Modeling Staphylococcus epidermidis-Induced Non-Unions: Subclinical and Clinical Evidence in Rats

S. epidermidis is one of the leading causes of orthopaedic infections associated with biofilm formation on implant devices. Open fractures are at risk of S. epidermidis transcutaneous contamination leading to higher non-union development compared to closed fractures. Although the role of infection in delaying fracture healing is well recognized, no in vivo models investigated the impact of subclinical low-grade infections on bone repair and non-union. We hypothesized that the non-union rate is directly related to the load of this commonly retrieved pathogen and that a low-grade contamination delays the fracture healing without clinically detectable infection. Rat femurs were osteotomized and stabilized with plates. Fractures were infected with a characterized clinical-derived methicillin-resistant S. epidermidis (10³, 10⁵, 10⁸ colony forming units) and compared to uninfected controls. After 56 days, bone healing and osteomyelitis were clinically assessed and further evaluated by micro-CT, microbiological and histological analyses. The biofilm formation was visualized by scanning electron microscopy. The control group showed no signs of infection and a complete bone healing. The 10³ group displayed variable response to infection with a 67% of altered bone healing and positive bacterial cultures, despite no clinical signs of infection present. The 10⁵ and 10⁸ groups showed severe signs of osteomyelitis and a non-union rate of 83–100%, respectively. The cortical bone reaction related to the periosteal elevation in the control group and the metal scattering detected by micro-CT represented limitations of this study. Our model showed that an intra-operative low-grade S. epidermidis contamination might prevent the bone healing, even in the absence of infectious signs. Our findings also pointed out a dose-dependent effect between the S. epidermidis inoculum and non-union rate. This pilot study identifies a relevant preclinical model to assess the role of subclinical infections in orthopaedic and trauma surgery and to test specifically designed diagnostic, prevention and therapeutic strategies.     

Computational Modeling of Open-Irrigated Electrodes for Radiofrequency Cardiac Ablation Including Blood Motion-Saline Flow Interaction

Radiofrequency catheter ablation (RFCA) is a routine treatment for cardiac arrhythmias. During RFCA, the electrode-tissue interface temperature should be kept below 80°C to avoid thrombus formation. Open-irrigated electrodes facilitate power delivery while keeping low temperatures around the catheter. No computational model of an open-irrigated electrode in endocardial RFCA accounting for both the saline irrigation flow and the blood motion in the cardiac chamber has been proposed yet. We present the first computational model including both effects at once. The model has been validated against existing experimental results. Computational results showed that the surface lesion width and blood temperature are affected by both the electrode design and the irrigation flow rate. Smaller surface lesion widths and blood temperatures are obtained with higher irrigation flow rate, while the lesion depth is not affected by changing the irrigation flow rate. Larger lesions are obtained with increasing power and the electrode-tissue contact. Also, larger lesions are obtained when electrode is placed horizontally. Overall, the computational findings are in close agreement with previous experimental results providing an excellent tool for future catheter research.     

Transgenic mice recapitulate the phenotypic heterogeneity of genetic prion diseases without developing prion infectivity: Role of intracellular PrP retention in neurotoxicity

Genetic prion diseases are degenerative brain disorders caused by mutations in the gene encoding the prion protein (PrP). Different PrP mutations cause different diseases, including Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker (GSS) syndrome and fatal familial insomnia (FFI). The reason for this variability is not known. It has been suggested that prion strains with unique self-replicating and neurotoxic properties emerge spontaneously in individuals carrying PrP mutations, dictating the phenotypic expression of disease. We generated transgenic mice expressing the FFI mutation, and found that they developed a fatal neurological illness highly reminiscent of FFI, and different from those of similarly generated mice modeling genetic CJD and GSS. Thus transgenic mice recapitulate the phenotypic differences seen in humans. The mutant PrPs expressed in these mice are misfolded but unable to self-replicate. They accumulate in different compartments of the neuronal secretory pathway, impairing the membrane delivery of ion channels essential for neuronal function. Our results indicate that conversion of mutant PrP into an infectious isoform is not required for pathogenesis, and suggest that the phenotypic variability may be due to different effects of mutant PrP on intracellular transport.     

Proteomics Analysis of Thermoplasma Quinone Droplets

A novel type of lipid droplet/lipoprotein (LD/LP) particle from Thermoplasma acidophilum has been identified recently, and based on biochemical evidences, it was named Thermoplasma Quinone Droplet (TaQD). The major components of TaQDs are menaquinones, and to some extent polar lipids, and the 153 amino acid long Ta0547 vitellogenin‐N domain protein. In this paper, the aim is to identify TaQD proteome components with 1D‐SDS‐PAGE/LC–MS/MS and cross reference them with Edman degradation. TaQD samples isolated with three different purification methods—column chromatography, immunoprecipitation, and LD ultracentrifugation—are analyzed. Proteins Ta0093, Ta0182, Ta0337, Ta0437, Ta0438, Ta0547, and Ta1223a are identified as constituents of the TaQD proteome. The majority of these proteins is uncharacterized and has low molecular weight, and none of them is predicted to take part in lipid metabolism. Bioinformatics analyses does not predict any interaction between these proteins, however, there are indications of interactions with proteins taking part in lipid metabolism. Whether if TaQDs provide platform for lipid metabolism and the interactions between TaQD proteins and lipid metabolism proteins occur in the reality remain for further studies.     

Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts

Mitochondria participate in key metabolic reactions of the cell and regulate crucial signaling pathways including apoptosis. Although several approaches are available to study mitochondrial function in situ are available, investigating functional mitochondria that have been isolated from different tissues and from cultured cells offers still more unmatched advantages. This protocol illustrates a step-by-step procedure to obtain functional mitochondria with high yield from cells grown in culture, liver and muscle. The isolation procedures described here require 1-2 hours, depending on the source of the organelles. The polarographic analysis can be completed in 1 hour.     

High-pass filtering of input signals by the Ih current in a non-spiking neuron, the retinal rod bipolar cell

Hyperpolarization-activated cyclic nucleotide-sensitive (HCN) channels mediate the I(f) current in heart and I(h) throughout the nervous system. In spiking neurons I(h) participates primarily in different forms of rhythmic activity. Little is known, however, about its role in neurons operating with graded potentials as in the retina, where all four channel isoforms are expressed. Intriguing evidence for an involvement of I(h) in early visual processing are the side effects reported, in dim light or darkness, by cardiac patients treated with HCN inhibitors. Moreover, electroretinographic recordings indicate that these drugs affect temporal processing in the outer retina. Here we analyzed the functional role of HCN channels in rod bipolar cells (RBCs) of the mouse. Perforated-patch recordings in the dark-adapted slice found that RBCs exhibit I(h), and that this is sensitive to the specific blocker ZD7288. RBC input impedance, explored by sinusoidal frequency-modulated current stimuli (0.1-30 Hz), displays band-pass behavior in the range of I(h) activation. Theoretical modeling and pharmacological blockade demonstrate that high-pass filtering of input signals by I(h), in combination with low-pass filtering by passive properties, fully accounts for this frequency-tuning. Correcting for the depolarization introduced by shunting through the pipette-membrane seal, leads to predict that in darkness I(h) is tonically active in RBCs and quickens their responses to dim light stimuli. Immunohistochemistry targeting candidate subunit isoforms HCN1-2, in combination with markers of RBCs (PKC) and rod-RBC synaptic contacts (bassoon, mGluR6, Kv1.3), suggests that RBCs express HCN2 on the tip of their dendrites. The functional properties conferred by I(h) onto RBCs may contribute to shape the retina's light response and explain the visual side effects of HCN inhibitors.     

Genetic basis for dosage sensitivity in Arabidopsis thaliana

Aneuploidy, the relative excess or deficiency of specific chromosome types, results in gene dosage imbalance. Plants can produce viable and fertile aneuploid individuals, while most animal aneuploids are inviable or developmentally abnormal. The swarms of aneuploid progeny produced by Arabidopsis triploids constitute an excellent model to investigate the mechanisms governing dosage sensitivity and aneuploid syndromes. Indeed, genotype alters the frequency of aneuploid types within these swarms. Recombinant inbred lines that were derived from a triploid hybrid segregated into diploid and tetraploid individuals. In these recombinant inbred lines, a single locus, which we call SENSITIVE TO DOSAGE IMBALANCE (SDI), exhibited segregation distortion in the tetraploid subpopulation only. Recent progress in quantitative genotyping now allows molecular karyotyping and genetic analysis of aneuploid populations. In this study, we investigated the causes of the ploidy-specific distortion at SDI. Allele frequency was distorted in the aneuploid swarms produced by the triploid hybrid. We developed a simple quantitative measure for aneuploidy lethality and using this measure demonstrated that distortion was greatest in the aneuploids facing the strongest viability selection. When triploids were crossed to euploids, the progeny, which lack severe aneuploids, exhibited no distortion at SDI. Genetic characterization of SDI in the aneuploid swarm identified a mechanism governing aneuploid survival, perhaps by buffering the effects of dosage imbalance. As such, SDI could increase the likelihood of retaining genomic rearrangements such as segmental duplications. Additionally, in species where triploids are fertile, aneuploid survival would facilitate gene flow between diploid and tetraploid populations via a triploid bridge and prevent polyploid speciation. Our results demonstrate that positional cloning of loci affecting traits in populations containing ploidy and chromosome number variants is now feasible using quantitative genotyping approaches.