Objectives were to determine the effect of supplementing 2 sources of vitamin D, cholecalciferol (CH) or calcidiol (CA), at 1 (1mg) or 3 mg/d (3mg) prepartum on concentrations of vitamin D metabolites in plasma, measures of innate immune function, and leukocyte mRNA expression. Parous Holstein cows (n = 99) were assigned to a daily treatment administered as top-dress containing either 1 or 3 mg of CH (CH1 or CH3) or of CA (CA1 or CA3) from 250 d of gestation until calving. Plasma concentrations of vitamin D, immune cell population in blood, cell adhesion markers, and granulocyte phagocytosis and oxidative burst were evaluated pre- and postpartum. The mRNA expression in leukocytes was determined at 270 d of gestation and 3 d postpartum for genes involved in cell migration, pathogen recognition receptors, cell signaling, cytokines, antimicrobial mechanisms, oxidative burst, and Ca and vitamin D metabolism. Concentrations of vitamin D₃ increased in cows fed CH, whereas those of 25-hydroxyvitamin D₃ increased in cows fed CA. Percentage of granulocytes from total leukocytes differed with amount of vitamin D pre- (1mg = 24.5 vs. 3mg = 37.9%) and postpartum (1mg = 22.0 vs. 3mg = 31.0%), thus shifting mononuclear cells in the opposite direction pre- (1mg = 75.5 vs. 3mg = 62.1%) and postpartum (1mg = 78.0 vs. 3mg = 69.0%). Granulocytes displaying phagocytosis (1mg = 69.0 vs. 3mg = 62.9%) and intensity of phagocytosis prepartum (1mg = 7.46 vs. 3mg = 7.28) tended to be less in cows fed 3mg compared with 1mg. During prepartum, CA increased mRNA expression of genes related to cell adhesion and migration (CD44, ICAM1, ITGAL, ITGB1, LGALS8, SELL), pathogen recognition receptor (NOD2, TLR2, TLR6), cell signaling (FOS, JUN, NFKB2), cytokine signaling (IL1B, IL1R1, IL1RN), antimicrobial mechanisms (CTSB, LYZ), and Ca metabolism (ATP2B1, STIM1, TRPV5) compared with CH. Similarly, postpartum, CA increased mRNA expression of genes related to cell adhesion and migration (CXCR2, SELL, TLN1), cell signaling (AKT2), cytokines (CCL2, IL1R1, ILRN), antimicrobial mechanisms (DEFB3), oxidative burst (RAC2), and calcium metabolism (CALM3) compared with CH. Feeding additional vitamin D in the last 3 wk of gestation changed the profile of blood leukocytes and attenuated granulocyte phagocytosis during the transition period, whereas supplementing CA prepartum increased mRNA expression of genes involved in immune cell function, including genes related to pathogen recognition and antimicrobial effects of leukocytes.

Keywords: calcidiol; dairy cow; immunity; vitamin D.

Background: Plenty of macrophages are recruited to the injured nerve to play key roles in the immunoreaction and engulf the debris of degenerated axons and myelin during Wallerian degeneration, thus creating a conducive microenvironment for nerve regeneration. Recently, drugs targeting the RhoA pathway have been widely used to promote peripheral axonal regeneration. However, the role of RhoA in macrophage during Wallerian degeneration and nerve regeneration after peripheral nerve injury is still unknown. Herein, we come up with the hypothesis that RhoA might influence Wallerian degeneration and nerve regeneration by affecting the migration and phagocytosis of macrophages after peripheral nerve injury.

Methods: Immunohistochemistry, Western blotting, H&E staining, and electrophysiology were performed to access the Wallerian degeneration and axonal regeneration after sciatic nerve transection and crush injury in the Lyz^Cre+/-; RhoA^flox/flox (cKO) mice or LyzCre+/- (Cre) mice, regardless of sex. Macrophages' migration and phagocytosis were detected in the injured nerves and the cultured macrophages. Moreover, the expression and potential roles of ROCK and MLCK were also evaluated in the cultured macrophages.

Results: 1. RhoA was specifically knocked out in macrophages of the cKO mice; 2. The segmentation of axons and myelin, the axonal regeneration, and nerve conduction in the injured nerve were significantly impeded while the myoatrophy was more severe in the cKO mice compared with those in Cre mice; 3. RhoA knockout attenuated the migration and phagocytosis of macrophages in vivo and in vitro; 4. ROCK and MLCK were downregulated in the cKO macrophages while inhibition of ROCK and MLCK could weaken the migration and phagocytosis of macrophages.

Conclusions: Our findings suggest that RhoA depletion in macrophages exerts a detrimental effect on Wallerian degeneration and nerve regeneration, which is most likely due to the impaired migration and phagocytosis of macrophages resulted from disrupted RhoA/ROCK/MLCK pathway. Since previous research has proved RhoA inhibition in neurons was favoring for axonal regeneration, the present study reminds us of that the cellular specificity of RhoA-targeted drugs is needed to be considered in the future application for treating peripheral nerve injury.

Keywords: Macrophage; Nerve regeneration; Peripheral nerve injury; RhoA; Wallerian degeneration.

Background and purpose: The immune response subsequent to an ischemic stroke is a crucial factor in its physiopathology and outcome. It is known that TLR4 is implicated in brain damage and inflammation after stroke and that TLR4 absence induces neutrophil reprogramming toward a protective phenotype in brain ischemia, but the mechanisms remain unknown. We therefore asked how the lack of TLR4 modifies neutrophil function and their contribution to the inflammatory process.

Methods: In order to assess the role of the neutrophilic TLR4 after stroke, mice that do not express TLR4 in myeloid cells (TLR4^loxP/Lyz-cre) and its respective controls (TLR4^loxP/loxP) were used. Focal cerebral ischemia was induced by occlusion of the middle cerebral artery and infarct size was measured by MRI. A combination of flow cytometry and confocal microscopy was used to assess different neutrophil characteristics (circadian fluctuation, cell surface markers, cell complexity) and functions (apoptosis, microglia engulfment, phagocytosis, NETosis, oxidative burst) in both genotypes.

Results: As previously demonstrated, mice with TLR4 lacking-neutrophils had smaller infarct volumes than control mice. Our results show that the absence of TLR4 keeps neutrophils in a steady youth status that is dysregulated, at least in part, after an ischemic insult, preventing neutrophils from their normal circadian fluctuation. TLR4-lacking neutrophils showed a higher phagocytic activity in the basal state, they were preferentially engulfed by the microglia after stroke, and they produced less radical oxygen species (ROS) in the first stage of the inflammatory process.

Conclusions: TLR4 is specifically involved in neutrophil dynamics under physiological conditions as well as in stroke-induced tissue damage. This research contributes to the idea that TLR4, especially when targeted in specific cell types, is a potential target for neuroprotective strategies.

Keywords: TLR4; inflammation; myeloid; neuroprotection; neutrophil; stroke.

Muscle-invasive bladder carcinoma (MIBC) accounts for 25% of newly diagnosed bladder carcinomas (BCs) and presents a high risk of progression and metastasis. This study aimed to identify reliable biomarkers associated with muscle invasion and prognosis to identify potential therapeutic targets for MIBC. Four gene datasets were downloaded from the Gene Expression Omnibus, and the integrated differentially expressed genes (DEGs) were then subjected to gene ontology (GO) terms and pathway enrichment analyses. Correlation analysis between the expression of the top-ranking DEGs and pathological T stages was performed to identify the genes associated with early muscle invasion. The corresponding prognostic values were evaluated, and co-expressed genes mined in the cBioPortal database were loaded into ClueGo in Cytoscape for pathway enrichment analysis. Using data mining from the STRING and TCGA databases, protein-protein interaction and competitive endogenous RNA networks were constructed. In total, 645 integrated DEGs were identified and these were mainly enriched in 26 pathways, including cell cycle, bladder cancer, DNA replication, and PPAR signaling pathway. S100A7 expression was significantly increased from the T2 stage and showed significantly worse overall survival and disease-specific survival in patients with BC. In total, 144 genes co-expressed with S100A7 in BC were significantly enriched in the IL-17 pathway. S100A7 was predicted to directly interact with LYZ, which potentially shows competitive binding with hsa-mir-140 to affect the expression of six lncRNAs in MIBC. In conclusion, high S100A7 expression was predicted to be associated with early muscle invasion and poor survival in patients with BC.

Keywords: Bioinformatics; Bladder carcinoma; Muscle invasion; Prognosis; S100A7.

Background: Probiotics are beneficial in intestinal disorders. However, the benefits of Lactobacillus johnsonii in experimental colitis remain unknown.

Objectives: This study aimed to investigate the benefits of L. johnsonii against Citrobacter rodentium-induced colitis.

Methods: Thirty-six 5-wk-old female C57BL/6J mice were randomly assigned to 3 groups (n = 12): control (Ctrl) group, Citrobacter rodentium treatment (CR) group (2 × 109 CFU C. rodentium), and Lactobacillus johnsonii and Citrobacter rodentium cotreatment (LJ + CR) group (109 CFU L. johnsonii with C. rodentium). Colon length, mucosal thickness, proinflammatory cytokine genes, and endoplasmic reticulum stress were tested.

<strong class="sub-title"> Results: </strong> The CR group had greater spleen weight, mucosal thickness, and Ki67+ cells (0.4-4.7 times), and a 23.8% shorter colon length than the Ctrl group, which in the LJ + CR group were 22.4%-77.6% lower and 30% greater than in the CR group, respectively. Relative to the Ctrl group, serum proinflammatory cytokines and immune cell infiltration were greater by 0.3-1.6 times and 6.2-8.8 times in the CR group, respectively; relative to the CR group, these were 19.9%-61.9% and 69.5%-84.2% lower in the LJ + CR group, respectively. The mRNA levels of lysozyme (<em>Lyz</em>) and regenerating islet-derived protein III were 22.7%-36.5% lower and 1.5-2.7 times greater in the CR group than in the Ctrl group, respectively, whereas they were 22.2%-25.7% greater and 57.2%-76.9% lower in the LJ + CR group than in the CR group, respectively. Cell apoptosis was 11.9 times greater in the CR group than in the Ctrl group, and 87.4% lower in the LJ + CR group than in the CR group. Consistently, the protein abundances of C/EBP homologous protein (CHOP), cleaved caspase 1 and 3, activating transcription factor 6α (ATF6A), and phospho-inositol-requiring enzyme 1α (P-IRE1A) were 0.3-2.1 times greater in the CR group and 31.1%-60.4% lower in the LJ + CR group. All these indexes did not differ between the Ctrl and LJ + CR groups, except for CD8+ T lymphocytes and CD11b+ and F4/80+ macrophages (1-1.5 times greater in LJ + CR) and mRNA concentration of <em>Lyz</em>2 (20.1% lower in LJ + CR).

Conclusions: L. johnsonii supplementation is a promising nutritional strategy for preventing C. rodentium-induced colitis in mice.

Keywords: Citrobacter rodentium; Lactobacillus johnsonii; endoplasmic reticulum stress; inflammatory response; probiotic.

Lysozyme (Lyz) is a naturally occurring enzyme that operates against Gram-positive bacteria and leads to cell death. This antimicrobial enzyme forms the part of the innate defense system of nearly all animals and exists in their somatic discharges such as milk, tears, saliva and urine. Increased Lyz level in serum is an important indication of several severe diseases and so, precise diagnosis of Lyz is an urgent need in biosensing assays. Up to know, various traditional and modern techniques have been introduced for Lyz determination. Although the traditional methods suffer from some significant limitations such as time-consuming, arduous, biochemical screening, bacterial colony isolation, selective enrichment and requiring sophisticated instrumentation or isotope labeling, some new modern approaches like aptamer-based biosensors (aptasensors) and quantum dot (QD) nanomaterials are the main goal in Lyz detection. Electrochemical and optical sensors have been highlighted because of their adaptability and capability to decrease the drawbacks of common methods. Using an aptamer-based biosensor, sensor selectivity is enhanced due to the specific recognition of the analyte. Thereby, in this review article, the recent advances and achievements in electrochemical and optical aptasensing detection of Lyz based on different QD nanomaterials and detection methods have been discussed in detail.

Keywords: Aptasensing platform; Lysozyme; Quantum dots.

Lysozyme (Lyz) is an important antibacterial protein that exists widely in nature. In recent years, the application of graphene oxide (GO) in the field of biotechnology electronics, optics, chemistry and energy storage has been extensively studied. However, due to the unique properties of GO, the mechanism of its interaction with biomacromolecule proteins is very complex. To further explore the interaction between GO and proteins we explore the influence of different pH and heat treatment conditions on the interaction between GO and Lyz, the GO (0-20 μg/mL) was added at a fixed Lyz concentration (0.143 mg/mL) under different pHs. The structure and surface charge changes of Lyz were measured by spectroscopic analysis and zeta potential. The results showed that the interaction between GO and Lyz depends on temperature and pH, significant changes have taken place in its tertiary and secondary structures. By analyzing the UV absorption spectrum, it was found that lysozyme and GO formed a stable complex, and the conformation of the enzyme was changed. In acidic pH conditions (i.e., pH < pI), a high density of Lyz were found to adsorb on the GO surface, whereas an increase in pH resulted in a progressive decrease in the density of the adsorbed Lyz. This pH-dependent adsorption is ascribed to the electrostatic interactions between the negatively charged GO surface and the tunable ionization of the Lyz molecules. The secondary structure of Lyz adsorbed on GO was also found to be highly dependent on the pH. In this paper, we investigated the exact mechanism of pH-influenced GO binding to lysozyme, which has important guidance significance for the potential toxicity of GO biology and its applications in biomedical fields such as structure-based drug design.

Keywords: Binding interaction; Electrostatic interactions; Fluorescence quenching; Graphene oxide; Lysozyme.

This study evaluated the growth performance, immune responses, and disease resistance of Nile tilapia upon pistachio hulls derived polysaccharide (PHDP) and Pediococcus acidilactici (PA) separately or as synbiotic. Fish received four types of diets: T1, control; T2, PHDP (0.1%); T3, PA (0.2%); T4, PHDP (0.1%) +PA (0.2%) for 56 days. The results showed that final weight and weight gain were markedly higher in fish fed T4 diet than that given T1 and T2 diets (P ≤ 0.05). In addition, a significantly greater specific growth rate was obtained by the T4 diet compared to the control. Fish survival was significantly improved in all supplemented diets compared to the control. On the other hand, the activities of lipase, protease, and amylase showed significant increases in the T4 group compared with other feeding groups. The total leucocytes and lymphocytes proportion significantly elevated in T3 and T4 than remaining groups (P ≤ 0.05). Further, fish fed T3 diet presented significantly higher serum total protein, total immunoglobulin, lysozyme activity (LYZ), alternative complement activity (ACH50), and alkaline phosphatase activity compared to fish fed T1 and T2 diets, while the mentioned indices were found significantly highest in T4 group than others. Fish received T3 and T4 diets had higher skin mucus LYZ and ACH50 than those fed T1 and T2 diets (P ≤ 0.05). The malondialdehyde levels were significantly declined in T3 and T4 when compared to the control. Fish fed T3 and T4 diets demonstrated significantly enhanced superoxide dismutase, catalase, and glutathione peroxidase activities compared to the control. The intestinal propionic acid significantly increased by T2 and T4 diets, while the highest levels of acetic acid detected in fish given T4 diet. The expression levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin 10 (IL-10) were significantly affected by T3 and T4 supplements. The efficacy of T4 diet against Aeromonas hydrophila infection was documented by a significantly lower mortality rate. In conclusion, the combination of PHDP and PA presented promising results as a synbiotic feed additive for Nile tilapia.

Keywords: Cytokine; Immunity; Polysaccharides; Prebiotic; Probiotic; Synbiotic.

The clinical manifestations of diabetic kidney disease (DKD) are more heterogeneous than those previously reported, and these observations mandate the need for the recruitment of patients with biopsy-proven DKD in biomarker research. In this study, using the public gene expression omnibus (GEO) repository, we aimed to identify urinary mRNA biomarkers that can predict histological severity and disease progression in patients with DKD in whom the diagnosis and histologic grade has been confirmed by kidney biopsy. We identified 30 DKD-specific mRNA candidates based on the analysis of the GEO datasets. Among these, there were significant alterations in the urinary levels of 17 mRNAs in patients with DKD, compared with healthy controls. Four urinary mRNAs-LYZ, C3, FKBP5, and G6PC-reflected tubulointerstitial inflammation and fibrosis in kidney biopsy and could predict rapid progression to end-stage kidney disease independently of the baseline eGFR (tertile 1 vs. tertile 3; adjusted hazard ratio of 9.68 and 95% confidence interval of 2.85-32.87, p < 0.001). In conclusion, we demonstrated that urinary mRNA signatures have a potential to indicate the pathologic status and predict adverse renal outcomes in patients with DKD.

Keywords: biomarker; diabetic kidney disease; mRNA; renal pathology; urine.

The current study addressed to investigate the effect of lycopene (LYC) on blood physiology, digestive-antioxidant enzyme activity, specific-nonspecific immune response, and inflammatory gene transcriptional regulation (cytokines, heat shock proteins, vitellogenins) in spotted snakehead (Channa punctata) against Pseudomonas aeruginosa. In unchallenged and challenged fish treated with 200 mg LYC enriched diet the growth performance and digestive-antioxidant enzymes increased after 30 days, whereas with inclusion of 100 or 400 mg LYC in the diets, the increase manifested on or after 45 days. No mortality in fish treated with any LYC diet against P. aeruginosa was revealed. In the unchallenged and challenged fish the phagocytic (PC) activity in head kidney (HK) and spleen were significantly enhanced when fed the control diet or other LYC diets, whereas the respiratory burst (RB) activity and nitric oxide (NO) production significantly increased when fed the 200 mg diet for 45 and 60 days. Similarly, the lysozyme (Lyz) activity in the HK and spleen, and total Ig content in serum were significantly higher in both groups fed the 200 mg LYC diet for 15, 45, and 60 days. Heat shock protein (Hsp 70) was significantly improved in the uninfected group fed the 200 mg LYC diet for 45 and 60 days, but Hsp27 did not significantly change among the experimental groups at any time points. TNF-α and IL-6 mRNA pro-inflammatory cytokine expression significantly increased in both groups fed the 200 mg LYC diet after 45 and 60 days, while the IL-12 mRNA expression was moderate in both groups fed the same diet for 60 days. The IL-10 did not significant mRNA expression between groups at any sampling. The iNOS and NF-κB mRNA expression was pointedly high in both groups fed the 200 mg LYC diet on day 45 and 60. Vitellogenin A (VgA) mRNA was significantly higher in the uninfected fish fed the 100 and 200 mg LYC diets for 45 and 60 days, but VgB did not reveal significant difference between the treatment groups at any time points. The present results suggest that supplementation of LYC at 200 mg significantly modulate the blood physiology, digestive-antioxidant enzymes, specific-nonspecific immune parameters, and cytokines, Hsp, and vitellogenins in spotted snakehead against P. aeruginosa.

Keywords: Antioxidant activity; Channa punctata; Innate-adaptive immune response; Lycopene (LYC); Pseudomonas aeruginosa.

Soya saponin (SS) is a natural active substance of leguminous plant, which could improve lipid metabolism and regulate immune function. Intestinal flora might play a key role in the biological functions of SS. The objective of this study was to measure the effects of dietary SS on immune function, lipid metabolism, and intestinal flora of laying hens with or without antibiotic treated. The experiment was designed as a factorial arrangement of 3 dietary SS treatments × 2 antibiotic treatments. Birds were fed a basal diet (CON) or a low-SS diet (50 SS) containing 50 mg/kg SS, or a high-SS diet (500 SS) containing 500 mg/kg SS. Birds were cofed with or without antibiotics. The growth experiment lasted for 10 wk. Results showed that birds fed the 50 mg/kg SS diet tended to have lower abdominal fat rate. The gene expression of liver X receptor-α (LxR-α) in liver and serum total cholesterol (TC) were dropped, and the gene expression of acyl-CoA thioesterase 8 (ACOT8) in liver were upregulated. Compared with CON group, the levels of lysozyme, IL-10, and transforming growth factor (TGF-β) in the serum were elevated as along with gene expression of IL-10, TGF-β, and LYZ in ileum of both 50 and 500 SS group. However, the level of secretory immunoglobulin A (sIgA) and Mucin-2 in the ileum were downregulated in the 500 SS group. Additionally, Lactobacillus and Lactobacillus gasseri were the dominant bacteria in the 50 SS group, whereas the relative abundance of Lactobacillus was dropped in the 500 SS group. With combined antibiotics treatment, the α-diversity of bacteria was reduced, and the biological effects of SS were eliminated. In conclusion, the lipid metabolism, immune function, and intestinal flora of the laying hens were improved with the dietary supplementation of 50 mg/kg SS. But dietary 500 mg/kg SS had negative effects on laying hens.

Keywords: intestinal health; laying hen; lipid metabolism; soya saponin.

Protein-drug binding study addresses a broad domain of biological problems associating molecular functions to physiological processes composing and modifying safe and coherent drug therapeutics. Comparison of the binding and thermodynamic aspect of sulfa drugs, sulfamethazine (SMZ) and sulfadiazine (SDZ) with the protein, lysozyme (Lyz) was carried out using spectroscopic, molecular docking, and dynamic simulation study. The fluorescence quenching and apparent binding constant for the binding reaction were calculated to be in the order of 10⁴ M^-1 , slightly higher for SMZ as compared to that of SDZ and the binding stoichiometry values show 1:1 drug binding with each protein molecule. The binding was an enthalpy-driven spontaneous exothermic reaction favored by a negative enthalpy and a positive entropy contribution for both the complexes. The binding from the fluorescence quenching data suggests a static quenching mechanism dominated by non-polyelectrolytic components. Synchronous fluorescence denoted a conformational change in the tryptophan moiety of the protein. Molecular docking and dynamic simulation study provided a clearer view of the interaction pattern, where the drug resides on the binding pocket of the protein structure. Overall the protein, Lyz binding of SMZ was slightly more favored over SDZ.

Keywords: Molecular Docking; Molecular Dynamics; Protein-drug binding; Spectroscopy; Thermodynamics.

Background: Resveratrol (RSV) plays a vital role in alleviating various stresses and improving intestinal health. The current study was conducted to explore whether RSV alleviates weaning stress through improving gut health in a weaning mouse model. Forty 21-day-old weaned mice were randomly assigned to a control group without RSV treatment and three treatment groups with 10, 20, and 50 mg/kg RSV for 28 days.

Results: The results showed that RSV at a dose of 20 mg/kg improved total body weight, intestinal morphology (villus length and the ratio of villus length to crypt depth), and the levels of intestinal barrier proteins (claudin-1 and occludin), but had little effect on the food intake, crypt depth, and serum free amino acids of mice. Compared with the control group, mice supplemented with RSV had decreased mRNA expression of genes related to inflammatory cytokines (IL-6 and IL-1β), but increased mRNA expression of genes related to host defense peptides (Defa3, Defa5, Defa20, and Lyz) and short-chain fatty acids (SCFAs) production (propionic acid, isobutyric acid, butyric acid, and isovaleric acid). In addition, 16S rRNA sequencing results showed that RSV supplementation increased the richness indices of intestinal microbiota (Chao, ACE) and shaped the composition of intestinal microbiota (e.g., increased β-diversity of intestinal microbiota community). Meanwhile, RSV supplementation increased genes of Butyricicoccus, Ruminococcus_1, and Roseburia, which are producers of SCFAs. Furthermore, RSV supplementation significantly influenced the metabolism of intestinal microbiota, namely, amino acids metabolism, lipid metabolism, and defense mechanisms.

Conclusion: RSV can improve growth performance and intestinal morphology in weaning mice, possibly through improving gut immune response and microbiota function.

Keywords: growth performance; intestinal morphology; microbiota community; resveratrol; weaning stress.

Background: Previous studies have identified many immune pathways which are consistently altered in humans and model organisms as they age. Dairy cows are often culled at quite young ages due to an inability to cope adequately with metabolic and infectious diseases, resulting in reduced milk production and infertility. Improved longevity is therefore a desirable trait which would benefit both farmers and their cows. This study analysed the transcriptome derived from RNA-seq data of leukocytes obtained from Holstein cows in early lactation with respect to lactation number.

Results: Samples were divided into three lactation groups for analysis: i) primiparous (PP, n = 53), ii) multiparous in lactations 2-3 (MP 2-3, n = 121), and iii) MP in lactations 4-7 (MP > 3, n = 55). Leukocyte expression was compared between PP vs MP > 3 cows with MP 2-3 as background using DESeq2 followed by weighted gene co-expression network analysis (WGCNA). Seven modules were significantly correlated (r ≥ 0.25) to the trait lactation number. Genes from the modules which were more highly expressed in either the PP or MP > 3 cows were pooled, and the gene lists subjected to David functional annotation cluster analysis. The top three clusters from modules more highly expressed in the PP cows all involved regulation of gene transcription, particularly zinc fingers. Another cluster included genes encoding enzymes in the mitochondrial beta-oxidation pathway. Top clusters up-regulated in MP > 3 cows included the terms Glycolysis/Gluconeogenesis, C-type lectin, and Immunity. Differentially expressed candidate genes for ageing previously identified in the human blood transcriptome up-regulated in PP cows were mainly associated with T-cell function (CCR7, CD27, IL7R, CAMK4, CD28), mitochondrial ribosomal proteins (MRPS27, MRPS9, MRPS31), and DNA replication and repair (WRN). Those up-regulated in MP > 3 cows encoded immune defence proteins (LYZ, CTSZ, SREBF1, GRN, ANXA5, ADARB1).

Conclusions: Genes and pathways associated with lactation number in cows were identified for the first time to date, and we found that many were comparable to those known to be associated with ageing in humans and model organisms. We also detected changes in energy utilization and immune responses in leukocytes from older cows.

Keywords: Ageing; Cow; Leukocytes; Longevity; Multiparous; Primiparous.

Deoxynivalenol (DON) is a threatening mycotoxin primarily present in the agricultural environment, especially in food commodities and animal forages, and exerts significant global health hazards. Lycopene (LYC) is a potent antioxidant carotenoid mainly present in tomatoes and other fruits with enormous health benefits. The present study was designed to ascertain whether LYC could protect DON-induced intestinal epithelium oxidative injury by regulating Keap1/Nrf2 signaling in the intestine of mice. A total of forty-eight mice were randomly distributed into four groups (n = 12), Control (CON), 10 mg/kg BW LYC, 3 mg/kg BW DON, and 3 mg/kg DON + 10 mg/kg LYC BW (DON + LYC). The experimental groups were treated by intragastric administration for 11 days. Our results showed that LYC significantly increased average daily feed intake (ADFI), average daily gain (ADG), and repaired intestinal injury and barrier dysfunction, as evident by increased trans-epithelial electrical resistance (TEER) and decreased diamine oxidase (DAO) activity, as well as up-regulated tight junction proteins (occludin, claudin-1) under DON exposure. Furthermore, LYC treatment stabilized the functions of intestinal epithelial cells (Lgr5, PCNA, MUC2, LYZ, and Villin) under DON exposure. Additionally, LYC alleviated DON-induced oxidative stress by reducing ROS and MDA accumulation and enhancing the activity of antioxidant enzymes (CAT, T-SOD, T-AOC, and GSH-Px), which was linked with the activation of Nrf2 signaling and degradation of Keap1 expression. Conclusively, our findings demonstrated that LYC protects intestinal epithelium from oxidative injury by modulating the Keap1/Nrf2 signaling pathway under DON exposure. These novel findings could lead to future research into the therapeutic use of LYC to protect the DON-induced harmful effects in humans and/or animals.

Keywords: Keap1/Nrf2 signaling; bioactive compound; deoxynivalenol; intestinal injury; lycopene; oxidative stress.

Sodium alginate-bacterial cellulose (SA-BC) is a nanocomposite hydrogel with multi-layered porous surfaces fabricated using an in-situ biosynthesis modification method. The enzymatic hydrolysate (EH) of glycerol-pretreated Moso bamboo (MBEH) was the carbon source for glucose substitution to generate SA-bamboo-BC. SA, a natural biological polysaccharide, was combined with BC at dosages of 0.25%, 0.5%, 0.75% and 1% through hydrogen bonding. Compared to the native BC, the addition of 0.75% SA, termed as SA-bamboo-BC-0.75, enhanced the thermal properties. The dynamic swelling/de-swelling were pH-dependent, with an increased swelling ratio (SR) of 613% observed at pH 7.4 but a lower SR of 366% observed at pH 1.2. These differences were attributable to the electrostatic repulsion of -COO^-. Two protein-based model drugs were compared to estimate their drug-release properties. Bovine serum albumin (BSA) was adsorbed on lignin from MBEH through hydrophobic interactions, resulting in poor drug release. Lysozyme (LYZ) exhibited a higher drug release rate (92.79%) over 60 h at pH 7.4 due to the static attraction between LYZ and -COO^- of SA-bamboo-BC-0.75. As such, SA-bamboo-BC nanocomposite hydrogel was shown to possess sufficient swelling, drug-release and biocompatibility for substrate use.

Keywords: Bacterial cellulose; Bamboo enzymatic hydrolysate; Drug delivery; In-situ biosynthesis; Nanocomposite hydrogel; Sodium alginate.

Lead (Pb) widely exists in the water environment and has severe toxic effects on aquatic organisms. The yellow catfish (Pelteobagrus fulvidraco) is one of the most important commercial species in China, and moreover, its natural populations are declining with the degradation of environmental water quality. However, little is known about the toxic effects of Pb on its immune organs. This study was performed to determine waterborne Pb exposure on bioaccumulation, histomorphology, antioxidant status, apoptotic and immune response in the head kidney and spleen of yellow catfish. Experimental fish were randomly allocated into twelve tanks (3 tanks per group), and the Pb concentrations of the four groups were 0, 5, 50, and 500 μg/L, respectively. The results reflected that the Pb bioaccumulation of the head kidney and spleen increased with increasing Pb exposure dose and time. Severe histological alterations in the head kidney and spleen were observed at concentration 500 ug/L. With increasing Pb exposure concentrations, the plasma activity of superoxide dismutase (SOD) and catalase (CAT) significantly increased after exposure 7 days and 14 days, and the levels significantly decreased after exposure 28 days. The change trend of glutathione (GSH) levels was opposite to that of SOD and CAT at corresponding exposure time. The plasma malondialdehyde (MDA) levels together with the activities of plasma alkaline phosphatase (AKP) and acid phosphatase (ACP) increased significantly with the increasing Pb concentrations. In contrast, the levels of lysozyme (LYZ), complement 3 (C3) and immunoglobulin M (IgM) decreased significantly with increasing Pb concentrations. Moreover, Pb exposure induced transcriptional upregulation of heat shock protein 70 (hsp70), metallothionein (mt), sod, cat, interleukin-10 (il-10), transforming growth factor-β (tgf-β), and tumor necrosis factor-α (tnf-α), bcl-2-associated X protein (bax), and cysteinyl aspartate specific proteinase -9 (caspase-9), genes in the head kidney and spleen tissues, while downregulating the levels of the lyz, c3, igm and B-cell lymphoma-2 (bcl-2) genes. Our data provide evidence that Pb impaired immune function and tissue integrity in yellow catfish through oxidative stress, inflammatory and apoptosis, and the results can serve as reference data to better protect water environments from Pb eco-toxicants.

Keywords: Apoptosis; Immunosuppression; Inflammatory; Oxidative stress; Pb; Pelteobagrus fulvidraco.

Effect of dietary with 100, 200, and 300 mg kg^-1 glycyrrhizic acid (GA) on growth enhancer, blood physiology, digestive-antioxidant enzyme ability, innate-adaptive defense, and inflammatory cytokines induction was studied in silver carp, Hypophthalmichthys molitrix against vibriosis caused by Vibrio alginolyticus. Significant weight gain (WG), specific growth rate (SGR), and 100% survival rate (SR) was attained non-infected health (NiH) fish fed in control or all GA diets on 30, 45, and 60 days. Both NiH and V. alginolyticus challenged (VaC) fish treated with 200 mg GA diet significantly (P < 0.05) exhibited an enhancement in leucocytes value on 30, 45, and 60 days. Albumin (AB) or total proteins (TP) levels were significantly (P < 0.05) better in both groups fed 200 GA on 45 and 60 days. Malondialdehyde (MDA) and superoxide dismutase (SOD) activities were also substantial (P < 0.05) in both groups fed 200 mg GA on days 30, 45, and 60; whereas glutathione peroxidase (GPx) and catalase (CAT) activities were significantly (P < 0.05) better in both groups received 200 mg GA on days 45 and 60. Phagocytic (PC) and lysozyme (Lyz) activities significantly enhanced in both groups fed 200 or 300 mg GA on 45 and 60 days. Respiratory burst (RB), reactive oxygen species (ROS) and immunoglobulin (Ig) production significantly (P < 0.05) increased in both groups administered 200 or 300 mg GA. Growth hormone (GH) mRNA was up regulated in 200 mg GA trial on 45 days and in 200 or 300 mg GA treatments on 60 days. The IL-8 cytokine mRNA expression was up-regulated in both groups 200 and 300 mg GA on days 45 and 60, whereas TNF-α mRNA expression was increased in 200 mg GA. In addition, IL-10 cytokine mRNA expression was up regulated in 200 mg GA on 45 days whereas it was increased in both 200 mg and 300 mg GA trial on 60 days. The present study revealed that feeding fish 200 mg GA per kg diet demonstrated a better growth, digestive-antioxidant activity, innate-adaptive defense, and inflammatory cytokines induction than lower or higher dosage of GA in H. molitrix against V. alginolyticus.

Keywords: Antioxidant activity; Glycyrrhizic acid (GA); Hypophthalmichthys molitrix; Innune response; Vibrio alginolyticus.

Daphnia similis chitin and its derivative chitosan were prepared as immunostimulants to boost the immune response and determine the ability to control infectious disease caused by Vibrio alginolyticus in white shrimp, Litopenaeus vannamei. Three experimental diets supplemented with 0% chitin or chitosan (control) and 0.4% chitin or 0.4% chitosan were fed to shrimp for 56 days. Dietary inclusion of 0.4% chitosan accelerated shrimp growth compared to chitin and control. The survival and disease resistance of shrimp increased significantly when fed chitin and chitosan diets, after pathogenic injection, as indicated by the up-regulated immune responses in respiratory burst (RB), superoxide dismutase (SOD), and phagocytic activity (PA). There were no significant differences in the total haemocyte count (THC), phenoloxidase (PO)activity, and lysozyme (LYZ) activity among the groups. No significant differences were observed for prophenoloxidase system-related gene expressions among groups. However, shrimp fed chitin, and chitosan expressed significantly higher levels of antimicrobial proteins (penaeidin 3a, crustin, and anti-lipopolysaccharide factor 2) in the haemocytes than in control. The gene expressions of catalase and heat shock protein 70 increased in the hepatopancreas of shrimp fed chitosan diet compared to the chitin and control diet. The O-linked N-acetylglucosamine transferase (ogt) was significantly higher in the haemocytes of shrimp fed chitosan and chitin than the control, but ogt was only significantly higher in the hepatopancreas of shrimp fed chitosan. Dietary chitin and chitosan also showed positive effects on the transcription of peritrophin-like protein. These findings suggest that both chitin and chitosan from D. similis are efficacious at boosting the immunity of shrimp by preventing and controlling infectious diseases caused by Vibrio and have great potential to be used as a feasible immunostimulant that significantly contributes to the circular economy.

Keywords: Chitin; Chitosan; Circular economy; Daphnia similis; Disease prevention; White shrimp.

A quantitative and qualitative comparison of the antimicrobial and hemolytic activities of silver in various states, in the form of ions, nanoparticles, and bioconjugates with the antimicrobial protein lysozyme stabilized in an inert zeolite matrix, has been carried out. A synthetic zeolite with a β structure was chosen as a zeolite matrix. Using the ion-exchange method, the method of chemical reduction, and treating the matrix with a silver hydrosol with specified characteristics, samples of zeolites with the same silver content in various forms (Ag⁺, Ag° - Ag°/<em>Lyz</em>) in the amounts of 0.8 and 5 wt % have been synthesized. The samples obtained were studied by a complex of physicochemical research methods: X-ray diffraction, UV absorption spectroscopy, low-temperature nitrogen adsorption, electron microscopy, and atomic absorption. Antimicrobial activity was assessed against antibiotic-resistant Gram-negative microbe (e.g., <i>Escherichia coli</i> <i>ML-35</i>, <i>Pseudomonas aeruginosa</i> <i>522/17</i> <i>MDR</i>, <i>Klebsiella pneumoniae</i> <i>ESBL</i> <i>344</i>) and Gram-positive microbe (e.g., <i>Staphylococcus aureus</i> <i>1399/17</i>). The hemolytic activity in relation to human erythrocytes was estimated. The results obtained showed significant antimicrobial activity with a simultaneously high hemolytic activity of ionic silver. Silver nanoparticles have a lower level of antimicrobial activity and toxicity. Bioconjugates of silver nanoparticles and lysozyme showed an optimal combination of antimicrobial properties and lack of hemolytic activity.

Background: Green nanoparticles are subjected as an immunostimulant against bacterial pathogens.

Methods: Murraya koenigii berry extract-based synthesized zinc oxide nanoparticles (Mb-ZnO NPs) and selenium nanoparticles (Mb-Se NPs) were relatively analyzed for immunostimulation in serum and mucus fish Oreochromis mossambicus against Aeromonas hydrophila infections. Initial minimum inhibitory concentration (MIC) was determined for both Mb-ZnO NPs and Mb-Se NPs followed by specific growth rate (SGR), antioxidant level (Superoxide dismutase activity (SOD), Catalase activity (CA), and Glutathione peroxidase activity (GPx)), and immune parameters Myeloperoxidase activity (MPO), Respiratory burst activity (RBA), Lysozyme activity (LYZ), Alkaline phosphatase activity (ALP), Serum antiprotease activity and Natural complement activity (NAC).

Results: The potential bacterial inhibition property of Mb-ZnO NPs and Mb-Se NPs exhibited the most negligible concentration of 25 and 15 μg mL^-1, respectively, against A. hydrophila. In addition, Mb-ZnO NPs and Mb-Se NPs exhibited 70-80 % and 90-95 % diminished biofilm activity at 50 μg mL^-1 that was viewed under an inverted research microscope and confocal laser scanning microscopy (CLSM). Protein leakage and nucleic acid leakage assay quantified oozed out protein and nucleic acid from A. hydrophila that confirms Mb-Se NPs exhibited vigorous antibacterial activity than Mb-ZnO NPs at tested concentrations. Oreochromis mossambicus fed with Mb-ZnO NPs and Mb-Se NPs supplemented diet at different concentrations (0.5 mg/kg, 1 mg/kg and 2 mg/kg) improved SGR along with a rise in the immune response of those fishes against A. hydrophila infection. Serum and mucus of fish fed with Mb-Se NPs supplemented diet exhibited a significant rise in antioxidant level SOD, CA and GPx at a dosage of 2 mg/kg. Likewise, lipid peroxidation assay detected significantly diminished oxidative stress in the serum and mucus of fish fed with Mb-Se NPs supplemented diet (2 mg/kg). Enhanced immune parameters in serum and mucus of fish fed with Mb-Se NPs supplemented diet determined by MPO, RBA, LYZ, ALP, Serum antiprotease activity and NAC.

Conclusion: Thus O. mossambicus fed with Mb-Se NPs supplemented diet was less prone to become infected by aquatic pathogen A. hydrophila established by challenge study. On the whole, Mb-Se NPs supplemented diet ensured the rise in antioxidant response that boosts the immune responses and reduces the chance of getting infected against A. hydrophila infections.

Keywords: A. hydrophila; Immunostimulant; M. koenigii berry; Nanoparticles; O. mossambicus; Supplemented diet.

Biofilms represent a common and increasingly challenging problem in healthcare practices worldwide, producing persistent and difficult to manage infections. Researchers have started developing antibiotic-free treatment alternatives in order to decrease the risk of resistant microbial strain selection and for the efficient management of antibiotic tolerant biofilm infections. The present study reports the fabrication and characterization of magnetite-based nanostructured coatings for producing biofilm-resistant surfaces. Specifically, magnetite nanoparticles (Fe₃O₄) were functionalized with chitosan (CS) and were blended with lysozyme (LyZ) and were deposited using the matrix-assisted pulsed laser evaporation (MAPLE) technique. A variety of characterization techniques were employed to investigate the physicochemical properties of both nanoparticles and nanocoatings. The biological characterization of the coatings assessed through cell viability and antimicrobial tests showed biocompatibility on osteoblasts as well as antiadhesive and antibiofilm activity against both Gram-negative and Gram-positive bacterial strains and no cytotoxic effect against human-cultured diploid cells.

Keywords: antibiofilm activity; antimicrobial properties; laser processing; lysozyme; magnetite-based coatings; nanostructured bioactive coatings.

Tartrazine (TZ) is an azo dye widely used in foods, cosmetics, beverages, textile, and leather. In recent years, there are reports on detecting azo dyes in the aquatic environment, so the impact of these compounds on aquatic organisms could not be ignored. In this study, we aimed to evaluate the adverse effects of TZ exposure on teleosts' embryo development and juvenile's health by using crucian carp (Carassius carassius) as the experimental fish. The results showed that embryos exposed to TZ (0.19, 0.76 and 1.5 mM) exhibited a deformity, delayed egg resorption and decreased fertilization and hatching rate. When the juvenile fish were exposed to TZ at a level higher than those present in water for 30 days caused severe histopathological damages of the gill, intestine, kidney and liver. Antioxidant enzymes (CAT, SOD and GSH-Px) activities in the gill, intestine and liver, exhibited a decreasing trend after TZ exposure, while MDA contents elevated. TZ exposure also resulted in the upregulation of pro-inflammatory cytokines (il1 and il6), lysozymes (lyz), complement component 3 (c3), and β-defensin 3 (defb3). In addition, TZ exposure also affected the intestinal microbiota structure. In summary, the data in the present study indicated that TZ exposure reduce the embryo fertilization and hatching rate; cause histopathological damage of tissues, trigger oxidative stress, innate immune disorders and dysbiosis of gut microbiota in juvenile crucian carp. Therefore, it is necessary to be informed about the hazards of TZ exposure and the discharge of the dye into waters should be strictly administrated to prevent environmental pollution.

Keywords: Inflammatory response; Intestinal microbiota dysbiosis; Oxidative stress; Tartrazine exposure; juvenile crucian carp (Carassius carassius).

Background: Coronary heart disease (CHD) is the most prevalent disease with an unelucidated pathogenetic mechanism and is mediated by complex molecular interactions of exosomes. Here, we aimed to identify differentially expressed exosome genes for the disease development and prognosis of CHD.

Method: Six CHD samples and 32 normal samples were downloaded from the exoRbase database to identify the candidate genes in the CHD. The differentially expressed genes (DEGs) were identified. And then, weighted gene correlation network analysis (WGCNA) was used to investigate the modules in coexpressed genes between CHD samples and normal samples. DEGs and the module of the WGCNA were intersected to obtain the most relevant exosome genes. After that, the function enrichment analyses and protein-protein interaction network (PPI) were performed for the particular module using STRING and Cytoscape software. Finally, the CIBERSORT algorithm was used to analyze the immune infiltration of exosome genes between CHD samples and normal samples.

Result: We obtain a total of 715 overlapping exosome genes located at the intersection of the DEGs and key modules. The Gene Ontology enrichment of DEGs in the blue module included inflammatory response, neutrophil degranulation, and activation of CHD. In addition, protein-protein networks were constructed, and hub genes were identified, such as LYZ, CAMP, HP, ORM1, and LTF. The immune infiltration profiles varied significantly between normal controls and CHD. Finally, we found that mast cells activated and eosinophils had a positive correlation. B cell memory had a significant negative correlation with B cell naive. Besides, neutrophils and mast cells were significantly increased in CHD patients.

Conclusion: The underlying mechanism may be related to neutrophil degranulation and the immune response. The hub genes and the difference in immune infiltration identified in the present study may provide new insights into the diagnostic and provide candidate targets for CHD.