. The results showed that zinc-binding proteins enriched in zinc with relative molecular weights distributed at 6 kDa and 3 kDa. Protein-binding zinc is predisposed to separate from proteins' interiors and converge on proteins' surface after being baked, and its structure tends to be crystalline. Especially -COO, -C-O, and -C-N played vital roles in the sites of zinc-binding proteins. However, baking did not affect protein-binding zinc's bioavailability which was superior to that of ZnSO4 and C12H22O14Zn. They were digested in the intestine, zinc-binding complexes that were easily transported and uptaken by Caco-2 cells, with transport and uptake rates as high as 62.15% and 15.85%. Consequently, baking can alter the conformation of zinc-binding proteins without any impact on protein-binding zinc's bioavailability which is superior to that of ZnSO4 and C12H22O14Zn.

Short communication: The boring sponge (Pione vastifica, Hancock, 1849) induces oxidative stress in the Pacific oyster (Magallana gigas, Thunberg, 1793).

Boring sponge infection affects growth, development and reduces the soft tissue weight of oysters. In this study, we investigated the effects of boring sponge on the activity of three antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GP)) in the mantle, and the production of reactive oxygen species (ROS) and potential genotoxicity in hemocytes of the Pacific oyster Magallana gigas. Our results showed a significant increase in ROS production and DNA damage in hemocytes. Notably, the activity of SOD, CAT, and GP in the mantle was not significantly affected by boring sponge infection. Collectively, these results suggest that sponge invasion may cause oxidative stress in Pacific oyster hemocytes through ROS overproduction.

Characterization of Oyster Protein Hydrolysate-Iron Complexes and their In Vivo Protective Effects against Iron Deficiency-Induced Symptoms in Mice.

Iron is one of the trace mineral elements, and iron deficiency is a common phenomenon that negatively influences human health. Food-derived iron supplements were considered excellent candidates for improving this syndrome. In this work, oyster-protein hydrolysates (OPH) and ferrous chloride successfully formed the OPH-Fe complex (6 mg/mL, 40 °C, 30 min), where the main binding sites involved were the carboxyl and amino groups. The OPH-Fe complex showed no obvious changes in the secondary structure, while the iron changed the morphological appearance and also showed fluorescence quenching, an ultraviolet shift, and an increase in size distribution. The OPH-Fe complex showed better dynamic absorption of iron (64.11 µmol/L) than ferrous sulfate (46.90 µmol/L), and the medium dose had better protective effects against iron-deficiency anemia in vivo. Three representative peptides (DGKGKIPEE, FAGDDAPRA, and VLDSGDGVTH) that were absorbed intact were identified. This experiment provided a theoretical foundation for further study of the digestion and absorption of the OPH-Fe complex.

Oyster reefs' control of carbonate chemistry-Implications for oyster reef restoration in estuaries subject to coastal ocean acidification.

Globally, oyster reef restoration is one of the most widely applied coastal restoration interventions. While reefs are focal points of processes tightly linked to the carbonate system such as shell formation and respiration, how these processes alter reef carbonate chemistry relative to the surrounding seawater is unclear. Moreover, coastal systems are increasingly impacted by coastal acidification, which may affect reef carbonate chemistry. Here, we characterized the growth of multiple constructed reefs as well as summer variations in pH and carbonate chemistry of reef-influenced seawater (in the middle of reefs) and ambient seawater (at locations ~50 m outside of reefs) to determine how reef chemistry was altered by the reef community and, in turn, impacts resident oysters. High frequency monitoring across three subtidal constructed reefs revealed reductions of daily mean and minimum pH (by 0.05-0.07 and 0.07-0.12 units, respectively) in seawater overlying reefs relative to ambient seawater (p < .0001). The proportion of pH measurements below 7.5, a threshold shown to negatively impact post-larval oysters, were 1.8×-5.2× higher in reef seawater relative to ambient seawater. Most reef seawater samples (83%) were reduced in total alkalinity relative to ambient seawater samples, suggesting community calcification was a key driver of modified carbonate chemistry. The net metabolic influence of the reef community resulted in reductions of CaCO3 saturation state in 78% of discrete samples, and juvenile oysters placed on reefs exhibited slower shell growth (p < .05) compared to oysters placed outside of reefs. While differences in survival were not detected, reef oysters may benefit from enhanced survival or recruitment at the cost of slowed growth rates. Nevertheless, subtidal restored reef communities modified seawater carbonate chemistry in ways that likely increased oyster vulnerability to acidification, suggesting that carbonate chemistry dynamics warrant consideration when determining site suitability for oyster restoration, particularly under continued climate change.

Immediate and delayed effects of a heatwave and Prorocentrum lima ((Ehrenberg) Stein 1878) bloom on the toxin accumulation, physiology, and survival of the oyster Magallana gigas (Thunberg, 1793).

Warming could facilitate the intensification of toxic algal blooms, two important stressors for marine organisms that are predicted to co-occur more frequently in the future. We investigated the immediate and delayed effects of a heatwave and a simulated bloom (3 × 106 cells L-1) of the diarrhetic shellfish toxin (DST)-producing benthic dinoflagellate Prorocentrum lima on the survival, physiology (oxygen consumption rate, condition index, immune parameters), and toxin accumulation in the Pacific rock oyster Magallana (Crassostrea) gigas. Oysters exposed to both stressors contained higher mean DST concentrations (mean ± 1 SE: 173.3 ± 19.78 µg kg-1 soft tissue) than those exposed to P. lima bloom alone (120.4 ± 20.90 µg kg-1) and exceeded the maximum permitted levels for human consumption. Exposure to individual stressors and their combination modified the physiology of M. gigas. Oysters exposed to heatwave alone had significantly higher oxygen consumption rates (0.7 ± 0.06 mg O2 h-1 g-1) than the control (0.3 ± 0.06 mg O2 h-1 g-1). However, this was not observed in oysters exposed to both heatwave and P. lima (0.5 ± 0.06 mg O2 h-1 g-1). This alteration of the metabolic response to warming in the presence of P. lima may affect the ability of rock oysters to adapt to environmental stressors (i.e., a heatwave) to ensure survival. Immunomodulation, through changes in total hemocyte count, was observed in oysters exposed to P. lima alone and in combination with warming. Individual stressors and their combination did not influence the condition index, but one mortality was recorded in oysters exposed to both stressors. The findings of this study highlight the vulnerability of rock oysters to the predicted increased frequency of heatwaves and toxic algal blooms, and the increased likelihood of shellfish containing higher than regulatory levels of DST in warming coasts.

Oyster ferritin can efficiently alleviate ROS-mediated inflammation attributed to its unique micro-environment around three-fold channels.

The conversion of toxic Fe2+ into non-toxic Fe3+ stored in the inner cavity of ferritin nanocage could effectively reduce the occurrence of the Fenton reaction and inhibit the formation of harmful reactive oxygen species (ROS). In this study, we reveal that oyster ferritin (GF1) can rely on its high catalytic activity (7.7 times that of rHuHF) and high binding ability of Fe2+ (9.1 times that of rHuHF) to reduce the precursors of Fenton reaction, thus inhibiting the occurrence of Fenton reaction and slowing down reactive oxygen species-mediated inflammation. The above significant advantage of GF1 can be attributed to the Asp at the position 120th, which could increase the negatively charged area of three-fold channels from 37.8% (rHuHF) to 67.8% and then enhance its oxidation rate and ability of GF1. The findings are of great value in advancing novel nanoparticle drug design based on crystalline structure.

What do oysters smell? Electrophysiological evidence that the bivalve osphradium is a chemosensory organ in the oyster, Magallana gigas.

The sensing of chemical cues is essential for several aspects of bivalve biology, such as the detection of food and pheromones. However, little is known about chemical communication systems in bivalves or the possible role of the osphradium as a chemosensory organ. To address this, we adapted an electrophysiological technique extensively used in vertebrates-the electro-olfactogram-to record from the osphradium in the Pacific oyster, Magallana gigas. This technique was validated using amino acids as stimulants. The osphradium proved to be sensitive to most proteinogenic L-amino acids tested, evoking tonic, negative, concentration-dependent 'electro-osphradiogram' (EOsG) voltage responses, with thresholds of detection in the range of 10- 6 to 10- 5 M. Conversely, it was insensitive to L-arginine and L-glutamic acid. The current study supports the hypothesis that the osphradium is, indeed, a chemosensory organ. The 'electro-osphradiogram' may prove to be a powerful tool in the isolation and characterization of pheromones and other important chemical cues in bivalve biology.

Early gonadal differentiation is associated with the antagonistic action of Foxl2 and Dmrt1l in the Pacific oyster.

As the second largest phylum in the zoological kingdom next to arthropods, the mechanism of gonadal differentiation in mollusca is quite complex. Currently, although much has been carried out on gonadal differentiation in the Pacific oyster, there is still unknown information that needs to be further explored. Here, analysis of the Foxl2 and Dmrt1l expression in samples at different development periods of male and female gonads as well as in annual gonad samples revealed that Log10 (Foxl2/Dmrt1l) values were an effective method for sex identification in oysters. In differentiated gonadal tissue, Log10 (Foxl2/Dmrt1l) values greater than 2 were females and less than 1 for males. Subsequent sequential sampling of the same individuals verified that Log10 (Foxl2/Dmrt1l) values greater than 2 for resting gonads would develop as females and less than 1 would develop as males in the future. Relative expression analysis of Foxl2 and Dmrt1l in the annual samples revealed a negative correlation between Log10 (Foxl2) and Log10 (Dmrt1l). Double fluorescence reporter validation results showed that DMRT1L protein was able to bind the Foxl2 promoter and repress its activity with a weak dosage effect. Antagonism between Dmrt1l and Foxl2 is therefore not restricted to vertebrates, and the competing regulatory networks are of great significance in the maintenance of gonadal sex in oysters after sexual differentiation. This study provides novel ideas and insights into the study of early gonadal differentiation in the adult oyster.

Application of bovine bone meal and oyster shell meal to heavy metals polluted soil: Vegetable safety and bacterial community.

The development of efficient, environmentally friendly soil amendments is necessary in order to minimize the risk of metal contaminants (Cd, Pb, Cu, and Zn) to the soil ecosystem. As soil amendments, bovine bone meal (BM) and oyster shell meal (OS) reduced the mobility and bioavailability of metals primarily by increasing soil pH. Soil geochemical properties (pH, EC, CEC, Ca, P, and K) after amendment supplementation were more likely to affect metal migration than enzyme activity. Furthermore, BM and OS were found to suppress the Cd and Pb uptake by water spinach, keeping them below international standards for safe utilization. The protein and sugar content and peroxidase (POD) activity showed a significant negative correlation with the amount of metal in water spinach, whereas superoxide dismutase (SOD), ascorbate peroxidase (APX) activities and malondialdehyde (MDA) content exhibited a positive correlation with metal content in water spinach. We also found that BM and OS had less perturbation to phylum-level and genus-level bacterial composition during the remediation of heavy metals contaminated soil. Based on the above, we assume that BM and OS are eco-friendly soil amendments, which could improve soil nutrients contents, stabilize heavy metals and regulate bacterial community structure. Our research contributes to resource utilization of waste and holds promise for widespread application in current agricultural systems.

In silico analysis of the C-terminal domain of big defensin from the Pacific oyster.

Defensins are antimicrobial peptides consisting of intramolecular disulphide bonds in a complex folded arrangement of two or three antiparallel ß-sheets with or without an α-helical structure. They are produced by a vast range of organisms being constitutively expressed or induced in various tissues against different stimuli like infection, injury or other inflammatory factors. Two classes of invertebrate defensin exist, namely CS-αß and big defensin, the latter being predominantly present in molluscs. Intriguingly, an invertebrate big defensin gene has been hypothesized as the most probable ancestor of vertebrate ß-defensins. Here, conserved residues were identified for both big defensin and ß-defensin. In silico mutation on conserved amino acid positions of the ß-defensin-like domain of big defensin from Crassostrea gigas was carried out to understand the effects of mutation on the structure and function of the protein. R64A and E71A have been identified as deleterious as well as destabilizing for the protein. Changes in amino acid network and aggregation propensity were also observed upon mutating these two charged residues. 100 ns molecular dynamics simulations of wild-type, R64A and E71A structures revealed significant conformational changes in the case of mutants. Furthermore, molecular docking highlighted the significance of R64 in ligand interaction. In conclusion, these results provide the first in-depth understanding of the structural and functional importance imparted by two conserved charged residues in the C-terminal region of big defensin. It also enhances the existing knowledge about this antimicrobial peptide for application in therapeutics and other aspects of protein engineering.Communicated by Ramaswamy H. Sarma.