Tandem Mass Tag-based proteomic analysis of protein changes in superchilled crayfish (Procambarus clarkii) presoaked with carrageenan oligosaccharides.

To assess the effectiveness of carrageenan oligosaccharides (COs) in enhancing superchilling storage of crayfish, the physicochemical features of muscle and protein abundance in the refrigerated sample (RS), superchilled sample (SS) and COs soaked superchilled sample (CS) were evaluated. Microstructural and SDS-PAGE analyses suggested that CS exhibited fewer pores, with a microstructure and protein subunits distribution more similar to RS. Tandem Mass Tags quantitative proteomic analysis revealed 66 up-regulated differentially abundant proteins (DAPs) in the CS vs. SS batch, including myosin light chain 2, neural cadherin, integrin beta, lectin-like protein, toll-1, reticulon-1, and moesin/ezrin/radixin homolog 1, which facilitate cells adhesion and maintain membrane/cytoskeleton integrity. Eukaryotic Clusters of Orthologous Groups results confirmed that COs treatment increased the stability of crayfish myofibrillar proteins by up-regulating DAPs, which were concentrated in functional categories such as "posttranslation modification, protein turnover, chaperones", "signal transduction mechanisms", "energy production and conversion", and "cytoskeleton".

A transporter that allows phosphate ions to control the polymorph of exoskeletal calcium carbonate biomineralization.

The SLC20A2 transporter supplies phosphate ions (Pi) for diverse biological functions in vertebrates, yet has not been studied in crustaceans. Unlike vertebrates, whose skeletons are mineralized mainly by calcium phosphate, only minute amounts of Pi are found in the CaCO3-mineralized exoskeletons of invertebrates. In this study, a crustacean SLC20A2 transporter was discovered and Pi transport to exoskeletal elements was studied with respect to the role of Pi in invertebrate exoskeleton biomineralization, revealing an evolutionarily conserved mechanism for Pi transport in both vertebrates and invertebrates. Freshwater crayfish, including the study animal Cherax quadricarinatus, require repeated molt cycles for their growth. During the molt cycle, crayfish form transient exoskeletal mineral storage organs named gastroliths, which mostly contain amorphous calcium carbonate (ACC), an unstable polymorph long-thought to be stabilized by Pi. RNA interference experiments via CqSLC20A2 dsRNA injections reduced Pi content in C. quadricarinatus gastroliths, resulting in increased calcium carbonate (CaCO3) crystallinity and grain size. The discovery of a SLC20A2 transporter in crustaceans and the demonstration that knocking down its mRNA reduced Pi content in exoskeletal elements offers the first direct proof of a long-hypothesized mechanism by which Pi affects CaCO3 biomineralization in the crustacean exoskeleton. This research thus demonstrated the distinct role of Pi as an amorphous mineral polymorph stabilizer in vivo, suggesting further avenues for amorphous biomaterial studies. STATEMENT OF SIGNIFICANCE: • Crustaceans exoskeletons are hardened mainly by CaCO3, with Pi in minute amounts • Pi was hypothesized to stabilize exoskeletal amorphous mineral forms in vivo • For the first time, transport protein for Pi was discovered in crayfish • Transport knock-down resulted in exoskeletal CaCO3 crystallization and reduced Pi.

An improved withdrawal interval calculation and risk assessment of doxycycline in crayfish (Procambarus clarkii) in the natural cultured environment.

Doxycycline is an important medicine in aquaculture for treating fish diseases. However, its excess use causes residue exceeding to threaten human health. So, this study aimed to estimate a reliable withdrawal time (WT) of doxycycline (DC) in crayfish (Procambarus clarkii) based on statistical approaches and conduct a risk assessment for human health in the natural environment. Samples were collected at predetermined time points and determined by high-performance liquid chromatography. A novel statistical method was used to process the data of residue concentration. The homogeneity and linearity of the regressed line of data were evaluated by Bartlett's, Cochran's, and F tests. Outliers were excluded by establishing the standardized residual versus their cumulative frequency distribution on a normal probability scale. The calculated WT was 43 days in muscle of crayfish based on China and European stipulations. After 43 days, estimated daily intakes of DC were ranged from 0.022 to 0.052 µg/kg/d. Hazard Quotients were ranged from 0.007 to 0.014, which were far less than 1. These results indicated that established WT could avoid health risks for humans resulting from DC's residue in crayfish.

Integrating transcriptomic and metabolomic analysis to understand muscle qualities of red swamp crayfish (Procambarus clarkii) under transport stress.

This study investigated the transport stress (crowding stress and duration) on the physicochemical properties, energy metabolism and antioxidant enzyme activities of the red swamp crayfish (Procambarus clarkii) tail muscle (CTM). Besides, transcriptomic and metabolomic were conducted to elucidate the possible mechanism of CTM alternations during transport stress. The survival rate of crayfish gradually decreased with the external crowding stress and crowding time increasing. The transport stress also led to the increased distance among muscle fibers, water mobility and energy consumption, and the decreased of water holding capacity (WHC), hardness of CTM. The hepatopancreas exhibited more sensitive to crowding stress than muscle. The multi-omics analysis revealed that transport stress could interfere the translation and protein folding functions of ribosomal proteins, fatty acid metabolism and degradation, physiological functions of mitochondria in CTM. This study could provide critical information to increase the understanding of the regulation mechanism of crayfish when subjected to transport stress.

Effect of dietary Bacillus subtilis supplement on Cd toxicokinetics and Cd-induced immune and antioxidant impairment of Procambarus clarkii.

Cadmium (Cd), a non-biodegradable contaminant in freshwater ecosystems, can pose a serious threat to aquatic animals at high levels. In this study, the Cd toxicokinetics and the immune and antioxidant defense were explored in Procambarus clarkii exposed to different levels of Cd (0, 0.1, 1.0 mg Cd/L) or treated with 1.0 mg Cd/L and dietary Bacillus subtilis supplementation (1 × 107 cfu/g). Results from the 21-day uptake and depuration experiment revealed that Cd exposure elicited a dose- and time-dependent uptake in all crayfish tissues, and the rank order of Cd concentration was gill > hepatopancreas > exoskeleton > muscle. The one-compartment model demonstrated that gills had the highest uptake rate (ku) value after Cd aqueous exposure and the ku and elimination rate (kd) values in gill, hepatopancreas, and exoskeleton of the group with 1.0 mg Cd/L were higher than those of the group at alow Cd concentration (0.1 mg Cd/L). However, B. subtilis could decrease Cd ku and increase Cd kd in hepatopancreas, resulting in the reduction of bioconcentration factors (BCF), steady-state concentrations (Css), and biological half-life (Tb1/2). A positive correlation was found between aqueous Cd concentration and the severity of hepatopancreas histopathological injury, while B. subtilis could ameliorate the pathological damage in the high Cd group. Similarly, aqueous exposure to Cd elevated malonaldehyde (MDA) content and suppressed the activities of lysozyme (LZM), acid phosphatase (ACP) in hepatopancreas and alkaline phosphatase (AKP) in hemolymph. The activities of superoxide dismutase (SOD) and catalase (CAT) in hepatopancreas were also inhibited. Nevertheless, they were all recovered with the dietary addition of B. subtilis. In conclusion, our results indicated that exposure to Cd significantly increased Cd accumulation and toxic damages in crayfish hepatopancreas, while dietary administration of B. subtilis to crayfish significantly decreased Cd accumulation and improved the immune and antioxidant defense, leading to the prevention in toxic effects of Cd.

Characterization of chitinase from Exiguobacterium antarcticum and its bioconversion of crayfish shell into chitin oligosaccharides.

Crayfish have become a heavily consumed food and its chitin-rich shell is of great value in terms of waste conversion. This study found a novel chitinase (EaChi40) from a marine bacterium Exiguobacterium antarcticum. The gene was cloned and expressed as a soluble protein of 40 kDa, having optimal activity at pH 6.0 and 30 °C. EaChi40 showed good stability and high specific activity, and kinetic studies found Km and Vmax were 0.86 mg/mL and 13.66 µmol/min/mg. For conversion crayfish shell into oligosaccharides, ball milling and ultrasound-assisted hydrogen peroxide decolorization were applied to pretreat crayfish shell to facilitate its hydrolysis. After the enzymatic conversion, the hydrolysis products of chitobiose and N-acetyl-D-glucosamine were 9.09 mg/mL and 9.21 mg/mL, respectively. EaChi40 efficiently degraded crayfish with a high hydrolysis rate of 76.1%. It is expected to be a good candidate for the production of chitin oligosaccharides in the food and biological fields.

A QuEChERS-HPLC-MS/MS Method with Matrix Matching Calibration Strategy for Determination of Imidacloprid and Its Metabolites in Procambarus clarkii (Crayfish) Tissues.

We developed a method for determination of imidacloprid and its metabolites 5-hydroxy imidacloprid, olefin imidacloprid, imidacloprid urea and 6-chloronicotinic acid in Procambarus clarkii (crayfish) tissues using quick, easy, cheap, effective, rugged, and safe (QuEChERS) and high-performance liquid chromatography-triple quadrupole mass spectrometry. Samples (plasma, cephalothorax, hepatopancrea, gill, intestine, and muscle) were extracted with acetonitrile containing 0.1% acetic acid and cleaned up using a neutral alumina column containing a primary secondary amine. The prepared samples were separated using reverse phase chromatography and scanned in the positive and negative ion multiple reaction-monitoring modes. Under the optimum experimental conditions, spiked recoveries for these compounds in P. clarkii samples ranged from 80.6 to 112.7% with relative standard deviations of 4.2 to 12.6%. The limits of detection were 0.02-0.5 µg·L-1, the limits of quantification were 0.05-2.0 µg·L-1 and the method of quantification was 0.05-2.0 µg·kg-1. The method is rapid, simple, sensitive and suitable for rapid determination and analysis of imidacloprid and its metabolites in P. clarkii tissues.

Recycling biofloc waste as novel protein source for crayfish with special reference to crayfish nutritional standards and growth trajectory.

Screening of novel feedstuffs, that too for data-deficient (nutritionally) animals, is somewhat ambiguous or problematic. Through systematic meta-analyses, the present study formulated most up-to-date crayfish nutritional standards, against which a recyclable waste (biofloc biomass, BM) from intensive aquaculture systems was assessed as a novel protein source. Growth trajectory dependencies and thermal growth coefficient qualifying for good growth in crayfish (TGC 0.5-0.64 units) were benchmarked. Using these standards and a 7-week growth trial, BM's suitability as a novel protein source for red swamp crayfish Procambarus clarkii was evaluated through its graded inclusions in a commercial feed. Results suggest that BM can elevate growth at 33-66% inclusion in existing feed formulations. Beyond 66% inclusion, BM can deteriorate growth in crayfish due to high ash content (exceeding physiological limit > 14%), arginine deficiency (~ 14-20% lower than an optimum requirement), and insufficient non-protein energy: protein ratio (3.7 cal mg-1). Arginine is perhaps the most critical amino acid in dietary protein for crayfish, and deficient in BM. Although no critical bioaccumulation levels of heavy metals were breached by feeding 100% BM to crayfish, a mineral and heavy metal (Hg) stress seemed plausible. Crayfish raised solely on biofloc may not realize full growth potential.

Ionophore Toxin Maduramicin Produces Haff Disease-Like Rhabdomyolysis in a Mouse Model.

Maduramicin is a toxic ionophore antibiotic that is isolated from Streptomyces, frequently occurring in an aquatic environment. To understand the potential role of maduramicin in crayfish consumption related Haff disease, a mouse model was established in this study. Two exposure routes of maduramicin in the abdominal muscle and the hepatopancreas tissue homogenates of crayfish were given intragastrically to mice in different doses for seven days. Action changes, clinical symptoms, feed consumption, body weight, blood biochemistry, and histopathology examination of mice were observed and analyzed. In the natural exposure group, relatively low concentration of maduramicin in crayfish muscle and hepatopancreas had no obvious effects on mental state, body weight, blood biochemical indexes, or histologic appearance. However, in the artificial exposure group, with increasing concentrations, maduramicin in crayfish muscle and hepatopancreas homogenates both induced mental sluggishness and weight loss of mice. Blood biochemical examination showed that 3.5 mg·kg-1 and 7 mg·kg-1 maduramicin in crayfish tissue homogenates significantly increased levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), lactate dehydrogenase (LDH), and creatine kinase (CK). Additionally, histopathological examination showed that multiple organs were damaged by maduramicin, including degeneration of liver cells, shedding of renal epithelial cells, and disturbance and partial lysis of myocardial and skeletal muscle filaments in the mice. In summary, maduramicin may not cause Haff disease through contamination of the aquatic environment under normal conditions. Maduramicin can be used as a potential toxin tool to establish a rhabdomyolysis disease animal model for drug development.

Red color-related proteins from the shell of red swamp crayfish (Procambarus clarkii): Isolation, identification and bioinformatic analysis.

Two water-soluble red color-related proteins with the molecular masses of 24 and 73 kDa were purified from the shell of Procambarus clarkii. Initial color changes of these two proteins were detected at 30 °C and the large amount of red precipitate were obtained at 80 °C. PAGE analysis showed that the 24 kDa protein was the monomer, while the 73 kDa protein was the trimer. Identification revealed that these two proteins belonged to the hemocyanin subunit 2 family. With respect to the amino acid sequence similarity, the red color-related proteins shared the highest sequence identity with the hemocyanin derived from giant freshwater prawn (Macrobrachium rosenbergii). The phylogenetic tree analysis also clearly supported this finding. The shell-derived red color-related proteins show potential use as the edible thermal-sensitive indicator in food processing field.

Nutritional Value and Contaminant Risk Assessment of Some Commercially Important Fishes and Crawfish of Lake Trasimeno, Italy.

The aim of our study was to describe the balance between health benefits and risks associated with the consumption of crawfish and nine fish species from lake Trasimeno. We thus determined both fatty acid profiles (particularly, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids) and chemical pollutants (some polychlorinated biphenyls, pesticides, and heavy metals) in fish muscle tissues. The contents of all fatty acids varied significantly among species. Sand smelt, carp, and tench, which have a high fat content, contained considerable amounts of EPA and DHA; lean fish, like perch, pike, and largemouth bass, which have relatively high percentages of the predominant n-3 fatty acids EPA and DHA, showed lower amounts of these fatty acids because of their low lipid contents. Some species contributed strongly to the Dietary Reference Intake (RDI) of EPA and DHA. The contribution of lean fish to the RDI of EPA and DHA was more limited. The concentrations of all contaminants in fish muscle tissues were lower than the regulatory limits, demonstrating the safety of the environmental conditions of the lake. The contribution to health-based reference values and benefit-risk quotients indicated that the health benefits of consumption of fish from lake Trasimeno outweigh the potential risks.

Ultrasound treatment of frozen crayfish with chitosan Nano-composite water-retaining agent: Influence on cryopreservation and storage qualities.

The synergistic impact of ultrasound and Nano-water retaining agent on the cryoprotective effect on crayfish during frozen storage was investigated. The samples soaked in water-retaining agent (WRA), ultrasonic 60 W treatment combined with water-retaining agent (WRA-US60W) and ultrasonic 80 W treatment combined with water-retaining agent (WRA-US80W) were frozen storage at -18 °C. The indices of frozen storage in 0, 7, 14, 21, 28, 35 and 42 days were measured. The results showed that there was a significant difference in the soaking weight gain, thawing loss, water content and water activity between control group and WRA groups (P < .05). The ultrasound combined with WRA treatment showed better water retention effect. The water holding capacity of heat induced gel decreased continuously during the frozen storage period, and the WRA-US60W group exhibited significantly higher values than that of other treatment groups (P < .05). The texture characteristics of hardness, elasticity and chewiness had a significant change (P < .05). The development of total volatile base nitrogen content, myofibrillar protein content and Ca2+-ATPase activity of muscle protein were significantly delayed by WRA-US60W treatment, maintaining the integrity of tissue structure. Therefore, WRA-US60W treatment was found effectively improving the quality of crayfish during frozen storage.

The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of astacin metallopeptidases.

Human fetuin-B plays a key physiological role in human fertility through its inhibitory action on ovastacin, a member of the astacin family of metallopeptidases. The inhibitor consists of tandem cystatin-like domains (CY1 and CY2), which are connected by a linker containing a "CPDCP-trunk" and followed by a C-terminal region (CTR) void of regular secondary structure. Here, we solved the crystal structure of the complex of the inhibitor with archetypal astacin from crayfish, which is a useful model of human ovastacin. Two hairpins from CY2, the linker, and the tip of the "legumain-binding loop" of CY1 inhibit crayfish astacin following the "raised-elephant-trunk mechanism" recently reported for mouse fetuin-B. This inhibition is exerted by blocking active-site cleft sub-sites upstream and downstream of the catalytic zinc ion, but not those flanking the scissile bond. However, contrary to the mouse complex, which was obtained with fetuin-B nicked at a single site but otherwise intact, most of the CTR was proteolytically removed during crystallization of the human complex. Moreover, the two complexes present in the crystallographic asymmetric unit diverged in the relative arrangement of CY1 and CY2, while the two complexes found for the mouse complex crystal structure were equivalent. Biochemical studies in vitro confirmed the differential cleavage susceptibility of human and mouse fetuin-B in front of crayfish astacin and revealed that the cleaved human inhibitor blocks crayfish astacin and human meprin α and ß only slightly less potently than the intact variant. Therefore, the CTR of animal fetuin-B orthologs may have a function in maintaining a particular relative orientation of CY1 and CY2 that nonetheless is dispensable for peptidase inhibition.

Determination of pendimethalin in water, sediment, and Procambarus clarkii by high performance liquid chromatography-triple quadrupole mass spectrometry.

We established a high-performance liquid chromatography-triple quadrupole mass spectrometry method for the analysis of pendimethalin residues in water, sediments, and Procambarus clarkii (Louisiana crayfish) tissues. Water samples were concentrated on a HLB solid-phase extraction column and eluted with dichloromethane and acetone (1:1). After drying under a stream of nitrogen gas, the sample volume was adjusted to 1 mL with the mobile phase solvent methanol/water/acetic acid (8:20:0.1). Pendimethalin was extracted with ethyl acetate containing 0.1% acetic acid, after rotary evaporation to dryness at 35 °C, the residue was dissolved in mobile phase solvent, purified by a neutral alumina column and graphitized carbon black powder (0.1 g). The mass characterization was conducted in positive ion mode, and the corresponding ions were detected in multi-reaction monitoring mode. The linear equations were y = 1 × 106x + 14275, at pendimethalin levels of 0.05-20 µg L-1 and y = 691029 × - 414368 for 20-200 µg L-1. The detection limits of pendimethalin in water, sediments, and P. clarkii tissues were 1.0 × 10-4µg L-1 , 5.0 × 10-3µg kg -1 and 5.0 × 10-3 µg kg -1, respectively. The spiked recoveries ranged from 81.6 to 106.3%, and the relative standard deviations ranged from 4.58 to 13.6% (n = 6). The method provided an efficient and low-cost extraction and purification procedure that enabled a sensitive determination of pendimethalin in water as well as complex matrices.

Desorption of calcium-rich crayfish shell biochar for the removal of lead from aqueous solutions.

In this study, crayfish shell was pyrolyzed at 600 °C to obtain a calcium-rich biochar (CS600). The biochar was saturated with Pb2+ in batch adsorption experiment. Six desorption reagents including HCl, HNO3, H2SO4, NaOH, EDTA and EDTA-2NaCa were then used to desorb Pb2+ from the post-adsorption CS600 to determine the desorption mechanism. Lead adsorption experiments were performed to determine the adsorption capacity of the regenerated CS600. HCl, HNO3, EDTA and EDTA-2NaCa presented good desorption effect with desorption rates all over 80%. Most of the regenerated biochars lost their lead adsorption ability due to the destruction of CaCO3 mineral on their surface. Only the one desorbed by EDTA-2NaCa retained a preferable lead adsorption capacity. EDTA-2NaCa thus is a promising desorption reagent of CS600 for the removal of lead from aqueous solutions.

Cu accumulation, detoxification and tolerance in the red swamp crayfish Procambarus clarkii.

Copper is an essential metal but potentially toxic to aquatic animals at high levels. The present study investigated physiologically adaptive responses to waterborne Cu2+ exposure (0, 0.03, 0.30, 3.00 mg/L) in a representative species of crustaceans, the red swamp crayfish (Procambarus clarkii) for 7 d, followed by a 7-d depuration period. The tissue-specific distribution of Cu showed that crayfish hepatopancreas was the primary accumulating site among internal tissues. During Cu2+ exposure, crayfish repressed the expression level of Cu homeostasis genes (Ctr1, Atox1, copper-transporting ATPase 2, MTF-1/2, and MT) in hepatopancreas to inhibit intracellular Cu transporting. Cu2+-exposed crayfish increased activities of GPx and GST, GSH contents, and mRNA expression of antioxidative enzyme genes (Cu/Zn-sod, cat, gpx, gst) to cope with the Cu2+-induced oxidative stress which accompanied by an increased MDA content. Additionally, after a 7-d depuration, crayfish effectively eliminated excess Cu from hepatopancreas by up-regulating expression level of Cu homeostasis genes, and recovered from oxidative damage by enhancing antioxidative enzyme gene expression (Cu/Zn-sod, cat, gpx, gst) and consuming more GSH, which thereby caused a return of the MDA level to the control value. Overall, our study provided new insights into the regulatory mechanisms of cellular Cu homeostasis system and antioxidative system, contributing to Cu detoxification and tolerance ability exhibited by crayfish under Cu2+ stress and after withdrawal of Cu2+ stress.

Preparation and antioxidant properties of crayfish (Procambarus clarkii) By-products protein hydrolysates and ultra filtration fractions.

Protein isolate from crayfish by-products (CBPI) were hydrolyzed using Alcalase, neutrase, pancreatin and bromelain. Hydrolysis by Alcalase had more remarkable digesting efficiency on crayfish by-products protein than that by the other enzymes. Therefore, protein hydrolysate from Alcalase digestion (CBPHa) was selected to be fractionated by ultrafiltration according to molecular weight into three fractions F1 (MW <1kDa), F2 (MW 1-3kDa) and F3 (MW 3-10kDa). The amino acid determination revealed that CBPI had essential amino acid (EAA) close to that required for human protein synthesis. In vitro activity experiments showed that CBPHa and its fractions possessed considerable antioxidant activity. F1 exhibited the highest DPPH, superoxide radicals scavenging activities and Fe2+ chelating ability, whereas F2 showed the best hydroxyl radicals scavenging capacity and reducing power. In addition, all the fractions showed higher super oxide radical scavenging activity than the crude hydrolysates. Our findings suggest that CBPHa and their ultra filtration fractions have the potential for use in nutraceutical and functional food industries to maximize the use of crayfish processing by-products.

Simultaneous fermentation and hydrolysis to extract chitin from crayfish shell waste.

Chitin is the second-most abundant bioresource and widely used in the food, agricultural, textile, biomedical, and pharmaceutical industries. However, an efficient, environmentally friendly, and economically feasible process for chitin extraction from shellfish waste remains to be explored. This study aimed to extract chitin from crayfish shell waste powder (CSP) by removing Ca2+ and protein, using Bacillus coagulans LA204 and proteinase K. A simultaneous enzymatic hydrolysis and fermentation process was conducted at 50 °C with 5% (w/v) CSP, 5% (w/v) glucose, 1000 U proteinase k g-1 CSP, and 10% inoculation of B. coagulans LA204 in a 5-L bioreactor under non-sterile conditions. After 48 h of fermentation, the deproteinization efficiency, demineralization efficiency, and chitin recovery reached 93%, 91%, and 94%, respectively. 1 mol additional glucose efficiently removed 0.91 mol calcium carbonate and 93% of the removed protein was hydrolyzed to acid-soluble protein. Simultaneous enzymatic hydrolysis and fermentation was a new strategy and a competitive biological method for chitin extraction.

Anti-proliferative effect of chitosan nanoparticles (extracted from crayfish Procambarus clarkii, Crustacea: Cambaridae) against MDA-MB-231 and SK-BR-3 human breast cancer cell lines.

Actually, the most common cancer in women is the breast cancer which is the second most widespread cancer overall. In 2018, there were over two million new cases of women breast cancer. Particularly, we tried to extract chitosan from crayfish Procambarus clarkii, Crustacea: Cambaridae, by N-deacetylation of chitin. The chemical structure of chitosan was characterized by Fourier transform infrared (FT-IR) spectroscopy. Also DDA was calculated from FT-IR and ultraviolet spectrophotometry data. Chitosan nanoparticles were prepared using a ball-milling technique. The as-prepared chitosan nanoparticles were characterized by transmission electron microscopy, dynamic light scattering as well as zeta potential. The cytotoxicity of chitosan and its nanoparticles (50 and 100 µg/mL) against human breast cancer (SK BR3 and MDA-MB-231 cell lines) was evaluated. MTT assay asserts the significant inhibitory action of both chitosan and its nanoparticles on the proliferation of human breast cancer cells in vitro. Chitosan nanoparticles had more anti-proliferative effects on MDA-MB-231 and SK-BR-3 cell lines than its corresponding chitosan. Although, chitosan nanoparticles, that has higher DDA, had a higher cytotoxic activity against human breast cancer MDA-MB-231 and SK-BR-3 cell lines in vitro. Eventually, chitosan and its nanoparticles can be considered as a promising natural compounds in human breast cancer treatment.

[Determination of 39 veterinary drug residues in Procambarusclarkii by ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry].

An ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) method was developed for determination of 39 veterinary drugs in Procambarusclarkii. Samples were extracted with acidified acetonitrile and purified with enhanced matrix removal-lipid (EMR-Lipid) adsorbent and graphitized multi-walled carbon nanotubes (GMWNTs). Extracts were analyzed by UHPLC-Q-TOF-MS. Target compounds were screened according to accurate mass, isotopic ratio, and retention time, and confirmed by library search. Target analytes were quantified using the chromatographic peak area of each compound. For each of the drugs of interest, calibration curves were linear in their respective ranges with correlation coefficients greater than 0.99. The limits of quantification (LOQs) were 3-15 µg/kg. The recoveries for target compounds spiked in Procambarusclarkii ranged from 62.4% to 105.8%, with relative standard deviations (RSDs) ranging from 2.5% to 13.5%. This method is simple, rapid, accurate, and suitable for simultaneous determination of 39 veterinary drugs, including sulfonamides, quinolones, and triphenylmethane, extracted from Procambarusclarkii.