Laser-structured spike surface shows great bone integrative properties despite infection in vivo.

Implant associated infections can result in devastating consequences for patients. One major cause is the formation of bacterial biofilms, which result in increased resistance against antimicrobial therapeutics. A reduction of implant associated infections can be achieved by functionalization of implant surfaces. The generation of three dimensional surface structures by femtosecond laser ablation is one method to fabricate bacterial repellent large scaled surfaces without altering the material chemical composition. The challenge is to reduce bacterial growth while improving cellular ongrowth. For this purpose, spike structures were created as small as possible by used fabrication method on cubic Ti90/Al6/V4-rods and their effectiveness against bacterial colonization was compared to unstructured Ti90/Al6/V4-rods. Rods were implanted in the rat tibia and infected intraoperatively with 103 CFU of Staphylococcus aureus. Besides clinical behaviour and lameness, the vital bacterial biomass, morphological appearance and the volume of eukaryotic cells were determined on the implant surface after 21 days. Bone alterations were examined by radiological and histological techniques. Unexpectedly, the laser-structured implants did not show a lower bacterial load on the implant surface and less severe infection related bone and tissue alterations compared to the group without structuring. Simultaneously, a better bony integration and a higher cellular colonization with eukaryotic cells was detected on the laser-structured implants. These findings don't support the previous in vitro results. Nevertheless, the strong integration into the bone is a powerful argument for further surface modifications focussing on the improvement of the antibacterial effect. Additionally, our results underline the need for in vivo testing of new materials prior to clinical use.

Zidovudine-Mediated Autophagy Inhibition Enhances Mitochondrial Toxicity in Muscle Cells.

Nucleoside reverse transcriptase inhibitors (NRTI), such as zidovudine (AZT), are constituents of HIV-1 therapy and are used for the prevention of mother-to-child transmission. Prolonged thymidine analogue exposure has been associated with mitochondrial toxicities to heart, liver, and skeletal muscle. We hypothesized that the thymidine analogue AZT might interfere with autophagy in myocytes, a lysosomal degradation pathway implicated in the regulation of mitochondrial recycling, cell survival, and the pathogenesis of myodegenerative diseases. The impact of AZT and lamivudine (3TC) on C2C12 myocyte autophagy was studied using various methods based on LC3-green fluorescent protein overexpression or LC3 staining in combination with Western blotting, flow cytometry, and confocal and electron microscopy. Lysosomal and mitochondrial functions were studied using appropriate staining for lysosomal mass, acidity, cathepsin activity, as well as mitochondrial mass and membrane potential in combination with flow cytometry and confocal microscopy. AZT, but not 3TC, exerted a significant dose- and time-dependent inhibitory effect on late stages of autophagosome maturation, which was reversible upon mTOR inhibition. Inhibition of late autophagy at therapeutic drug concentrations led to dysfunctional mitochondrial accumulation with membrane hyperpolarization and increased reactive oxygen species (ROS) generation and, ultimately, compromised cell viability. These AZT effects could be readily replicated by pharmacological and genetic inhibition of myocyte autophagy and, most importantly, could be rescued by pharmacological stimulation of autophagolysosomal biogenesis. Our data suggest that the thymidine analogue AZT inhibits autophagy in myocytes, which in turn leads to the accumulation of dysfunctional mitochondria with increased ROS generation and compromised cell viability. This novel mechanism could contribute to our understanding of the long-term side effects of antiviral agents.

Development and Characterization of a Porcine Mitral Valve Scaffold for Tissue Engineering.

Decellularized scaffolds represent a promising alternative for mitral valve (MV) replacement. This work developed and characterized a protocol for the decellularization of whole MVs. Porcine MVs were decellularized with 0.5% (w/v) SDS and 0.5% (w/v) SD and sterilized with 0.1% (v/v) PAA. Decellularized samples were seeded with human foreskin fibroblasts and human adipose-derived stem cells to investigate cellular repopulation and infiltration, and with human colony-forming endothelial cells to investigate collagen IV formation. Histology revealed an acellular scaffold with a generally conserved histoarchitecture, but collagen IV loss. Following decellularization, no significant changes were observed in the hydroxyproline content, but there was a significant reduction in the glycosaminoglycan content. SEM/TEM analysis confirmed cellular removal and loss of some extracellular matrix components. Collagen and elastin were generally preserved. The endothelial cells produced newly formed collagen IV on the non-cytotoxic scaffold. The protocol produced acellular scaffolds with generally preserved histoarchitecture, biochemistry, and biomechanics.

The Caenorhabditis elegans GARP complex contains the conserved Vps51 subunit and is required to maintain lysosomal morphology.

In yeast the Golgi-associated retrograde protein (GARP) complex is required for tethering of endosome-derived transport vesicles to the late Golgi. It consists of four subunits--Vps51p, Vps52p, Vps53p, and Vps54p--and shares similarities with other multimeric tethering complexes, such as the conserved oligomeric Golgi (COG) and the exocyst complex. Here we report the functional characterization of the GARP complex in the nematode Caenorhabditis elegans. Furthermore, we identified the C. elegans Vps51 subunit, which is conserved in all eukaryotes. GARP mutants are viable but show lysosomal defects. We show that GARP subunits bind specific sets of Golgi SNAREs within the yeast two-hybrid system. This suggests that the C. elegans GARP complex also facilitates tethering as well as SNARE complex assembly at the Golgi. The GARP and COG tethering complexes may have overlapping functions for retrograde endosome-to-Golgi retrieval, since loss of both complexes leads to a synthetic lethal phenotype.

Polyphosphate at the Streptomyces lividans cytoplasmic membrane is enhanced in the presence of the potassium channel KcsA.

The distribution of polyphosphate (polyP) within the cytoplasmic membrane of Streptomyces lividans hyphae or protoplasts has been determined at high spatial resolution by elemental mapping using energy-filtered electron microscopy (EFTEM). The results revealed that polyP was best traceable after its interaction with lead ions followed by their precipitation as lead sulphide. Concomitant studies of the S.lividans wildtype (WT) strain and its co-embedded mutant DeltaK (lacking a functional kcsA gene) were conducted by labelling as the surface matrix of either one was labelled by cationic colloidal thorium dioxide. Within the WT strain, additional polyP was found to accumulate distinctly at the inner face of the cytoplasmic membrane. After removal of the cell wall (within protoplasts), the polyP-derived lead-sulphide (PbS) precipitate formed clusters of fibrillar material extending up to 50 nm into the cytoplasm. This feature was absent in the DeltaK mutant strain. Together the results revealed that the presence of the KcsA channel and the structured polyP coincide.

Defining the mycoplasma 'cytoskeleton': the protein composition of the Triton X-100 insoluble fraction of the bacterium Mycoplasma pneumoniae determined by 2-D gel electrophoresis and mass spectrometry.

After treating Mycoplasma pneumoniae cells with the nonionic detergent Triton X-100, an undefined, structured protein complex remains that is called the 'Triton X-100 insoluble fraction' or 'Triton shell'. By analogy with eukaryotic cells and supported by ultrastructural analyses it is supposed that this fraction contains the components of a bacterial cytoskeleton-like structure. In this study, the composition of the Triton X-100 insoluble fraction was defined by electron microscopic screening for possible structural elements, and by two-dimensional (2-D) gel electrophoresis and MS to identify the proteins present. Silver staining of 2-D gels revealed about 100 protein spots. By staining with colloidal Coomassie blue, about 50 protein spots were visualized, of which 41 were identified by determining the mass and partial sequence of tryptic peptides of individual proteins. The identified proteins belonged to several functional categories, mainly energy metabolism, translation and heat-shock response. In addition, lipoproteins were found and most of the proteins involved in cytadherence that were previously shown to be components of the Triton X-100 insoluble fraction. There were also 11 functionally unassigned proteins. Based on sequence-derived predictions, some of these might be potential candidates for structural components. Quantitatively, the most prevalent proteins were the heat-shock protein DnaK, elongation factor Tu and subunits alpha and beta of the pyruvate dehydrogenase complex (PdhA, PdhB), but definite conclusions regarding the composition of the observed structures can only be drawn after specific proteins are assigned to them, for example by immunocytochemistry.