Comparison and evaluation of experimental mediastinitis models: precolonized foreign body implants and bacterial suspension inoculation seems promising

BACKGROUND: Post-sternotomy mediastinitis (PSM) is a devastating surgical complication affecting 1–3% of patients that undergo cardiac surgery. Staphylococcus aureus is one of the most commonly encountered bacterial pathogen cultured from mediastinal samples obtained from patients with PSM. A component of the membrane of the gram positive bacteria, lipoteichoic acid, stimulates the blood monocytes and macrophages to secrete cytokines, radicals and nitrogen species leading to oxido-inflammatory damage. This seems to be responsible for the high mortality rate in PSM. For the evaluation of the pathogenesis of infection or for the investigation of alternative treatment models in infection, no standard model of mediastinitis seems to be available. In this study, we evaluated four mediastinitis models in rats. METHODS: The rats were divided into four groups to form different infection models. Group A: A suspension of 1 × 10(7 )colony-forming units Staphylococcus aureus in 0,5 mL was inoculated from the right second intercostal space into the mediastinum. Group B: A hole was created in the right second intercostal space and a piece of stainless-steel implant with a length of 0.5 cm was inserted into the mediastinum and a suspension of 1 × 10(7 )cfu bacteria in 0,5 mL was administered via the tail vein. Group C: Precolonized stainless-steel implant was inserted into the mediastinum. Group D: Precolonized stainless-steel implant was inserted into the mediastinum and the bacteria suspension was also injected into the mediastinum. On the 10(th )day, rats were sacrificed and the extension of infection in the mediastenae was evaluated by quantitative cultures. Myeloperoxidase activity (MPO) and malondialdehyde (MDA) levels were determined in the sera to evaluate the neutrophil activation and assess the inflammatory oxidation. RESULTS: The degree of infection in group C and D were 83.3% and 100% respectively (P < 0.001). MDA levels were significantly higher in these two groups than the others (P < 0.001). CONCLUSION: Infected implants and high bacterial concentration administration were the two important components that played a significant role in the outcome of a successful infection in mediastinum in a rat model

Host Cell Egress and Invasion Induce Marked Relocations of Glycolytic Enzymes in Toxoplasma gondii Tachyzoites

Apicomplexan parasites are dependent on an F-actin and myosin-based motility system for their invasion into and escape from animal host cells, as well as for their general motility. In Toxoplasma gondii and Plasmodium species, the actin filaments and myosin motor required for this process are located in a narrow space between the parasite plasma membrane and the underlying inner membrane complex, a set of flattened cisternae that covers most the cytoplasmic face of the plasma membrane. Here we show that the energy required for Toxoplasma motility is derived mostly, if not entirely, from glycolysis and lactic acid production. We also demonstrate that the glycolytic enzymes of Toxoplasma tachyzoites undergo a striking relocation from the parasites' cytoplasm to their pellicles upon Toxoplasma egress from host cells. Specifically, it appears that the glycolytic enzymes are translocated to the cytoplasmic face of the inner membrane complex as well as to the space between the plasma membrane and inner membrane complex. The glycolytic enzymes remain pellicle-associated during extended incubations of parasites in the extracellular milieu and do not revert to a cytoplasmic location until well after parasites have completed invasion of new host cells. Translocation of glycolytic enzymes to and from the Toxoplasma pellicle appears to occur in response to changes in extracellular [K+] experienced during egress and invasion, a signal that requires changes of [Ca2+]c in the parasite during egress. Enzyme translocation is, however, not dependent on either F-actin or intact microtubules. Our observations indicate that Toxoplasma gondii is capable of relocating its main source of energy between its cytoplasm and pellicle in response to exit from or entry into host cells. We propose that this ability allows Toxoplasma to optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for growth and replication of intracellular parasites

Inhibition of the Mitochondrial Enzyme ABAD Restores the Amyloid-β-Mediated Deregulation of Estradiol

Alzheimer's disease (AD) is a conformational disease that is characterized by amyloid-β (Aβ) deposition in the brain. Aβ exerts its toxicity in part by receptor-mediated interactions that cause down-stream protein misfolding and aggregation, as well as mitochondrial dysfunction. Recent reports indicate that Aβ may also interact directly with intracellular proteins such as the mitochondrial enzyme ABAD (Aβ binding alcohol dehydrogenase) in executing its toxic effects. Mitochondrial dysfunction occurs early in AD, and Aβ's toxicity is in part mediated by inhibition of ABAD as shown previously with an ABAD decoy peptide. Here, we employed AG18051, a novel small ABAD-specific compound inhibitor, to investigate the role of ABAD in Aβ toxicity. Using SH-SY5Y neuroblastoma cells, we found that AG18051 partially blocked the Aβ-ABAD interaction in a pull-down assay while it also prevented the Aβ42-induced down-regulation of ABAD activity, as measured by levels of estradiol, a known hormone and product of ABAD activity. Furthermore, AG18051 is protective against Aβ42 toxicity, as measured by LDH release and MTT absorbance. Specifically, AG18051 reduced Aβ42-induced impairment of mitochondrial respiration and oxidative stress as shown by reduced ROS (reactive oxygen species) levels. Guided by our previous finding of shared aspects of the toxicity of Aβ and human amylin (HA), with the latter forming aggregates in Type 2 diabetes mellitus (T2DM) pancreas, we determined whether AG18051 would also confer protection from HA toxicity. We found that the inhibitor conferred only partial protection from HA toxicity indicating distinct pathomechanisms of the two amyloidogenic agents. Taken together, our results present the inhibition of ABAD by compounds such as AG18051 as a promising therapeutic strategy for the prevention and treatment of AD, and suggest levels of estradiol as a suitable read-out

Monoclonal Antibodies against Accumulation-Associated Protein Affect EPS Biosynthesis and Enhance Bacterial Accumulation of Staphylococcus epidermidis

Because there is no effective antibiotic to eradicate Staphylococcus epidermidis biofilm infections that lead to the failure of medical device implantations, the development of anti-biofilm vaccines is necessary. Biofilm formation by S. epidermidis requires accumulation-associated protein (Aap) that contains sequence repeats known as G5 domains, which are responsible for the Zn2+-dependent dimerization of Aap to mediate intercellular adhesion. Antibodies against Aap have been reported to inhibit biofilm accumulation. In the present study, three monoclonal antibodies (MAbs) against the Aap C-terminal single B-repeat construct followed by the 79-aa half repeat (AapBrpt1.5) were generated. MAb18B6 inhibited biofilm formation by S. epidermidis RP62A to 60% of the maximum, while MAb25C11 and MAb20B9 enhanced biofilm accumulation. All three MAbs aggregated the planktonic bacteria to form visible cell clusters. Epitope mapping revealed that the epitope of MAb18B6, which recognizes an identical area within AapBrpt constructs from S. epidermidis RP62A, was not shared by MAb25C11 and MAb20B9. Furthermore, all three MAbs were found to affect both Aap expression and extracellular polymeric substance (EPS, including extracellular DNA and PIA) biosynthesis in S. epidermidis and enhance the cell accumulation. These findings contribute to a better understanding of staphylococcal biofilm formation and will help to develop epitope-peptide vaccines against staphylococcal infections

A fast and flexible panoramic virtual reality system for behavioural and electrophysiological experiments

Ideally, neuronal functions would be studied by performing experiments with unconstrained animals whilst they behave in their natural environment. Although this is not feasible currently for most animal models, one can mimic the natural environment in the laboratory by using a virtual reality (VR) environment. Here we present a novel VR system based upon a spherical projection of computer generated images using a modified commercial data projector with an add-on fish-eye lens. This system provides equidistant visual stimulation with extensive coverage of the visual field, high spatio-temporal resolution and flexible stimulus generation using a standard computer. It also includes a track-ball system for closed-loop behavioural experiments with walking animals. We present a detailed description of the system and characterize it thoroughly. Finally, we demonstrate the VR system’s performance whilst operating in closed-loop conditions by showing the movement trajectories of the cockroaches during exploratory behaviour in a VR forest

Polycomb Group Protein Bmi1 Is Required for Growth of RAF Driven Non-Small-Cell Lung Cancer

Background: We have previously described a RAF oncogene driven transgenic mouse model for non small cell lung cancer (NSCLC). Here we examine whether tumor initiation and growth requires the stem cell self-renewal factor Bmi1. Principal Findings: In order to evaluate Bmi1 function in NSCLC two founder lines that differ in incidence and latency of tumor formation were compared. Ablation of Bmi1 expression in both lines had a dramatically decreased tumor growth. As the line with shorter latency matched the life span of Bmi1 knock out mice, these mice were chosen for further study. The absence of Bmi1 did not decrease the number of tumor initiation in these mice as only the size and not the number of tumors decreased. Reduction in tumor growth resulted from an increase in cell death and decrease in cell cycle progression that corresponded with up-regulation of the p16 INK4a and p19 ARF. Significance: The data identifies Bmi1 as an important factor for expansion but not initiation of RAF driven NSCLC

Alveolar proteins stabilize cortical microtubules in Toxoplasma gondii

Single-celled protists use elaborate cytoskeletal structures, including arrays of microtubules at the cell periphery, to maintain polarity and rigidity. The obligate intracellular parasite Toxoplasma gondii has unusually stable cortical microtubules beneath the alveoli, a network of flattened membrane vesicles that subtends the plasmalemma. However, anchoring of microtubules along alveolar membranes is not understood. Here, we show that GAPM1a, an integral membrane protein of the alveoli, plays a role in maintaining microtubule stability. Degradation of GAPM1a causes cortical microtubule disorganisation and subsequent depo-lymerisation. These changes in the cytoskeleton lead to parasites becoming shorter and rounder, which is accompanied by a decrease in cellular volume. Extended GAPM1a depletion leads to severe defects in division, reminiscent of the effect of disrupting other alveolar proteins. We suggest that GAPM proteins link the cortical microtubules to the alveoli and are required to maintain the shape and rigidity of apicomplexan zoites

A miRNA Signature of Prion Induced Neurodegeneration

MicroRNAs (miRNAs) are small, non-coding RNA molecules which are emerging as key regulators of numerous cellular processes. Compelling evidence links miRNAs to the control of neuronal development and differentiation, however, little is known about their role in neurodegeneration. We used microarrays and RT-PCR to profile miRNA expression changes in the brains of mice infected with mouse-adapted scrapie. We determined 15 miRNAs were de-regulated during the disease processes; miR-342-3p, miR-320, let-7b, miR-328, miR-128, miR-139-5p and miR-146a were over 2.5 fold up-regulated and miR-338-3p and miR-337-3p over 2.5 fold down-regulated. Only one of these miRNAs, miR-128, has previously been shown to be de-regulated in neurodegenerative disease. De-regulation of a unique subset of miRNAs suggests a conserved, disease-specific pattern of differentially expressed miRNAs is associated with prion–induced neurodegeneration. Computational analysis predicted numerous potential gene targets of these miRNAs, including 119 genes previously determined to be also de-regulated in mouse scrapie. We used a co-ordinated approach to integrate miRNA and mRNA profiling, bioinformatic predictions and biochemical validation to determine miRNA regulated processes and genes potentially involved in disease progression. In particular, a correlation between miRNA expression and putative gene targets involved in intracellular protein-degradation pathways and signaling pathways related to cell death, synapse function and neurogenesis was identified

agr-Mediated Dispersal of Staphylococcus aureus Biofilms

The agr quorum-sensing system of Staphylococcus aureus modulates the expression of virulence factors in response to autoinducing peptides (AIPs). Recent studies have suggested a role for the agr system in S. aureus biofilm development, as agr mutants exhibit a high propensity to form biofilms, and cells dispersing from a biofilm have been observed displaying an active agr system. Here, we report that repression of agr is necessary to form a biofilm and that reactivation of agr in established biofilms through AIP addition or glucose depletion triggers detachment. Inhibitory AIP molecules did not induce detachment and an agr mutant was non-responsive, indicating a dependence on a functional, active agr system for dispersal. Biofilm detachment occurred in multiple S. aureus strains possessing divergent agr systems, suggesting it is a general S. aureus phenomenon. Importantly, detachment also restored sensitivity of the dispersed cells to the antibiotic rifampicin. Proteinase K inhibited biofilm formation and dispersed established biofilms, suggesting agr-mediated detachment occurred in an ica-independent manner. Consistent with a protease-mediated mechanism, increased levels of serine proteases were detected in detaching biofilm effluents, and the serine protease inhibitor PMSF reduced the degree of agr-mediated detachment. Through genetic analysis, a double mutant in the agr-regulated Aur metalloprotease and the SplABCDEF serine proteases displayed minimal extracellular protease activity, improved biofilm formation, and a strongly attenuated detachment phenotype. These findings indicate that induction of the agr system in established S. aureus biofilms detaches cells and demonstrate that the dispersal mechanism requires extracellular protease activity

Study protocol of the iMPaCT project : A longitudinal cohort study assessing psychological determinants, sexual behaviour and chlamydia (re)infections in heterosexual STI clinic visitors

Acknowledgements We are grateful to the staff at the STI clinics of Amsterdam, Kennemerland, Hollands Noorden, Twente, who are involved in the recruitment and data collection of participants, and Marlous Ratten and Klazien Visser from Soapoli-online, who are involved in the coordination of laboratory testing of the home-based sampling kits at six-month follow-up. We also thank the staff at the STI department at the National Institute for Public Health and the Environment, especially Birgit van Benthem. Funding This project is funded by the Strategic Programme (SPR) of the National Institute for Public Health and the Environment (RIVM) (project number S/113004/01/IP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Availability of data and materials The dataset (anonymised) generated during this study will be made available for interested parties on request.Peer reviewedPublisher PD