The acyl-biotinyl exchange (ABE) is widely used for detection of S-palmitoylated proteins by replacing palmitic acid with biotin, which is a common method for detecting S-palmitoylated proteins. In this study, the effects of acetone precipitation and methanol-chloroform precipitation on the detection of S-palmitoylation proteins in acyl-biotin exchange method were compared, and the S-palmitoylated proteins in mouse cardiac tissue were analyzed. First, N-ethylmaleimide (NEM) was used to block free sulfhydryls within protein molecules. Then, biotinylation reagent (HPDP-Biotin) was used to label the newly produced cysteine thiols that were resulted from treatment by hydroxylamine (HA) in mouse heart tissue. During the ABE reaction, excess unreacted NEM, HA and HPDP-Biotin were removed by precipitation of the proteins. Then, the S-palmitoylated proteins from heart tissue were labeled with ABE reaction based on different precipitation methods, and the S-palmitoylated proteins labeled with biotin were enriched by streptavidin agarose beads. The enriched proteins were analyzed by mass spectrometry, and 50 S-palmitoylated proteins were identified. Specifically, 23 S-palmitoylated proteins were identified in acetone precipitation assay group, and 37 S-palmitoylated proteins in the methanol-chloroform precipitation assay group were identified. 10 palmitoylated proteins were identified in both groups. The results showed that the combination of different precipitation methods could be helpful for the identification of palmitoylated proteins.

Previous studies have demonstrated that T cells and microglia could fight against cerebral Listeria monocytogenes (Listeria); however, their synergistic anti-Listeria mechanisms remain unknown. Following Listeria infection in a culture system, we found that microglia, but not nerve cells, could release extracellular traps (ETs) which originated from microglial vesicles. Specific inhibitor analysis showed that extracellular DNA (eDNA), matrix metallopeptidases (MMP9 and MMP12), citrullinated histone H3, and peptidyl arginine deiminase 2 were the major components of microglial ETs (MiETs) and were also the components of vesicles. Systematic analysis indicated that Listeria-induced MiETs were cytosolic reactive oxygen species (ROS)- and NADPH oxidase (NOX)-dependent and involved ERK. MiETs were exhibited in Listeria-infected mouse brain and might protected against Listeria infection via bacterial killing in a mouse meningitis model, and MiETs existed in cerebrospinal fluid (CSF) from Listeria meningitis patients in vivo and in vitro. Additionally, interferon-γ could induce MiET formation in Listeria-infected microglia in vitro that was mediated by NOX, and there was a positive relationship between the elevated level of IFN-γ and eDNA and nucleosomes in the brain homogenates and CSF of Listeria meningitis model mice and in the CSF before treatment in clinical Listeria meningitis patients. Together, this is the first report of MiET formation, these findings pave the way for deeper exploration of the innate immune response to pathogens in CNS.

The successful treatment of bacterial infections is the achievement of a synergy between the host’s immune defences and antibiotics. Here, we examined whether fosfomycin (FOM) could improve the bactericidal effect of phagocytes and investigated the potential mechanisms. FOM enhanced the phagocytosis and extra- or intracellular killing of S. aureus by phagocytes. And FOM enhanced the extracellular killing of S. aureus in macrophage (MФ) and in neutrophils mediated by extracellular traps (ETs). ET production was related to NADPH oxidase-dependent reactive oxygen species (ROS). Additionally, FOM increased the intracellular killing of S. aureus in phagocytes, which was mediated by ROS through the oxidative burst process. Our results also showed that FOM alone induced S. aureus producing hydroxyl radicals in order to kill the bacterial cells in vitro . In a mouse peritonitis model, FOM treatment increased the bactericidal extra- and intracellular activity in vivo and FOM strengthened ROS and ET production from peritoneal lavage fluid ex vivo . An IVIS imaging system assay further verified the observed in vivo bactericidal effect of the FOM treatment. This work may provide a deeper understanding of the role of the host’s immune defences and antibiotic interactions in microbial infections.

Staphylococcus aureus is one of the most important pathogens in humans and animals. The formation of biofilm by S. aureus is considered an important mechanism of antimicrobial resistance. Therefore, finding effective drugs against the biofilm produced by S. aureus has been a high priority. Licochalcone A (LAA), a natural plant product, was reported to have antibacterial activities and showed good activity against all 21 tested strains of S. aureus biofilm and planktonic cells. To detect the possible molecular mechanism of LAA against S. aureus biofilm or planktonic cells, Affymetrix GeneChips were used to determine the global comparative transcription of S. aureus biofilm and planktonic cells triggered by treatment with sub-bactericidal and sub-inhibitory concentrations of LAA, respectively. LAA significantly altered (greater than a 2- or less than −2-fold change) the expression of 693 genes in planktonic cells and 817 genes in biofilm. The levels of genes encoding autolysis-associated proteins, cell wall proteins, pathogenic factors, protein synthesis genes, and enzymes involved in capsule synthesis were significantly altered in LAA-treated S. aureus. Furthermore, some differences observed in the microarray analysis were verified by real-time RT–PCR. To our knowledge, this is the first observation of phenotype and expression profiles of S. aureus biofilm and planktonic cells in response to LAA treatment.

To investigate the antimicrobial activity of imipenem and rifampicin alone and in combination against clinical isolates of Acinetobacter baumannii grown in planktonic and biofilm cultures. Minimum inhibitory concentrations were determined for each isolate grown in suspension and in biofilm using a microbroth dilution method. Chequerboard assays and the agar disk diffusion assay were used to determine synergistic, indifferent or antagonistic interactions between imipenem and rifampicin. We used the tissue culture plate method for A. baumannii biofilm formation to measure the percentage of biofilm inhibition and the amount of extracellular DNA after the treatment. To understand the synergistic mechanisms, we conducted hydroxyl radical formation assays. The results were verified by confocal laser scanning microscopy. Imipenem and rifampicin showed effective antimicrobial activity against suspensions and biofilm cultures of A. baumannii, respectively. Synergistic antimicrobial effects between imipenem and rifampicin were observed in 13 and 17 of the 20 clinical isolates when in suspension and in biofilms, respectively. Imipenem and rifampicin alone and in combination generated hydroxyl radicals, which are highly reactive oxygen forms and the major components of bactericidal agents. Furthermore, treatment with imipenem and rifampicin individually or in combination has obvious antibiofilm effects. The synergistic activity of imipenem and rifampicin against clinical isolates of A. baumannii (in suspension and in biofilms) was observed in vitro. Therefore, we conclude that imipenem combined with rifampicin has the potential to be used as a combinatorial therapy for the treatment of infectious diseases caused by A. baumannii.

Nisin has been widely used as a natural preservative in foods including dairy products, but the emergence of the nisin-resistant strains could compromise its use to control food-borne pathogens. It has been reported that coenzyme Q (CoQ) is a powerful antioxidant, but a small number of studies have described the antibacterial activity of CoQ, One of the most abundant forms of CoQ is CoQ(10). CoQ(0) is chosen for this study, which is the water-soluble homolog of CoQ(10). In the present study, the in vitro interaction of nisin and CoQ(0) against 15 food-borne isolates of Staphylococcus aureus was assessed using a checkerboard microdilution method. Synergism was observed in strains tested, the FICI values ranging from 0.25 to 0.375. No antagonistic interaction between nisin and CoQ(0) occurred. The positive interactions were verified confirmed using the time-killing test and agar diffusion assay. Our discovery of efficacy of combining nisin with CoQ(0) might pave the way for a novel solution for spoilage problem in food industry and provide an alternative approach to overcome antimicrobial drug resistance. (C) 2014 Elsevier Ltd. All rights reserved.

Reactive oxygen species (ROS) elicited by oxidative stress are widely recognized as a major initiator in the dege-neration of dopaminergic neurons distinctive of Parkinson's disease (PD). The interaction of ROS with mitochondria triggers sequential events in the mitochondrial cell death pathway, which is thought to be responsible for ROS-mediated neurodegeneration in PD. α-lipoic acid (LA) is a pleiotropic compound with potential pharmacotherapeutic value against a range of pathophysiological insults. Its protective actions against oxidative damage by scavenging ROS and reducing production of free radicals have been reported in various in vitro and in vivo systems. This study analyzed the ability of LA to protect PC12 neuronal cells from toxicity of 1-methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which is known to kill dopaminergic neurons selectively and to cause severe parkinsonism-like symptoms in humans and primate animals. Our results demonstrate that the apoptosis of PC12 cells elicited by MPP+ could be significantly prevented by pretreatment with LA for 1 h. In addition, LA inhibits intercellular ROS levels and the mitochondrial transmembrane permeability, the key players in the pathogenesis of PD, thereby protecting dopaminergic neuronal cells against oxidative damage.

Staphylococcus aureus is one of the most common pathogens of infectious diseases with a significant morbidity and mortality. Antimicrobial activity of azithromycin against S. aureus were determined for each microorganism grown in suspension or biofilm using microbroth dilution method, and influence of azithromycin on biofilm formation and secretion of virulence factors by S. aureus was assessed by biofilm formation assay, hemolysin assay, Western blot and real-time RT-PCR. The results showed that azithromycin had better antimicrobial activity against S. aureus grown in suspension or biofilm, and effective inhibition of biofilm formation; subinhibitory concentration of azithromycin reduced the production of α-haemolysin and enterotoxin A by S. aureus in dosedependent manner. In particular, 1/2MIC of azithromycin almost completely suppresses the production of α-haemolysin and enterotoxin A. This study might be especially helpful for the treatment of S. aureus infections.