Frank Steiniger

Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation

Bacteria have evolved elaborate communication strategies to co‐ordinate their group activities, a process termed quorum sensing (QS). Pseudomonas aeruginosa is an opportunistic pathogen that utilizes QS for diverse activities, including disease pathogenesis. P. aeruginosa has evolved a novel communication system in which the signal molecule 2‐heptyl‐3‐hydroxy‐4‐quinolone (Pseudomonas Quinolone Signal, PQS) is trafficked between cells via membrane vesicles (MVs). Not only is PQS packaged into MVs, it is required for MV formation. Although MVs are involved in important biological processes aside from signalling, the molecular mechanism of MV formation is unknown. To provide insight into the molecular mechanism of MV formation, we examined the interaction of PQS with bacterial lipids. Here, we show that PQS interacts strongly with the acyl chains and 4′‐phosphate of bacterial lipopolysaccharide (LPS). Using PQS derivatives, we demonstrate that the alkyl side‐chain and third position hydroxyl of PQS are critical for these interactions. Finally, we show that PQS stimulated purified LPS to form liposome‐like structures. These studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non‐signalling functions.

Lecithin-Based Novel Cationic Nanocarriers (Leciplex) II: Improving Therapeutic Efficacy of Quercetin on Oral Administration†

The objective of the present investigation was to evaluate ability of the novel self-assembled phospholipid- based cationic nanocarriers (LeciPlex) in improving the therapeutic efficacy of a poorly water-soluble natural polyphenolic agent, quercetin (QR), on oral administration. Quercetin loaded LeciPlex (QR-LeciPlex) were successfully fabricated using a biocompatible solvent Transcutol HP. The QR-LeciPlex were characterized for particle size, encapsulation efficiency, zeta potential, and particle morphology by cryo-TEM. UV and fluorescence spectral characterization was carried out to find out the association of QR with LeciPlex. Small angle neutron scattering studies (SANS) were carried out to understand the internal structure of Leciplex and to evaluate the influence of the incorporation of QR in the LeciPlex. Anti-inflammatory and antitumorigenic activity of QR-LeciPlex was determined in comparison to QR suspension to evaluate the potential of LeciPlex in improving oral delivery of QR. QR-LeciPlex exhibited a particle size of ∼400 nm and had excellent colloidal stability. The QR-LeciPlex had a zeta potential greater than +30 mV and exhibited very high encapsulation efficiency of QR (>90%). UV and fluorescence spectral characterization indicated the interaction/association of QR with LeciPlex components. Cryo-TEM studies showed that LeciPlex and QR-LeciPlex have a unilamellar structure. SANS confirmed the unilamellar structure of LeciPlex and indicated that the incorporation of QR does not have any effect on the internal structure of the LeciPlex. QR-LeciPlex exhibited significantly higher anti-inflammatory and antitumorigenic activity (p < 0.01) as compared to that of QR suspension on oral administration.

Belt-like localisation of caveolin in deep caveolae and its re-distribution after cholesterol depletion

Caveolae are specialised vesicular microdomains of the plasma membrane. Using freeze-fracture immunogold labelling and stereoscopic imaging, the distribution of labelled caveolin 1 in caveolae of 3T3-L1 mouse fibroblast cells was shown. Immunogold-labelled caveolin structures surrounded the basolateral region of deeply invaginated caveolae like a belt whereas in the apical region distal to the plasma membrane, the caveolin labelling was nearly absent. Shallow caveolar membranes showed a dispersed caveolin labelling. After membrane cholesterol reduction by methyl-ß-cyclodextrin treatment, a dynamic re-distribution of labelled caveolin 1 and a flattening of caveolar structures was found. The highly curved caveolar membrane got totally flat, and the initial belt-like caveolin labelling disintegrated to a ring-like structure and later to a dispersed order. Intramembrane particle-free domains were still observable after cholesterol depletion and caveolin re-distribution. These results indicate that cholesterol interacting with caveolin structures at the basolateral part of caveolae is necessary for the maintenance of the deeply invaginated caveolar membranes.

Spontaneous crowding of ribosomes and proteins inside vesicles: a possible mechanism for the origin of cell metabolism.

One of the open questions in the origin of life is the spontaneous formation of primitive cell‐like compartments from free molecules in solution and membranes. “Metabolism‐first” and “replicator‐first” theories claim that early catalytic cycles first evolved in solution, and became encapsulated inside lipid vesicles later on. “Compartment‐first” theories suggest that metabolism progressively occurred inside compartments. Both views have some weaknesses: the low probability of co‐entrapment of several compounds inside the same compartment, and the need to control nutrient uptake and waste release, respectively. By using lipid vesicles as early‐cell models, we show that ribosomes, proteins and lipids spontaneously self‐organise into cell‐like compartments to achieve high internal concentrations, even when starting from dilute solutions. These findings suggest that the assembly of cell‐like compartments, despite its low probability of occurrence, is indeed a physically realistic process. The spontaneous achievement of high local concentration might provide a rational account for the origin of primitive cellular metabolism.

Bacterial invasion of dentinal tubules beneath apparently intact but hypomineralized enamel in molar teeth with molar incisor hypomineralization

BACKGROUND The most common problems for a patient with molar incisor hypomineralization (MIH) are the collapse of enamel and cavitations, loss of fillings, and secondary caries, but most of all, severe hypersensitivity. OBJECTIVE The aim of this paper was therefore to histologically study possible bacterial invasion of dentinal tubules beneath apparently intact, but hypomineralized enamel in permanent molars with MIH. MATERIAL AND METHODS Five extracted permanent first molars diagnosed with MIH were fixated, demineralized, and sagittally serially sectioned in a bucco-lingual direction in a microtome with a thickness of 4-5 microm. Sections were stained with a modified Brown and Benn staining for bacteria, unstained sections were analysed in field emission SEM. RESULTS Stained sections from the cuspal areas, below the hypomineralized enamel, the staining indicated the presence of bacteria in the dentinal tubules. The HTX staining showed that the pulp in sections without any findings was normal and free from bacteria or infiltrates from inflammatory cells. In sections where bacteria were found in the cuspal areas or deeper in the dentin, a zone of reparative dentin was found, and in sections from one tooth, the coronal pulp showed an inflammatory reaction with inflammatory cells. In sections adjacent to those without any bacterial staining, the SEM analyses revealed empty dentinal tubules without any odontoblast processes or signs of bacteria. When odontoblast processes were found, the dentinal tubules were filled with bacteria located on the surface of the odontoblast processes. In some areas, a large number of tubules were found with bacteria. No bacteria were found close to the pulp. The odontoblast processes appeared larger in areas where bacteria were found. CONCLUSIONS The presence of bacteria in the dentinal tubules and inflammatory reactions in the pulp indicate that oral bacteria may penetrate through the hypomineralized enamel into the dentin, thus possibly contribute to hypersensitivity of teeth with MIH.

Neonatal lines in the enamel of primary teeth—A morphological and scanning electron microscopic investigation

OBJECTIVE The neonatal line (NNL) is in principle found in all primary teeth and the line represents the time of birth. Earlier findings of the appearance of the NNL in light microscope and in microradiographs have shown not only changes in the prism direction of the enamel, but that the NNL has a hypomineralized character. METHODS The neonatal line was analyzed in un-decalcified sections of primary lower and central incisors, collected from individuals of different ages utilizing polarized light microscopy, microradiography, scanning electron microscopy (SEM) and X-ray analysis (XRMA). RESULTS In polarized light the NNL appeared to have a more porous structure than the enamel in general. The appearance of the NNL as a dark line in microradiographs is interpreted as the NNL being less mineralized than neighbouring enamel. Analysis with ImageJ visualized the reduction of the amount of grey value, indicating that the NNL is less mineralized. Analysis of the NNL in SEM showed a reduction of the diameter of enamel prisms, the more narrow diameters continued through the postnatal enamel. A change of the growth direction of the prisms was also observed at the NNL. In a three-dimensional image the NNL appeared as a grove, however, in non-etched enamel no grove was seen. The elemental analyses with XRMA showed no marked changes in the content of C, Ca, P, N, O or S in the area around the NNL. CONCLUSIONS The NNL is an optical phenomenon due to alterations in height, and degree of mineralization of the enamel prisms.

The shape of caveolae is omega-like after glutaraldehyde fixation and cup-like after cryofixation

Caveolae were defined as flask- or omega-shaped plasma membrane invaginations, abundant in adipocytes, fibroblasts, endothelial and smooth muscle cells. The major protein component of caveolar membranes is an integral membrane protein named caveolin. We compared the freeze-fracture behavior of caveolae in glutaraldehyde-fixed and cryofixed mouse fibroblast cells and found distinct differences. In glutaraldehyde-fixed cells almost all caveolae were cross-fractured through their pore and only very few caveolar membranes were membrane-fractured. We found the reverse situation in rapid frozen cells without any chemical fixation where most of the caveolae were membrane-fractured, showing different degrees of invagination from nearly flat to deeply invaginated. In ultrathin sections of glutaraldehyde-fixed heart endothelial cells, caveolae exhibit the well known omega-like shape. In high-pressure frozen, freeze-substituted and low temperature embedded heart endothelial cells, the caveolae frequently exhibit a cup-like shape without any constriction or pore. The cup-like caveolar shape could also be shown by tilt series analysis of freeze-fracture replicas obtained from cryofixed cells. Freeze-fracture immunolabeling of caveolin-1 revealed a lateral belt-like caveolin alignment. These findings point out that the constricted “neck” region of caveolae in most cases is an effect that is caused and intensified by the glutaraldehyde fixation. Our data indicate that caveolae in vivo show all degrees of invagination from nearly flat via cup-like depressed to in a few cases omega-like.

Magnetic and fluorescent core-shell nanoparticles for ratiometric pH sensing.

This paper describes the preparation of nanoparticles composed of a magnetic core surrounded by two successive silica shells embedding two fluorophores, showing uniform nanoparticle size (50-60 nm in diameter) and shape, which allow ratiometric pH measurements in the pH range 5-8. Uncoated iron oxide magnetic nanoparticles (∼10 nm in diameter) were formed by the coprecipitation reaction of ferrous and ferric salts. Then, they were added to a water-in-oil microemulsion where the hydrophilic silica shells were obtained through hydrolysis and condensation of tetraethoxyorthosilicate together with the corresponding silylated dye derivatives-a sulforhodamine was embedded in the inner silica shell and used as the reference dye while a pH-sensitive fluorescein was incorporated in the outer shell as the pH indicator. The magnetic nanoparticles were characterized using vibrating sample magnetometry, dynamic light scattering, transmission electron microscopy, x-ray diffraction and Fourier transform infrared spectroscopy. The relationship between the analytical parameter, that is, the ratio of fluorescence between the sensing and reference dyes versus the pH was adjusted to a sigmoidal fit using a Boltzmann type equation giving an apparent pK(a) value of 6.8. The fluorescence intensity of the reference dye did not change significantly (∼3.0%) on modifying the pH of the nanoparticle dispersion. Finally, the proposed method was statistically validated against a reference procedure using samples of water and physiological buffer with 2% of horse serum, indicating that there are no significant statistical differences at a 95% confidence level.

The physical state of lipid nanoparticles influences their effect on in vitro cell viability

Although lipid nanoparticles represent potent drug carriers, for many formulations toxicity data are rare. Thus, in this study, the effect of different lipid nanoparticles on the cell viability of L929 mouse fibroblasts was systematically investigated using the MTT assay. The formulations were composed of trimyristin, tristearin or cholesteryl myristate stabilized with poloxamer 188, polysorbate 80, polyvinyl alcohol or a blend of soybean phospholipid and sodium glycocholate. Depending on lipid and storage conditions, the nanoparticles were prepared in different physical states or crystal modifications leading to different particle shapes. The cell viability was influenced considerably by the physical state of the particle matrix with crystalline nanoparticles causing a stronger decrease in viability than the corresponding liquid or liquid crystalline particles. Effects on the cell viability were also related to the type of matrix lipid, stabilizer and the particle shape. However, the effects of differently shaped particles of different polymorphic modifications of crystalline tristearin were comparable. The low viability caused by poloxamer 188-stabilized particles could be correlated with a strong cell uptake which was investigated by confocal laser scanning microscopy.

Morphological aspects of dental hard tissues in primary teeth from preterm infants.

BACKGROUND Preterm children with very low birth weight suffer from several neonatal and post-natal complications that may affect the mineralization of the teeth. Clinical studies have shown enamel aberrations in both dentitions. AIMS The aims of this study were to describe enamel histo-morphology in primary teeth, and investigate the relationship between medical history and morphological appearance. DESIGN Dental enamels in 44 exfoliated primary teeth, from 14 children with a gestational age below 29 weeks and with a very low birth weight, were investigated, using polarized light microscopy (POLMI) and under a scanning electron microscope (SEM). RESULTS The neonatal line was found in 1/3 of the sections located coronally of the crown. In the post-natal enamel, 31 teeth showed a degree of porosity higher than 5% with a varying extension. More than half of the teeth showed one or more increment lines. The SEM analysis confirmed the POLMI findings with irregular prisms covered with a structure-less film. CONCLUSIONS Enamel from primary teeth of preterm children was found to have a high frequency of mineralization disturbances found in POLMI and SEM. The morphological features of the enamel from preterm children do not reflect the disturbances on general growth and development occurred during the neonatal period.