The adhesion force of Notch with Delta and the rate of Notch signaling.

Notch signaling is repeatedly used during animal development to specify cell fates. Using atomic force microscopy on live cells, chemical inhibitors, and conventional analyses, we show that the rate of Notch signaling is linked to the adhesion force between cells expressing Notch receptors and Delta ligand. Both the Notch extracellular and intracellular domains are required for the high adhesion force with Delta. This high adhesion force is lost within minutes, primarily due to the action of Presenilin on Notch. Reduced turnover or Delta pulling accelerate this loss. These data suggest that strong adhesion between Notch and Delta might serve as a booster for initiating Notch signaling at a high rate.

XPS analysis of chemical functions at the surface of Bacillus subtilis.

The surface chemical composition of nine strains of Bacillus subtilis was determined by X-ray photoelectron spectroscopy. Regressions between elemental concentrations and concentrations associated with different components of C1s, N1s, and O1s peaks provided a more precise validation of the procedure used for peak decomposition and allowed the assignment of the peak components to be completed or strengthened. The component of the O1s peak appearing around 531.2 eV was shown to contain a contribution of oxygen from phosphate groups (PO, PO-), the other contribution being due to oxygen involved in amide functions. The surface negative charge may be fully attributed to phosphate groups, despite the observation of two types of zeta potential vs pH curves. The strains exhibiting a sharp variation of the zeta potential (range of -35 to -55 mV) between pH 2 and 4.7 were characterized by a high phosphate surface concentration and by an excess (about 25%) of phosphate with respect to the sum of potassium, an exchangeable cation, and protonated nitrogen, attributed to protein or to alanine involved in teichoic acids.

Delta activity independent of its activity as a ligand of Notch.

BACKGROUND: Delta, Notch, and Scabrous often function together to make different cell types and refine tissue patterns during Drosophila development. Delta is known as the ligand that triggers Notch receptor activity. Scabrous is known to bind Notch and promote Notch activity in response to Delta. It is not known if Scabrous binds Delta or Delta has activity other than its activity as a ligand of Notch. It is very difficult to clearly determine this binding or activity in vivo as all Notch, Delta, and Scabrous activities are required simultaneously or successively in an inter-dependent manner. RESULTS: Using Drosophila cultured cells we show that the full length Delta promotes accumulation of Daughterless protein, fringe RNA, and pangolin RNA in the absence of Scabrous or Notch. Scabrous binds Delta and suppresses this activity even though it increases the level of the Delta intracellular domain. We also show that Scabrous can promote Notch receptor activity, in the absence of Delta. CONCLUSION: Delta has activity that is independent of its activity as a ligand of Notch. Scabrous suppresses this Delta activity. Scabrous also promotes Notch activity that is dependent on Delta's ligand activity. Thus, Notch, Delta, and Scabrous might function in complex combinatorial or mutually exclusive interactions during development. The data reported here will be of significant help in understanding these interactions in vivo.

Aerosolization properties, surface composition and physical state of spray-dried protein powders.

Powder aerosols made of albumin, dipalmitoylphosphatidylcholine (DPPC) and a protein stabilizer (lactose, trehalose or mannitol) were prepared by spray-drying and analyzed for aerodynamic behavior, surface composition and physical state. The powders exited a Spinhaler inhaler as particle aggregates, the size of which depending on composition, spray-drying parameters and airflow rate. However, due to low bulk powder tap density (<0.15 g/cm3), the aerodynamic size of a large fraction of aggregates remained respirable (<5 microm). Fine particle fractions ranged between 21% and 41% in an Andersen cascade impactor operated at 28.3 l/min, with mannitol and lactose providing the most cohesive and free-flowing powders, respectively. Particle surface analysis by X-ray photoelectron spectroscopy (XPS) revealed a surface enrichment with DPPC relative to albumin for powders prepared under certain spray-drying conditions. DPPC self-organized in a gel phase in the particle and no sugar or mannitol crystals were detected by X-ray diffraction. Water sorption isotherms showed that albumin protected lactose from moisture-induced crystallization. In conclusion, a proper combination of composition and spray-drying parameters allowed to obtain dry powders with elevated fine particle fractions (FPFs) and a physical environment favorable to protein stability.

Biofilm cohesiveness measurement using a novel atomic force microscopy methodology.

Biofilms can be undesirable, as in those covering medical implants, and beneficial, such as when they are used for waste treatment. Because cohesive strength is a primary factor affecting the balance between growth and detachment, its quantification is essential in understanding, predicting, and modeling biofilm development. In this study, we developed a novel atomic force microscopy (AFM) method for reproducibly measuring, in situ, the cohesive energy levels of moist 1-day biofilms. The biofilm was grown from an undefined mixed culture taken from activated sludge. The volume of biofilm displaced and the corresponding frictional energy dissipated were determined as a function of biofilm depth, resulting in the calculation of the cohesive energy. Our results showed that cohesive energy increased with biofilm depth, from 0.10 +/- 0.07 nJ/microm(3) to 2.05 +/- 0.62 nJ/microm(3). This observation was reproducible, with four different biofilms showing the same behavior. Cohesive energy also increased from 0.10 +/- 0.07 nJ/microm(3) to 1.98 +/- 0.34 nJ/microm(3) when calcium (10 mM) was added to the reactor during biofilm cultivation. These results agree with previous reports on calcium increasing the cohesiveness of biofilms. This AFM-based technique can be performed with available off-the-shelf instrumentation. It could therefore be widely used to examine biofilm cohesion under a variety of conditions.

Effect of protein, polysaccharide, and oxygen concentration profiles on biofilm cohesiveness.

It is important to control biofilm cohesiveness to optimize process performance. In this study, a membrane-aerated biofilm reactor inoculated with activated sludge was used to grow mixed-culture biofilms of different ages and thicknesses. The cohesions, or cohesive energy levels per unit volume of biofilm, based on a reproducible method using atomic force microscopy (F. Ahimou, M. J. Semmens, P. J. Novak, and G. Haugstad, Appl. Environ. Microbiol. 73:2897-2904, 2007), were determined at different locations within the depths of the biofilms. In addition, the protein and polysaccharide concentrations within the biofilm depths, as well as the dissolved oxygen (DO) concentration profiles within the biofilms, were measured. It was found that biofilm cohesion increased with depth but not with age. Level of biofilm cohesive energy per unit volume was strongly correlated with biofilm polysaccharide concentration, which increased with depth in the membrane-aerated biofilm. In a 12-day-old biofilm, DO also increased with depth and may therefore be linked to polysaccharide production. In contrast, protein concentration was relatively constant within the biofilm and did not appear to influence cohesion.

The Notch amino terminus regulates protein levels and Delta-induced clustering of Drosophila Notch receptors.

Notch signaling is required for the development of almost all animal tissues. It is a cell surface receptor that generates intracellular signals in response to Delta binding its extracellular domain. Notch response to Delta is affected by mutations in its extracellular domain outside of the Delta binding region. One such region is the Notch amino terminus. Mutations in this region are associated with developmental defects. How a mutation in the Notch amino terminus affects Notch function is unknown. We explored this issue in Drosophila melanogaster. We report that Notch receptors mutated in the amino terminus accumulate to abnormal levels, are deficient in Delta induced receptor clustering, and exhibit reduced rate of internalization and signaling. Notch receptors lacking the whole or the carboxy-terminal half of the intracellular domain are defective in internalization but not in clustering or accumulation. None of the other mutated Notch receptors showed defects in clustering, accumulation, or internalization. These observations suggest that the Notch amino terminus regulates Notch levels and clustering, which could affect the rate of Notch signaling and down-regulation.

Real-time imaging of the surface topography of living yeast cells by atomic force microscopy.

Atomic force microscopy (AFM) was used to image the surface topography of living Saccharomyces cerevisiae cells at high resolution and to monitor enzyme digestion of the cell wall in real time. Apart from the presence of bud scars, the surface of native cells imaged in aqueous solution was homogeneous and smooth. Topographic images of the surface were recorded to a lateral resolution of 2 nm without significant modification of the surface morphology. Successive images of single cells were collected at fixed time intervals following addition of protease and amyloglucosidase solutions. Protease caused a progressive increase of surface roughness. Large depressions surrounded by protruding edges, approximately 50 nm in height, were formed and attributed to the erosion of the mannoprotein outer layer. By contrast, no modification of the cell surface was noted upon addition of amyloglucosidase, which was consistent with the cell wall biochemical composition. These results indicate that AFM is a complementary tool to electron microscopy in that it allows the surface of living cells to be explored directly in real time.