Glyphosate is a broad-spectrum herbicide frequently used world widely in agricultural and non-agricultural areas to control unwanted plants. Health risk of chronic and subchronic exposure of glyphosate on animals and humans has received increasing attention in recent years. The aim of this study was to evaluate the effects of glyphosate on the immunoglobulin M (IgM), complement C3 (C3), and lysozyme (LYZ) in the kidney of common carp exposed to 52.08 or 104.15mgL(-1) of glyphosate for 168h. The results showed that the transcriptions of IgM, C3, or LYZ were altered due to glyphosate-exposure, for example, IgM and C3 initially increased at 24h later it decreased (except for a increase of C3 in higher dose group at 24h) while the expression of G-type LYZ were not affected at 24h, then increased at 72h, but decreased at the end of test, however C-type LYZ expression was initially up-regulated (24-72h) but down-regulated at the end of exposure (168h). However, glyphosate-exposure generally decreased the contents of IgM and C3 or inhibited LYZ activity in the kidney of common carp. In addition, glyphosate-exposure also caused remarkable histopathological damage, mainly including vacuolization of the renal parenchyma and intumescence of the renal tubule in fish kidney. The results of this study indicate that glyphosate causes immunotoxicity on common carp via suppressing the expressions of IgM, C3, and LYZ and also via damaging the fish kidney.

Under mechanical ventilation with high-inspired oxygen concentration, diffuse alveolar damage was found to take place in some patients. To clarify the molecular pathophysiology of this condition, we investigated the time course of gene expression changes induced by hyperoxia exposure in mouse lung using real-time quantitative polymerase chain reaction (qPCR). Our results normalized by glyceraldehyde 3-phosphate dehydrogenase showed that mRNA levels of cysteine rich protein 61 (CYR61) and connective tissue growth factor (CTGF) were significantly upregulated, while those of surfactant-associated protein C (SFTPC), cytochrome P450, 2F2 (CYP2F2), Claudin 1, (CLDN1), membrane-associated zonula occludens protein-1 (ZO-1), lysozyme (LYZS), and P lysozyme structural (LZP-S) were significantly downregulated. Increasing level of mRNAs, each encoding CYR61 and CTGF, suggests a serious risk of fibrosing alveolitis. Decrease in levels of mRNAs for SFTPC, CYP2F2, CLDN1, ZO-1, LYZS, and LZP-S suggests alveolar dysfunction and disruption of the immune system. Moreover, we confirmed apoptotic conditions, such as significant upregulations of mRNA levels in Myc and Galectin-3. Hyperoxic condition probably yielded reactive oxygen species (ROS), which resulted in a malignant cycle of ROS production by Myc overexpression.

Bare FePtCu nanoparticles (NPs) are first prepared for laser desorption/ionization mass spectroscopy (LDI-MS) analysis as affinity probes to selectively trap oppositely charged analytes from a sample solution. Our present results demonstrate bare FePtCu NPs to be a potentially useful matrix for surface-assisted laser desorption/ionization mass spectroscopy (SALDI-MS), for the analysis of small proteins and peptides. The upper detectable mass range of peptides was approximately 5 kDa, and the detection limit for peptides approximately 5 fmol. Sulfonate group-modified FePtCu nanoparticles (FePtCu-SO(3)(-) NPs), with ionization being independent of the solution pH, can interact with a positively charged analyte, and the analyte-bound NPs can be separated from the reaction supernatant by centrifugation or an external magnetic field. An oligopeptide, Gly-Gly-Tyr-Arg (GGYR) from an oligopeptide mixture containing Asp-Asp-Asp-Asp (DDDD), Gly-Gly-Gly-Gly (GGGG) and GGYR, was detected using SALDI-MS with FePtCu-SO(3)(-) NPs employing electrostatic interaction. Furthermore, FePtCu-SO(3)(-) NPs can detect lysozyme (Lyz) in human serum through the electrostatic attraction between positively charged Lyz and FePtCu-SO(3)(-) NPs at pH 8, while detection of negatively charged albumin in human serum is not possible.

The kinetics of O2*- reaction with semi-oxidized tryptophan radicals in lysozyme, Trp*(Lyz) have been investigated at various pHs and conformational states by pulse radiolysis. The Trp*(Lyz) radicals were formed by Br2*- oxidation of the 3-4 exposed Trp residues in the protein. At pH lower than 6.2, the apparent bimolecular rate is about 2 x 10(8) M(-1) s(-1); but drops to 8 x 10(7) M(-1) s(-1) or less above pH 6.3 and in CTAC micelles. Similarly, the apparent bimolecular rate constant for the intermolecular Trp*(Lyz) + Trp*(Lyz) recombination reaction is about (4-7 x 10(6) M(-1) s(-1)) at/or below pH 6.2 then drops to 1.3-1.6 x 10(6) M(-1) s(-1) at higher pH or in micelles. This behavior suggests important conformational and/or microenvironmental rearrangement with pH, leading to less accessible semi-oxidized Trp* residues upon Br2*- reaction. The kinetics of Trp*(Lyz) with ascorbate, a reducing species rather larger than O2*- have been measured for comparison. The well-established long range intramolecular electron transfer from Tyr residues to Trp radicals--leading to the repair of the semi-oxidized Trp*(Lyz) and formation of the tyrosyl phenoxyl radical is inhibited by the Trp*(Lyz) + O2*- reaction, as is most of the Trp*(Lyz) + Trp*(Lyz) reaction. However, the kinetic behavior of Trp*(Lyz) suggests that not all oxidized Trp residues are involved in the intermolecular recombination or reaction with O2*-. As the kinetics are found to be quite pH sensitive, this study demonstrates the effect of the protein conformation on O2*- reactivity. To our knowledge, this is the first report on the kinetics of a protein-O2*- reaction not involving the detection of change in the redox state of a prosthetic group to probe the reactivity of the superoxide anion.

Bacterial infections remain a significant challenge in biomedicine and environment safety. Increasing worldwide demand for point-of-care techniques and increasing concern on their safe development and use, require a simple and sensitive bioanalysis for pathogen detection. However, this goal is not yet achieved. A design for fluorescein isothiocyanate-labeled lysozyme (FITC-LYZ), which provides quantitative binding information for gram-positive bacteria, Micrococcus luteus, and detects pathogen concentration, is presented. The functional lysozyme is used not only as the pathogenic detection platform, but also as a tracking reagent for microbial population in antibacterial tests. A nonlinear relationship between the system response and the logarithm of the bacterial concentration was observed in the range of 1.2×10(2)-1.2×10(5) cfu mL(-1). The system has a potential for further applications and provides a facile and simple method for detection of pathogenic bacteria. Meanwhile, the fluorescein isothiocyanate -labeled lysozyme is also employed as the tracking agent for antibacterial dynamic assay, which show a similar dynamic curve compared with UV-vis test.

The bactericidal and bacteriolytic effects of lysolecithin (LL) and egg-white lysozyme (LYZ) on Staph. aureus and group A streptococci and the solubilization of phospholipids from the bacterial membranes by these agents was studied. Low concentrations of lysolecithin (1--10 microgrames/ml) are highly bactericidal for Steph. aureus and group A streptococci, but induce neither bacteriolysis nor solubilization of a substantial amount of membrane phospholipids. On the other hand, while LL at greater than 50 micrograms/ml causes substantial lipid release, a combination of LL and LYZ is absolutely needed to solubilize lipids from streptococci. This combination is, however, not bacteriolytic for this microrganism. The solubilization of lipids from staphylococci by LL is much faster than that induced in streptococci by LL + LYZ. The solubilization of the bulk of membrane lipids from staphylococci can also be achieved by Triton X-100 and by sodium lauryl sulfate and from group A streptococci by Triton X-100 plus LYZ. A variety of other detergents (e.g., Cetavlon, sodium taurocholate, cetyl pyrdinium chloride) have no lipid-releasing properties even in the presence of LYZ. The release of lipids by LYZ (in the presence of LL) from group A streptococci is related to its enzymatic activity, on a still unknown substrate, but not to its cationic nature as this muramidase cannot be replaced by a variety of cation substances (histone, polylysin, leukocyte cationic proteins, polymyxin B, and spermidine). The release of lipids from staphylococci by LL is not inhibited by a variety of anionic and cationic polyelectrocytes (heparin, liquoid, chondroitin sulfate, DNA histone, and polylysine) which markedly inhibit the release of lipids from group A streptococci by LL and LYZ. Streptococci that had been cultivated in the presence of subinhibitory concentrations of penicillin G lose their membrane phospholipids to a larger extent and by much smaller concentrations of LL and LYZ, as compared to controls, suggesting that the interference with the synthesis of the peptidoglycan increases the accessibility of the cell membrane to the lipid-releasing agents. The mechanism by which LL collaborates with LYZ in lipid release is still not known. The possible role of bacterial lipids and lyso compounds in the control of bacterial survival in inflammatory sites is briefly discussed.

BACKGROUND Essential thrombocythemia (ET) is a form of chronic myeloproliferative neoplasm (MPN), and thrombosis is an important complication. This study aimed to use bioinformatics analysis to identify differentially expressed genes (DEGs) in ET associated thrombosis. MATERIAL AND METHODS Two datasets were identified from the Gene Expression Omnibus (GEO) database to investigate the expression profiles in ET. The GSE103176 dataset included 24 patients with ET and 15 healthy individuals with samples from CD34+ bone marrow cells. The GSE54644 dataset included 47 patients with ET and 11 healthy individuals with samples from peripheral neutrophils. GEO2R was used to screen DEGs, followed by over-representation analysis. Protein-protein interaction (PPI) network analysis and module analysis were performed using the STRING database and Cytoscape software. Hub genes were identified using the cytoHubba Cytoscape plugin, and maximal clique centrality (MCC) was identified. The MCODE Cytoscape plugin was used to identify network clusters, or highly interconnected regions. RESULTS There were 586 and 392 DEGs identified from the GSE103176 and GSE54644 datasets, respectively. The upregulated DEGs for CD34+ cells were predominantly enriched for granulocyte activation or related pathways for biological process (BP), and secretory vesicle for the cellular component (CC). The top hub genes within CD34+ cells included CXCL1, CAMP, HP, MMP8, PTX3, ORM1, LYZ, LTF, PGLYRP1, and OLFM4. CONCLUSIONS Bioinformatics analysis identified DEGs and hub genes that interacted with CD34+ cells and neutrophils that may predict an increased risk of thrombosis in patients with ET. These preliminary findings should be validated using next-generation sequencing (NGS) and clinical studies.

Lysozymes are important proteins to bivalve in the innate immune responses against bacterial infections, and provide nutrition as digestion enzymes. A new LYZ1 from the freshwater mussel Cristaria plicata was cloned by rapid amplification of cDNA ends (RACE) and nested PCR method. The full-length cDNA sequence of CpLYZ1 was 763 bp. The cDNA contained a 5'-terminal untranslated region (UTR) of 21 bp, a 3'- terminal UTR of 259 bp with a 29 bp poly(A) tail, a tailing signal (AATAAA) and the open reading frame of 483 bp. The CpLYZ1 cDNA encoded a polypeptide of 160 amino acids with a predicted molecular mass of 17.8 kDa, and a theoretical isoelectric point of 6.07. The comparison of the deduced amino acid sequences with LYZs from other species showed that the enzyme belonged to i-type lysozyme. The mRNA transcript of CpLYZ1 could be detected in all the examined tissues with the highest expression level in hepatopancreas. The expression levels of CpLYZ1 in hemocytes, hepatopancreas and gill significantly increased after Aeromonas hydrophila challenge. The expression level of CpLYZ1 in hemocytes sharply decreased from 6 h to 24 h and significantly increased at 48 h, and was the highest level in hepatopancreas at 24 h, and was the maximum level in gill at 48 h. Furthermore, the recombinant CpLYZ1 was induced to be expressed as an inclusion body form by IPTG at 37 °C for 4 h, and then was purified by using the Ni(2+) affinity chromatography. The relative enzyme activity of the recombinant CpLYZ1 was influenced on pH and temperature. The optimal pH and temperature was 5.5 and 50 °C, respectively. Against Escherichia coli, A. hydrophila, Staphyloccocus aureus, Bacillus subtilis, Streptococcus sp. and Staphylococcus epidermidis, the recombinant CpLYZ1 had bacteriolytic activity.

Use of a highly sensitive, selective capacitive biosensor is reported for label-free, real-time, easy and rapid detection of trypsin by using the microcontact imprinting method. Real-time trypsin detection was performed with trypsin-imprinted (trypsin-MIP) capacitive electrodes using standard trypsin solutions in the concentration range of 1.0×10(-13)-1.0×10(-7)M with a detection limit of 3.0×10(-13)M. Selectivity and cross-reactivity of the system were tested by using competing proteins including chymotrypsin (chy), bovine serum albumin (BSA), lysozyme (lyz) and cytochrome c (cyt c) in singular and competitive manner and the selectivity of the system was determined with the selectivity coefficients of approximately 705.1, 6.5, 6.4 and 5.1 for chy, BSA, lyz and cyt c, respectively. The trypsin-MIP capacitive electrode was used for ~80 assays during 2 months and retained its binding property during all that time with a decrease of approximately 2.3% in the signal amplitude. In the last step, trypsin activity was measured by using Nα-Benzoyl-D, l-arginine 4-nitroanilide hydrochloride (BAPNA) as the substrate with spectrophotometer at 410nm. The trypsin activity was measured as 9mU/mL by spectrophotometer while the amount of captured enzyme calculated from the capacitive system was 7.9mU/mL which shows the correlation between two methods. From the comparison it is obvious that the new method is an attractive alternative for assaying trypsin and the developed capacitive system might be used successfully to monitor label-free, real-time enzymatic activity of different proteases in a sensitive, rapid, cost-effective manner for different applications.

There is a growing interest in repurposing mycobacterial efflux pump inhibitors, such as verapamil, for tuberculosis (TB) treatment. To aid in the design of better analogs, we studied the effects of verapamil on macrophages and Mycobacterium tuberculosis-specific T cells. Macrophage activation was evaluated by measuring levels of nitric oxide, tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and gamma interferon (IFN-γ). Since verapamil is a known autophagy inducer, the roles of autophagy induction in the antimycobacterial activities of verapamil and norverapamil were studied using bone marrow-derived macrophages from ATG5(flox/flox) (control) and ATG5(flox/flox) Lyz-Cre mice. Our results showed that despite the well-recognized effects of verapamil on calcium channels and autophagy, its action on intracellular M. tuberculosis does not involve macrophage activation or autophagy induction. Next, the effects of verapamil and norverapamil on M. tuberculosis-specific T cells were assessed using flow cytometry following the stimulation of peripheral blood mononuclear cells from TB-skin-test-positive donors with M. tuberculosis whole-cell lysate for 7 days in the presence or absence of drugs. We found that verapamil and norverapamil inhibit the expansion of M. tuberculosis-specific T cells. Additionally, three new verapamil analogs were found to inhibit intracellular Mycobacterium bovis BCG, and one of the three analogs (KSV21) inhibited intracellular M. tuberculosis replication at concentrations that did not inhibit M. tuberculosis-specific T cell expansion. KSV21 also inhibited mycobacterial efflux pumps to the same degree as verapamil. More interestingly, the new analog enhances the inhibitory activities of isoniazid and rifampin on intracellular M. tuberculosis. In conclusion, KSV21 is a promising verapamil analog on which to base structure-activity relationship studies aimed at identifying more effective analogs.

The direct correlation between disease and lysozyme (LYZ) levels in human body fluids makes the sensitive and convenient detection of LYZ the focus of scientific research. Fluorescent molecularly imprinted polymer has emerged as a new alternative for LYZ detection in order to resolve the limitation of immunoassays, which are expensive, unstable, require complex preparation, and are time consuming. In this study, a novel fluorescence molecularly imprinted polymer based on Navicula sp. frustules (FITC-MIP) has been synthesized via post-imprinting treatment for LYZ detection. Navicula sp. frustules were used as supported material because of their unique properties of moderate surface area, reproducibility, and biocompatibility, to address the drawbacks of nanoparticle core material with low adsorption capacity. The FITC acts as recognition signal and optical readout, whereas MIP provides LYZ selectivity. The synthesized FITC-MIP showed a response time as short as 5 min depending on the concentration of LYZ. It is found that the LYZ template can significantly quench the fluorescence intensity of FITC-MIP linearly within a concentration range of 0 to 0.025 mg mL(-1), which is well described by Stern-Volmer equation. The FITC-MIP can selectively and sensitively detect down to 0.0015 mg mL(-1) of LYZ concentration. The excellent sensing performance of FITC-MIP suggests that FITC-MIP is a potential biosensor in clinical diagnosis applications.

Progranulin (PGRN) is a multi-functional growth factor that mediates cell proliferation, survival, migration, tumorigenesis, wound healing, development, and anti-inflammation activity. A novel alternatively spliced transcript from the short-form PGRN1 gene encoding a novel, secreted GRN peptide composed of 20-a.a. signal peptide and 41-a.a. GRN named GRN-41 was identified to be abundantly expressed in immune-related organs including spleen, head kidney, and intestine of Mozambique tilapia. The expression of GRN-41 and PGRN1 were further induced in the spleen of tilapia challenged with Vibrio vulnificus at 3 h post infection (hpi) and 6 hpi, respectively. In this study, we established three transgenic zebrafish lines expressing the secreted GRN-41, GRN-A and PGRN1 of Mozambique tilapia specifically in muscle. The relative percent of survival (RPS) was enhanced in adult transgenic zebrafish expressing tilapia GRN-41 (68%), GRN-A (32%) and PGRN1 (36%) compared with control transgenic zebrafish expressing AcGFP after challenge with V. vulnificus. It indicates tilapia GRN-41 is a potent peptide against V. vulnificus infection. The secreted tilapia GRN-41 can induce the expression of innate immune response-related genes, such as TNFa, TNFb, IL-8, IL-1β, IL-6, IL-26, IL-21, IL-10, complement C3, lysozyme (Lyz) and the hepatic antimicrobial peptide hepcidin (HAMP), in adult transgenic zebrafish without V. vulnificus infection. The tilapia GRN-41 peptide can enhance the innate immune response by further elevating TNFb, IL-1β, IL-8, IL-6, and HAMP expression in early responsive time to the V. vulnificus challenge in transgenic zebrafish. Our results suggest that the novel GRN-41 peptide generated from alternative splicing of the tilapia PGRN1 gene is a potent peptide that defends against V. vulnificus in the transgenic zebrafish model by modulation of innate immunity.

OBJECTIVE

To investigate disease-specific gene expression profiles of peripheral blood mononuclear cells (PBMCs) from Crohn's disease (CD) patients in clinical remission.

METHODS

Patients with CD in clinical remission or with very low disease activity according to the Crohn's disease activity index were genotyped regarding nucleotide-binding oligomerization domain 2 (NOD2), and PBMCs from wild-type (WT)-NOD2 patients, patients with homozygous or heterozygous NOD2 mutations and healthy donors were isolated for further analysis. The cells were cultured with vitamin D, peptidoglycan (PGN) and lipopolysaccharide (LPS) for defined periods of time before RNA was isolated and subjected to microarray analysis using Clariom S assays and quantitative real-time PCR. NOD2- and disease-specific gene expression profiles were evaluated with repeated measure ANOVA by a general linear model.

RESULTS

Employing microarray assays, a total of 267 genes were identified that were significantly up- or downregulated in PBMCs of WT-NOD2 patients, compared to healthy donors after challenge with vitamin D and/or a combination of LPS and PGN (P < 0.05; threshold: ≥ 2-fold change). For further analysis by real-time PCR, genes with known impact on inflammation and immunity were selected that fulfilled predefined expression criteria. In a larger cohort of patients and controls, a disease-associated expression pattern, with higher transcript levels in vitamin D-treated PBMCs from patients, was observed for three of these genes, CLEC5A (P < 0.030), lysozyme (LYZ; P < 0.047) and TREM1 (P < 0.023). Six genes were found to be expressed in a NOD2-dependent manner (CD101, P < 0.002; CLEC5A, P < 0.020; CXCL5, P < 0.009; IL-24, P < 0.044; ITGB2, P < 0.041; LYZ, P < 0.042). Interestingly, the highest transcript levels were observed in patients with heterozygous NOD2 mutations.

CONCLUSIONS

Our data identify CLEC5A and LYZ as CD- and NOD2-associated genes of PBMCs and encourage further studies on their pathomechanistic roles.

Sepsis is associated with bacterial invasion and inflammation and has a high mortality rate. Previous studies have demonstrated that tripartite motif 59 (TRIM59) was involved in NF-κB signaling and could promote phagocytosis of macrophages, but the role of TRIM59 in sepsis is still unknown. In our study, we found that TRIM59 was downregulated in lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDMs). In the cecal ligation and puncture (CLP) sepsis mice model, the mortality of Trim59^flox/floxLyz-Cre (Trim59-cKO) mice was higher, the immune cell infiltration and damage of liver and lung were more severe, and bacteria burden was increased. We also found that TRIM59 altered the production of pro-inflammation cytokines, as well as macrophage phagocytosis ability. Further analysis indicated that NF-κB signal pathway and Fcγ receptors might be involved in these regulations. Our study demonstrated for the first time that TRIM59 protects mice from sepsis by regulating inflammation and phagocytosis in macrophages.

Defective autophagic machinery, such as that in Crohn's disease patients homozygous for ATG16L1 risk allele, is associated with alteration of resident gut bacterial communities. However, whether or not host autophagy responds to changes in the resident gut microbial community is not known. Here, we investigated the effect of antibiotic-induced disruption of the gut microbiome (dysbiosis) on autophagy gene expression and the expression of antimicrobial peptides/protein (AMP) over time.

To test the hypothesis that antibiotic treatment may cause time-dependent changes in gut bacterial density, autophagy genes, and antimicrobial protein/peptide gene expression.

Mice (n = 8 per group) were treated with antibiotic cocktail and sacrificed at different intervals of recovery (days 3, 7, 10, 14, 21, 28, 35, and 42) post-antibiotics. DNA and RNA were extracted from small intestinal tissues. Bacterial density, expression of host autophagy genes, and AMP genes were analyzed by relative quantitative PCR. Fold change difference in comparison with untreated control group was calculated using 2-ΔΔCt method. Statistical analysis was performed using nonparametric Mann-Whitney test.

Gut bacterial density changed in a time-dependent fashion in response to antibiotic treatment. These changes were concurrent with upregulation of autophagy genes and antimicrobial peptide/protein gene expression. We further showed that an oral gavage of a resident microbe Desulfovibrio, which bloomed in antibiotic-treated animals, induced Atg5 and lysozyme (Lyz) gene expression.

Autophagy genes respond to dysbiosis induced by antibiotics. This response may be a host mechanism to detect and possibly correct dysbiosis by activating antimicrobial peptides/proteins that control the microbial load in the gut.

Streptococcus agalactiae is an important pathogen causing bovine mastitis. The aim of this study was to develop a simple and specific method for direct detection of S. agalactiae from milk products. Propidium monoazide (PMA) and sodium dodecyl sulfate (SDS) were utilized to eliminate the interference of dead and injured cells in qPCR. Lysozyme (LYZ) was adopted to increase the extraction efficiency of target bacteria DNA in milk matrix. The specific primers were designed based on cfb gene of S. agalactiae for qPCR. The inclusivity and exclusivity of the assay were evaluated using 30 strains. The method was further determined by the detection of S. agalactiae in spiked milk. Results showed significant differences between the SDS-PMA-qPCR, PMA-qPCR and qPCR when a final concentration of 10 mg/ml (R² = 0.9996, E = 95%) of LYZ was added in DNA extraction. Viable S. agalactiae was effectively detected when SDS and PMA concentrations were 20 μg/ml and 10 μM, respectively, and it was specific and more sensitive than qPCR and PMA-qPCR. Moreover, the SDS-PMA-qPCR assay coupled with LYZ was used to detect viable S. agalactiae in spiked milk, with a limit of detection of 3 × 10³ cfu/ml. Therefore, the SDS-PMA-qPCR assay had excellent sensitivity and specificity for detection of viable S. agalactiae in milk.

Nonfouling materials such as neutral poly(ethylene glycol) (PEG) and zwitterionic poly(sulfobetaine methacrylate) (pSBMA) are ideal biocompatible materials for drug, especially protein drug delivery. The interaction behavior of protein between the nonfouling materials could cause great impact on their future applications, such as controlled release drug delivery systems. In this work, we investigated the diffusion behavior of the fluorescence-labeled model proteins (bovine serum albumin (BSA) and lysozyme (LYZ)) in nonfouling PEG, pSBMA and mixed PEG-SBMA hydrogels (SBMA:PEG 4:1, SBMA:PEG 1:4). It was observed that these four hydrogels showed varied diffusion behavior for either negatively charged BSA or positively charged LYZ due to protein-polymer interaction and the free water content in hydrogel matrix. The relatively stronger interaction between protein-PEG than protein-pSBMA could increase protein loading efficiency and control release rate by changing ratio of PEG to SBMA in the hybrid hydrogel. Moreover, it is further demonstrated the free water (freezable water) content in low cross-linked hydrogel, not the equilibrium water content (EWC), is a more accurate parameter to reflect the diffusion behavior of protein molecules. Thus, these results together provide new insights of the interactions between protein molecules and nonfouling polymers as well as the bio applications of the nonfouling polymeric hydrogels.

This work shows that the relative stronger interaction between protein-PEG than protein-pSBMA could increase protein loading efficiency and control release rate by the change ratio of PEG to SBMA in the hydrogel, while the free water (freezable water) content in low cross-linked hydrogel, not the equilibrium water content (EWC), is a more accurate parameter to reflect the diffusion behavior of protein molecules. The impact of this work (i) gains some new insights of the interactions between protein molecules and nonfouling polymer matrixes for protein drug delivery; (ii) prompts to apply the weak PEG-protein interactions to protein drug loading and release; (iii) provides a new fundamental understanding of free water in hydrogel for protein diffusion.

Intestinal alkaline phosphatase (IAP) regulates bicarbonate secretion, detoxifies lipopolysaccharide (LPS), regulates gut microbes, and dephosphorylates proinflammatory nucleotides. IAP also exhibits anti-inflammatory effects in a Toll-like Receptor-4 (TLR-4) dependent manner. However, it is not known whether IAP induces autophagy. We tested the hypothesis that IAP may induce autophagy which may mediate the anti-inflammatory effects of IAP. We found that exogenous IAP induced autophagy in intestinal epithelial cells and in macrophages. TLR4INC34 (C34), a TLR4 signaling inhibitor, suppressed IAP-induced autophagy. IAP also inhibited LPS-induced IL-1β mRNA expression and activation of NF-κB. When autophagy was blocked by 3-methyladenine (3MA) or by Atg5 siRNA, IAP failed to block LPS-mediated effects. IAP also upregulated autophagy-related gene expression in small intestine in mice. We administered either vehicle or IAP (100 U/ml) in drinking water for 14 days in C57BL/6 mice. Mice were sacrificed and ileal tissues collected. Increased expression of Atg5, Atg16, Irgm1, Tlr4, and Lyz genes was observed in the IAP treated group compared to the vehicle treated group. Increase in Atg16 protein expression and fluorescence intensity of LC3 was also observed in IAP-treated tissues compared to the vehicle-treated tissues. Thus, our study lays the framework for investigating how IAP and autophagy may act together to control inflammatory conditions.

The four experimental groups were carried out to test the response of crucian carp Carassius auratus to ammonia toxicity and taurine: group 1 was injected with NaCl, group 2 was injected with ammonium acetate, group 3 was injected with ammonium acetate and taurine, and group 4 was injected with taurine. Fish in group 2 had the highest ammonia and glutamine contents, and the lowest glutamate content in liver and brain. Serum superoxide dismutase (SOD), glutathione (GSH) activities, red cell count (RBC), white cell count (WBC), lysozyme (LYZ) activity, complement C3 content of fish in group 2 reflected the lowest, but malondialdehyde content was the highest. Importantly, serum SOD and GSH activites, RBC, WBC, and LYZ activity, C3, C4 and total immunoglobulin contents of fish in group 3 were significantly higher than those of fish in group 2. This study indicates that ammonia exerts its toxic effects by interfering with amino acid transport, inducing ROS generation, leading to malondialdehyde accumulation and immunosuppression of crucian carp. The exogenous taurine could mitigate the adverse effect of high ammonia level on fish physiological disorder.

Toll-like receptors, the best known pattern recognition receptors, play important roles in recognizing non-self molecules and binding pathogen-associated molecular patterns in the innate immune system. In the present research, the cDNA and protein characterization of the TLR signalling pathway genes including IRAK4, TRAK6 and IKKα (named CsIRAK4, CsTRAF6 and CsIKKα, respectively) with the typical motifs from Cyclina sinensis showed significant similarity with their homologues from other shellfish. Furthermore, the mRNA transcripts of these three genes are ubiquitously expressed in all tissues tested and are dominantly expressed in C. sinensis haemocytes (P < 0.05). Moreover, IRAK4, TRAK6 and IKKα cDNA expression levels were all up-regulated after injection with Vibrio anguillarum, Micrococcus luteus and poly I:C (P < 0.01) as shown by quantitative real-time PCR, indicating that they were involved in responding to pathogenic stimulation. We explored the function of the TLR13-MyD88-NF-κB signalling pathway in the innate immune responses of C. sinensis by RNA interference and immune challenges. The results suggested the mRNA expression patterns of CsMyD88, CsIRAK4, CsTRAF6, CsIKKα, CsIκB, CsNF-κB, CsC-LYZ and CsAMP were all down-regulated (P < 0.01) in normal and stimulated C. sinensis haemocytes, revealing the involvement of the TLR13-MyD88-NF-κB signalling pathway in innate immunity by positively adjusting internal signalling factors and immune-related genes. In summary, a TLR13-MyD88-NF-κB signalling pathway exists and plays vital roles in innate immune responses in C. sinensis. These findings collectively lay the foundation for studying the functional characterization of internal signalling factors and establishing a regulatory network for the TLR signalling pathway in molluscs.

Infertility is a longstanding limitation in livestock production with important economic impact for the cattle industry. Female reproductive traits are polygenic and lowly heritable in nature, thus selection for fertility is challenging. Beef cattle operations leverage estrous synchronization in combination with artificial insemination (AI) to breed heifers and benefit from an early and uniform calving season. A couple of weeks following AI, heifers are exposed to bulls for an opportunity to become pregnant by natural breeding (NB), but they may also not become pregnant during this time period. Focusing on beef heifers, in their first breeding season, we hypothesized that: a- at the time of AI, the transcriptome of peripheral white blood cells (PWBC) differs between heifers that become pregnant to AI and heifers that become pregnant late in the breeding season by NB or do not become pregnant during the breeding season; and b- the ratio of transcript abundance between genes in PWBC classifies heifers according to pregnancy by AI, NB, or failure to become pregnant.

We generated RNA-sequencing data from 23 heifers from two locations (A: six AI-pregnant and five NB-pregnant; and B: six AI-pregnant and six non-pregnant). After filtering out lowly expressed genes, we quantified transcript abundance for 12,538 genes. The comparison of gene expression levels between AI-pregnant and NB-pregnant heifers yielded 18 differentially expressed genes (DEGs) (ADAM20, ALDH5A1, ANG, BOLA-DQB, DMBT1, FCER1A, GSTM3, KIR3DL1, LOC107131247, LOC618633, LYZ, MNS1, P2RY12, PPP1R1B, SIGLEC14, TPPP, TTLL1, UGT8, eFDR≤0.02). The comparison of gene expression levels between AI-pregnant and non-pregnant heifers yielded six DEGs (ALAS2, CNKSR3, LOC522763, SAXO2, TAC3, TFF2, eFDR≤0.05). We calculated the ratio of expression levels between all gene pairs and assessed their potential to classify samples according to experimental groups. Considering all samples, relative expression from two gene pairs correctly classified 10 out of 12 AI-pregnant heifers (P = 0.0028) separately from the other 11 heifers (NB-pregnant, or non-pregnant).

The transcriptome profile in PWBC, at the time of AI, is associated with the fertility potential of beef heifers. Transcript levels of specific genes may be further explored as potential classifiers, and thus selection tools, of heifer fertility.

Environmental pollutants may cause adverse effects on the immune system of aquatic organisms. However, the cellular effects of pollutants on fish immune system are largely unknown. Here, we exploited the transgenic zebrafish Tg(lysC:DsRed2) larva as a preliminary screening system to evaluate the potential inflammatory effects of environmental pollutants. Tg(lysC:DsRED2) larvae aged 7-day-postfertilization (7 dpf) were treated with selected environmental chemicals for 24 h (24 h) and the number of neutrophils were quantified using both image analysis and fluorescence activated cell sorting (FACS). We found that the numbers of neutrophils in the Tg(lysC:DsRED2) larvae were significantly increased by most of the organic chemicals tested, including E2 (17β-estradiol), BPA (Bisphenol-A), NDEA (N-nitrosodiethylamine), 4-NP (4-Nitrophenol) and Lindane (γ-hexachlorocyclohexane). Neutrophil numbers were also increased by all the metals tested (Na2HAsO4· 7H2O, Pb(NO3)2, HgCl2, CdCl2, CuSO4·5H2O, ZnSO4, and K2Cr2O7). The only exception was TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), which significantly reduced the number of neutrophils after exposure. Additionally, the transcription of genes (lyz, mpo, tnfα and il8) related to fish immune system were significantly modulated upon exposure to some of the selected chemicals such as E2, TCDD, Cu and Cd. This study revealed that representatives of major categories of environmental pollutants could cause an acute inflammatory response in zebrafish larvae as shown by alterations in the neutrophils, which may imply a common immunotoxicity mechanism for most environmental pollutants. This study has also demonstrated that Tg(lyz:DsRed2) transgenic zebrafish is an excellent tool for screening environmental chemicals with potential inflammatory effects through FACS-facilitated neutrophil counting.