A 10-week feeding trial was conducted to investigate the effects of dietary carbohydrate-to-lipid (CHO:L) ratios on glycogen content, hematological indices, liver, and intestinal enzyme activity of sub-adult grouper Epinephelus coioides. Five iso-nitrogenous (496.0 g kg^-1 protein) and iso-energetic (21.6 KJ g^-1 gross energy) diets with varying CHO: L ratios of 0.65 (D1), 1.31 (D2), 2.33 (D3), 4.24 (D4), and 8.51 (D5), respectively, were fed to triplicate groups of 20 fish (average 275.1 ± 1.86 g). Results showed that the weight gain rate (WGR), specific growth rate (SGR), and protein efficiency ratio (PER) of sub-adult grouper increased and then stable when dietary CHO:L ratios reach D4 (CHO:L = 4.24). The trend of feed conversion ratio (FCR) was opposite to PER. Along with the dietary CHO:L ratios, the liver and muscle glycogen level increased gradually. Plasma triglycerides (TG) and glucose (GLU) were all maximized at D5 (CHO:L = 8.51) group, cholesterol (CHOL) at D4 (CHO:L = 4.24) group. Digestive enzyme activities were significantly affected by dietary CHO:L ratios. Liver hexokinase (HK), alkaline phosphatase (AKP), and glucose-6-phosphate dehydrogenase (G6PDH) activity increased significantly as CHO:L ratios increased. Liver lysozyme (LYZ) and superoxide dismutase (SOD) activity of sub-adult grouper fed the D4 diet was significantly higher than that of the D2 (CHO:L = 1.31) diet. The trend of acid phosphatase (ACP) is opposite to AKP. The regression model analysis showed that the most suitable dietary CHO:L ratio to reach the highest SGR is 6.06.

The aim of this experiment was used to investigate the effects of different contents of dietary vitamin D₃ on the growth performance and antioxidant and innate immune responses in juvenile black carp Mylopharyngodon piceus. Black carp juveniles were fed six levels of dietary vitamin D₃ (VD₃) (96, 220, 412, 840, 1480, and 3008 IU/Kg) for 9 weeks. Results showed that highest weight gain (WG) and special growth ratio (SGR) were obtained at 534.2 IU/Kg dietary VD₃ according to the second-order polynomial regression model. The protein efficiency ratio (PER) of black carp could be significantly increased by 412, 840, and 1480 IU/Kg dietary VD₃ (p < 0.05), while the feed conversion ratio (FCR) were reduced by 412, 840, and 1480 IU/Kg dietary VD₃ (p < 0.05). Adequate dietary VD₃ content (412, 840, and 1480 IU/Kg) could significantly upregulate expression levels of lipoxygenase 5 (LPO 5); increase the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GR); and improve GSH contents and total antioxidant capacities (T-AOC) in the liver of black carp. However, glutathione S-transferase (GST) activities and malondialdehyde (MDA) levels were significantly reduced by adequate dietary VD₃ content (412, 840, and 1480 IU/Kg) in the fish liver. In addition, 412, 840, and 1480 IU/Kg dietary VD₃ could significantly upregulate the mRNA expression levels of interferon-α (IFN-α), lysozyme (LYZ), hepcidin (HEPC), natural resistance-associated macrophage protein (NRAMP), and complement component 3 (C3) and C9 in the hemocytes and liver of black carp juveniles compared with the VD₃-deficient diet (96 IU/Kg). Meanwhile, higher contents of dietary VD₃ could increase serum LYZ and ACP activities and C3 and C4 contents in black carp juveniles compared with the groups fed VD₃-deficient diet. In conclusion, these results suggest that adequate dietary VD₃ could increase growth performances, improve antioxidant capacities, and then enhance innate immune parameters in black carp juveniles.

Keywords: Antioxidant capacities; Growth performance; Innate immune responses; Mylopharyngodon piceus; Vitamin D3.

Fish nocardiosis is a chronic granulomatous bacterial disease and three pathogens have been reported so far, including Nocardia asteroids, N. seriolae and N. salmonicida. However, the absence of antigen markers is a bottleneck for developing effective vaccines against fish nocardiosis. In this study, the antigenicity of whole-cell protein of these three pathogenic Nocardia species were profiled by immunoproteomic analysis and 7 common immunogenic proteins were identified as follows: molecular chaperone DnaK (DnaK), molecular chaperone GroEL (GroEL), 30S ribosomal protein S1 (RpsA), TerD family protein (TerD), FHA domain-containing protein (FHA), 50S ribosomal protein L7/L12 (RplL) and PspA/IM30 family protein (PspA). Furthermore, the DNA vaccine encoding FHA gene against fish nocardiosis was developed and its efficacy was investigated in hybrid snakehead. The results suggested that it needed at least 7 d to transport pcDNA-FHA DNA vaccine from injected muscle to head kidney, spleen and liver and stimulate host's immune system for later protection. In addition, non-specific immunity paraments (serum lysozyme (LYZ), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (AKP) and superoxide dismutase (SOD) activities), specific antibody (IgM) titers production and immune-related genes (MHCIα, MHCIIα, CD4, CD8α, IL-1β and TNFα) were used to evaluate the immune response induced in pcDNA-FHA vaccinated hybrid snakehead, it proved that all these mentioned immune activities were significantly enhanced after immunization. The results also showed hybrid snakehead vaccinated with pcDNA-FHA had higher survival rate (79.33%) compared with the controls after challenge with N. seriolae, indicating that the pcDNA-FHA DNA vaccine can supply immune protection against N. seriolae infection. Taken together, this study may warrant further development of these common immunogenic proteins as the antigens for vaccine or diagnosis and facilitate the prevention and treatment of fish nocardiosis.

We have studied the ability of peptide anxiolytic selank (Thr-Lyz-Pro-Arg-Pro-Gly-Pro) to compensate for mnestic dysfunction caused by the administration of actinomycin D, which inhibits protein synthesis by blocking DNA-dependent RNA polymerase. The experiments were performed on white rats with acquired adaptive ability of spatial visual orientation in a 16-door labyrinth. The learning was based on the avoidance of electric skin irritation at alternating sites of escape reaction (site reflex). Selank (0.5 mg/kg, i.p.) prevented or compensated for actinomycin D (250 mg/kg, i.p.) induced violation of the process of acquisition, improvement, and consolidation of memory trace during the development of a complex site reflex. The drug administration also reduced the time required for acquisition of the adaptive ability of spatial visual orientation in the labyrinth and restored the actinomycin D violated process of re-learning upon a change in the alternation of escape sites under free-choice conditions.

Aeromonas veronii is a pathogen capable of infecting humans, livestock and aquatic animals, resulting in serious economic losses. In this study, two recombinant Lactobacillus casei expressing flagellin A (FlaA) of A. veronii, Lc-pPG-1-FlaA (surface-displayed) and Lc-pPG-2-FlaA (secretory) were constructed. The immune responses in fish administered with recombinant L. casei were evaluated. The two recombinant L. casei were orally administered to common carp, which stimulated high serum IgM and induced higher ACP, AKP, SOD and LYZ activity. Using qRT-PCR, the expression of IL-10, IL-8, IL-1β, TNF-α and IFN-γ in the tissue of fish immunized with recombinant L. casei was significantly (p < 0.05) upregulated, which indicated that recombinant L. casei could activate the innate immune system to trigger the cell immune response and inflammatory response. Furthermore, recombinant L. casei was able to survive the intestinal environment and colonize in intestine mucosal. The study showed that after being challenged by A. veronii, fish administered with Lc-pPG-1-FlaA (70%) and Lc-pPG-2-FlaA (50%) had higher survival rates compared to Lc-pPG and PBS, indicating that recombinant L. casei might prevent A. veronii infection by activating the immune system to trigger immune responses. We demonstrated that flagellin as an antigen of vaccine, is acceptable for preventing A. veronii infection in fish. The recombinant L. casei expressing FlaA may be a novel mucosal vaccine for treating and controlling A. veronii.

Fish nocardiosis is a widespread chronic granulomatous disease in aquatic environment, which was particularly caused by Nocardia seriolae. The phage shock protein A (PspA) and tellurium resistance protein D (TerD) were identified to be the immunodominant antigens of the wild-type N. seriolae strain ZJ0503 in our previous study. In an attempt to develop effective DNA vaccines against this pathogen, PspA and TerD were used as candidates to ligate with pcDNA3.1-Flag plasmids, respectively. In addition, the abilities of these two DNA vaccines to elicit various immune responses in hybrid snakehead and supply protective efficacy against artificial challenge with N. seriolae were determined in the present study. The results showed that intramuscular injection with pcDNA-PspA and pcDNA-TerD did not exhibit cytotoxic activities in hybrid snakehead via histopathological examination. Besides, hybrid snakehead immunization with pcDNA-PspA and pcDNA-TerD could increase several non-specific immune paraments in serum, including LYZ, POD, ACP, AKP and SOD activities. Meanwhile, the pcDNA-TerD DNA vaccine could induce strongly specific antibody (IgM) titer in hybrid snakehead with a relative percent of survival (RPS) value of 83.14% against N. seriolae, while that of pcDNA-PspA DNA vaccine was displayed comparably low IgM titer with RPS value of 57.83%. Furthermore, quantitative real-time PCR assays presented that the expression of immune-related genes (MHCIα, MHCIIα, CD4, CD8α, IL-1β and TNFα) were up-regulated to various degrees after vaccination with pcDNA-PspA or pcDNA-TerD, indicating that these two DNA vaccines were able to boost humoral and cell-mediated immune responses in hybrid snakehead. Taken together, both the pcDNA-PspA and pcDNA-TerD DNA vaccines were proved to be safe, immunogenic and effective in protecting hybrid snakehead against N. seriolae infection, which can promote the development and application of DNA vaccines to control fish nocardiosis in aquaculture.

Keywords: DNA vaccine; Hybrid snakehead; Nocardia seriolae; Phage shock protein A (PspA); Tellurium resistance protein D (TerD).

Lysozyme is a well-characterized protein in terms of its structure, dynamics, and functions. It has thus emerged as a potential target to understand protein-drug interactions. The aim of our study is to gain a biophysical outlook on the interaction of lysozyme (Lyz), a well-known model protein, with Noscapine, a potent tubulin-binding anticancer drug. Noscapine (Nos) is effective against a wide range of cancer and shows low toxicity and few side effects. We report the underlying mechanism of complex formation between Nos and Lyz using spectroscopic and advanced computational avenues. The spectroscopic techniques, that is, absorption and steady-state and time-resolved fluorescence, proved that Lyz-Nos forms a complex, and the quenching mechanism was of the static type. The binding constant was in the order of 10³ indicative of moderate binding, while the stoichiometry of the protein-drug complex was 1:1 at 298 K. The secondary structural analysis using CD and UV thermal denaturation further confirmed the conformational changes in the protein upon binding with Nos. Molecular dynamics simulation studies confirmed the stable binding with minimum deviations in RMSD. The above conclusions are significant to the development of the pharmacokinetics and pharmacodynamic properties of Nos, and its successful interaction with a versatile protein like Lyz will help in overcoming its previous limitations.

Among vanadium compounds with potential medicinal applications, [V^IVO(acac)₂] is one of the most promising for its antidiabetic and anticancer activity. In the organism, however, interconversion of the oxidation state to +III and +V and binding to proteins are possible. In this report, the transformation of V^III(acac)₃, V^IVO(acac)₂, and V^VO₂(acac) <math><msubsup><mrow></mrow> <mrow><mn>2</mn></mrow> <mrow><mo>-</mo></mrow> </msubsup> </math> after the interaction with two model proteins, lysozyme (<em>Lyz</em>) and ubiquitin (Ub), was studied with ESI-MS (ElectroSpray Ionization-Mass Spectroscopy), EPR (Electron Paramagnetic Resonance), and computational (docking) techniques. It was shown that, in the metal concentration range close to that found in the organism (15-250 μM), V^III(acac)₃ is oxidized to V^IVO(acac)⁺ and V^IVO(acac)₂, which-in their turn-interact with proteins to give <i>n</i>[V^IVO(acac)]-Protein and <i>n</i>[V^IVO(acac)₂]-Protein adducts. Similarly, the complex in the +IV oxidation state, V^IVO(acac)₂, dissociates to the mono-chelated species V^IVO(acac)⁺ which binds to <em>Lyz</em> and Ub. Finally, V^VO₂(acac) <math><msubsup><mrow></mrow> <mrow><mn>2</mn></mrow> <mrow><mo>-</mo></mrow> </msubsup> </math> undergoes complete dissociation to give the 'bare' V^VO <math><msubsup><mrow></mrow> <mrow><mn>2</mn></mrow> <mrow><mo>+</mo></mrow> </msubsup> </math> ion that forms adducts <i>n</i>[V^VO₂]-Protein with <i>n</i> = 1-3. Docking calculations allowed the prediction of the residues involved in the metal binding. The results suggest that only the V^IVO complex of acetylacetonate survives in the presence of proteins and that its adducts could be the species responsible of the observed pharmacological activity, suggesting that in these systems V^IVO²⁺ ion should be used in the design of potential vanadium drugs. If V^III or V^VO₂ potential active complexes had to be designed, the features of the organic ligand must be adequately modulated to obtain species with high redox and thermodynamic stability to prevent oxidation and dissociation.

Keywords: anticancer action; antidiabetic action; drug design; metal drugs; proteins; transport in the organism; vanadium.

In the present work, biophysical insight into the binding interactions of the protein, hen egg white (HEW) lysozyme (Lyz) with an anticancer drug, 6-mercaptopurine (6-MP)' was investigated by using a combination of spectroscopic and computational tools. 6-MP, a synthetic analog of natural purines, is a well-known anticancer drug and antiviral agent that inhibits the synthesis of RNA, DNA, and proteins. Lysozyme is a single-chain protein that can combine with endogenous and exogenous substances to exert its antiviral, antibacterial, and antitumor effects. The intrinsic fluorescence of lysozyme was quenched with the increased addition of 6-MP. The quenching mechanism was found to be static in nature as shown by the fluorescence lifetime and excitation spectrum measurements. The conformational changes of Lyz in the presence of 6-MP were monitored both at the ensemble and single-molecule level by using synchronous fluorescence spectroscopy, circular dichroism (CD), and fluorescence correlation spectroscopy (FCS). Molecular docking results predicted the probable binding sites for 6-MP on Lyz. The experimental findings are in good agreement with the results obtained by the molecular dynamics (MD) simulation study. Graphical abstract.

A 8-week feeding trial was conducted to evaluate the effect of different berberine-dietary feeding modes on growth, non-specific immune responses and disease resistance of blunt snout bream, Megalobrama amblycephala. Fish (average initial weight 4.70 ± 0.02 g) were fed two fat levels (5% and 10%) diets in three berberine-feeding modes (supplementing 50 mg/kg berberine continuously, two-week or four-week intervals) with four replicates, respectively. Then, fish were challenged by Aeromonas hydrophila and mortality was recorded for the next 96 h after feeding trial. The results showed that different feeding modes of berberine significantly influenced growth, innate immunity and antioxidant capability of fish. Fish fed normal diet with 50 mg/kg berberine at two-week interval mode reflected remarkably (P < 0.05) high weight gain (WG). Plasma TC and TG contents were significantly (P < 0.05) decreased. The lysozyme (LYZ) activities, complement component 3 (C3) and complement component 4 (C4) concentrations were significantly (P < 0.05) increased. Fish not only exhibited relatively low hepatopancreas malondialdehyde (MDA) and lipid peroxide (LPO) contents, but also significantly (P < 0.05) improved superoxide dismutase (SOD) and catalase (CAT) activities. Fish mortality after challenged by Aeromonas hydrophila was decreased. Same results were also presented in fish fed high-fat diet with 50 mg/kg berberine at two-week, four-week intervals or continuous feeding modes. Based on fish healthy improvement and feeding cost saving, blunt snout bream fed normal diet with 50 mg/kg berberine at two-week interval or fed high-fat diet with berberine at two-week or four-week intervals were optimal feeding mode, respectively.

This study evaluates the effects of dietary inclusion of grape pomace flour (GPF) on growth, antioxidant, anti-inflammatory, innate-adaptive immunity, and immune genes expression in Labeo rohita against Flavobacterium columnaris. In both normal and challenged fish the growth rate, hematology and biochemical parameters significantly increased when fed with 200 and 300 mg GPF enriched diets; similarly the activities of antioxidants and innate-adaptive immune parameters, such as malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione (GSH), phagocytic (PC), respiratory burst (RB), alternative pathway complement (ACP), lysozyme (Lyz), and total immunoglobulin M (IgM) significantly increased in both groups. Similarly, the immune, antioxidant, and anti-inflammatory-related gene mRNA expression was significantly up-regulated in head kidney (HK) tissues. The challenged fish fed without GPF always exhibited lower values of all the studied parameters. The results indicate that both normal and challenged fish treated with 200 mg GPF inclusion diet had significantly enhanced growth rate, antioxidant status, and immune defense mechanisms than with 300 mg GPF diet in L. rohita against F. columnaris.

Keywords: Antioxidant; Flavobacterium columnaris; Grape pomace flour (GPF); Innate-adaptive immune response; Labeo rohita.

Non-typhoidal Salmonella is one of the most common causes of bacterial foodborne disease and consumption of contaminated poultry products, including turkey, is one source of exposure. Minimizing Salmonella colonization of commercial turkeys could decrease the incidence of Salmonella-associated human foodborne illness. Understanding host responses to these bacteria is critical in developing strategies to minimize colonization and reduce food safety risk. In this study, we evaluated bacterial load and blood leukocyte transcriptomic responses of 3-week-old turkeys challenged with the Salmonella enterica serovar Typhimurium (S. Typhimurium) UK1 strain. Turkeys (n = 8/dose) were inoculated by oral gavage with 10⁸ or 10¹⁰ colony forming units (CFU) of S. Typhimurium UK1, and fecal shedding and tissue colonization were measured across multiple days post-inoculation (dpi). Fecal shedding was 1-2 log₁₀ higher in the 10¹⁰ CFU group than the 10⁸ CFU group, but both doses effectively colonized the crop, spleen, ileum, cecum, colon, bursa of Fabricius and cloaca without causing any detectable clinical signs in either group of birds. Blood leukocytes were isolated from a subset of the birds (n = 3-4/dpi) both pre-inoculation (0 dpi) and 2 dpi with 10¹⁰ CFU and their transcriptomic responses assayed by RNA-sequencing (RNA-seq). At 2 dpi, 647 genes had significant differential expression (DE), including large increases in expression of immune genes such as CCAH221, IL4I1, LYZ, IL13RA2, IL22RA2, and ACOD1. IL1β was predicted as a major regulator of DE in the leukocytes, which was predicted to activate cell migration, phagocytosis and proliferation, and to impact the STAT3 and toll-like receptor pathways. These analyses revealed genes and pathways by which turkey blood leukocytes responded to the pathogen and can provide potential targets for developing intervention strategies or diagnostic assays to mitigate S. Typhimurium colonization in turkeys.

Keywords: Colonization; Foodborne pathogen; Leukocyte; RNA-seq; Salmonella enterica serovar Typhimurium; Turkey.

Background: The cellular origin and molecular mechanisms of Barrett's esophagus (BE) are still controversial. Trans-differentiation is a mechanism characterized by activation of the intestinal differentiation program and inactivation of the squamous differentiation program.

Aims: Renal capsule grafting (RCG) was used to elucidate whether CDX2 overexpression on the basis of P63 deficiency in the esophageal epithelium may generate intestinal metaplasia.

Methods: P63^-/-;Villin-Cdx2 embryos were generated by crossing P63^+/- mice with Villin-Cdx2 mice. E18.5 esophagus was xenografted in a renal capsule grafting (RCG) model. At 1, 2, or 4 weeks after RCG, the mouse esophagus was immunostained for a proliferation marker (BrdU), squamous transcription factors (SOX2, PAX9), squamous differentiation markers (CK5, CK4, and CK1), intestinal transcription factors (CDX1, HNF1α, HNF4α, GATA4, and GATA6), intestinal columnar epithelial cell markers (A33, CK8), goblet cell marker (MUC2, TFF3), Paneth cell markers (LYZ and SOX9), enteroendocrine cell marker (CHA), and Tuft cell marker (DCAMKL1).

Results: The P63^-/-;Villin-Cdx2 RCG esophagus was lined with proliferating PAS/AB+ cuboidal cells and formed an intestinal crypt-like structure. The goblet cell markers (TFF3 and MUC2) and intestinal transcription factors (CDX1, HNF1α, HNF4α, GATA4, and GATA6) were expressed although no typical morphology of goblet cells was observed. Other intestinal cell markers including enteroendocrine cell marker (CHA), Paneth cell markers (LYZ and Sox9), and intestinal secretory cell marker (UEA/WGA) were also expressed in the P63^-/-;Villin-Cdx2 RCG esophagus. Squamous cell markers (PAX9 and SOX2) were also expressed, suggesting a transitional phenotype.

Conclusion: CDX2 overexpression on the basis of P63 deficiency in esophageal epithelial cells induces Barrett's-like metaplasia in vivo. Additional factors may be needed to drive this transitional phenotype into full-blown BE.

Keywords: Barrett’s esophagus; CDX2; Esophagus; P63; Renal capsule grafting.

The synthesis and characterization of one oxidoethoxidovanadium(V) [V^VO(L¹)(OEt)] (<b>1</b>) and two nonoxidovanadium(IV) complexes, [V^IV(L^2-3)₂] (<b>2</b> and <b>3</b>), with aroylhydrazone ligands incorporating naphthalene moieties, are reported. The synthesized oxido and nonoxido vanadium complexes are characterized by various physicochemical techniques, and their molecular structures are solved by single crystal X-ray diffraction (SC-XRD). This revealed that in <b>1</b> the geometry around the vanadium atom corresponds to a distorted square pyramid, with a O₄N coordination sphere, whereas that of the two nonoxido V^IV complexes <b>2</b> and <b>3</b> corresponds to a distorted trigonal prismatic arrangement with a O₄N₂ coordination sphere around each "bare" vanadium center. In aqueous solution, the V^VO moiety of <b>1</b> undergoes a change to V^VO₂ species_, yielding [V^VO₂(L¹)]^- (<b>1'</b>), while the nonoxido V^IV-compounds <b>2</b> and <b>3</b> are partly converted into their corresponding V^IVO complexes, [V^IVO(L^2-3)(H₂O)] (<b>2'</b> and <b>3'</b>). Interaction of these V^VO₂, V^IVO, and V^IV systems with two model proteins, ubiquitin (Ub) and lysozyme (<em>Lyz</em>), is investigated through docking approaches, which suggest the potential binding sites: the interaction is covalent for species <b>2'</b> and <b>3'</b>, with the binding to Glu16, Glu18, and Asp21 for Ub, and His15 for <em>Lyz</em>, and it is noncovalent for species <b>1'</b>, <b>2</b>, and <b>3</b>, with the surface residues of the proteins. The ligand precursors and complexes are also evaluated for their <i>in vitro</i> antiproliferative activity against ovarian (A2780) and prostate (PC3) human cancer cells and in normal fibroblasts (V79) to check the selectivity of the compounds for cancer cells.

The development of novel biocompatible and cost effective cryogel membrane which shows enhanced antimicrobial properties in order to use for several approaches such as wound dressing, scaffold or food packaging was aimed in this study. A super macro porous lysozyme imprinted cryogel membranes showing antibacterial effect against both Gram-positive and Gram-negative bacteria were prepared by using molecular imprinting technique. N-methacryloyl-(L)-histidine methyl ester (MAH) was used as the pseudo specific ligand and complexed with Cu⁺⁺ in order to provide metal ion coordination between MAH and template molecule (lysozyme). Comparing the antibacterial activity of different lysozyme concentrations, cryogel membranes were prepared in three different concentrations. To synthesize Poly (hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine methylester) P(HEMA-MAH) cryogel membrane, free radical polymerization initiated by N, N, N', N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) was carried out at -12 °C. The characterization of the lysozyme imprinted cryogel membrane was accomplished by using scanning electron microscopy (SEM), swelling degree measurements and Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) spectroscopy. The cytotoxicity test of produced membrane was performed by using mouse fibroblast cell line L929. The antibacterial activity of P(HEMA-MAH) lysozyme molecular imprinted [P(HEMA-MAH) Lyz-MIP] cryogel membranes against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were determined by Kirby-Bauer membranes diffusion and viable cell counting methods. When the antibacterial effect of P(HEMA-MAH) Lyz-MIP cryogel membranes were evaluated, it was found that P(HEMA-MAH) Lyz-MIP cryogel membranes had stronger antibacterial effects against Gram-negative E. coli bacteria even in low lysozyme concentrations. In addition, 100% bacterial inhibition was detected for both of two bacteria at increasing lysozyme concentrations.

Keywords: Lysozyme; P(HEMA-MAH) cryogel; antibacterial; molecular imprinting.

Herein we report the binding interactions between lysozyme (Lyz) and an anthracycline drug, epirubicin hydrochloride (EPR), through an extensive spectroscopic approach at both ensemble average and single molecular resolution. Our steady-state and time-resolved fluorescence spectroscopy reveals that the drug-induced fluorescence quenching of the protein proceeds through a static quenching mechanism. Isothermal titration calorimetry (ITC) and steady-state experiments reveal almost similar thermodynamic signatures of the drug-protein interactions. The underlying force that plays pivotal roles in the said interaction is hydrophobic in nature, which is enhanced in the presence of a strong electrolyte (NaCl). Circular dichroism (CD) spectra indicate that there is a marginal increase in the secondary structure of the native protein (α-helical content increases from 26.9 to 31.4% in the presence of 100 μM EPR) upon binding with the drug. Fluorescence correlation spectroscopy (FCS) was used to monitor the changes in structure and conformational dynamics of Lyz upon interaction with EPR. The individual association (K_ass = 0.33 × 10⁶ ms^-1 M^-1) and dissociation (K_diss = 1.79 ms^-1) rate constants and the binding constant (K_b = 1.84 × 10⁵ M^-1) values, obtained from fluctuations of fluorescence intensity of the EPR-bound protein, have also been estimated. AutoDock results demonstrate that the drug molecule is encapsulated within the hydrophobic pocket of the protein (in close proximity to both Trp62 and Trp108) and resides ∼20 Å apart from the covalently labelled CPM dye. Förster resonance energy transfer (FRET) studies proved that the distance between the donor (CPM) and the acceptor (EPR) is ∼22 Å, which is very similar to that obtained from molecular docking analysis (∼20 Å). The system also shows temperature-dependent reversible FRET, which may be used as a thermal sensor for the temperature-sensitive biological systems.

Ozone nanobubble (NB-O₃) is a promising technology for improving dissolved oxygen and reducing bacterial concentration in aquaculture systems. Here, we investigated the effects of NB-O₃ on the innate immunity of fish by monitoring the expression levels of nonspecific immune-related genes (IL-1β, IL-2β, TNF-α), heat-shock protein genes (HSP70, HSP90-α), and a bacteriolytic enzyme, C-type lysozyme, gene (LYZ) post-treatment with this technology. Following exposure to NB-O₃, the different tissues of Nile tilapia (Oreochromis niloticus) were collected over time for quantitative real-time PCR (qPCR) analysis. The expression of all the genes evaluated in the gills, the head kidney, and the spleen of the NB-O₃ treated group was significantly up-regulated compared to that in the untreated control group. The expression levels were the highest (approx. 2 to 4-fold) at 15 min and 3 h post-exposure and then decreased from 6 to 24 h. These findings suggested that NB-O₃ could switch on the innate immunity genes of Nile tilapia. Thus, we hypothesized that the NB-O₃-immune-activated fish would respond more effectively to subsequent bacterial infections, thereby improving survivability compared to that of untreated fish. To test this hypothesis, 3 h post NB-O₃ exposed fish and unexposed fish were challenged with a lethal dose of Streptococcus agalactiae. Interestingly, the survival rate of the NB-O₃ group was significantly higher than that of the non-treated controls, with a relative percent survival (RPS) of 60-70%. Together, these findings indicate, for the first time, that NB-O₃ may trigger the nonspecific defense system of the fish, thereby improving fish survivability during subsequent bacterial infections. This research identified another potential benefit of NB-O₃ in aquaculture for preventing infectious bacterial diseases.

Keywords: Gene expression; Innate immunity; Nile tilapia; Ozone nanobubble; Streptococcus agalactiae.

Lysozyme (Lyz) is an antimicrobial peptide, a safe adjunct, and it has been indicated that Lyz can promote vibrissae follicle growth by enhancing the hair-inductive capacity of dermal papilla cells in mice. The present study produced a new type of minoxidil (Mx)-coated antifungal Lyz-shelled microbubble (LyzMB) for inhibiting bacteria and allergies on the oily scalp. The potential of Mx-coated LyzMBs (Mx-LyzMBs) combined with ultrasound (US) and the role of LyzMB fragments in enhancing hair follicle growth were investigated. Mx grafted with LyzMBs were synthesized and the loading efficiency of Mx on cationic LyzMBs was 20.3%. The biological activity of Lyz in skin was determined using an activity assay kit and immunohistochemistry expression, and the activities in the US+Mx-LyzMBs group were 65.8 and 118.5 μU/mL at 6 and 18 h, respectively. In hair follicle cell culture experiments, the lengths of hair follicle cells were significantly enhanced in the US+Mx-LyzMBs group (108.2 ± 11.6 μm) compared to in the US+LyzMBs+Mx group (44.3 ± 9.8 μm) and the group with Mx alone (79.6 ± 12.0 μm) on day 2 (p < 0.001). During 21 days of treatment in animal experiments, the growth rates at days 10 and 14 in the US+Mx-LyzMBs group increased by 19.4 and 65.7%, respectively, and there were significant differences (p < 0.05) between the US+Mx-LyzMBs group and the other four groups. These findings indicate that 1-MHz US (applied at 3 W/cm², acoustic pressure = 0.266 MPa) for 1 min combined with Mx-LyzMBs can significantly increase more penetration of Mx and LyzMB fragments into skin and enhance hair growth than Mx alone.

Keywords: cavitation; hair follicle; lysozyme-shelled microbubbles; minoxidil; ultrasound.

The rapid emergence of drug-resistant bacteria has raised a great social concern together with the impetus for exploring advanced antibacterial ways. NIR-triggered antimicrobial photodynamic therapy (PDT) by virtue of lanthanide-doped upconversion nanoparticles (UCNP) as energy donor exhibits the advantages of high tissue penetration, broad antibacterial spectrum and less acquired resistance, but is still limited by its low efficacy. Herein, we designed a novel bio-inorganic nanohybrid and for the first time combined lysozyme (LYZ) with UCNP-PDT system to enhance the efficiency against resistant bacteria. Benefiting from the rapid adhesion to bacteria, intelligently bacteria-responsive LYZ release and synergistic LYZ-PDT effect, the nanoplatform achieves an exceptionally strong bactericidal capacity and conspicuous bacteriostasis on methicillin-resistant S. aureus . These findings pave the way for designing efficiently antibacterial nanomaterials and provide a new synergistic strategy for combating deep-tissue bacterial infection.

Keywords: Antibacterial agent; enzyme; mesoporous materials; photodynamic therapy; upconversion nanoparticle.

Avian pathogenic Escherichia coli (APEC) is the causative agent of avian colibacillosis. Baicalin (BA) possesses multiple pharmacological effects, but the mechanism underlying its activity in APEC-induced intestinal injury remains unknown. This study aims to investigate the protective effects and possible mechanism of BA against APEC-induced intestinal injury. Sixty 1-day-old chicks were randomly divided into 4 groups: the control group (basal diet), E. coli group (basal diet), BAI10 group (10 mg/kg BA), and BAI20 group (20 mg/kg BA). After pretreatment with BA for 15 d and subsequent induction of APEC infection by pectoralis injection, the ileum was collected and analyzed. The results showed that BA-pretreatment demonstrated an alleviation of chicks in diarrhea rate, mortality, and histopathological changes in intestinal tissues after APEC infection. Additionally, following APEC infection, BA improved the intestinal barrier by elevating zona occludens (ZO)s (ZO-1, 2, 3), Claudins (Claudin1, 2, 3), Occludin, avian β-defensin (AvBD)s (AvBD1, 2, 4), lysozyme (Lyz) mRNA levels and ZO-1, Claudin1, and Occludin protein levels. Besides, the activities of total superoxide dismutase (T-SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) and the SOD-1 and CAT mRNA levels and SOD-1 protein level were elevated by BA pretreatment. BA pretreatment also decreased the malondialdehyde (MDA) content, heme oxygenase-1 (HO-1) and NADH quinone oxidoreductase 1 (NQO1) mRNA levels, and HO-1 protein level after APEC infection. BA alleviated the APEC-induced inflammatory response, including downregulating the mRNA levels of proinflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin [IL]-1β, IL-6, IL-8) and upregulating the mRNA levels of anti-inflammatory cytokines (IL-4, IL-10, IL-13, transforming growth factor-β [TGF-β]). Furthermore, BA decreased the mRNA and protein levels of phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), and nuclear factor kappa-B (NF-κB) as well as the expression of the phosphorylated forms of these proteins after APEC infection. Collectively, our findings indicate that BA exerts a protective effect against APEC-induced intestinal injury in chicks by inhibiting the PI3K/AKT-mediated NF-κB pathway, suggesting that BA may be a potential therapeutic approach for avian colibacillosis.

Keywords: Baicalin; PI3K/AKT/NF-κB, chick; avian pathogenic Escherichia coli; intestinal injury.

As a cytoplasmic tyrosine kinase in the Tec family, Bruton's tyrosine kinase (Btk) participates in various biological processes, including cell growth, differentiation, and apoptosis. Although recent studies have indicated that Btk is involved in pro-inflammatory cytokine production, the underlying impact of Btk on the development and pathogenesis of diabetic nephropathy (DN) has not been elucidated. The aim of this study was to determine whether Btk knockout (KO) could reduce inflammation and kidney injury in DN. First, diabetic mice models were established via an intraperitoneal injection of streptozotocin. Thereafter, the underlying mechanism was explored by comparing Btk ^flox/flox Lyz-Cre mice to wild-type (C57BL/6N) mice. Albuminuria was significantly reduced, and kidney injuries were attenuated in Btk conditional deletion diabetic mice. More importantly, these changes were demonstrated to be associated with decreased levels of pro-inflammatory cytokines owing to the downregulation of the MAPK and NF-κB signaling pathways. Collectively, these findings indicate that Btk plays a critical role in the regulation of kidney inflammation and provides a prospective therapeutic strategy for the treatment of DN.

Keywords: Bruton’s tyrosine kinases; Diabetic nephropathy; inflammation; macrophage.

We have designed and synthesized a novel pyrene-naphthalene sulphonyl conjugate, 1-((1Z)-(4-((Z)-4-(pyrene-1-yl)methyleneamino)phenylsulfonyl)phenylimino)methyl)naphthalene-2-ol (PSN) through a facile two-step reactions. It was characterized by various spectral techniques. Fluorescence spectral studies showed that compound PSN featured fluorescence enhancement upon increasing the water content in THF. This can be attributed to the phenomena of aggregated induced emission (AIE), which is confirmed by SEM and AFM studies, due to the restriction of CHN isomerization of PSN. The anion sensing of PSN was examined with various anions. Among these anions, H₂PO₄^- and F^- ions were selectively sensing with a low detection limit of 3.52 × 10^-7 M and 7.23 × 10^-7 M, respectively, and an obvious color change from yellow to orange was observed by the naked eye. The mechanism of sensing involved the formation of hydrogen bonding interaction between O-H group of PSN and H₂PO₄^-/ F^- ions. The binding of PSN with LYZ was also examined by docking studies, which shows that H-bonding and hydrophobic interactions play crucial roles for the interaction of LYZ toward PSN.

Keywords: Aggregation induced emission; Anion sensor; Lysozyme; Naphthalene; Pyrene.

Renal ischemia-reperfusion injury (IRI) contributes to acute kidney injury (AKI), increases morbidity and mortality, and is a significant risk factor for chronic kidney disease (CKD). Macrophage infiltration is a common feature after renal IRI, and infiltrating macrophages can be polarized into the following two distinct types: M1 macrophages, i.e., classically activated macrophages, which can not only inhibit infection but also accelerate renal injury, and M2 macrophages, i.e., alternatively activated macrophages, which have a repair phenotype that can promote wound healing and subsequent fibrosis. The role of TSC1, which is a negative regulator of mTOR signaling that regulates macrophage polarization in inflammation-linked diseases, has been well documented, but whether TSC1 contributes to macrophage polarization in the process of IRI is still unknown. Here, by using a mouse model of renal ischemia-reperfusion, we found that myeloid cell-specific TSC1 knockout mice (termed Lyz-TSC1 cKO mice) had higher serum creatinine levels, more severe histological damage, and greater proinflammatory cytokine production than wild-type (WT) mice during the early phase after renal ischemia-reperfusion. Furthermore, the Lyz-TSC1 cKO mice showed attenuated renal fibrosis during the repair phase of IRI with decreased levels of M2 markers on macrophages in the operated kidneys, which was further confirmed in a cell model of hypoxia-reoxygenation (H/R) in vitro. Mechanistically, by using RNA sequencing of sorted renal macrophages, we found that the expression of most M1-related genes was upregulated in the Lyz-TSC1 cKO group (Supplemental Table 1) during the early phase. However, C/EBPβ and CD206 expression was decreased during the repair phase compared to in the WT group. Overall, our findings demonstrate that the expression of TSC1 in macrophages contributes to the whole process of IRI but serves as an inflammation suppressor during the early phase and a fibrosis promoter during the repair phase.

Keywords: fibrosis; ischemia-reperfusion (IR); kidney; macrophage polarization; tuberous sclerosis complex 1 (TSC1).

Food dyes, or color additives, are often added into foods, cosmetics and beverages during processing to improve the sensory attributes of the final products. However, the toxicity of tartrazine (TZ), one of the most common azo-dyes, is still unclear, and needs to be ascertained by further study. Hence, in the present study, we aimed to evaluate the effects of TZ consumption on health by using a teleost, crucian carp (Carassius auratus) as the experimental fish. TZ consumption (1.4, 5.5 and 10 mg/kg bwt/day) could cause severe histopathological and cellular alterations in intestine and liver. The height of intestinal villus, thickness of intestinal muscle, and microvilli density were also affected. With the increasing of TZ concentrations, the activities of antioxidant enzymes (CAT, SOD and GSH-Px), exhibited a decreasing trend, while the contents of MDA elevated. Upregulations of pro-inflammatory cytokines (il6 and tnfα), anti-inflammatory cytokines (il8, and il10) and other immune related genes (complement component 3 (c3), lysozymes (lyz), β-defensin 3 (defb3)), were observed after TZ uptake. In addition, TZ consumption also affected the community structure of the microbiota in the intestine of crucian carp. The amount of some probiotic bacteria (Roseomonas, Rhodococcus and Bacillus) and the bacteria (Bacteroides and Clostridium), producing short chain fatty acids, were significantly reduced, and some pathogenetic microorganisms (e.g. Bdellovibrio and Shewanella) were significantly increased after TZ uptake. In summary, the data in the present study indicate that TZ consumption, even at a low concentration, may lead to adverse effects on fish health. Therefore, in aquaculture, it is necessary to be informed about the hazardous effects of TZ, and more attentions should be focused on using natural substitutes.

Keywords: Crucian carp (Carassius auratus); Inflammatory response; Intestinal microbiome; Oxidative stress; Tartrazine.