Mapping the pathways to staphylococcal pathogenesis by comparative secretomics.

The gram-positive bacterium Staphylococcus aureus is a frequent component of the human microbial flora that can turn into a dangerous pathogen. As such, this organism is capable of infecting almost every tissue and organ system in the human body. It does so by actively exporting a variety of virulence factors to the cell surface and extracellular milieu. Upon reaching their respective destinations, these virulence factors have pivotal roles in the colonization and subversion of the human host. It is therefore of major importance to obtain a clear understanding of the protein transport pathways that are active in S. aureus. The present review aims to provide a state-of-the-art roadmap of staphylococcal secretomes, which include both protein transport pathways and the extracytoplasmic proteins of these organisms. Specifically, an overview is presented of the exported virulence factors, pathways for protein transport, signals for cellular protein retention or secretion, and the exoproteomes of different S. aureus isolates. The focus is on S. aureus, but comparisons with Staphylococcus epidermidis and other gram-positive bacteria, such as Bacillus subtilis, are included where appropriate. Importantly, the results of genomic and proteomic studies on S. aureus secretomes are integrated through a comparative "secretomics" approach, resulting in the first definition of the core and variant secretomes of this bacterium. While the core secretome seems to be largely employed for general housekeeping functions which are necessary to thrive in particular niches provided by the human host, the variant secretome seems to contain the "gadgets" that S. aureus needs to conquer these well-protected niches.

Endoscope disinfection and its pitfalls--requirement for retrograde surveillance cultures.

BACKGROUND AND STUDY AIMS: Several endoscopy-related outbreaks of infection have been reported in recent years. For early recognition of inadequate disinfection of endoscopes we designed a microbiological surveillance system to evaluate the efficacy of the cleaning and disinfection procedure, and to trace disinfection problems to individual endoscopes or washer-disinfectors. METHODS: Our surveillance protocol included anterograde and retrograde sampling, a decision algorithm, genetic fingerprinting, and scanning electron microscopy. RESULTS: Over a period of 29 months we found an increasing number of patient-ready endoscopes testing positive for Candida species other than albicans, especially C. parapsilosis. These yeasts were also isolated from the washer-disinfectors. The number of positive tests for Candida species varied from 1 out of 21 to 14 out of 27 samples from nine frequently used endoscopes. The number of colony-forming units per milliliter ranged from 1 - 10 to 3000 for endoscopes and 0.002 to 0.06 for the washer disinfectors. DNA fingerprinting was not able to discriminate different strains within C. parapsilosis. CONCLUSIONS: Our protocol was able to detect a structural problem in the endoscope disinfection process. Retrograde sampling was crucial for this purpose, because it has much higher sensitivity than anterograde sampling. Endoscopes with damaged working channels are probably the source of the contamination problem with Candida species.

Perovskites in the comb roof base of hornets: their possible function.

On the ceiling of the Oriental hornet comb cell, there are mineral granules of polycrystalline material known to belong to the group of perovskites. In a comb cell intended to house a worker hornet, the roof base usually carries one or several such perovskite granules containing titanium (Ti), whereas in the roof base of a cell housing a developing queen, there are usually several granules containing a high percentage of silicon (Si), aluminum (Al), calcium (Ca), and iron (Fe), but very little if any Ti. In worker comb cells, Ti usually appears as ilmenite (FeTiO3). Besides documenting the above-mentioned facts, this report discusses possible reasons for the appearance of ilmenite crystals in worker cells only and not in queen cells.

Electronmicroscopical evaluation of short-term nerve regeneration through a thin-walled biodegradable poly(DLLA-epsilon-CL) nerve guide filled with modified denatured muscle tissue.

The aim of this study was to evaluate short-term peripheral nerve regeneration across a 15-mm gap in the sciatic nerve of the rat, using a thin-walled biodegradable poly(DL-lactide-epsilon-caprolactone) nerve guide filled with modified denatured muscle tissue (MDMT). The evaluation was performed using transmission electron microscopy and morphometric analysis. Evaluation times ranged from 3 to 12 weeks after reconstruction. Already, 3 weeks after reconstruction, myelinated nerve fibers could be observed in the distal nerve stump. Twelve weeks after reconstruction, the number of (non)myelinated nerve fibers had significantly increased in the distal nerve stump. From this study, we can conclude that a thin-walled biodegradable poly(DL-lactide-epsilon-caprolactone) nerve guides filled with MDMT can be successfully applied in the reconstruction of severed nerves in the rat model. Furthermore, we showed fast nerve regeneration across the 15-mm nerve gap and found that the use of MDMT functioned as a mechanical support preventing a collapse of this thin-walled nerve guide.

Superhydrophobic modification fails to improve the performance of small diameter expanded polytetrafluoroethylene vascular grafts.

To determine whether superhydrophobic modification of small diameter expanded polytetrafluoroethylene (ePTFE) vascular grafts improves the performance of these grafts, we assessed neointima formation and platelet deposition in standard and superhydrophobic modified ePTFE grafts. Standard and superhydrophobic vascular grafts were implanted in the carotid arteries of two rabbits and two pigs. Furthermore, standard and superhydrophobic vascular patches were implanted in the carotid arteries of seven pigs. After 4 weeks of implantation all patches were removed and histomorphometric data were analyzed. The early thrombotic effect of superhydrophobic modification was examined by quantifying platelet glycoprotein receptor IIIa deposition onto each type of vascular graft after 15 min of in vitro circulation with human blood. All superhydrophobic and standard ePTFE vascular grafts occluded 15 min to 1 h after implantation in both rabbit and pig carotid arteries. All implanted patches remained patent and were completely covered by endothelium. Superhydrophobic modification of ePTFE vascular grafts did not lead to less neointima formation and resulted in significantly more platelet deposition than did standard ePTFE vascular grafts. Thus, superhydrophobic modification does not improve the performance of small diameter ePTFE vascular grafts.

Heterogeneity of surfaces of subgingival bacteria as detected by zeta potential measurements.

Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans (A.a.) are Gram-negative bacteria which are implicated in various forms of periodontal disease. The Gram-positive Peptostreptococcus micros may also play an important role. For investigation of the possible adhesion and colonization mechanisms of these organisms, the charge properties of the outermost layers of bacterial cell surfaces were studied through the measurement of zeta potentials at various pH values. Eleven fresh clinical isolates, representing the four species, and one laboratory strain, P. gingivalis W83, were examined. Eleven of the 12 strains displayed heterogeneity with respect to pH-dependent zeta potentials. Within single cultures of each of these strains, two distinct populations of cells were found, one which was more negatively charged than the other. For the Gram-negative strains, the more negatively charged subpopulation was in the majority, while the P. micros strains appeared to be composed mainly of a less-negatively-charged subpopulation. Vesicles prepared from two strains displayed the same pH dependence and heterogeneity of zeta potentials as the parent cells. An A.a. strain which was passaged several times in fluid medium had lost its fimbriae and became homogeneous with respect to charge.

A transmission electron microscopy study of the adsorption patterns of early developing artificial pellicles on human enamel.

Adsorption patterns of pellicles formed in a flow cell system under a moderate shear rate of 21 s-1, were studied with a replica technique. Characteristic features of the bare enamel surface disappeared within seconds after exposure to reconstituted saliva, indicating the adsorption of a homogeneous film. After that, three to four distinct homogeneous films were adsorbed on top of each other within 10 s and an uneven, knotted structure developed with stalky projections on the outer surface. This heterogeneous adsorption pattern determined the structure of the pellicle surface for at least 2 h.

A micro-Raman spectroscopic study of hydrazine-treated human dental calculus.

Hydrazine has been used to remove organic components and to isolate the mineral(s) from human calculus. Micro-Raman measurements were performed on the mineral phase. After the hydrazine-treatment, not only a large reduction in fluorescence but also an increase in Raman signal was observed. The treatment was essential in minimizing thermally-induced chemical changes which could otherwise occur to the original calculus mineral due to the intense laser light. The Raman spectral features of the mineral were nearly all identical among the Raman spectra obtained at many randomly-selected sites by the micro-Raman microbe with a lateral resolution of approximately 1 micron, and were consistent with those of impure hydroxyapatite containing CO3(2-) and HPO4(2-). The spectra contained typical hydroxyapatite bands including PO4(3-)bands of the v1, v2, v3 and v4 modes and one OH- stretch band. Other minor bands due to the CO3(2-) v1 and v3 modes and bands possibly due to the HPO4(2-) v1, v2 and v4 modes were observable by the technique despite the hydrazine-treatment that could in principle remove the HPO4 and CO3 ions from the mineral. In comparison with pure synthetic hydroxyapatite, the intensity of the OH- stretch band relative to that of the PO4(3-) v1 band was approximately 70% weaker, and the bandwidth of the phosphate v1 band was 200% broader, reflecting various crystal imperfections presumably present in the calculus mineral.

Combined Raman and SEM study on CaF2 formed on/in enamel by APF treatments.

Raman spectra containing the distinct band at 322 cm-1 due to CaF2 or CaF2-like material formed in/on fluoridated bovine enamel were recorded using a micro-Raman spectrograph. Due to increasing levels of background fluorescence with increasing thickness of enamel, the Raman measurements were carried out on thin regions of wedged enamel sections. The distribution of the CaF2 or CaF2-like material was estimated using a simple model. The results indicate that 1/3 of the total CaF2 was concentrated within the narrow depth < 2 microns with high CaF2 concentrations (> 10 wt%), and that the majority of the CaF2 was distributed over the depths up to 26 microns (1 wt% CaF2). SEM observations on fractured fluoridated enamel confirmed that morphological changes were present in the depth range comparable to that of the high CaF2 concentration region expected from the Raman analysis. In deeper regions where lower concentration (< 10%) but a large amount of CaF2 was still expected, the SEM images failed to distinguish between the normal and fluoridated enamel. After KOH treatment, the Raman spectra did not show evidence of the CaF2 peak and the SEM micrographs also confirmed the removal of globules. The peak position of the Raman band of the CaF2 formed by the fluoridation was identical to that of pure CaF2. However, the linewidth was 23 cm-1 (FWHM) and a factor of 2 broader than that of pure CaF2 (12 cm-1). This implies that the lattice dynamics of the CaF2 formed by fluoridation is different from of pure CaF2, and that the material formed is 'CaF2-like' or 'disordered CaF2'.

The diameter of dentinal tubules in human coronal dentine after demineralization and air drying. A combined light microscopy and SEM study.

The diameter of dentinal tubules after demineralization and/or air drying has been quantified using light microscopy and scanning electron microscopy (SEM). The tubule diameter was assessed at a distance of about 1.5 mm (range 1.0-2.0 mm) from the pulp. Tubule diameter was measured in the wet state, after 10-min and 12-hour air drying as well as after critical point drying (CO2) by SEM. The results show that drying effects on tubule diameter are small in sound tissue, but are sizeable in demineralized dentine. Comparing light microscopic (wet) and SEM observations show that the differences in tubule diameter are small for sound but substantial for demineralized dentine. An important result is that in the wet state the diameters of the tubules (being 1.3 +/- 0.2 microns in sound dentine) are 2.5 +/- 0.3, 2.2 +/- 0.3 and 1.7 +/- 0.2 microns after 1, 2 and 3 weeks of demineralization, respectively. The decreased tubule diameter with increasing demineralization may be important for permeability and transport phenomena in dentine caries and presumably in hypersensitivity.

The effects of glycol methacrylate as a dehydrating agent on the dimensional changes of liver tissue.

The dimensional changes of liver sections during the course of processing with glycol methacrylate (GMA) or with ethanol are described. Tissue processing with ethanol served as a control. During prolonged processing steps (24 h each), linear shrinkage of tissue specimens dehydrated with GMA at room temperature was 13.2%. Subsequent infiltration with GMA resulted in trivial swelling, and polymerization in slight shrinkage (2.3%). In comparison, processing with cold GMA resulted in shrinkage during dehydration (about 10.8%), a slight swelling in pure GMA, followed by shrinkage during polymerization (2.2%). Short routine processing schedules resulted in similar shrinkage/swelling patterns, although precise values differed slightly. In all experiments, ethanolic dehydration resulted in smaller dimensional tissue changes than did GMA dehydration. The dimensional changes of tissue sections during stretching on water, mounting and drying compensated for the major part of the shrinkage manifested during processing.

Influence of radial TEM distortion on the accuracy of lattice parameter determinations of apatites: a comparison of four electron microscopes.

Lattice fringe distances of synthetic hydroxyapatite were measured quantitatively in four different TEM microscopes as a function of the position with respect to the optical axis. Using the accurately known "a" value of this apatite from X-ray diffraction, the maximum radial distortion in the image planes can be determined in various positions. For the Philips EM 400, EM 300 (1) and EM 300 (2) and JEOL 200 CX the maximum radial distortion was found to be 1.0%, 0.8%, 1.5% and 0.5%, respectively. In accurate lattice parameter determinations of mineral and mineral phases in biological tissues by means of TEM; this radial distortion is an important parameter to take into account.

Quantitative cement solubility experiments in vivo.

The dissolution and dissolution rate of dental luting cements was measured longitudinally. Cement samples in enamel were placed in a full prosthesis and worn in the mouth for up to 6 months. At given intervals, replicas of the cement surfaces were made and quantified by means of SEM stereographic photographs. With a stereometer the distance between cement surface and original enamel surface can be determined quantitatively. The results show in vivo solubility rates for zinc-phosphate and glass-ionomer cement of about 80 microns week-1 and 2 microns week-1 respectively.

Morphology and origin of arachnoid cysts. Scanning and transmission electron microscopy of three cases.

Three surgically removed primary arachnoidal cysts were studied with scanning electron microscopy (SEM) and two of the cases with transmission electron microscopy (TEM). The cells lining the cyst cavity had microvilli at the surface, true cilia were absent. In the cytoplasm multivesicular bodies, many pinocytotic vesicles, some large vacuoles and strands of tonofilaments were prominent features. The cells were interconnected by desmosome-like junctions and were separated from the surrounding tissue by a distinct but sometimes incomplete basal membrane. Based on these findings it is concluded that arachnoid cysts are derived from the outer arachnoid cells (subdural neurothelium), the formation of the cysts being attributabel to secretory capacity of the subdural neurothelium.

A comparative study of thin coatings of Au/Pd, Pt and Cr produced by magnetron sputtering for FE-SEM.

Visualization of structural details of specimens in field emission scanning electron microscopy (FE-SEM) requires optimal conductivity. This paper reports on the differences in conductive layers of Au/Pd, Pt and Cr, with a thickness of 1.5­3.0 nm, deposited by planar magnetron sputtering devices. The coating units were used under standard conditions for source­substrate distance, current, HT and argon pressure. Carbon films, deposited by high-vacuum evaporation on small, freshly cleaved pieces of mica, were used as substrate and mounted on copper grids for TEM and SEM inspection. Au/Pd, Pt and, to a lesser extent, Cr coatings varied in particle density, size and shape. Au/Pd coatings have a slightly more granular appearance than Cr and Pt coatings, but this is strongly dependent on the type of sputtering device employed. In FE-SEM images there is almost no difference in contrast and particle size between the Au/Pd layer and the Pt layers of a similar thickness. The nuclei of Au/Pd are rather small with almost no growth to the sides or in height, making Au/Pd coatings a good alternative to chromium and platinum for FE-SEM of biological tissues because of its higher yield of secondary electrons.

The remineralization of EDTA-treated human dentine.

The remineralization of whole human dentine treated with a neutral EDTA solution was investigated: the treatment periods were 15, 30 and 120 min. From the literature it is known that EDTA removes noncollagenous proteins (NCPs) from dentine powders. In order to extract more phosphoproteins from dentine lesions, in part of this work samples were also treated with 1 M NaCl or 4 M guanidine chloride solutions. All the dentine samples after the treatments mentioned were immersed subsequently in a remineralizing solution without fluoride for 2 weeks and microradiographed. To investigate the effects of fluoride, samples treated with EDTA for 120 min were also remineralized with 2 ppm fluoride in solution. The results presented show that: (1) Measurable remineralization did not occur when fluoride was free in the remineralizing solution. Because remineralization occurred neither at the lesion front nor in the nonmineral part of the surface-softened tissue, presumably the NCPs (inhibitors) of underlying dentine diffused into the tissue during the remineralization period. (2) 2 ppm fluoride caused remineralization at the lesion front. In this case we assume fluoride either acted as nucleating agent or overcame the NCP effects at the lesion front.

Magnetic minerals. Keystone-like crystals in cells of hornet combs.

The hexagonal brood-rearing cells inside the nest combs of the hornet Vespa orientalis are uniform in both their architecture and orientation. We have discovered that each cell contains a minute crystal that projects down from the centre of its domed roof and has a composition typical of the magnetic mineral ilmenite. These tiny crystals form a network that may act like a surveyor's spirit-level, helping the hornets to assess the symmetry and balance of the cells and the direction of gravity while they are building the comb.

Changes of ventricular ependyma and choroid plexus in experimental hydrocephalus, as observed by scanning electron microscopy.

Hydrocephalus was induced in rats by the injection of silicone oil or kaolin suspension into the cisterna magna. One to 5 weeks later the walls of the lateral ventricles were studied with the scanning electron microscope after killing the animals by perfusion fixation. In contrast to controls, the hydrocephalic animals killed 1 or 2 weeks after injection showed degeneration of ependymal cilia and infestation of the ependymal and choroid plexus surface with reactive cells, which presumably may be identified as Kolmer phagocytic cells by their ultrastructural features as studied by the transmission electron microscope. A coating of debris on the surface of the choroid plexus in the hydrocephalic animals possibly bears upon the ciliary degeneration with consequent deficiency of the clearing effect of ciliary movement. In the longer surviving hydrocephalic animals regeneration of cilia seemed to have occurred.

Non-coating fixation techniques or redundancy of conductive coating, low kV FE-SEM operation and combined SEM/TEM of biological tissues.

Non-coating fixation methods, in particular the tannic acid/arginine/osmium tetroxide procedure, are employed for a number of reasons on the guinea-pig organ of Corti hair cell stereocilia glycocalyx and the imprints of the stereocilia at the bottom side of the tectorial membrane, and on the rat and cat intestinal epithelial microvilli glycocalyx and mucus-producing goblet cells. These methods are used firstly to confirm that non-coating prepared specimens can be embedded for TEM observation at 60-100 kV without loss of detail information, and these images can be compared with cryo-FE-SEM images of the same structure/tissue. Secondly, they show that specimens treated according non-coating techniques become optimally preserved and electrically conductive, so that no external conductive coating is required. In this way a comparison of images of subsequent fresh fracture faces is possible without a decrease in information on detail, which otherwise could happen after subsequent coating layers required after standard fixation. Thirdly, they show that non-coating methods can be used quite well with low accelerating voltages because the osmium-tannic acid complex in the specimen surface produces a large number of backscattered and secondary electrons in the surface layer, showing in particular surface phenomena. Fourthly, they show that with an optimal non-coating preservation, in combination with a well-balanced pre-fixation mixture, preparation artefacts due to extraction and even dehydration and drying are minimized. This is compared with images of the organ of Corti hair cells treated with a so-called three-aldehyde pre-fixation mixture, which causes disrupted stereocilia to cling onto the bottom side of the tectorial membrane.

Amount and surface structure of albumin adsorbed to solid substrata with different wettabilities in a parallel plate flow cell.

In this article we studied the adsorption of serum albumin to substrata with a broad range of wettabilities from solutions with protein concentrations between 0.03 and 3.00 mg.mL-1 in a parallel-plate flow cell. Wall shear rates were varied between 20 and 2000 s-1. The amount of albumin adsorbed in a stationary state was always highest on PTFE, the most hydrophobic material employed and decreased with increasing wettability of the substrata. Increasing stationary amounts of adsorbed albumin were observed with increasing wall shear rates at the lowest protein concentration. Inverse observations were made at the highest protein concentration. Transmission electron micrographs of replicas from the albumin-coated substrata showed that proteins were mostly adsorbed in islandlike structures on the hydrophobic substrata. The tendency to form islandlike structures was shear rate- and concentration-dependent and disappeared gradually going to more hydrophilic substrata. On glass, the most hydrophilic material employed, a homogeneous, well distributed, fine knotted, reticulated structure was found. In conclusion, this study demonstrates that both the amount of adsorbed albumin as well as the surface structure of the adsorbed proteins are regulated by the substratum wettability. This observation may well account for the fact that substratum properties can be transferred by an adsorbed protein film to the interface with adhering cells or microorganisms.