Lysozyme (LYZ) sensors have attracted increased attention because rapid and sensitive detection of LYZ is highly desirable for various diseases associated with humans. In this research, we designed L-cysteine-protected ultra small photoluminescent (PL) copper nanoclusters (CuNCs) conjugated with β-cyclodextrin (β-CD) for rapid detection of LYZ in human serum samples at room temperature. The proposed β-CD-CuNCs exhibited excellent water solubility, ultrafine size, good dispersion, bright photoluminescence, and good photostability. The β-CD-CuNCs exhibit an excitation and emission maximum at 370 nm and 492 nm, respectively, with an absolute quantum yield (QY) of 54.6%. β-CD-CuNCs showed a fluorescence lifetime of 12.7 ns. The addition of LYZ would result in PL quenching from β-CD-CuNCs. The lowest detectable LYZ concentration was 50 nM for the naked eye and the limit of detection (LOD) and limit of quantification (LOQ) were 0.36 nM and 1.21 nM, respectively, by emission measurement observed in the LYZ concentration range from 30 to 100 nM. It is important to note that the PL β-CD-CuNC strategy possessed great selectivity toward LYZ relative to other biomolecules. The proposed nanosensor was efficiently applied to determine the LYZ level in human serum sample average recoveries from 96.15 to 104.05% and relative standard deviation (RSD) values lower than 3.0%. Moreover, the proposed sensing system showed many advantages, including high speed, high sensitivity, high selectivity, low cost, and simple preparation.

Keywords: CuNCs; Cysteine; Lysozyme; Naked eye detection; Photoluminescence; β-CD.

Graphene aerogel (GA)-supported metal-organic framework (MOF) particles with a three-dimensional (3D) architecture were fabricated for the first time via a facile template-free "sol-cryo" method. The prepared MOFs@graphene hybrid aerogels exhibit a 3D interconnected macroporous framework of graphene sheets with uniform dispersion of MOF particles. We also report the first attempt at using the hybrid aerogels as adsorbents for the solid-phase extraction (SPE) of non-steroidal anti-inflammatory drugs (NSAIDs) and the selective enrichment of proteins. The macroporous skeletons of GA provide both low backpressure and rapid mass transfer in SPE application, thus overcoming the obstacle of high backpressure caused by directly packing submicron or micron sized MOF particles into SPE cartridges. Excellent performances including satisfactory recoveries, high sensitivity and good reproducibility were achieved in the extraction of five NSAIDs. The hybrid aerogels also showed an interesting ability for selective enrichment of ribonuclease A (RNase A) and simultaneous exclusion of cytochrome C (Cyt C) and lysozyme (Lyz), which could be attributed to the electrostatic interactions between proteins and the positively charged coordinatively unsaturated metal sites (CUS) in MIL-101. We believe that this work will promote the application of MOFs in adsorption and separation, and our synthetic strategy could be further extended to develop other graphene-based hybrid aerogels.

In this study, we demonstrate the enhanced disease resistance and positive immunomodulation of novel pectin isolated from Spirulina maxima (SmP) in zebrafish model. Zebrafish larvae exposed to SmP had significantly (p < 0.05) higher cumulative percent survival (CPS) at 25 (44.0%) and 50 μg/mL (67.0%) against Edwardsiella piscicida compared to the control. However, upon Aeromonas hydrophila challenge, SmP exposed larvae at 50 μg/mL had slightly higher CPS (33.3%) compared to control group (26.7%). SmP supplemented zebrafish exhibited the higher CPS against E. piscicida (93.3%) and A. hydrophila (60.0%) during the early stage of post-infection (<18 hpi). qRT-PCR results demonstrated that exposing (larvae) and feeding (adults) of SmP, drive the modulation of a wide array of immune response genes. In SmP exposed larvae, up-regulation of the antimicrobial enzyme (lyz: 3.5-fold), mucin (muc5.1: 2.84, muc5.2: 2.11 and muc5.3: 2.40-fold), pro-inflammatory cytokines (il1β: 1.79-fold) and anti-oxidants (cat: 2.87 and sod1: 1.82-fold) were identified. In SmP fed adult zebrafish (gut) showed >2-fold induced pro-inflammatory cytokine (il1β) and chemokines (cxcl18b, ccl34a.4 and ccl34b.4). Overall results confirmed the positive modulation of innate immune responses in larval stage and it could be the main reason for developing disease resistance against E. piscicida and A. hydrophila. Thus, non-toxic, natural and biodegradable SmP could be considered as the potential immunomodulatory agent for sustainable aquaculture.

Structural and molecular properties extracted from circular dichroism (CD), tryptophan fluorescence and 1-anilino-8-napthalene sulfonate (ANS) binding experiments suggest that the high concentration of synthetic crowding agents (dextran 40, dextran 70 and ficoll 70) stabilizes and refolds the base-denatured ferricytochrome c (Ferricyt c) and lysozyme (Lyz) at pH 12.9 (±0.1) to molten globule (MG) states (CB-states). These results further revealed that the CB-states resemble the generic properties of MG-states. Thermodynamic analysis of thermal denaturation curves of base-denatured Ferricyt c and Lyz at pH 12.9 (±0.1) under variable concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) revealed that the crowder presence increases the thermal stability of base-denatured proteins and also prevents the cold denaturation of Ferricyt c. The results further showed that the nature, size and shape of crowder influence the crowding-mediated increase in secondary structure stabilization and thermal stability of base-denatured Ferricyt c and Lyz. Analysis of kinetic and thermodynamic parameters measured for CO association reaction of alkaline ferrocytochrome c (Ferrocyt c) at pH 12.9 (±0.1) under variable concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) revealed that the crowder presence reduces the level of structural fluctuation of M80-containing Ω-loop that control CO association to alkaline Ferrocyt c.

In this present work, a detailed investigation of the effect of an anticancer drug, 5-Fluorouracil (5-FU), on conformation, stability and activity of lysozyme (Lyz) was reported. The interaction between Lyz and 5-FU was reflected in terms of intrinsic fluorescence quenching and change in secondary structure of Lyz. The mode of quenching mechanism involved was evaluated by the steady-state and time-resolved fluorescence measurements. Synchronous and Circular Dichroism (CD) results revealed the conformational changes induced in Lyz upon complexation with 5-FU. Additionally, the effect of temperature and chemical denaturant on the stability of Lyz-5FU complex was carried out. As well as the activity of Lyz in the absence and presence of 5-FU were measured using Micrococcus luteus strain. To support our experimental findings, in vitro interaction between Lyz and 5-FU was done by theoretical studies. The current study will provide a better understanding on the nature of the interactions possible between proteins and drug molecules, which might create a bench mark in medical science in terms of the toxic effect or biological benefits of drug molecules on protein structure and conformation.

Proteins represent a versatile biopolymer material for the preparation of nanoparticles due to their biocompatibility, biodegradability, and low immunogenicity. This study presents a protein-based nanoparticle system consisting of high surface PEGylated lysozyme polyethylene glycol-modified lysozyme (LYZmPEG ). This protein modification leads to a solubility switch, which allows a nanoparticle preparation using a mild double emulsion method without the need of surfactants. The method allows the encapsulation of large hydrophilic payloads inside of the protein-based nanoparticle system. Native lysozyme (LYZ) was chosen as payload because of its innate activity as natural antibiotic. The mild particle preparation procedure retains the structure and activity of the enzyme which was successfully tested against the gram-positive bacteria strain M. Luteus. In comparison, the particle system shows no toxicity to human cells. This first report of a full protein-based particle material for the transport of large hydrophilic payloads opens up new therapeutic applications for biopolymer-based delivery systems.

We studied the early protein profile in the ocular tissue extracted after LASIK and SMILE surgery. SMILE and LASIK was performed in contralateral eyes and stromal tissue samples were collected from 10 eyes of 5 donors. The stromal tissue samples were analyzed using label free quantification approach and ITRAQ labelling approach in LC-MS/MS. Combined functional analysis revealed many differentially expressed proteins which were involved in important biological processes. About 117 unique differentially expressed proteins were identified using two different proteomic approaches. Collagens, proteoglycans, corneal crystallins were enriched and showed differential expression in SMILE and LASIK as compared to the non-surgical control. Apart from these, 14-3-3 class of proteins, Lysozyme (LYZ), Macrophage inhibitory factor protein (MIF), Pigment Epithelial Derived Factor (PEDF) were differentially expressed when compared between LASIK and SMILE. Peroxiredoxin 1 (PRDX1) expression was found to be reduced in LASIK as compared to SMILE. The expression of Lysozyme C and macrophage inhibitory factor inflammatory response was found to be less in SMILE as compared to LASIK. Western blot validation of specific markers such as collagen IV (COL4), keratocan (KERA), lumican (LUM), aldehyde dehydrogenase 3 A1 (ALDH3A1), lysozyme C (LYZC) confirmed the differences in the protein levels observed in SMILE and LASIK operated tissues as compared to non-surgical controls. In conclusion, this study revealed the early molecular changes occurring in the cornea resulting from these two surgical procedures which may have implications on managing post-operative complications.

The present study was performed to investigate the immunomodulatory and health promoting effects of combined or singular administration of apple cider vinegar (ACV) and Lactobacillus casei in common carp (Cyprinus carpio) diet. An 8-week feeding trial was designed with following treatments: Control (basal diet), Pro (contains 107 CFU g-1L. casei), LACV (contains 1% ACV), HACV (contains 2% ACV), Pro + LACV (contains 107 CFU g-1L. casei plus 1% ACV) and Pro + HACV (contains 107 CFU g-1L. casei plus 2% ACV). Evaluation of skin mucus revealed notable increase of total Ig level and lysozyme activity in Pro + LACV and Pro + HACV treatments compared other groups (P < 0.05). Similarly, serum total Ig and lysozyme activity in HACV, Pro + LACV and Pro + HACV fed carps was remarkably higher than other groups (P < 0.05). However, regarding serum alternative complement (ACH50) activity significant difference was observed just between Pro + HACV and control treatment (P < 0.05). The highest expression of immune related (LYZ, TNF-alpha, IL1b, IL8) and antioxidant enzymes genes (GSR, GST) were observed in carps fed Pro + HACV and Pro + LACV. The expression of GH gene expression in Pro, LACV and HACV treatments was significantly higher than those in control group (P < 0.05). The highest expression level of GH and IGF1 was observed in fish fed combined Pro and ACV (P < 0.05). These results indicated that co-administration of ACV boosted immunomodulatory and health promoting effects of L. casei and can be considered as a promising immunostimulants in early stage of common carp culture.

Intracellular contents and serum levels of neutrophilic granule protein components, including myeloperoxidase (MPO), lactoferrin (LF) and lysozyme (LYZ), were investigated in 30 cases of myelodysplastic syndromes (MDS), with 59 healthy adult donors as controls. Both neutrophilic MPO and LF decreased significantly, suggesting the transformation of abnormal clones. Both serum levels of LF and LYZ exhibited a considerable fluctuation which may reflect reflect granulopoiesis in the bone marrow. We are of the opinion that measurement of intracellular MPO, LF, LYZ and their serum levels may aid in the diagnosis and prognosis of MDS and is proved to be of great clinical significance.

Graphene oxide (GO) nanosheets can be functionalized with reactive pentafluorophenyl ester via esterification of the carboxylic groups. The resulting reactive GO nanosheets provide a versatile platform for grafting of amino-containing polymers or biomolecules via ester-amine coupling. Coupling of poly[(9,9-dioctylfluorene)-alt-(4-amino-phenylcarbazole)] (PFCz-NH(2) ), amino-terminated hyperbranched polyglycerol (HPG-NH(2) ), and lysozyme (Lyz) was illustrated. The Al/GO-g-PFCz/ITO sandwich thin-film device exhibits bistable electrical switching and rewritable memory effects. The GO-g-Lyz nanohybrids exhibit high bactericidal efficacy against S. aureus and E. coli, while the GO-g-HPG nanohybrids exhibit reduced cytotoxicity toward 3T3 fibroblasts.

Colloidal-chemical characteristics of block/branched cationic and non-ionic polyamphiphiles containing poly(fluorine-alkyl methacrylate) (poly(FMA)) block and their intermolecular complexes with biopolymers were studied. The dependences of their surface activity and micelle size on the length of hydrophobic and hydrophilic blocks, as well as the length of side fluorine-alkyl branches were established. Poly(FMA)-block-poly(DMAEMA) was used for formation of interpolyelectrolyte complexes with plasmid DNA (pDNA) via their electrostatic interaction. Novel non-viral polyplexes were tested as gene delivery systems for mammalian cells. The results of DLS, TEM and MALDI-ToF studies demonstrated disaggregation of lysozyme (LYZ) aggregates in the presence of poly(FMA)-block-poly(NVP) and formation of the polyamphiphile…LYS complex possessing antibacterial action.

Binding interactions between the drug Juglone (JUG) and Lysozyme (LYZ) have been explored in details from spectroscopic studies aided by in silico calculations. UV-Vis, steady state and time resolved fluorescence spectroscopic studies indicate the formation of LYZ-JUG complex in the ground state. Quenching of corrected fluorescence spectra of LYZ in presence of JUG at varied concentrations in different temperature range have been estimated from Stern-Volmer (SV) plots. Time resolved fluorescence spectroscopic studies confirm the mechanism of quenching to be of static type. Binding constant associated with the LYZ-JUG complex has been estimated from Scatchard plot. The number of binding sites, thermodynamic parameters and the modes of interaction are also estimated. Synchronous fluorescence spectra monitored at two discrete wavelength windows confirm the prominent role of Tryptophan residues towards quenching of fluorescence in LYZ. The circular dichroism (CD) spectra signify alterations in the population of α-helical content within the secondary structure of LYZ in presence of JUG molecules. REES of LYZ in the presence of JUG further signify definite impact of the drug JUG molecule on the Trp residues of the protein. The experimental observations are supported by in silico molecular docking and molecular dynamics simulations.

In this study, a procedure for quantifying the surface deposition of proteins in crossflow ultrafiltration has been developed. The procedure consists of determining the protein adsorption behavior onto the membrane surface from a few dynamic measurements performed in a nonfiltration and a filtration mode, and evaluating the concentration polarization (CP) layer thickness based on the adsorption data. To predict the interdependence between the protein adsorption and CP, a simplified mathematical model has been formulated. The model was used to assess the protein adsorption and thus yield reduction in the ultrafiltration process at different protein concentration in the solution. As a case study, ultrafiltration of aqueous solutions of BSA and lysozyme (LYZ) was examined on a polyethersulfone membrane with the molecular weight cutoff of 10 or 100 kDa. The protein concentration in the solutions varied within a relatively low concentration range, i.e. below 10 mg mL^-1, characteristic for solvent exchange between sequential operations of protein purification by chromatography and extraction. Both proteins markedly differed in the mechanism of surface deposition; for BSA hydrophobic interactions were suggested to be dominant, whereas in case of LYZ electrostatic interactions contributed the most to the deposition mechanism. The effect of additives of the protein solutions, i.e. inorganic salts, PEG, and urea depended on the adsorption mechanism and was also specific for each protein. Nevertheless, the proposed procedure performed well in the evaluation of surface deposition and yield reduction, regardless of the protein type and its solvent environment.

Keywords: BSA; Concentration polarization; Lysosyme; Protein adsorption; Ultrafiltration.

The retention behavior of polyethylene glycol (PEG) on different types of hydrophobic interaction chromatography (HIC) resins containing butyl, octyl, and phenyl ligands was analyzed. An incomplete elution or splitting of the polymer peak into two parts was observed, where the first one was eluted at the dead time of the column, whereas the second one was strongly retained. The phenomenon was attributed to conformation changes of the polymer upon its adsorption on hydrophobic surface. The effect enhanced with increasing molecular weight of the polymer and hydrophobicity of the HIC media. Addition of PEG to the mobile phase reduced binding of proteins to HIC resins, which was demonstrated with two model systems: lysozyme (LYZ) and immunoglobulin G (IgG), and their mixtures. In case of LYZ, the presence of PEG caused reduction in the protein retention, whereas for IgG-a decrease in efficiency of the protein capture. The effect depended on the adsorption pattern of PEG; it was pronounced in the systems in which conformational changes of the polymer were suggested to occur.

Background: In silico deconvolution of invasive immune cell infiltration in bulk breast tumors helps characterize immunophenotype, expands treatment options, and influences survival endpoints. In this study, we identify the differential expression (DE) of the LM22 signature to classify immune-rich and -poor breast tumors and evaluate immune infiltration by receptor subtype and lymph node metastasis.

Methods: Using publicly available data, we applied the CIBERSORT algorithm to estimate immune cells infiltrating the tumor into immune-rich and immune-poor groups. We then tested the association of receptor subtype and nodal status with immune-rich/poor phenotype. We used DE to test individual signature genes and over-representation analysis for related pathways.

Results: CCL19 and CXCL9 expression differed between rich/poor signature groups regardless of subtype. Overexpression of CHI3L2 and FES was observed in triple negative breast cancers (TNBCs) relative to other subtypes in immune-rich tumors. Non-signature genes, LYZ, C1QB, CORO1A, EVI2B, GBP1, PSMB9, and CD52 were consistently overexpressed in immune-rich tumors, and SCUBE2 and GRIA2 were associated with immune-poor tumors. Immune-rich tumors had significant upregulation of genes/pathways while none were identified in immune-poor tumors.

Conclusions: Overall, the proportion of immune-rich/poor tumors differed by subtype; however, a subset of 10 LM22 genes that marked immune-rich status remained the same across subtype. Non-LM22 genes differentially expressed between the phenotypes suggest that the biologic processes responsible for immune-poor phenotype are not yet well characterized.

Keywords: breast cancer; microenvironment; transcriptomics; tumor-infiltrating immune cells.

The development of new strategies for thermal stability and storage of enzymes is very important, considering the nonretention of catalytic activity by enzymes under harsh conditions of temperature. Following this, herein, a new approach based on the interfacial adsorption of lysozyme (LYZ) at nanointerfaces of ionic liquid (IL)-based microemulsions, for enhanced thermal stability of LYZ, is reported. Microemulsions (MEs) composed of dialkyl imidazolium-based surface active ILs (SAILs) as surfactants, ILs as the nonpolar phase, and ethylene glycol (EG) as the polar phase, without any cosurfactants, have been prepared and characterized in detail. Various regions corresponding to polar-in-IL, bicontinuous, and IL-in-polar phases have been characterized using conductivity measurements. Dynamic light scattering (DLS) measurements have provided insights into the size distribution of microdroplets, whereas temperature-dependent DLS measurements established the thermal stability of the MEs. Nanointerfaces formed by SAILs with EG in thermally stable reverse MEs act as fluid scaffolds to adsorb and provide thermal stability, up to 120 °C, to LYZ. Thermally treated LYZ upon extraction into a buffer shows enzyme activity owing to negligible change in the active site of LYZ, as marked by retention of microenvironment of Trp residues present in the active site of LYZ. The present work is expected to establish a new platform for the development of novel nanointerfaces utilizing biobased components for other biomedical applications.

Introduction: Antimicrobial peptides and proteins (AMPs) constitute the first line of defense against pathogenic microorganisms in the airway. The association between AMPs and chronic rhinosinusitis with nasal polyps (CRSwNP) requires further investigations. This study is aimed at investigating the expression and regulation of major dysregulated AMPs in the nasal mucosa of CRSwNP.

Methods: The expression of AMPs was analyzed in nasal tissue from patients with eosinophilic (E) CRSwNP and nonECRSwNP and healthy subjects using RNA sequencing. The 10 most abundant AMPs expressed differentially in CRSwNP patients were verified by real-time PCR, and of these, the expression and regulation of secretory leukoprotease inhibitor (SLPI) and clusterin (CLU) were investigated further.

Results: The 10 most abundant AMPs expressed differentially in CRSwNP compared to healthy control, regardless of subtypes, included BPIFA1, BPIFB1, BPIFB2, CLU, LTF, LYZ, and SLPI, which were downregulated, and S100A8, S100A9, and HIST1H2BC, which were upregulated. ELISA and immunofluorescence confirmed the decreased expression of SLPI and CLU levels in CRSwNP. SLPI is expressed in both nasal epithelial cells and glandular cells, whereas CLU is mainly expressed in glandular cells. AB/PAS staining further demonstrated that both SLPI and CLU were mainly produced by mucous cells in submucosal glands. Furthermore, the numbers of submucosal glands were significantly decreased in nasal polyp tissue of CRSwNP compared to nasal tissue of controls. SLPI was downregulated by TGF-βin vitro, while CLU expression was inhibited by TGF-βLYZ. Additionally, budesonide significantly increased the expression of SLPI and CLU in cultured nasal tissues.

Conclusion: The expression of major antimicrobial proteins is significantly decreased in nasal tissue of CRSwNP. The expression of SLPI and CLU is correlated with the numbers of submucosal glands and regulated by inflammatory cytokines and glucocorticoids.

Diabetic nephropathy (DN) is the main cause of end stage renal disease (ESRD). Glomerulus damage is one of the primary pathological changes in DN. To reveal the gene expression alteration in the glomerulus involved in DN development, we screened the Gene Expression Omnibus (GEO) database up to December 2020. Eleven gene expression datasets about gene expression of the human DN glomerulus and its control were downloaded for further bioinformatics analysis. By using R language, all expression data were extracted and were further cross-platform normalized by Shambhala. Differentially expressed genes (DEGs) were identified by Student's t-test coupled with false discovery rate (FDR) (P < 0.05) and fold change (FC) ≥1.5. DEGs were further analyzed by the Database for Annotation, Visualization, and Integrated Discovery (DAVID) to enrich the Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. We further constructed a protein-protein interaction (PPI) network of DEGs to identify the core genes. We used digital cytometry software CIBERSORTx to analyze the infiltration of immune cells in DN. A total of 578 genes were identified as DEGs in this study. Thirteen were identified as core genes, in which LYZ, LUM, and THBS2 were seldom linked with DN. Based on the result of GO, KEGG enrichment, and CIBERSORTx immune cells infiltration analysis, we hypothesize that positive feedback may form among the glomerulus, platelets, and immune cells. This vicious cycle may damage the glomerulus persistently even after the initial high glucose damage was removed. Studying the genes and pathway reported in this study may shed light on new knowledge of DN pathogenesis.

Keywords: bioinformatics; diabetic nephropathy; glomerulus; immune cell; pathogenesis; platelet.

To develop multifunctional protein imprinted materials, a cobalt-iron double ion-BSA directional chelation-assisted thermo-sensitive surface-imprinted hollow nanocage (Co-Fe@CBMA-MIPs) with excellent specificity is developed on the surface of ZIF-67@Co-Fe in this study by synergizing the advantages of surface imprinting, metal ion chelation, anti-protein adsorption segments, and thermo-sensitive components. Beyond previous research, well-designed multifunctional protein-imprinted materials possess high binding capacity, fast adsorption kinetics, and outstanding selectivity. When the adsorption is carried out at 32 °C, the adsorption capacity of Co-Fe@CBMA-MIPs for BSA reaches 520.35 mg/g within 50 min. The imprinting factor is 8.55. The selectivity factors of Co-Fe@CBMA-MIPs for HSA, Bhb, OVA, and Lyz are 3.72, 6.09, 4.10, and 8.41, respectively. More significantly, Co-Fe@CBMA-MIPs could specifically recognize BSA from mixed proteins and actual samples and exhibit excellent repeated use stability. Based on the above advantages, the development of this research provides an effective means to improve the recognition specificity of molecularly imprinted polymers.

Keywords: MOFs; anti-protein segment; protein imprinting; specific adsorption; thermo-responsive.

Aquaculture plays a pivotal role in covering dietary animal protein demands and restocking endangered fish populations. However, high mortality takes place at the earliest life stages: prior and immediately after hatching. Improving growth and health parameters by immunostimulants is widely used in older fish, but rarely studied in larvae. Fulvic acids (FAs) are natural substances found in soil and water. Using zebrafish as a model organism, we evaluated the effects of exposure to a FA at concentrations ranging from 1 to 500 mg C/L (mg dissolved organic carbon per liter) on embryonic development. Furthermore, the concentration of reactive oxygen species (ROS) inside the larvae as well as the molecular mechanisms involved in growth, immune response, and antioxidative protection were determined at 5, 50, and 500 mg C/L. 20 to 200 mg C/L accelerated the hatching, which was mediated by increased expression of ifg-1, gh, and he1-α. Furthermore, lyz and mpx were significantly increased at 5 and 50 mg C/L. A concentration of 500 mg C/L induced genes involved in the protection against ROS (nrf-2, keap-1, cat, sod-1), increased the concentration of ROS inside the larvae and caused tissue damage and mortality. Interestingly, 50 mg C/L activated ROS protection as well (nrf-2, sod-2), while no increase of ROS was found in the larvae. Our results show, that FA at low to medium concentrations can increase the health of larvae, but becomes detrimental at higher concentrations.

Keywords: Embryonic development; Humic substance; Oxidative stress; Reactive oxygen species (ROS); Zebrafish (Danio rerio).

Yellow catfish (Pelteobagrus fulvidraco) is an important economic cultured fish in China. Here we report antioxidative activity and immune regulation in head kidney using a central composite design based on water temperature (20-34 °C) and dietary lipid (2-17%). Response values were optimized using response surface methodology to maximize the immune response and relieve oxidative stress. The experiment was conducted under laboratory conditions and lasted for seven weeks. The results showed that the linear effects of lipid level on superoxide dismutase (SOD, and lysozyme (LYZ) activity, and malondialdehyde (MDA) content in head kidney, respiratory burst activity (RBA) of head kidney macrophages, and cumulative mortality of fish infected by Streptococcus iniae (S. iniae) were significant (P < 0.05). Similarly, the linear effects of water temperature on SOD activity, MDA content, and cumulative mortality were significant (P < 0.05). In addition, the quadratic effects of water temperature and lipid level on all experimental response values were significant (P < 0.05), and no interactive effect was found between water temperature and lipid level (P>> 0.05). High water temperature and high lipid diet significantly reduced the antioxidative activity and immune response in head kidney, and increased MDA content, which caused increased mortality of the S. iniae-infected fish. The adjusted R2 values for SOD activity, MDA content, LYZ activity, RBA, phagocytic activity, and cumulative mortality regression models were 0.76, 0.85, 0.87, 0.79, 0.64, and 0.87, respectively. The optimal combination of water temperature and lipid level was 26.9 °C and 7.7%, at which good antioxidative activity and immune regulation were achieved, with reliability of 0.878. This combination was close to the optimal combination of water temperature and lipid level for growth performance (27.5 °C and 9.2%) reported previously. Thus, the optimal combination may not only promote growth, but also enhance antioxidant and immune levels.

Intestine in fish is a complex multifunctional organ, not only plays roles in digestion and absorption of nutrient, but also has critical role in immunity. The present study evaluated the effects of different levels of dietary sodium butyrate [Butirex^® C4 (Butirex)] on intestinal immune-,antioxidant-and tight junction-related gene expression injuvenile rainbow trout(Oncorhynchusmykiss). 240 healthy rainbow trout were dispensed in 12 fiberglass tanks appointed to four treatments [0 (control), 1.5 (B1.5), 2.5 (B2.5) and 5 (B5)g Butirex per kg diet]. After a 45-day feeding trial, the fish fed with the Butirex-supplemented diets showed higher intestinal lysozyme (LYZ), complement(ACH50) and bactericidal activities; the elevations in ACH50 and bactericidal activities depended on Butirex levels (P < 0.05). The Butirex-supplemented groups, particularly the B2.5 group, had significantly higher LYZ gene expression compared to the control group (P < 0.05). Butirex at 2.5 and 5 g/kg levels led to significantly higher IL-1β gene expression. B2.5 and B5 had significantly lower and higher TNF-α gene expression compared to the control group (P < 0.05). The B2.5 group had significantly higher TGF-B, and significantly lower IL-8 compared to the control group (P < 0.05). The B1.5 and B2.5 group had significantly higher IL-10 gene expression compared to the control group (P < 0.05). The B2.5 and B5 groups had significantly higher SOD gene expression compared to the other groups; the highest expression was related to the B2.5 group (P < 0.05). Dietary Butirex supplementation significantly up-regulated CAT and GPx genes expression compared to the control group; the highest expression as related to the B2.5 and B5 groups (P < 0.05). The B2.5 group had significantly lower CLD12 gene expression compared to the control group (P < 0.05). The B2.5 and B5 groups had significantly higher CLD3, OCLD and ZO-1 gene expression compared to the control. The highest CLD3, ZO-1 gene expressions was related to the B2.5, and B5 groups respectively (P < 0.05). After challenge with Streptococcus iniae, B2.5 and B5 had significantly higher survival compared to the control group (55.6 ± 7.70 and 68.9 ± 10.2 vs. 33.3 ± 6.67). In conclusion, Butirex is efficient immune stimulant and health booster in rainbow trout, which augments the fish resistance to disease. Modulation of immune components, cytokines, antioxidant system and intestinal integrity might involve in improving disease resistance in Butirex-treated fish. Although most of the examined genes were modulated by 2.5 g/kg Butirex under normal conditions, 5 g/kg level is recommended under pathogenic state to mitigate mortality.

A major user of carbon black is the pigment and dyes industry, where carbon black is incorporated into paints, inks, printers, and plastics. However, little is known about the mechanism underlying the toxicity of carbon black to antioxidant proteins. Carbon black can cause oxidative stress to organisms after they invade into the body. Antioxidant proteins play a key role in keeping the organism from nanoparticle-induced oxidative damage and tend to bind with nanoparticles immediately after their invading into the biological environment, so it is meaningful to elucidate the toxicity of nanoparticles on the antioxidant proteins. In this study, the toxicity of carbon black (SB100) on three different antioxidant proteins (TF (transferrin), SOD (superoxide dismutase), and LYZ (lysozyme)) were investigated. The multi-spectra studies indicated that SB100 interacted with these three proteins and changed their structure in different ways. SB100 changed the microenvironment of fluorophores in SOD and LYZ by quenching the fluorescence spectra of the two enzymes, while changed that of TF by increasing the fluorescence intensity of TF. SB100 changed the secondary structure of these three proteins by decreasing the α-helix content of TF and increasing that of SOD and LYZ. Moreover, SB100 changed the hydrophobicity of the three proteins in different ways as well. And SOD exhibits a more severe activity inhibition than LYZ after exposed to SB100. In summary, SB100 caused different structural and functional changes to these three antioxidant enzymes.

Fish nocardiosis is a chronic granulomatous bacterial disease mainly caused by three pathogenic bacteria, including Nocardia seriolae, N. asteroids and N. salmonicida. Molecular chaperone DnaK and GroEL were identified to be the common antigens of the three pathogenic Nocardia species in our previous studies. To evaluate the immune protective effect of two DNA vaccines encoding DnaK or GroEL against fish nocardiosis, hybrid snakehead were vaccinated and the immune responses induced by these two vaccines were comparatively analyzed. The results suggested it needed at least 7 d to transport DnaK or GroEL gene from injected muscle to head kidney, spleen and liver and stimulate host's immune system for later protection after immunization by DNA vaccines. Additionally, non-specific immunity parameters (serum lysozyme (LYZ), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (AKP) and superoxide dismutase (SOD) activities), specific antibody (IgM) production and immune-related genes (MHCIα, MHCIIα, CD4, CD8α, IL-1β and TNFα) were used to evaluate the immune responses induced in vaccinated hybrid snakehead. It proved that all the above-mentioned immune activities were significantly enhanced after immunization with these two DNA vaccines. The protective efficacy of pcDNA-DnaK and pcDNA-GroEL DNA vaccines, in terms of relative percentage survival (RPS), were 53.01% and 80.71% respectively. It demonstrated that these two DNA vaccines could increase the survival rate of hybrid snakehead against fish nocardiosis, albeit with variations in immunoprotective effects. Taken together, these results indicated that both pcDNA-DnaK and pcDNA-GroEL DNA vaccines could boost the innate, humoral and cellular immune response in hybrid snakehead and show highly protective efficacy against fish nocardiosis, suggesting that DnaK and GroEL were promising vaccine candidates. These findings will promote the development of DNA vaccines against fish nocardiosis in aquaculture.