Selective protein adsorption is a key challenge for the development of biosensors, separation technologies, and smart materials for medicine and biotechnologies. In this work, a strategy was developed for selective protein adsorption, based on the use of mixed polymer brushes composed of poly(ethylene oxide) (PEO), a protein-repellent polymer, and poly(acrylic acid) (PAA), a weak polyacid whose conformation changes according to the pH and ionic strength of the surrounding medium. A mixture of lysozyme (Lyz), human serum albumin (HSA), and human fibrinogen (Fb) was used to demonstrate the success of this strategy. Polymer brush formation and protein adsorption were monitored by quartz crystal microbalance, whereas protein identification after adsorption from the mixture was performed by time-of-flight secondary ion mass spectrometry (ToF-SIMS) with principal component analysis and gel electrophoresis with silver staining. For the ToF-SIMS measurements, adsorption was first performed from single-protein solutions in order to identify characteristic peaks of each protein. Next, adsorption was performed from the mixture of the three proteins. Proteins were also desorbed from the brushes and analyzed by gel electrophoresis with silver staining for further identification. Selective adsorption of Lyz from a mixture of Lyz/HSA/Fb was successfully achieved at pH 9.0 and ionic strength of 10^-3 M, while Lyz and HSA, but not Fb, were adsorbed at ionic strength 10^-2 M and pH 9.0. The results demonstrate that by controlling the ionic strength, selective adsorption can be achieved from protein mixtures on PEO/PAA mixed brushes, predominantly because of the resulting control on electrostatic interactions. In well-chosen conditions, the selectively adsorbed proteins can also be fully recovered from the brushes by a simple ionic strength stimulus. The developed systems will find applications as responsive biointerfaces in the fields of separation technologies, biosensing, drug delivery, and nanomedicine.

A comprehensive insight into the interaction between proteins and poly(ethylene glycol) (PEG) is crucial to understand the behavior of PEG, which is widely used in pharmaceutical and medical applications. Although PEG is believed to be an excellent material to resist non-specific protein adsorption, there is a lack of quantitative information about the interactions between proteins and PEG. In this paper the interactions of bovine serum albumin (BSA) and lysozyme (LYZ) with different molecular weight (MW) PEGs were investigated through the T2 relaxation time of PEGs measured by low field nuclear magnetic resonance spectroscopy. The integrated signal intensity of PEGs was quantified under various conditions from the concentrations and MWs of PEG, and ionic strength of solutions, as well as the molar ratios of PEG to protein. The results show that a large number of PEG molecules could associate with protein molecules with association constants in the range ~10(4)-10(5) M(-1). The association constant is insensitive to the ionic strength change in the physiological range and the lowest associate constant occurs at the medium MW PEG with protein. This suggests that the interaction between PEG and protein molecules might not be negligible in investigations of the resistance to non-specific protein adsorption. Long chain PEG coatings might cause modest protein adsorption, which could interfere with any weak specific interaction between ligand and receptor. Thus, it is necessary to reconsider the popular accepted method of protecting nanoparticles (NP) in blood with long chain PEG coatings since these NPs might be surrounded by a layer of weakly adsorbed plasma protein in the circulatory system.

Biomedical sciences, and in particular biomarker research, demand efficient glycoprotein enrichment platforms. In this work, a facile and efficient method was developed to synthesize boronic acid polymer brushes immobilized on magnetic graphene oxide via surface initiated atom transfer radical polymerization (SI-ATRP) for the selective enrichment of glycoproteins from complex biological samples. The magnetic graphene oxide (GO@Fe₃O₄) nanocomposites were prepared by a solvothermal reaction, providing an ultrahigh surface area and allowing fast separation. Through the self-assembly procedure, the pyrene-based initiators (GO@Br) of SI-ATRP were easily functionalized on the GO sheet via noncovalent π-π interaction between pyrene and GO. Finally, the well-defined and high density poly(4-vinylphenylboronic acid) brushes (GO@PVPBA) via SI-ATRP were successfully fabricated. The morphology and structure of GO@Fe₃O₄, GO@Br, and GO@PVPBA nanocomposites were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), vibrating sample magnetometry (VSM), Fourier transform-infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). The selective recognition capability of GO@PVPBA nanocomposites was demonstrated by the selective enrichment of glycoproteins from a complex system consisting of standard proteins ovalbumin (OVA), transferrin (Trf), bovine serum albumin (BSA), and lysozyme (Lyz). Furthermore, the GO@PVPBA nanocomposite also exhibited a high binding capacity up to 514.8 and 445.9 mg g^-1 for OVA and Trf, respectively, and was applied to capture directly glycoproteins from the egg white samples.

Complex formation of poly(N-isopropylacrylamide) (PNIPA) having a weight-average molecular weight of 1,720,000g/mol with human serum albumin (HSA), ovalbumin (OVA) and lysozyme (LYZ) was studied in an aqueous medium containing 0.01 M NaCl and adjusted to pH 3. The polymer-protein mixtures at different molar ratios (r(m)) were examined by static light scattering (SLS). The analysis of SLS data using our own approach [Kokufuta et al., Langmuir 15 (1999) 940; Biomacromolecules 4 (2003) 728] showed that the molecular weight of each resulting complex is smaller than that of the interpolymer complex composed of two polymer chains plus one protein. This indicates the formation of an intrapolymer complex in all the polymer-protein systems studied. Thus, at each r(m) we calculated the number of bound proteins per polymer, the value of which was OVA>HSA>LYZ in order. These results were compared with the hydropathy profiles of each protein which are a good tool for obtaining an information about distribution of hydrophobic and hydrophilic segments in a protein. It has become apparent that the hydrophobic interaction between polymer and protein plays an important role in the intrapolymer complex formation.

Microplastics (MPs) are ubiquitous in the environment and pose substantial threats to the water ecosystem. However, the impact of natural aging of MPs on their toxicity has rarely been considered. This study found that visible light irradiation with hydrogen peroxide at environmentally relevant concentration for 90 days significantly altered the physicochemical properties and mitigated the toxicity of polyamide (PA) fragments to infantile zebrafish. The size of PA particles was reduced from ∼8.13 to ∼6.37 μm, and nanoparticles were produced with a maximum yield of 5.03%. The end amino groups were volatilized, and abundant oxygen-containing groups (e.g., hydroxyl and carboxyl) and carbon-centered free radicals were generated, improving the hydrophilicity and colloidal stability of degraded MPs. Compared with pristine PA, the depuration of degraded MPs mediated by multixenobiotics resistance was much quicker, leading to markedly lower bioaccumulation in fish and weaker inhibition on musculoskeletal development. By integrating transcriptomics and transgenic zebrafish [Tg(lyz:EGFP)] tests, differences in macrophages-triggered proinflammatory effects, apoptosis via IL-17 signaling pathway, and antioxidant damages were identified as the underlying mechanisms for the attenuated toxicity of degraded MPs. This work highlights the importance of natural degradation on the toxicity of MPs, which has great implications for risk assessment of MPs.

Nowadays, the research on the recognition and separation of proteins has attracted extensive attention in the fields of materials science, bioengineering and life science. Protein imprinted polymers are ideal recognition materials, due to its high selectivity, good stability, easy preparation, and low cost. Herein, novel surface imprinting biowaste-derived molecularly imprinted polymers (BD-MIPs) were synthesized for specific recognition and purification of lysozyme (Lyz). This is the first time that magnetic pomegranate rind-derived carbon was used as a carrier to immobilize Lyz. Then, with the self-polymerization of dopamine, a large number of biocompatible recognition sites were generated under mild conditions. The physical/chemical properties and surface morphologies of the synthetic BD-MIPs were characterized, indicating that the imprinted film was successfully synthesized, and the BD-MIPs had good thermal stability and magnetic property. To investigate the recognition performance of BD-MIPs, four adsorption experiments were performed. The results show that BD-MIPs had a high adsorption capacity of 301.87 mg g^-1, fast equilibrium time within 40 min, satisfactory selectivity and good reusability for Lyz. Furthermore, the practicability of BD-MIPs was confirmed by the isolation of Lyz from a biological sample. The good adsorption capacity and gentle one-step preparation make the BD-MIPs attractive for Lyz recognition, which shows potential values in basic biomedical research, industrial protein purification and clinical diagnostics.

Keywords: Agricultural waste; Biological application; Magnetic pomegranate rind carbon; Protein imprinted polymers; Protein recognition.

Molecularly imprinted polymers (MIPs) were applied for selective enrichment of proteins in MALDI-TOF MS analysis for the first time. MIPs were achieved simply via immobilization of template lysozyme (Lyz) on core-shell magnetic nanoparticles and self-polymerization of dopamine (DA).

A unique split-type photoelectrochemical (PEC) immunoassay has been constructed for detection of low-abundance biocompounds (lysozyme, Lyz, used in this case) via a new trigger strategy by disintegrating bioconjugates of dopamine-grafted silica nanospheres (DA@SiO2NSs) for signal amplification. The preferred electron donor assembly of DA@SiO2NSs is first used as a molecular printboard for positioning anti-Lyz secondary antibody (Ab2) through an amide reaction. With specific immunoreactions in a high-binding microplate, a sandwich immunoassay, the DA@SiO2NSs-based bioconjugate is achieved. By initiating the disintegration of the bioconjugates via acid etching, numerous electron donors of DA are released, thus efficiently triggering hole-trapping with amplified signals obtained. The smart integration of ZnIn2S4-based heterojunctions as photoactive material, a split-type detection mode, and a new trigger strategy by disintegrating the DA@SiO2NSs-based bioconjugate offer an attractive high-throughput signal-on PEC immunoassay for detection of Lyz. Such an unusual PEC sensor exhibits an outstanding linear response to the concentration in the range between 0.002 and 500 ng mL-1, and the detection limit is as low as 0.6 ppt ( S/ N = 3). The as-fabricated assay is cost-effective and sensitive. It has been successfully used for measuring Lyz in real samples, which demonstrates great promise for practical applications.

An efficient and selective glycoproteins enrichment platform is essential to glycoprotein biomarkers in early clinic diagnostics. In this work, the poly (ethylene glycol) methyl ether methacrylate (PEGMA) and 4-vinylphenylboronic acid (VPBA) copolymer brushes grafted magnetic carbon nanotubes composite MCNTs@p (PEGMA-co-VPBA) was prepared by surface-initiated atom transfer polymerization and applied for the selective enrichment of glycoproteins from the complex biological samples. The as-prepared MCNTs@p (PEGMA-co-VPBA) nanocomposite was characterized by Fourier transform-infrared spectroscopy (FTIR), transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and vibrating sample magnetometry (VSM). The MCNTs@p (PEGMA-co-VPBA) can recognize and bind specifically for glycoproteins via strong boronate affinity and excellent hydrophilicity and shows a really low non-specificity adsorption capability for non-glycoproteins. The adsorption capacity of MCNTs@p (PEGMA-co-VPBA) towards glycoproteins transferrin (Trf), horseradish peroxidase (HRP), and non-glycoproteins cytochrome c (Cyt C), lysozyme (Lyz) is 253.3 mg g^-1, 51.1 mg g^-1, 13.9 mg g^-1 and 14.5 mg g^-1, respectively. Furthermore, MCNTs@p (PEGMA-co-VPBA) can be applied to extract glycoproteins directly from egg white samples. These results demonstrated that MCNTs@p (PEGMA-co-VPBA) could be a potential affinity adsorbent in glycoprotein enrichment.

A hierarchical imprinting strategy was used to create protein imprints in a silicate film with a high binding capacity as well as selectivity toward the imprint protein and little specificity towards other proteins. In the first part of this work, rod-shaped bacteria were used as templates to create imprints in silica films of various thicknesses to open up the silica framework and increase the surface area exposed to solution. In the second part, the protein (e.g., cytochrome c (CYC) or green fluorescent protein (GFP)) was covalently attached to the surface of Bacillus subtilis and this protein-bacteria complex served as the imprint moiety. Atomic force microscopy and scanning electron microscopy were used to image the micron-size rod-shaped bacteria imprints formed on the silica surface. Fluorescence microscopy, which was used to follow the fabrication process with GFP as the representative protein, clearly demonstrated protein imprinting, protein removal and protein rebinding as well as protein specificity. Visible absorption spectroscopy using CYC as the imprint protein demonstrated relatively fast uptake kinetics and also good specificity against other proteins including bovine serum albumin (BSA), horseradish peroxidase (HRP), glucose oxidase (GOD), and lysozyme (LYZ). Collectively this work demonstrates a new surface bio-imprinting approach that generates recognition sites for proteins and provides a viable means to increase the binding capacity of such imprinted thin films.

This study describes the use of human serum albumin (HSA)-modified gold nanoparticles (HSA-AuNPs) for the selective extraction and enrichment of high-pI protein, lysozyme (Lyz) prior to analysis by capillary electrophoresis (CE) with UV detection. HSA-AuNPs are capable of extracting Lyz from a complex matrix because a HSA capping layer not only stabilizes gold nanoparticles in a high-salt environment but also exhibits strong electrostatic attraction with Lyz under neutral pH condition. Efficient separation of Lyz and other high-pI proteins has been successfully achieved by the filling of cationic polyelectrolyte, poly(diallydimethylammonium chloride) (PDDAC), to the background electrolyte. After capturing Lyz with HSA-AuNPs, PDDAC-filled CE can be directly used for the analysis of the extracted Lyz without the addition of the releasing agent into the extractor. The extraction efficiency relied on the pH of the solution and the concentration of HSA-AuNPs. Under optimal extraction conditions, the limit of detection at a signal-to-noise ratio of 3 for Lyz was down to 8 nM. The combination of HSA-AuNP extraction and PDDAC-filled CE has been applied the analyses of Lyz in hen egg white, human milk, and human tear. Also, this NP-based extraction can be coupled to matrix-assisted desorption/ionization time-of-flight mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A protein-based molecularly imprinted monolithic column was synthesized based on ionic liquids (ILs) and deep eutectic solvents (DESs) in a stainless steel column (50 mm × 4.6 mm id). An IL (1-allyl-3-butylimidazolium Br) and acrylamide were used as dual monomers. Another type of IL (1,2-bis [N,N'-vinylimidazolium] ethane bis Br) and N,N'-methylenebisacrylamide were used as dual cross-linking agents, and the DES (choline chloride : ethylene glycol 1 : 2) was used as a porogen in the preparation of a monolithic polymer. Bovine serum albumin (BSA) and lysozyme (Lyz), which differ greatly in molecular size, isoelectric point, and charge, were selected for imprinting templates to evaluate the recognition property of the green solvent-based MIP monolithic column. Some important factors, such as template-monomer molar ratio, total monomer concentration, and cross-linking density, were investigated systematically. Under optimal conditions, the MIP monolithic column obtained showed higher binding affinity for the templates than its corresponding non-imprinted (NIP) monolithic column.

Selenium (Se) is an essential trace element for aquatic animals. The aquatic plant Potamogeton maackianus is an important natural food of Chinese mitten crab (Eriocheir sinensis). The aim of this study was to determine whether the antioxidant and immune responses of Chinese mitten crab are affected by including Se-cultured P. maackianus in the diet. Three groups of P. maackianus were cultured at levels of 0.02 mg/kg Se, 8.83 mg/kg Se, and 16.92 mg/kg Se, and the plants in these groups were used in experimental diets fed to crabs (dietary Se content of 0.05, 0.43, and 0.82 mg/kg, respectively). Compared with crabs in the 0.05 mg/kg group, those in the 0.82 mg/kg group showed significantly increased specific growth rate, protease and lipase activities, triglyceride and cholesterol contents, and Se content in the hepatopancreas and muscle (P < 0.05); increased activities of glutathione peroxidase, glutathione reductase, and catalase in the antioxidant system; increased transcript levels of MT (encoding metallothionein); and decreased malondialdehyde content (P < 0.05). At the end of the rearing experiment, the crabs in the different groups were exposed to copper (Cu²⁺) stress for 96 h. All the juvenile crabs in the 0.43 and 0.82 mg/kg groups survived 96 h of Cu²⁺ stress. Crabs in the 0.82 mg/kg group showed enhanced antioxidant responses under Cu²⁺ stress, increased transcript levels of MT and LYZ, and increased resistance. Therefore, supplementation of the diet of Chinese mitten crab with increased levels of Se-cultured P. maackianus can reduce oxidative stress under Cu²⁺ exposure, activate the immune response, and benefit growth.

Keywords: Chinese mitten crab (Eriocheir sinensis); Cu2+ exposure; Potamogeton maackianus; immune regulation; oxidative stress; selenium.

Granulin is a pleiotropic protein involved in inflammation, wound healing, neurodegenerative disease, and tumorigenesis. These roles in human health have prompted research efforts to use granulin to treat rheumatoid arthritis and frontotemporal dementia and to enhance wound healing. But how granulin contributes to each of these diverse biological functions remains largely unknown. Here, we have uncovered a new role for granulin during myeloid cell differentiation. We have taken advantage of the tissue-specific segregation of the zebrafish granulin paralogues to assess the functional role of granulin in hematopoiesis without perturbing other tissues. By using our zebrafish model of granulin deficiency, we revealed that during normal and emergency myelopoiesis, myeloid progenitors are unable to terminally differentiate into neutrophils and macrophages in the absence of granulin a (grna), failing to express the myeloid-specific genes cebpa, rgs2, lyz, mpx, mpeg1, mfap4, and apoeb. Functionally, macrophages fail to recruit to the wound, resulting in abnormal healing. Our CUT&RUN experiments identify Pu.1, which together with Irf8, positively regulates grna expression. In vivo imaging and RNA sequencing experiments show that grna inhibits the expression of gata1, leading to the repression of the erythroid program. Importantly, we demonstrated functional conservation between the mammalian granulin and the zebrafish ortholog grna. Our findings uncover a previously unrecognized role for granulin during myeloid cell differentiation, which opens a new field of study that can potentially have an impact on different aspects of human health and expand the therapeutic options for treating myeloid disorders such as neutropenia or myeloid leukemia.

Objectives: The purpose of this study was to prepare carboxymethyl chitosan (CMC) and lysozyme nanogels that could encapsulate amorphous calcium phosphate (ACP) for achieving its controlled delivery, thus forming an aprismatic enamel-like layer on the demineralized enamel surface.

Methods: CMC/LYZ-ACP nanogels were developed, and the controlled delivery of ACP from the nanogels was induced by the presence of NaCl. The nanogel morphologies at various NaCl concentrations was measured by transmission electron microscopy (TEM). The particle sizes and zeta potentials (ζ-potential) of the samples were determined using a combined dynamic light scattering/particle electrophoresis instrument. Comparing the remineralization effect of the CMC/LYZ-ACP nanogels on the demineralized enamel surface with that of a fluoride treatment, the remineralization effect was examined by nanoindentation tests, X-ray diffraction (XRD), confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM).

Results: CMC/LYZ-ACP nanogels were negatively charged spherical structures with a particle size of approximately 300 nm. At high concentrations of NaCl (0.15 M), ACP was dissociated from the disassembled nanogels and transformed into hydroxyapatite (HAP). Groups treated with the CMC/LYZ-ACP nanogels showed the regeneration of an aprismatic enamel-like layer on an acid-etched enamel surface, which provided increased mechanical properties (P < 0.05) and a high impermeability (P < 0.01) compared to those of the fluoride-treated group.

Conclusions: This research provides a new idea for the stable and controllable delivery of ACP from CMC/LYZ-ACP nanogels, which can form an aprismatic enamel-like layerin situ on the surface of demineralized enamel. In regard to further clinical development, this material and method may be promising for treating early enamel caries.

Keywords: Amorphous Calcium Phosphate; Aprismatic enamel; Carboxymethyl Chitosan; Lysozyme; Nanogels; Remineralization.

L-amino acid oxidase (LAAO) is a recently discovered novel fish immune enzyme. To explore the role of LAAO in the immune system of bony fishes, we cloned the full-length coding sequence (CDS) of LAAO of the zebrafish Danio rerio (ZF-LAAO), conducted bioinformatics analysis of ZF-LAAO, and analyzed its expression profile in zebrafish infected with the pathogen Streptococcus agalactiae. The CDS of ZF-LAAO was 1,515 base pairs long, and the encoded protein of ZF-LAAO contained an 18 amino acid signal peptide. ZF-LAAO contained the conserved domains of the LAAO family (dinucleotide binding motif and GG-motif), 2 N-glycosylation sites, and 2 O-glycosylation sites, and it was a stable hydrophilic exocrine protein. Similarity of the amino acid sequence of ZF-LAAO with LAAOs of 14 other bony fish species was > 50% in all cases. The greatest similarity (79.45%) was with the LAAO of Anabarilius grahami, and these two LAAOs were grouped together in the phylogenetic tree. In wild-type zebrafish infected with S. agalactiae, changes in ZF-LAAO gene (zflaao) expression occurred mainly in the early stage of infection, and the changes in zflaao expression were more pronounced than those of the immune enzyme lysozyme (LYZ). The expression levels of both LYZ gene of zebrafish (zflyz) and zflaao were significantly elevated at 6 h after infection (p < 0.001), but zflyz expression in the spleen decreased at 12 h whereas zflaao expression in the liver and spleen peaked at 12 h. These results provided a reference for functional studies of the novel immune enzyme LAAO in bony fish.

Keywords: Danio rerio; Expression profile; L-amino acid oxidase; Streptococcus agalactiae.

Aiming at developing a safe and efficient alternative to traditional drinking water disinfection, this work successfully synthesized a novel antibacterial material with high surface area, ultra large pore size and tunable loading of immobilized lysozyme. The immobilized enzymes exhibit high antibacterial efficacy without forming carcinogenic disinfection byproducts. Critical immobilization parameters were optimized to keep the activity of the immobilized enzyme at a high level. The immobilization of lysozymes on 3DOm COOH could be confirmed by the characterizations of transmission electron microscopy, X-ray diffraction and Zeta-Potential. In addition, the structural stability of lysozymes on 3DOm COOH were studied by Fourier transform infrared spectroscopy. The antibacterial performance of 3DOm COOH-Lyz were specifically investigated based on the disinfection efficacy of Staphylococcus aureus in water. The results revealed that the immobilization capacity and relative activity of immobilized lysozyme were 814mg/g carrier and 80%, respectively, under the optimal immobilization conditions. And the antibacterial material with the initial mass ratio of lysozyme and 3DOm COOH as 3:1 exhibited maximum bacteria removal efficiency (98.1%) at pH 5. Moreover, the reusability test indicated that 3DOm COOH-Lyz has certain operational stability, and remains 82% bacterial removal efficiency even in the fifth cycle, which provides a promising application for safe and efficient drinking water disinfection in small-scale and emergency water treatment.

Fluoroquinolones (FQs) constitute an important class of biologically active broad-spectrum antibacterial drugs that are which are in contact with many biological fluids under different acidity conditions. We studied the reactivity of ciprofloxacin (Cpx) and levofloxacin (Lev) and their interaction with lysozyme (Lyz) at different pH values, using UV-visible absorption, fluorescence, infrared spectroscopies supported by DFT calculation and docking. In addition, by antimicrobial assays, the biological consequences of the interaction were evaluated. DFT calculation predicted that the FQ cationic species present at acid pH have lower stabilization energies, with an electric charge rearrangement because of their interactions with solvent molecules. NBO and frontier orbital calculations evidenced the role of two charged centers, NH₂⁺ and COO-, for interactions by electronic delocalization effects. Both FQs bind to Lyz via a static quenching with a higher interaction in neutral medium. The interaction induces a structural rearrangement in β-sheet content while in basic pH a protective effect against the denaturation of Lyz was inferred. The analysis of thermodynamic parameters and docking showed that hydrophobic, electrostatic forces and hydrogen bond are the responsible of Cpx-Lyz and Lev-Lyz associations. Antimicrobial assays evidenced an antagonist effect of Lyz in acid medium while in neutral medium the FQs' activities were not modified by Lyz.

Although substantial knowledge of mercury toxicity in fish has been assembled; until now, studies investigating the toxic impacts in Nile tilapia (Oreochromis niloticus) following dietary exposure to organic methyl mercury (MeHg) are less prolific. Accordingly, the current study aimed to evaluate the impacts of MeHg on neurobehavioral and immune integrity in Nile tilapia after dietary exposure. Two hundred and twenty-five juvenile Nile tilapia (19.99 ± 0.33 g) were allocated into five groups in triplicates (15 fish/replicate). G1, G2, G3, G4, and G5. O. niloticus were fed corresponding basal diets containing 0, 0.5, 1, 1.5, and 2 mg/kg diet MeHg chloride (MeHgCl) daily for 30 days, zero value represented the control G1 group. The results showed that MeHg induced significant alterations in O. niloticus behavior, the swimming behavior was significantly decreased, while scratching, biting, and fin tugging behaviors were significantly augmented. Moreover; chasing, mouth pushing, and butting behaviors were significantly increased in all the exposed groups. MeHg significantly decreased brain acetylcholine esterase (AChE) and serum immunoglobulin M (IgM) levels in all the exposed groups. Meanwhile, serum levels of lysozyme (LYZ), nitric oxide (NO), superoxide dismutase (SOD) malondialdehyde (MDA), protein carbonyl (PCO), and 8 hydroxy 2 deoxyguanosine (8OH2dG) were significantly elevated in all the exposed groups except for serum reduced glutathione (GSH) content was significantly decreased implying oxidative stress (OS), lipid peroxidation (LPO), protein, DNA damage and impaired immune response of the exposed tilapia. MeHg significantly altered transcriptional expression of immune-related genes including (TNF-α, IL-1β, and IL-8, and IL-10) in all the exposed groups. From the obtained outcomes, the present research is the premier to investigate that dietary MeHg exposure in O. niloticus significantly induced neurobehavioral and immune defense impairments in a dose-related manner. This study exhibits that dietary MeHg may pose a potential threat to the O. niloticus populations.

Keywords: Acetylcholine esterase; Immune related gene expression; Methyl mercury chloride; Neurobehavioral impairments; Oreochromis niloticus; Oxidative stress.

In this work, we developed an ultrasensitive "turn on-off" fluorescence nanosensor for lysozyme (Lyz) detection. The novel nanosensor was constructed with the carboxymethyl chitosan modified CdTe quantum dots (CMCS-QDs). Firstly, the CMCS-QDs were fabricated via the electrostatic interaction between amino groups in CMCS polymeric chains and carboxyl groups on the surface of QDs. In the fluorescence "turn-on" step, the strong binding ability between Zn(2+) and CMCS on the surface of QDs can enhance the photoluminescence intensity (PL) of QDs. In the following fluorescence "turn-off" step, the N-acetyl-glucosamine (NAG) section along the CMCS chains was hydrolyzed by Lyz. As a result, Zn(2+) was released from the surface of QDs, and the Lyz-QDs complexes were formed to quench the QDs PL. Under the optimal conditions, there was a good linear relationship between the PL of QDs and the Lyz concentration (0.1-1.2 ng/mL) with the detection limit of 0.031 ng/mL. The developed method was ultrasensitive, highly selective and fast. It has been successfully employed in the detection of Lyz in the serum with satisfactory results.

The easy and effective capture of a single protein from a complex mixture is of great significance in proteomics and diagnostics. However, adsorbing nanomaterials are commonly decorated with specific ligands through a complicated and arduous process. Fe3 O4 /carboxymethylated chitosan (Fe3 O4 /CMCS) nanoclusters are developed as a new nonligand modified strategy to selectively capture bovine hemoglogin (BHB) and other structurally similar proteins (i.e., lysozyme (LYZ) and chymotrypsin (CTP)). The ligand-free Fe3 O4 /CMCS nanoclusters, in addition to their simple and economical two-step preparation process, possess many merits, including uniform morphology, high negative charges (-27 mV), high saturation magnetization (60 emu g(-1) ), and high magnetic content (85%). Additionally, the ligand-free Fe3 O4 /CMCS nanoclusters are found to selectively capture BHB in a model protein mixture even within biological samples. The reason for selective protein capture is further investigated from nanomaterials and protein structure. In terms of nanomaterials, it is found that high negative charges are conducive to selectively adsorb BHB. In consideration of protein structure, interestingly, the ligand-free magnetic nanoclusters display a structure-selective protein adsorption capacity to efficiently capture other proteins structurally similar to BHB, such as LYZ and CTP, showing great potential of the ligand-free strategy in biomedical field.

Characterization of structural differences between coexisting conformational states of protein is difficult with conventional biophysical techniques. Hydrogen/deuterium exchange (HDX) coupled with top-down mass spectrometry (MS) allows different conformers to be deuterated to different extents and distinguished through gas-phase separation based on molecular weight distributions prior to determination of deuteration levels at local sites for each isolated conformer. However, application of this strategy to complex systems is hampered by the interference from conformers with only minor differences in overall deuteration levels. In this work, we performed differential HDX while the different conformers were separated according to their differing charge to size ratios in capillary electrophoresis. Mixtures of holo- and apo-myoglobin (Mb) and disulfide isomers of lysozyme (Lyz) were characterized in a conformer-specific fashion using this strategy, followed by conformation interrogation for the sequentially eluted ²H-labeled species in real-time using top-down MS. Under mildly denaturing conditions that minimize the charge difference, disulfide isomers of Lyz were differentially labeled with ²H during separation based on their disulfide-dependent sizes. The resulting differences in deuteration pattern between these isomers are in line with their difference in covalent structural constraints set by the disulfide patterns. Under physiologically relevant conditions, we identified the segments undergoing conformational changes of Mb in the absence of the heme group by comparing the deuteration patterns of holo- and apo-Mb.

To explore the role of calpain and its signaling pathway in lipopolysaccharide (LPS)-induced acute kidney injury (AKI), animal models of endotoxemia were established by administration of LPS to mice with endothelial-specific Capn4 knockout (TEK/Capn4^-/-), mice with calpastatin (an endogenous calpain inhibitor) overexpression (Tg-CAST) and mice with myeloid-specific Capn4 knockout (LYZ/Capn4^-/-). Mouse pulmonary microvascular endothelial cells (PMECs) were used as a model of the microvascular endothelium and were stimulated with LPS. Renal function, renal inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) expression, cellular apoptosis, plasma and renal levels of NO and reactive oxygen species (ROS), and phosphorylation of mitogen-activated protein kinase (MAPK) family members (p38, ERK1/2, and JNK1/2) were examined. Moreover, a calpain inhibitor, calpastatin overexpression adenoviruses and MAPK inhibitors were used. Significant renal dysfunction was induced by LPS stimulation, and recovery was observed in TEK/Capn4^-/- and Tg-CAST mice but not in LYZ/Capn4^-/- mice. Endothelial Capn4 knockout also abrogated the LPS-induced increases in renal iNOS expression, caspase-3 activity and apoptosis and plasma and renal NO and ROS levels but did not obviously affect renal eNOS expression. Moreover, LPS increased both calpain and caspase-3 activity, and only the expression of iNOS in PMECs was accompanied by increased phosphorylation of p38 and JNK. Inhibiting calpain activity or p38 phosphorylation alleviated the increased iNOS expression, NO/ROS production, and cellular apoptosis induced by LPS. These results suggest that endothelial calpain plays a protective role in LPS-induced AKI by inhibiting p38 phosphorylation, thus attenuating iNOS expression and further decreasing NO and ROS overproduction-induced endothelial apoptosis.

The search for clinically relevant molecular markers in classical Hodgkin lymphoma (cHL) is hampered by the histopathological complexity of the disease, resulting from the admixture of a small number of neoplastic Hodgkin and Reed-Sternberg (H-RS) cells with an abundant and heterogeneous microenvironment. In this study, we evaluated gene expression profiles of 11 selected genes previously proposed as a molecular score for adult cHL, aiming to validate its application in the pediatric setting. Assays were performed by RT-qPCR from formalin-fixed paraffin-embedded (FFPE) lymph nodes in 80 patients with cHL. Selected genes were associated with cell cycle (CENPF, CDK1, CCNA2, CCNE2, and HMMR), apoptosis (BCL2, BCL2L1, and CASP3), and monocytes/macrophages (LYZ and STAT1). Despite using controlled preanalytical and analytical strategies, we were not able to validate the 11-gene score to be applied in pediatric cHL. Principal component analysis (PCA) disclosed 3 components that accounted for 65.7% of the total variability. The second PC included microenvironment and apoptosis genes, from which CASP3 expression was associated with a short time of progression-free survival, which impact was maintained in the unfavorable risk group, Epstein-Barr virus-negative cases, and multivariate analysis (P < .05). Because this is a counterintuitive association, CASP3 active expression was assessed at the protein level in H-RS cells by double immunohistochemistry. In contrast to the association of mRNA levels with a poor therapeutic response, a high number of cleaved CASP3+ cells were associated with longer progression-free survival (P = .03) and overall survival (P = .002). Our results demonstrate the feasibility of using FFPE samples as RNA source for molecular prognostication, but argue against the concept of direct and wide applicability of molecular scores in cHL. We reinforce the potential of CASP3 as an interesting target to be explored in adult and pediatric cHL, and alert for its dual biological role in H-RS cells and tumor microenvironment.