Structural and Compositional Changes in Two Marine Shell Traditional Chinese Medicines: A Comparative Analysis Pre- and Post-Calcination.

BACKGROUND: Arcae concha and Meretricis concha cyclinae concha are two marine shellfish herbs with similar composition and efficacy, which are usually calcined and used clinically. OBJECTIVE: This study investigated variations in the inorganic and organic components of Arcae concha and Meretricis concha cyclinae concha from different production regions, both Arcae concha and Meretricis concha cyclinae concha. The aim was to enhance the understanding of these two types of marine shell traditional Chinese medicine (msTCM) and provide a foundation for their future development and application. METHOD: Spectroscopic techniques, including infrared spectroscopy, X-ray spectroscopy, and X-ray fluorescence spectroscopy, were used to analyze the calcium carbonate (CaCO3) crystal and trace elements. Thermogravimetric analysis was used to investigate the decomposition process during heating. The proteins were quantified using the BCA protein assay kit. Principal component analysis (PCA) was used to classify inorganic elements in the two marine shellfish traditional Chinese medicines. RESULTS: No significant differences were found among the various production regions. The crystal structure of CaCO3 in the raw products was aragonite, but it transformed into calcite after calcination. The contents of Ca, Na, Sr, and other inorganic elements were highest. The protein content was significantly reduced after calcination. Therefore, these factors cannot accurately reflect the internal quality of TCM, rendering qualitative identification challenging. CaCO3 dissolution in the decoction of Arcae concha and Meretricis concha cyclinae concha increased after calcination, aligning with the clinical application of calcined shell TCM. PCA revealed the inorganic elements in them, indicating that the variation in trace element composition among different drugs leads to differences in their therapeutic focus, which should be considered during usage. CONCLUSIONS: This study clarifies the composition and structure changes of corrugated and clam shell before and after calcining, and it lays the foundation for the comprehensive utilization of marine traditional Chinese medicine. HIGHLIGHTS: These technical representations reveal the differences between raw materials and processed products, which will provide support for the quality control of other shellfish TCM.

Chemical extraction of chitin from American lobster (Homarus americanus) shells optimized through response surface methodology.

Chitin extraction from the shells of American lobsters (Homarus americanus) was optimized through the use of response surface methodology (RSM). The demineralization step was optimized to minimize the ash content of shell samples and the deproteination step was optimized to minimize the protein content of the chitin product. At a laboratory scale, one set of optimized conditions for the demineralization step was 7.35 % w/w acetic acid at a 40 mL/g of powdered lobster shell ratio for 15 min; this lowered the ash content from 39.62 % to 0.41 ± 0.08 %. A set of optimized conditions for the deproteination step at a similar scale was 4 % w/w sodium hydroxide at a 43 mL/g demineralized shell ratio heated to 95 °C for 83 min. These conditions were indicated to entirely remove protein from the resultant chitin. Average yields under optimized conditions were 23.43 ± 1.75 % for demineralization and 30.33 ± 0.02 % for deproteination, though a demineralization reaction with larger biomass input had a higher yield at 40.31 %.

Effects of eggshell and seashell powder as natural dietary calcium supplements on growth, molting frequency, and carapace calcium composition of juvenile red claw crayfish, Cherax quadricarinatus.

The growth performance of red claw crayfish, Cherax quadricarinatus, fed diets with different sources and concentrations of natural calcium was evaluated. Formulated diets containing 30% protein and 8% lipid were prepared using supplementation of 0 (control), 3%, 6%, 9% eggshell, and 6% seashell. C. quadricarinatus juveniles with an average total weight of 0.21 ± 0.01 g and length of 25.0 ± 0.10 mm were obtained from ten ovigerous females bred in captivity. An aquarium with a size of 0.72 m² was divided into eight compartments with an area of 0.09 m² per compartment and was individually stocked with juvenile crayfish. A total of eight juvenile crayfish were used per dietary treatment. There were five dietary treatments in total and each of these treatments were replicated three times. The addition of eggshell and seashell powder exerted positive effects on the growth performance, molting frequency, and survival of the crayfish. Crayfish fed with a 6% eggshell-supplemented diet exhibited the best overall growth performance. The calcium percentage of the carapace increased with the increase in calcium content of the diets. Meanwhile, the performance of diets comprising eggshells and seashells was not significantly different (P > 0.05). Considering the low cost and abundance of eggshells, the addition of 6% of this product to the diet is recommended for the best growth of juvenile C. quadricarinatus.

Ocean acidification, warming and feeding impacts on biomineralization pathways and shell material properties of Magallana gigas and Mytilus spp.

Molluscs are among the organisms affected by ocean acidification (OA), relying on carbon for shell biomineralization. Metabolic and environmental sourcing are two pathways potentially affected by OA, but the circumstances and patterns by which they are altered are poorly understood. From previous studies, mollusc shells grown under OA appear smaller in size, brittle and thinner, suggesting an important alteration in carbon sequestration. However, supplementary feeding experiments have shown promising results in offsetting the negative consequences of OA on shell growth. Our study compared carbon uptake by δ13C tracing and deposition into mantle tissue and shell layers in Magallana gigas and Mytilus species, two economically valuable and common species. After subjecting the species to 7.7 pH, +2 °C seawater, and enhanced feeding, both species maintain shell growth and metabolic pathways under OA without benefitting from extra feeding, thus, showing effective acclimation to rapid and short-term environmental change. Mytilus spp. increases metabolic carbon into the calcite and environmental sourcing of carbon into the shell aragonite in low pH and high temperature conditions. Low pH affects M. gigas mantle nitrogen isotopes maintaining growth. Calcite biomineralization pathway differs between the two species and suggests species-specific response to OA.

Ontogeny, evolution and palaeogeographic distribution of the world's largest ammonite Parapuzosia (P.) seppenradensis (Landois, 1895).

The world's largest ammonite, Parapuzosia (P.) seppenradensis (Landois, 1895), fascinated the world ever since the discovery, in 1895, of a specimen of 1.74 metres (m) diameter near Seppenrade in Westfalia, Germany, but subsequent findings of the taxon are exceedingly rare and its systematic position remains enigmatic. Here we revise the historical specimens and document abundant new material from England and Mexico. Our study comprises 154 specimens of large (< 1 m diameter) to giant (> 1m diameter) Parapuzosia from the Santonian and lower Campanian, mostly with stratigraphic information. High-resolution integrated stratigraphy allows for precise cross-Atlantic correlation of the occurrences. Our specimens were analysed regarding morphometry, growth stages and stratigraphic occurrence wherever possible. Our analysis provides insight into the ontogeny of Parapuzosia (P.) seppenradensis and into the evolution of this species from its potential ancestor P. (P.) leptophylla Sharpe, 1857. The latter grew to shell diameters of about 1 m and was restricted to Europe in the early Santonian, but it reached the Gulf of Mexico during the late Santonian. P. (P.) seppenradensis first appears in the uppermost Santonian- earliest Campanian on both sides of the Atlantic. Initially, it also reached diameters of about 1 m, but gradual evolutionary increase in size is seen in the middle early Campanian to diameters of 1.5 to 1.8 m. P. (P.) seppenradensis is characterized by five ontogenetic growth stages and by size dimorphism. We therefore here include the many historic species names used in the past to describe the morphological and size variability of the taxon. The concentration of adult shells in small geographic areas and scarcity of Parapuzosia in nearby coeval outcrop regions may point to a monocyclic, possibly even semelparous reproduction strategy in this giant cephalopod. Its gigantism exceeds a general trend of size increase in late Cretaceous cephalopods. Whether the coeval increase in size of mosasaurs, the top predators in Cretaceous seas, caused ecological pressure on Parapuzosia towards larger diameters remains unclear.

Mechanical adaptation of brachiopod shells via hydration-induced structural changes.

The function-optimized properties of biominerals arise from the hierarchical organization of primary building blocks. Alteration of properties in response to environmental stresses generally involves time-intensive processes of resorption and reprecipitation of mineral in the underlying organic scaffold. Here, we report that the load-bearing shells of the brachiopod Discinisca tenuis are an exception to this process. These shells can dynamically modulate their mechanical properties in response to a change in environment, switching from hard and stiff when dry to malleable when hydrated within minutes. Using ptychographic X-ray tomography, electron microscopy and spectroscopy, we describe their hierarchical structure and composition as a function of hydration to understand the structural motifs that generate this adaptability. Key is a complementary set of structural modifications, starting with the swelling of an organic matrix on the micron level via nanocrystal reorganization and ending in an intercalation process on the molecular level in response to hydration.

Porous Biphasic Calcium Phosphate Granules from Oyster Shell Promote the Differentiation of Induced Pluripotent Stem Cells.

Oyster shells are rich in calcium, and thus, the potential use of waste shells is in the production of calcium phosphate (CaP) minerals for osteopathic biomedical applications, such as scaffolds for bone regeneration. Implanted scaffolds should stimulate the differentiation of induced pluripotent stem cells (iPSCs) into osteoblasts. In this study, oyster shells were used to produce nano-grade hydroxyapatite (HA) powder by the liquid-phase precipitation. Then, biphasic CaP (BCP) bioceramics with two different phase ratios were obtained by the foaming of HA nanopowders and sintering by two different two-stage heat treatment processes. The different sintering conditions yielded differences in structure and morphology of the BCPs, as determined by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Brunauer-Emmett-Teller (BET) surface area analysis. We then set out to determine which of these materials were most biocompatible, by co-culturing with iPSCs and examining the gene expression in molecular pathways involved in self-renewal and differentiation of iPSCs. We found that sintering for a shorter time at higher temperatures gave higher expression levels of markers for proliferation and (early) differentiation of the osteoblast. The differences in biocompatibility may be related to a more hierarchical pore structure (micropores within macropores) obtained with briefer, high-temperature sintering.

PU14, a Novel Matrix Protein, Participates in Pearl Oyster, Pinctada Fucata, Shell Formation.

Biomineralization is a widespread biological process, involved in the formation of shells, teeth, and bones. Shell matrix proteins have been widely studied for their importance during shell formation. In 2015, our group identified 72 unique shell matrix proteins in Pinctada fucata, among which PU14 is a matrix protein detected in the soluble fraction that solely exists in the prismatic layer. However, the function of PU14 is still unclear. In this study, the full-length cDNA sequence of PU14 was obtained and functional analyses of PU14 protein during shell formation were performed. The deduced protein has a molecular mass of 77.8 kDa and an isoelectric point of 11.34. The primary protein structure contains Gln-rich and random repeat units, which are typical characteristics of matrix protein and indicate its potential function during shell formation. In vivo and in vitro experiments indicated PU14 has prismatic layer functions during shell formation. The tissue expression patterns showed that PU14 was mainly expressed in the mantle tissue, which is consistent with prismatic layer formation. Notching experiments suggested that PU14 responded to repair and regenerate the injured shell. After inhibiting gene expression by injecting PU14-specific double-stranded RNA, the inner surface of the prismatic layer changed significantly and became rougher. Further, in vitro experiments showed that recombinant protein rPU14 impacted calcite crystal morphology. Taken together, characterization and functional analyses of a novel matrix protein, PU14, provide new insights about basic matrix proteins and their functions during shell formation.

Phosphorus Co-Existing in Water: A New Mechanism to Boost Boron Removal by Calcined Oyster Shell Powder.

The removal of boron (B) from water by co-precipitation with hydroxyapatite (HAP) has been extensively studied due to its low cost, ease of use and high efficiency. However, there is no explicit mechanism to express how resolved B was trapped by HAP. Thus, in this work, the process of removing B from water was studied using a low-cost calcium (Ca) precipitation agent derived from used waste oyster shells. The results showed that the removal rate of B in the simulated wastewater by calcined oyster shell (COS) in the presence of phosphorus (P) is up to more than 90%, as opposed to virtually no removal without phosphate. For B removal, the treated water needs to be an alkaline solution with a high pH above 12, where B is removed as [CaB(OH)4]+ but is not molecular. Finally, the synergistic mechanism of co-precipitation between HAP and dissolved B, occlusion co-precipitation, was explained in detail. The proposed method discovered the relationship between Ca, P and B, and was aimed at removing B without secondary pollution through co-precipitation.

Low concentrated phosphorus sorption in aqueous medium on aragonite synthesized by carbonation of seashells: Optimization, kinetics, and mechanism study.

Phosphorus (P) concentration beyond threshold limit can trigger eutrophication in stagnant water bodies nevertheless it is an indispensable macronutrient for aquatic life. Even in low P concentration (≤1 mg L-1), P can be detrimental for ecosystem's health, but this aspect has not been thoroughly investigated. The elimination of low P content is rather expensive or complex. Therefore, a unique and sustainable approach has been proposed in which valorized bivalve seashells can be used for the removal of low P content. Initially, acicular shaped aragonite particles (~21 µm) with an aspect ratio of around 21 have been synthesized through the wet carbonation process and used to treat aqueous solutions containing P in low concentration (P ≤ 1 mg L-1). Response surface methodology based Box-Behnken design has been employed for optimization study which revealed that with aragonite dosage (140 mg), equilibrium pH (~10.15), and temperature (45 °C), a phosphorus removal efficiency of ~97% can be obtained in 10 h. The kinetics and isotherm studies have also been carried out (within the range P ≤ 1 mg L-1) to investigate a probable removal mechanism. Also, aragonite demonstrates higher selectivity (>70%) towards phosphate with coexisting anions such as nitrate, chloride, sulfate, and carbonate. Through experimental data, elemental mapping, and molecular dynamic simulation, it has been observed that the removal mechanism involved a combination of electrostatic adsorption of Ca2+ ions on aragonite surface and chemical interaction between the calcium and phosphate ions. The present work demonstrates a sustainable and propitious potential of seashell derived aragonite for the removal of low P content in aqueous solution along with its unconventional mechanistic approach.

The Pleistocene-Holocene aquatic molluscs as indicators of the past ecosystem changes in Transbaikalia (Eastern Siberia, Russia).

Data on the historical change of the Transbaikalian malacofauna in the Neopleistocene and Holocene is presented. Aquatic mollusc shells from archaeological excavations of the ancient settlements dating from the Neolithic period to Medieval and also from a drill hole of the Neopleistocene alluvial deposits were collected. In total eight species of bivalve molluscs from the families Margaritiferidae, Unionidae, Lymnocardiidae, Glycymerididae [marine], and two gastropod species from families Viviparidae and Planorbidae were identified. These species were aged using radiocarbon dating. It was found that the species ranged in age from more than 50.000 to 2.080-1.210 years BP. Five species inhabited the Transbaikal region which are locally extirpated today. Their disjunctive ranges in the past included southern Europe and Western and Eastern Siberia to Transbaikalia and in the east to Far East and Primorye Territory of Russia. A remarkable finding is that of the bivalve genus Monodacna, which was found very far from its native range, the Ponto-Caspian region. The time of existence and extirpation of the thermophilic species of genera Monodacna, Planorbis, Lanceolaria and Amuropaludina corresponds to cycles of the warming and cooling in Pleistocene and Holocene according to regional climate chronological scales. These species can be used as palaeoclimate indicators. Change of the regional malacofaunal species composition is connected with the natural climatochron cycles in the Pleistocene and Holocene resulting in evidence for succession. In the course of this succession, these stenothermal species became extirpated on a regional level, decreasing their global ranges.

From sporadic single genes to a broader transcriptomic approach: Insights into the formation of the biomineralized exoskeleton in decapod crustaceans.

One fundamental character common to pancrustaceans (Crustacea and Hexapoda) is a mineralized rigid exoskeleton whose principal organic components are chitin and proteins. In contrast to traditional research in the field that has been devoted to the structural and physicochemical aspects of biomineralization, the present study explores transcriptomic aspects of biomineralization as a first step towards adding a complementary molecular layer to this field. The rigidity of the exoskeleton in pancrustaceans dictates essential molt cycles enabling morphological changes and growth. Thus, formation and mineralization of the exoskeleton are concomitant to the timeline of the molt cycle. Skeletal proteinaceous toolkit elements have been discovered in previous studies using innovative molt-related binary gene expression patterns derived from transcriptomic libraries representing the major stages comprising the molt cycle of the decapod crustacean Cherax quadricarinatus. Here, we revisited some prominent exoskeleton-related structural proteins encoding and, using the above molt-related binary pattern methodology, enlarged the transcriptomic database of C. quadricarinatus. The latter was done by establishing a new transcriptomic library of the cuticle forming epithelium and molar tooth at four different molt stages (i.e., inter-molt, early pre-molt, late pre-molt and post-molt) and incorporating it to a previous transcriptome derived from the gastroliths and mandible. The wider multigenic approach facilitated by the newly expanded transcriptomic database not only revisited single genes of the molecular toolkit, but also provided both scattered and specific information that broaden the overview of proteins and gene clusters which are involved in the construction and biomineralization of the exoskeleton in decapod crustaceans.

Thermal stable characteristics of acid- and pepsin-soluble collagens from the carapace tissue of Chinese soft-shelled turtle (Pelodiscus sinensis).

The carapace from the Chinese soft-shelled turtle (Pelodiscus sinensis) is used as a traditional Chinese medicine. Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from turtle carapace were isolated and characterized to screen novel collagen material in this study. Yields of 1.0% and 2.8% were obtained for ASC and PSC which contained glycine as the major amino acid and had high imino acid content. Both collagens had maximum ultraviolet absorption peaks of 220 nm. SDS-PAGE revealed that the structure of both collagens was similar, belonging to type I collagen. Relative viscosities of collagens were decreased as the temperature increased. Collagens showed minimum solubility at pH 8 and maximum solubility at a salt concentration of 3%. The denaturation temperature (Td) of PSC was higher whereas the melting temperature was lower than that of ASC. Both ASC and PSC appeared to be spongy like microstructure with fibrillar pores shown by scanning electron microscopy. The results suggest that collagens isolated from turtle carapace has high thermal stability with potential uses as new substitute for mammalian collagen in medicinal, food or biomaterial fields. However, their biological or pharmacological activities are needed to be further studied.

Calcarea carbonica treatment rescues lipopolysaccharide-induced inflammatory response in human mononuclear cells via downregulation of inducible cyclooxygenase pathway.

OBJECTIVE: Prolonged use of nonsteroidal anti-inflammatory drugs is associated with severe side effects and toxicity. Therefore, we studied the anti-inflammatory role of Calcarea carbonica which had minimal toxicity at the low doses. METHODS: THP-1 human mononuclear cells were treated with C. carbonica to evaluate the 50% cytotoxicity concentration (CC50) and 50% effective concentration (EC50). Cell survival was evaluated in lipopolysaccharide-stimulated C. carbonica-treated cells. Nitric oxide (NO) and tumor necrosis factor-α (TNF-α) were measured to evaluate the anti-inflammatory activity of C. carbonica. Cyclooxygenase-2 (COX-2) protein expression was determined by Western blotting analysis, and the interaction of C. carbonica with the COX-2 protein was evaluated using molecular docking simulation. RESULTS: The CC50 and EC50 of C. carbonica were found to be 43.26 and 11.99 µg/mL, respectively. The cell survival assay showed a 1.192-fold (P = 0.0129), 1.443-fold (P = 0.0009) and 1.605-fold (P = 0.0004) increase in cell survival at 24, 48 and 72 h after initiating C. carbonica treatment, respectively. C. carbonica-treated cells showed a reduction in NO levels by 2.355 folds (P = 0.0001), 2.181 folds (P = 0.0001) and 2.071 folds (P = 0.0001) at 24, 48 and 72 h, respectively. The treated cells also showed a reduction in TNF-α levels by 1.395 folds (P = 0.0013), 1.541 folds (P = 0.0005) and 1.550 folds (P = 0.0005) at 24, 48 and 72 h, respectively. In addition, a 1.193-fold reduction (P = 0.0126) in COX-2 protein expression was found in C. carbonica-treated cells. The molecular docking showed interaction of C. carbonica with the phenylalanine 367 residue present in active site of Cox-2. CONCLUSION: C. carbonica exhibited anti-inflammatory properties in lipopolysaccharide-stimulated cells by significantly reducing NO production and TNF-α level through downregulation of the COX-2 protein. This effect is probably mediated through interaction of C. carbonica with the phenylalanine 367 residue present in active site of Cox-2.

Bioshell calcium oxide (BiSCaO) for cleansing and healing Pseudomonas aeruginosa-infected wounds in hairless rats.

BACKGROUND: Scallop shell powder is called bioshell calcium oxide (BiSCaO), which is known to possess deodorizing properties and broad antimicrobial activity against various pathogenic microbes, including viruses, bacteria, spores, and fungi. OBJECTIVE: This study aims to investigate the applications of BiSCaO suspension cleansing in clinical situations, for instance for the prevention and treatment of infections in chronic wounds in healing-impaired patients, without delaying wound healing. METHODS: The bactericidal activities of 1000 ppm BiSCaO suspension; 500 ppm hypochlorous acid; 1000 ppm povidone iodine; and saline were compared to evaluate in vivo disinfection and healing of Pseudomonas aeruginosa-infected wounds in hairless rats. RESULTS: Cleansing of the infected wounds with BiSCaO suspension daily for 3 days significantly enhanced wound healing and reduced the in vivo bacterial counts, in comparison to hypochlorous acid, povidone iodine, and saline. Furthermore, histological examinations showed significantly advanced granulation tissue and capillary formation in the wounds cleansed with BiSCaO suspension than in those cleansed with the other solutions. CONCLUSIONS: This study suggested that the possibility of using BiSCaO suspension as a disinfectant for infected wounds and limiting disinfection to 3 days may be sufficient to avoid the negative effects on wound repair.

Toxicology study of fraxinellone as ovicidal agents against Mythimna separata Walker and Bombyx mori Linaeus.

Fraxinellone is a naturally occurring degraded limonoid isolated from many species of plants in Meliaceae and Rutaceae. Besides structural modification of the lead compounds, the toxicology study of the lead compounds is also a very important procedure to develop insecticidal agents. Herein the toxicology study of fraxinellone was carried out as the ovicidal agent against the eggs of two lepidopteran insects Mythimna separata Walker and Bombyx mori Linaeus. Fraxinellone selectively exhibited an ovicidal activity against the eggs of M. separata. After treatment with fraxinellone, the eggshells of M. separata were shrinked, whereas those of B. mori had no obvious change. The dynamic process of M. separata embryo development demonstrated that the distinct difference between the treated eggs and the control ones was obvious at the second day after treatment, especially, the control embryo finished blastokinesis, whereas the treated ones were still laid at pre-reversion status and a lot of yolk can be seen around the embryo. It ultimately resulted in the eggshell withered and the egg hatching inhibited.

Determination of four nitrofuran metabolites in gelatin Chinese medicine using dispersive solid phase extraction and pass-through solid phase extraction coupled to ultra high performance liquid chromatography-tandem mass spectrometry.

This study established a validated analytical method for the first time on the determination of nitrofuran metabolites, including semicarbazide (SEM), 1-aminohydantoin (AHD), 3-amino-2-oxazolidinone (AOZ) and 3-amino-5-morpholinomethyl-2-oxazolinone (AMOZ) in gelatin Chinese medicine. A C18 column with the mobile phase consisting of acetonitrile and 5 mmol/L ammonium acetate in water was used to separate these nitrofuran metabolites. The limit of detection of SEM, AHD, AOZ and AMOZ were found to be 0.2 µg/kg, 0.3 µg/kg, 0.2 µg/kg and 0.2 µg/kg, whereas their limit of quantification were 0.6 µg/kg, 0.8 µg/kg, 0.6 µg/kg and 0.5 µg/kg. These nitrofuran metabolites exhibited a good linear standard curve (regression coefficients above 0.99) with a concentration range of 2 µg/L to 100 µg/L. Regarding extraction procedure, gelatin Chinese medicine was pre-treated with pepsin and then extracted using 5% formic acid (v/v) in acetonitrile. The resultant extract was purified through dispersive solid phase extraction using 1000 mg anhydrous sodium sulfate, 300 mg octadecyl carbon silica gel sorbent absorbent and 500 mg ethylenediamine-N-propyl carbon silica gel absorbent, and then further purified on Oasis PRiME HLB cartridges. The matrix effect was effectively eliminated after the clean-up procedure as confirmed by comparing the ratio of standard curves prepared by standards dissolved in both matrix solvent and 5 mmol/L ammonium acetate in water: acetonitrile (95:5, v/v). The recoveries of these nitrofuran metabolites under the 1 µg/kg, 2 µg/kg and 10 µg/kg spiking levels were between 77.4% and 95.6%. These metabolites after the extraction were stable at 4 °C for 24 h. The validated method was used to analyze the residue level of these nitrofuran metabolites in 25 gelatin Chinese medicines. Results showed that only one Colla Corii Asini sample contained SEM (2.52 µg/kg) and AOZ (6.27 µg/kg), whereas one Testudinis Carapacis et Plastri sample had SEM (1.27 µg/kg) and AMOZ (9.53 µg/kg).

A Classification Method of Oracle Materials Based on Local Convolutional Neural Network Framework.

The classification of materials of oracle bone is one of the most basic aspects for oracle bone morphology. However, the classification method depending on experts' experience requires long-term learning and accumulation for professional knowledge. This article presents a multiregional convolutional neural network to classify the rubbings of oracle bones. First, we detected the "shield grain" and "tooth grain" on the oracle bone rubbings, then complete the division of multiple areas on an image of oracle bone. Second, the convolutional neural network is used to extract the features of each region and we complete the fusion of multiple local features. Finally, the classification of tortoise shell and animal bone was realized. Utilizing the image of oracle bone provided by experts, we conducted an experiment; the result show our method has better classification accuracy. It has made contributions to the progress of the study of oracle bone morphology.

Acoustic behaviour of male European lobsters (Homarus gammarus) during agonistic encounters.

Previous studies have demonstrated that male European lobsters (Homarus gammarus) use chemical and visual signals as a means of intraspecific communication during agonistic encounters. In this study, we show that they also produce buzzing sounds during these encounters. This result was missed in earlier studies because low-frequency buzzing sounds are highly attenuated in tanks, and are thus difficult to detect with hydrophones. To address this issue, we designed a behavioural tank experiment using hydrophones, with accelerometers placed on the lobsters to directly detect their carapace vibrations (i.e. the sources of the buzzing sounds). While we found that both dominant and submissive individuals produced carapace vibrations during every agonistic encounter, very few of the associated buzzing sounds (15%) were recorded by the hydrophones. This difference is explained by their high attenuation in tanks. We then used the method of algorithmic complexity to analyse the carapace vibration sequences as call-and-response signals between dominant and submissive individuals. Even though some intriguing patterns appeared for closely size-matched pairs (<5 mm carapace length difference), the results of the analysis did not permit us to infer that the processes underlying these sequences could be differentiated from random ones. Thus, such results prevented any conclusions about acoustic communication. This concurs with both the high attenuation of the buzzing sounds during the experiments and the poor understanding of acoustic perception by lobsters. New approaches that circumvent tank acoustic issues are now required to validate the existence of acoustic communication in lobsters.

Bioavailability and prebiotic potential of Carapax Trionycis, a waste from soft-shelled turtle processing.

BACKGROUND: Carapax Trionycis is the shell of the soft-shelled turtle. It is rich in minerals, amino acid, peptides, and other nutrients. Current processing and consumption of soft-shelled turtle leads to the waste of huge quantities of Carapax Trionycis in the form of spent materials. In this study, the bioavailability, prebiotic activity, and physicochemical properties of Carapax Trionycis using different processing methods were investigated. The vinegar-quenched Carapax Trionycis (V-CT), fine powders (D0.18, D0.10), and superfine powders (D0.05, D0.025) of Carapax Trionycis were prepared by the vinegar-quenching method, common grinding, and the superfine grinding method. RESULTS: The average particle sizes of D0.18, D0.10, D0.05, and D0.025 were 147.82, 77.35, 36.65, and 2.24 µm, respectively. Superfine grinding changed the surface morphology of Carapax Trionycis and promoted the release of active ingredients. D0.025 had the highest polypeptide (8.15%), polysaccharide (1.21%), total free amino acid (232.36 mg 100 g-1 ) and water-soluble extract content (10.74%), and showed the highest calcium release rate (55.64%) after in vitro digestion. The apparent permeability (PAPP ) of the resulting Carapax Trionycis samples in the dialysis tubing model and the everted intestinal sac model increased significantly with the decrease in the Carapax Trionycis particle sizes. Furthermore, the five Carapax Trionycis samples significantly stimulated the growth of the tested probiotics and increased lactic acids production after 48 h fermentation compared to the control. The Carapax Trionycis powder prepared by superfine grinding displayed better prebiotic activity than other samples as it significantly induced a greater proliferation of probiotic bacteria and higher production of lactic acid, as well as greater release of free calcium. CONCLUSIONS: The results showed that Carapax Trionycis superfine powder D0.025 had the highest active ingredient content, calcium bioavailability, and prebiotic activity. Our approach of developing Carapax Trionycis superfine powder as natural calcium supplement or potential prebiotic would therefore broaden the scope of soft-shelled turtle processing waste utilization in an eco-friendly, cost-effective, and sustainable approach in the future. © 2020 Society of Chemical Industry.