In this study we compared the proteomes of macrophages and heterophils isolated from the spleen 4 days after intravenous infection of chickens with Salmonella Enteritidis. Heterophils were characterized by expression of MMP9, MRP126, LECT2, CATHL1, CATHL2, CATHL3, LYG2, LYZ and RSFR. Macrophages specifically expressed receptor proteins, e.g. MRC1L, LRP1, LGALS1, LRPAP1 and a DMBT1L. Following infection, heterophils decreased ALB and FN1, and released MMP9 to enable their translocation to the site of infection. In addition, the endoplasmic reticulum proteins increased in heterophils which resulted in the release of granular proteins. Since transcription of genes encoding granular proteins did not decrease, these genes remained continuously transcribed and translated even after initial degranulation. Macrophages increased amounts of fatty acid elongation pathway proteins, lysosomal and phagosomal proteins. Macrophages were less responsive to acute infection than heterophils and an increase in proteins like CATHL1, CATHL2, RSFR, LECT2 and GAL1 in the absence of any change in their expression at RNA level could even be explained by capturing these proteins from the external environment into which these could have been released by heterophils.

Temperature influences fish's susceptibility to infectious disease through an immune response. However, the mechanism underlying this regulation is yet to be elucidated. In this study, we compared the susceptibility of crucian carp that were grown at 18°C and 33°C, respectively, to Aeromonas sobrial infection and found that crucian carp was more susceptible when grown at 33°C. These distinct susceptibilities of fish at different temperatures to infection may partially be explained by their differences in the metabolism as revealed by comparative metabolomics profiling: crucian carp demonstrated enhanced TCA cycle but reduced fatty acid biosynthesis; Our study also found that maltose was the most suppressed metabolite in fish grown at 33°C. Importantly, exogenous injection of maltose enhances crucian carp survival grown at 33°C by 30%. Further study showed that exogenous maltose downregulated the production of several cytokines but enhanced the lysozyme (lyz) and complement component c3, which involves the humoral innate immunity. Our results suggest that maltose promotes the survival of crucian carp likely through fine tuning the immune gene expression, and this finding provides a novel approach to manage bacterial infection.

Keywords: Aeromonas sobrial; Metabolomics; crucian carp; immune response; maltose.

Mercury (Hg) is a widespread environmental pollutant that can produce severe negative effects on fish even at very low concentrations. However, the mechanisms underlying inorganic Hg-induced oxidative stress and immunotoxicity in the early development stage of fish still need to be clarified. In the present study, zebrafish (Danio rerio) embryos were exposed to different concentrations of Hg2+ (0, 1, 4 and 16μg/L; added as mercuric chloride, HgCl2) from 2h post-fertilization (hpf) to 168hpf. Developmental parameters and total Hg accumulation were monitored during the exposure period, and antioxidant status and the mRNA expression of genes related to the innate immune system were examined at 168hpf. The results showed that increasing Hg2+ concentration and time significantly increased total Hg accumulation in zebrafish embryos-larvae. Exposure to 16μg/L Hg2+ caused developmental damage, including increased mortality and malformation, decreased body length, and delayed hatching period. Meanwhile, HgCl2 exposure (especially in the 16μg/L Hg2+ group) induced oxidative stress affecting antioxidant enzyme (CAT, GST and GPX) activities, endogenous GSH and MDA contents, as well as the mRNA levels of genes (cat1, sod1, gstr, gpx1a, nrf2, keap1, hsp70 and mt) encoding antioxidant proteins. Moreover, the transcription levels of several representative genes (il-1β, il-8, il-10, tnfα2, lyz and c3) involved in innate immunity were up-regulated by HgCl2 exposure, suggesting that inorganic Hg had the potential to induce immunotoxicity. Taken together, the present study provides evidence that waterborne HgCl2 exposure can induce developmental impairment, oxidative stress and immunotoxicity in the early development stage of fish, which brings insights into the toxicity mechanisms of inorganic Hg in fish.

The fabrication of montmorillonite (Mt) decorated with lysozyme-modified silver nanoparticles (Ag/lyz-Mt) was reported. The lysozyme (lyz) was served as both reducing and capping reagent. Coupling the bactericidal activity of the lyz with AgNPs, along with the high porous structure and large specific surface area of the Mt, prevented aggregation of AgNPs and promoted nanomaterial-bacteria interactions, resulting in a greatly enhanced bactericidal capability against both Gram positive and Gram negative bacteria. This paper systematically elucidated the bactericidal mechanisms of Ag/lyz-Mt. Direct contact between the Ag/lyz-Mt surface and the bacterial cell was essential to the disinfection. Physical disruption of bacterial membrane was considered to be one of the bactericidal mechanisms of Ag/lyz-Mt. Results revealed that Ag(+) was involved in the bactericidal activity of Ag/lyz-Mt via tests conducted using Ag(+) scavengers. A positive ROS (reactive oxygen species) scavenging test indirectly confirmed the involvement of ROS (O2(-), H2O2, and OH) in the bactericidal mechanism. Furthermore, the concentrations of individual ROS were quantified. Results showed that Ag/lyz-Mt nanomaterial could be a promising bactericide for water disinfection.

As typical perfluorinated compounds (PFCs), perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have been detected in various environmental media and their toxic effects have been extensively studied. Nevertheless, it remains unclear how PFCs cause cell apoptosis in healthy hepatocytes by inducing oxidative stress at the subcellular and molecular levels. In this study, the apoptotic pathways induced by PFOA and PFOS were explored. Besides, the effects of PFCs on the structure and function of lysozyme (LYZ) were investigated. After PFOA and PFOS exposure, the cell membrane and mitochondrial membrane potential were damaged. Further, PFOA and PFOS increased intracellular Ca²⁺ levels to 174.41 ± 1.70 and 158.91 ± 5.94%, respectively. Ultimately, caspase-3 was activated, causing cell apoptosis. As an indirect antioxidant enzyme, the molecular structure of LYZ was destroyed after interacting with PFOA and PFOS. Both PFOA and PFOS bound to the active center of LYZ, leading to the decrease of LYZ activity to 91.26 ± 0.78 and 76.01 ± 4.86%, respectively. This study demonstrates that PFOA and PFOS inhibit LYZ function, which can reduce the body's ability to resist oxidative stress, and then lead to mitochondria-mediated apoptosis.

Formalin-fixed paraffin-embedded (FFPE) tissues are invaluable sources of biological material for research and diagnostic purposes. In this study, we aimed to identify biological and technical variability in RT-qPCR TaqMan® assays performed with FFPE-RNA from lymph nodes of classical Hodgkin lymphoma samples. An ANOVA-nested 6-level design was employed to evaluate BCL2, CASP3, IRF4, LYZ and STAT1 gene expression. The most variable genes were CASP3 (low expression) and LYZ (high expression). Total variability decreased after normalization for all genes, except by LYZ. Genes with moderate and low expression were identified and suffered more the effects of the technical manipulation than high-expression genes. Pre-amplification was shown to introduce significant technical variability, which was partially alleviated by lowering to a half the amount of input RNA. Ct and Cy0 quantification methods, based on cycle-threshold and the kinetic of amplification curves, respectively, were compared. Cy0 method resulted in higher quantification values, leading to the decrease of total variability in CASP3 and LYZ genes. The mean individual noise was 0.45 (0.31 to 0.61 SD), indicating a variation of gene expression over ~1.5 folds from one case to another. We showed that total variability in RT-qPCR from FFPE-RNA is not higher than that reported for fresh complex tissues, and identified gene-, and expression level-sources of biological and technical variability, which can allow better strategies for designing RT-qPCR assays from highly degraded and inhibited samples.

Molecular imprinting is a method for making selective binding sites in synthetic polymers using a molecular template. The aim of this study is to prepare lysozyme-imprinted supermacroporous cryogels which can be used for the purification of lysozyme (Lyz) from egg white. N-Methacryloyl-(L)-histidinemethylester (MAH) was chosen as the metal-coordinating monomer. In the first step, Cu2+ was complexed with MAH and the lysozyme-imprinted poly(HEMA-MAH) [Lyz-MIP] cryogel were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) in an ice bath. After that, the template (i.e., lysozyme) was removed using 0.05 M phosphate buffer containing 1M NaCl (pH 8.0). The maximum lysozyme adsorption capacity was 22.9 mg/g polymer. The relative selectivity coefficients of Lyz-MIP cryogel for lysozyme/bovine serum albumin and lysozyme/cytochrome c were 4.6 and 3.2 times greater than non-imprinted poly(HEMA-MAH) (NIP) cryogel, respectively. Purification of lysozyme from egg white was also monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 94% with recovery about 86%. The Lyz-MIP cryogel could be used many times without decreasing the adsorption capacity significantly.

Di(2-ethylhexyl) phthalate (DEHP) mediates the immune system mainly by triggering the production of reactive oxygen species (ROS) and nitric oxide (NO) in higher animals. In the present study, spatial variation in the expression of immune-related genes in clam (Venerupis philippinarum) under acute short-term DEHP treatment was assessed by qPCR. The expression of six genes including glutamine synthetase (GS), IkB (IK), transcription factor activator protein-1 (AP-1), cyclophilin A-1 (CypA-1), heat shock protein 90 (HSP90) and superoxide dismutase (SOD) was dose-dependent. A negative correlation between expression and DEHP treatment was observed for big defensin (BD), glutathione S-transferase (GST), and thioredoxin peroxidase (TP). Surprisingly, lysozyme (LYZ) exhibited two distinct expression patterns at two DEHP doses. Significant differences between the experimental and control groups were observed for all tested genes at the various time points. Overall, our results revealed that DEHP mediates immune responses in clams by various means, and certain genes are promising candidate for biomarkers in DEHP monitoring.

The present study was conducted to evaluate the effects of marine polysaccharides from seaweed Enteromorpha on growth performance, immune responses, intestinal morphology and microbial community in the banana shrimp Fenneropenaeus merguiensis. Two thousand and four hundred juvenile shrimps with an average body weight of 2.18 ± 0.06 g were fed for 42 d with diets containing different levels of Enteromorpha polysaccharides (EPS): 0 (control), 1, 2 and 3 g/kg as treatment groups, each of group was replicated three times with two hundred shrimps per replicate. Dietary supplementation of 1 g/kg EPS showed a consistent improvement in the final weight, weight gain, average daily gain rate (ADGR) and specific growth rate (SGR) (P < 0.05), while showed a decrease in the feed conversion ratio (FCR) of shrimp (P < 0.05). Besides, the total anti-oxidative capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), lysozyme (Lyz), alkaline phosphatase (ALP), and phenoloxidase (PO) activities in hemolymph were enhanced by dietary supplementation of 1 g/kg EPS (P < 0.05), while it reduced the hemolymph MDA content (P < 0.05). Shrimp fed 1 g/kg EPS supplemented diets up-regulated FmLyz, FmSOD5 and FmCLAP gene expression level of hepatopancreas and gill (P < 0.05), and also improved the intestinal FmLC2, FmLyz, FmSOD5 and FmCLAP gene expression levels (P < 0.05). In addition, shrimp fed diets containing 1 g/kg EPS increased the villus width (P < 0.05) and resulted in a higher villus surface area (P < 0.05). According to 16S rRNA sequencing results, dietary supplementation of 1 g/kg EPS tended to increase the relative abundance of Firmicutes at phylum level (P = 0.07) and decrease the relative abundance of Vibrio at genus level (P = 0.08). There was a significant positive correlation between the relative abundance of Firmicutes and mRNA expression of intestinal immune-related genes (P < 0.05). These findings revealed that dietary 1 g/kg EPS could improve growth performance, enhance nonspecific immunity and modulate intestinal function of banana shrimp F. merguiensis.

Keywords: Fenneropenaeus merguiensis; Growth; Immune responses; Intestinal health; Marine polysaccharides.

BACKGROUND

The aim of this study was to examine the cardiac function and transcriptional response of the heart to propofol after ischemia-reperfusion.

METHODS

Rat hearts were Langendorff-perfused using the modified Krebs-Henseleit buffer, and took 20 min stabilizing periods, 40 min ischemia periods, and then 120 min reperfusion period. The hearts were divided into 5 groups;

METHODS

180 min perfusion after stabilization, Ischemic: 40 min global ischemia after stabilization, followed by 120 min reperfusion, Pre: 2 microM propofol treatment was preformed only before ischemia, Post: 2 microM propofol treatment was performed only during reperfusion after ischemia, Pre/Post: 2 microM propofol treatment was performed both before and after ischemia. The measurement for cardiac performances, such as left ventricular developed pressure (LVDP), rate of left ventricular pressure generation (dP/dt), heart rate, and coronary flow were obtained. The expression profiles of isolated mRNA were determined by using Agilent microarray and real time-polymerase chain reaction (RT-PCR) was used to confirm the microarray results for a subset of genes.

RESULTS

The Post group showed better LVDP and dP/dt than the Ischemic group. But there were no significant differences in heart rate and coronary flow among the groups. On the results of RT-PCR, the expressions of Abcc9, Bard1, and Casp4 were increased, but the expressions of Lyz, Casp8, and Timp1 were decreased in the Post group compared with the Ischemic group.

CONCLUSIONS

This study suggests that 2 microM propofol may provide cardioprotective effect, and modulate gene expression such as apoptosis, and K(ATP) ion channel related-genes during reperfusion in the isolated rat hearts.

We have found diffuse alveolar damage (DAD) has taken place in some patients under mechanical ventilation with high-inspired oxygen concentrations. To clarify the molecular pathophysiology of this, the time course of gene expression changes induced by hyperoxia exposure in mouse lungs was examined using real-time quantitative polymerase chain reaction (real-time qPCR). Our raw data and those normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) showed that: (1) there is a decrease in levels of mRNAs for surfactant-associated protein C (SFTPC), cytochrome P450, 2F2 (CYP2F2), Claudin 1 (CLDN1), membrane-associated zonula occludens protein-1 (ZO-1), lysozyme (LYZS), and this suggests alveolar dysfunction and a disruption of the immune system, (2) we confirmed apoptotic conditions, such as significant up-regulations of mRNA levels in Myc and Galectin-3, and (3) hyperoxic conditions probably yielded reactive oxygen species (ROS), which resulted in a malignant cycle of ROS production by Myc overexpression [Shimada I, Matsui K, Brinkmann B, Hohoff C, Hiraga K, Tabuchi Y, et al. Novel transcript profiling of diffuse alveolar damage induced by hyperoxia exposure in mice: normalization by glyceraldehyde 3-phosphate dehydrogenase. Int J Legal Med 2008;122:373-83]. In this experiment, GAPDH was up-regulated when hyperoxia exposure was continued. Therefore, we reexamined our data and found that: (1) mRNA levels of other housekeeping genes, including beta(2)-microglobulin (beta2M), ribosomal protein: large P2 (RPLP2), and importin 8 (IPO8) altered to a lesser extent, (2) mRNA levels of beta2M and IPO8 were down-regulated when hyperoxia exposure was continued, and (3) our previous work was validated by normalization with these three housekeeping genes.

Epithelial-specific Ets (ESE) transcription factors, consisting of ESE-1, ESE-2, and ESE-3, are constitutively expressed in distinct epithelia of mucosal tissues, including the lung. Each ESE member exhibits alternative splicing and yields at least two isoforms (a and b) with transcriptional targets largely unidentified. The studies described herein define a novel role for ESE transcription factors in transactivation of the human lysozyme gene (LYZ), an essential component of innate defense in lung epithelia. Of the six ESE isoforms, ESE-1a and ESE-1b transactivated LYZ promoter in reporter gene assays, whereas only ESE-1b dramatically upregulated transcription of endogenous LYZ in both nonpulmonary and pulmonary epithelial cells. Importantly, ESE-1a and ESE-1b could transactivate the LYZ promoter in cultured primary airway epithelial cells. ESE-2 and ESE-3 isoforms were unable to substantially transactivate the lysozyme promoter or upregulate transcription of endogenous LYZ. Two functional consensus Ets sites located in the proximal 130-bp LYZ promoter were responsive to ESE-1b as identified by site-directed mutagenesis and DNA binding assays. Short hairpin RNA attenuation of endogenous ESE-1b mRNA levels in lung epithelia resulted in decreased LYZ transcription. Furthermore, ESE-1 antibody specifically enriched the 130-bp proximal LYZ promoter in chromatin immunoprecipitation analyses. These findings define a novel role for ESE transcription factors in regulating lung innate defense and suggest distinct regulatory functions for ESE family members.

The adsorption and covalent immobilization of human immunoglobulin (HIgG) and lysozyme (LYZ) on surface-modified poly(tert-butyl methacrylate) PtBMA films have been evaluated using x-ray photoelectron spectroscopy (XPS), ellipsometry and atomic force microscopy (AFM). Surface modification of PtBMA (UV irradiation) afforded surfaces suitable for both the physical and covalent attachment of proteins. The XPS and ellipsometry results showed good correlation in terms of variable-dense/thickness protein layer formation between physisorbed and covalently bound proteins. The amount of physisorbed HIgG ranged from 23.0 +/- 1.6 ng mm(2) on PtBMA, with corresponding film thicknesses 17.0 +/- 1.2 nm. Covalent immobilization mediated through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysulfosuccinimide (sulfo-NHS) coupling chemistry, afforded 5.6-8 ng mm(2) of HIgG with a corresponding thickness of 5.9 +/- 0.6 nm on PtBMA. The attachment of LYZ to modified PtBMA surface was similarly translated, where adsorption yielded up to 15 ng mm(2), while covalent immobilization afforded typically 7-8 ng mm(2). The thickness of the adsorbed LYZ protein layer was 11.0 +/- 3.2 nm (PtBMA), suggesting the greater portion of protein adsorbs on surface-modified PtBMA.

The presence of micron aggregates in protein formulations has recently attracted increased interest from regulatory authorities, industry, and academia because of the potential undesired side effects of their presence. In this study, we characterized the micron aggregate formation of hen egg-white lysozyme (Lyz) and its diPEGylated (5 kDa) analog as a result of typical handling stress conditions. Both proteins were subjected to mechanical stress in the absence and presence of silicone oil (SO), elevated temperatures, and freeze-thaw cycles. Flow imaging microscopy showed that PEGylated Lyz formed approximately half as many particles as Lyz, despite its lower apparent thermodynamic stability and more loose protein fold. Further characterization showed that the PEGylation led to a change from attractive to repulsive protein-protein interactions, which may partly explain the reduced particle formation. Surprisingly, the PEGylated Lyz adsorbed an order of magnitude faster onto SO, despite being much larger in size, as determined by small-angle X-ray scattering and dynamic light scattering measurements. Thus, PEGylation may significantly reduce, but not prevent, micron aggregate formation of a protein during typical handling stresses.

The pentablock (PB) copolymers based composite nanosystems were designed to provide a long-term delivery of macromolecules to the back of the eye. A unique arrangement of each block (polyethylene glycol, polylactic acid, and polycaprolactone) with various molecular weights (PB-A and PB-B) was selected for the synthesis of nanoparticles (NPs) and thermosensitive gel (PB-C) by sequential ring-opening bulk copolymerization reaction. PB copolymers were characterized for their molecular weight and purity by 1H-NMR spectroscopy and crystallinity by PXRD. The macromolecule model drugs [lysozyme (Lyz ~ 14.5 kDa), IgG-Fab (~ 50 kDa), and IgG (~ 150 kDa)] were selected to delineate the effect of molecular weights on in vitro release profile of nanoformulations. Lyz-, Fab-, and IgG-encapsulated NPs were prepared by double emulsion solvent evaporation method. The entrapment efficiency (EE%) and drug loading (DL%) of macromolecules was higher for PB-B copolymers due to its higher molecular weight and hydrophobicity compare to PB-A. The particle size range of NPs was ~ 200-270 nm. In vitro release profiles of Lyz-, Fab-, and IgG-encapsulated in NPs alone and NPs suspended in gel (composite nanosystem) demonstrated a minimal burst release and drug release over a long period. The effect of hydrodynamic diameter of macromolecules and hydrophobicity of PB copolymers was investigated on the release profile of nanosystems. In vitro biocompatibility study showed negligible cytokine (IL-1, IL-6, and TNF-α) release, which confirmed the safety of the PB copolymers. Based on the results, it is anticipated that long-term ocular delivery of macromolecules can be achieved through composite nanosystems.

We demonstrate here a novel single-molecule, label-free bioanalytical system capable of sensing the presence of specific ssDNA oligomer sequences and proteins with high selectivity and sensitivity. An ssDNA concentration of 1 nM and a Lyz concentration of 0.65 nM could be detected.

A new type of molecularly imprinted polymer (MIP)-based fluorescent artificial receptor was developed by anchoring MIP on the surface of denatured bovine serum albumin (dBSA) modified CdTe quantum dots (QDs) using the surface molecular imprinting process. The approach combined the merits of molecular imprinting technology and the fluorescent property of the CdTe QDs. The dBSA was used not only to modify the surface defects of the CdTe QDs, but also as assistant monomer to create effective recognition sites. Three different proteins, namely lysozyme (Lyz), cytochrome c (Cyt) and methylated bovine serum albumin (mBSA), were tested as the template molecules and then the receptors were synthesized by sol-gel reaction (imprinting process). The results of fluorescence and binding experiments demonstrated the recognition performance of the receptors toward the corresponding template. Under optimum conditions, the linear range for Lyz was from 1.4×10(-8) to 8.5×10(-6) M, and the detection limit was 6.8 nM. Moreover, the new artificial receptors were applied to separate and detect Lyz in real samples. This fluorescent artificial receptor may serve as a starting point in the design of highly effective synthetic fluorescent receptor for recognition of target protein.

In this article the molecular conformation of xanthan chains in hydrogel films was investigated by means of circular dichroism, showing substantial differences between xanthan hydrogel prepared in the absence (XNT) and in the presence of citric acid (XCA). The xanthan chains in XNT hydrogels films presented ordered conformation (helixes), while in XCA they were in the disordered conformation (coils), exposing a larger number of carboxylate groups than XNT. The large charge density in XCA hydrogels was evidenced by their behavior under variable ionic strength. Studies about the application of XNT and XCA for loading and delivering of bovine serum albumin (BSA) and lysozyme (LYZ) showed that both events are controlled by hydrogels and proteins net charge, which can be triggered by pH. The preservation of LYZ native conformation after hydrogel loading explained the substantial bactericidal activity of LYZ loaded hydrogels and enables their use as active wound dressings.

Lysozyme hydrolyses bacterial cell walls and acts as a nonspecific innate immunity molecule against the invasion of bacterial pathogens. We cloned the cDNA of lysozyme from Fenneropenaeus chinensis and named Fc-lysozyme (FcLyz in short). The full length of the gene was of 709 bp, and the open reading frame (477 bp) encoded 158 amino acids. The predicted protein had a signal peptide (-1--18 residue) and molecular weight of the mature protein (residue 1-140) was of 16.2 kD. A Lyz 1 domain (residue 1-130) in the lysozyme was found by SMART analysis. The results of semiquantity RT-PCR showed that FcLyz was constitutively expressed in tested tissues in a low level in normal shrimp, and up-regulated in hemocytes, heart, hepatopancreas and gill of bacterial challenged shrimp. The DNA fragment of mature Fc-Lys was subcloned to pET-30a (+) expression vector, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and then induced by isopropylthio-beta-D-galactoside (IPTG). The antibacterial activity of the purified recombinant FcLys was analyzed and minimal inhibitory concentration (MIC) was assayed. The recombinant protein showed high antibacterial activity against some Gram-positive bacteria, and MIC reached 3.43 micromol/L, and relatively low activity against Gram-negative bacteria. All together, the Fc-Lys was regulated by pathogen infection and had antibacterial activity. This suggested that the FcLyz may be one of the important molecules against pathogens in innate immunity of the shrimp.

The present study investigates the effect of different levels of galactooligosaccharide (GOS) on innate immune parameters, immune related genes expression as well as growth performance in zebrafish (Danio rerio). Four hundred and twenty fish (mean weight 45 ± 0.1 mg) were supplied, randomly stocked in twelve aquaria assigned to four treatments. Zebrafish were fed with either control diet or control diet enriched with different levels (0.5, 1 and 2%) of GOS for eight weeks. At the end of feeding trial innate immune parameters (total immunoglobulin, total protein and alkaline phosphatase activity), immune related genes expression (interleukin 1 beta [il1b], Lysozyme [lyz], tumor necrosis factor alpha [tnf-alpha]) as well as growth performance were measured. Evaluation of immune parameters revealed significant (P < .05) increase of total protein and total Ig in zebrafish fed 1 or 2% GOS compared other treatments. However, in case of lysozyme activity no significant (P>> .05) differences were noticed between GOS fed fish and control group. Also, in case of ALP activity, significant increase (P < .05) was observed in 2% GOS treatment. Gene expression studies revealed significant upregulation (P < .05) of tnf-alpha and lyz genes in GOS fed fish. While no significant (P>> .05) difference was observed in case of il1b gene expression. Evaluation of growth performance at the end of feeding trial revealed no significant (P < .05) improvement in GOS fed groups. The present results revealed positive effects of GOS on innate immune parameters and related gene expression in zebrafish.

The present study investigated the effects of dietary Spirulina platensis supplementation on growth performance, hematological and serum biochemical parameters, hepatic antioxidant status, immune responses and resistance to the pathogen infection in Coral trout Plectropomus leopardus. The fish were fed for 8-week with diets containing different levels of S. platensis: 0% (C), 2% (SP2), 4% (SP4), 6% (SP6), 8% (SP8) and 10% (SP10) as treatment groups, followed by a Vibrio harveyi infection test for 14 d. The study indicated that dietary supplementation with Spirulina platensis could significantly improve growth performance, and the highest weight gain rate (WGR) and specific growth rate (SGR) were observed in group SP10 (P < .05). Red cell count (RBC), white cell count (WBC), hemoglobin (Hb) and total antioxidant capacity (T-AOC) in the S. platensis supplemented groups were significantly higher than those of group C (P < .05). However, the levels of cholesterol, triglyceride and malondialdehyde (MDA) contents, and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activities decreased with the increasing of dietary S. platensis levels. Compared with group C, the lysozyme (LYZ) and respiratory burst activities (RBA), and immunoglobulin (Ig) and complement contents in group SP4, SP6, SP8 and SP10 increased significantly than those of group C respectively (P < .05). After challenge with V. harveyi, the survival rate in group SP4, SP6, SP8 and SP10 was significantly higher than that of group C, and the highest survival rate was in group SP10 (P < .05). These results indicated that P. leopardus fed a diet supplemented with S. platensis (especially at 10%) could significantly promote its growth performance, improve its hepatic antioxidant status, and enhance its immune ability and resistance to V. harveyi infection.

The monogenean Dactylogyrus intermedius and the bacterium Flavobacterium columnare are 2 common pathogens in aquaculture. The objective of the present study was to examine the effect of prior parasitism by D. intermedius on the susceptibility of goldfish to F. columnare and to explore the potential immune mechanisms related to the parasite infection. A F. columnare challenge trial was conducted between D. intermedius-parasitized and non-parasitized goldfish. The F. columnare load in gill, kidney, spleen and liver were compared. The expression of immune-related genes (IL-1β2, TNF-α1, TGF-β, iNOS-a, C3 and Lyz) in gill and kidney of D. intermedius-only infected and uninfected control fish were evaluated. D. intermedius-parasitized goldfish exhibited higher mortality and significantly higher loads (3051 to 537,379 genome equivalents [GEs] mg(-1)) of F. columnare, which were 1.13 to 50.82-fold higher than non-parasitized fish (389 to 17,829 GEs mg(-1)). Furthermore, the immune genes IL-1β2, TNF-α1, iNOS-a and Lyz were up-regulated while the TGF-β and C3 were down-regulated in the gill and kidney of parasite-infected fish compared to the non-parasitized controls. The down-regulation TGF-β and C3 was especially noteworthy, as this might indicate the suppression of the host immune functions due to the parasitism by D. intermedius. Taken together, these data demonstrate that parasite infection can enhance bacterial invasion and presents a hypothesis, based on gene expression data, that modulation of host immune response could play a role.

BACKGROUND

The systematic mechanisms of acute intracerebral hemorrhage are still unknown and unverified, although many recent researches have indicated the secondary insults. This study was aimed to disclose the pathological mechanism and identify novel biomarker and therapeutic target candidates by plasma proteome.

METHODS

Patients with AICH (n = 8) who demographically matched healthy controls (n = 4) were prospectively enrolled, and their plasma samples were obtained. The TMT-LC-MS/MS-based proteomics approach was used to quantify the differential proteome across plasma samples, and the results were analyzed by Ingenuity Pathway Analysis to explore canonical pathways and the relationship involved in the uploaded data.

RESULTS

Compared with healthy controls, there were 31 differentially expressed proteins in the ICH group (P < 0.05), of which 21 proteins increased while 10 proteins decreased in abundance. These proteins are involved in 21 canonical pathways. One network with high confidence level was selected by the function network analysis, in which 23 proteins, P38MAPK and NFκB signaling pathways participated. Upstream regulator analysis found two regulators, IL6 and TNF, with an activation z-score. Seven biomarker candidates: APCS, FGB, LBP, MGMT, IGFBP2, LYZ, and APOA4 were found. Six candidate proteins were selected to assess the validity of the results by subsequent Western blotting analysis.

CONCLUSIONS

Our analysis provided several intriguing pathways involved in ICH, like LXR/RXR activation, acute phase response signaling, and production of NO and ROS in macrophages pathways. The three upstream regulators: IL-6, TNF, LPS, and seven biomarker candidates: APCS, APOA4, FGB, IGFBP2, LBP, LYZ, and MGMT were uncovered. LPS, APOA4, IGFBP2, LBP, LYZ, and MGMT are novel potential biomarkers in ICH development. The identified proteins and pathways provide new perspectives to the potential pathological mechanism and therapeutic targets underlying ICH.

The effect of acids on the structure of lysozyme (Lyz) during electrospray ionization (ESI) was studied by comparing the solution and gas-phase structures of Lyz. Investigation using circular dichroism spectroscopy and small-angle X-ray scattering demonstrated that the folded conformation of Lyz was maintained in pH 2.2 solutions containing different acids. On the other hand, analysis of the charge state distributions and ion mobility (IM) distributions, combined with molecular dynamics simulations, demonstrated that the gas phase structures of Lyz depend on the pKa of the acid used to acidify the protein solution. Formic acid and acetic acid, which are weak acids (pKa>> 3.5), induce unfolding of Lyz during ESI, presumably because the undissociated weak acids provide protons to maintain the acidic groups within Lyz protonated and prevent the formation of salt bridges. However, HCl suppressed the formation of the unfolded conformers because the acid is already dissociated in solution, and chloride anions within the ESI droplet can interact with Lyz to reduce the intramolecular electrostatic repulsion. These trends in the IM distributions are observed for all charge states, demonstrating the significance of the acid effect on the structure of Lyz during ESI.