Bioactive molecules for regenerative pulp capping.

Since the discovery of bioactive molecules sequestered in dentine, researchers have been exploring ways to harness their activities for dental regeneration. One specific area, discussed in this review, is that of dental-pulp capping. Dental-pulp caps are placed when the dental pulp is exposed due to decay or trauma in an attempt to enhance tertiary dentine deposition. Several materials are used for dental-pulp capping; however, natural biomimetic scaffolds may offer advantages over manufactured materials such as improved aesthetic, biocompatibility and success rate. The present review discusses and appraises the current evidence surrounding biomimetic dental-pulp capping, with a focus on bioactive molecules sequestered in dentine. Molecules covered most extensively in the literature include transforming growth factors (TGF-ßs, specifically TGF-ß1) and bone morphogenetic proteins (BMPs, specifically BMP-2 and BMP-7). Further studies would need to explore the synergistic use of multiple peptides together with the development of a tailored scaffold carrier. The roles of some of the molecules identified in dentine need to be explored before they can be considered as potential bioactive molecules in a biomimetic scaffold for dental-pulp capping. Future in vivo work needs to consider the inflammatory environment of the dental pulp in pulpal exposures and compare pulp-capping materials.

Patchiness of ion-exchanged mica revealed by DNA binding dynamics at short length scales.

The binding of double-stranded (ds) DNA to mica can be controlled through ion-exchanging the mica with divalent cations. Measurements of the end-to-end distance of linear DNA molecules discriminate whether the binding mechanism occurs through 2D surface equilibration or kinetic trapping. A range of linear dsDNA fragments have been used to investigate length dependences of binding. Mica, ion-exchanged with Ni(II) usually gives rise to kinetically trapped DNA molecules, however, short linear fragments (<800 bp) are seen to deviate from the expected behaviour. This indicates that ion-exchanged mica is heterogeneous, and contains patches or domains, separating different ionic species. These results correlate with imaging of dsDNA under aqueous buffer on Ni(II)-mica and indicate that binding domains are of the order of 100 nm in diameter. Shorter DNA fragments behave intermediate to the two extreme cases of 2D equilibration and kinetic trapping. Increasing the incubation time of Ni(II) on mica, from minutes to hours, brings the conformations of the shorter DNA fragments closer to the theoretical value for kinetic trapping, indicating that long timescale kinetics play a role in ion-exchange. X-ray photoelectron spectroscopy (XPS) was used to confirm that the relative abundance of Ni(II) ions on the mica surface increases with time. These findings can be used to enhance spatial control of binding of DNA to inorganic surfaces with a view to patterning high densities arrays.

Improvement of the pore trapping method to immobilize vital coccoid bacteria for high-resolution AFM: a study of Staphylococcus aureus.

Preparation of vital bacteria for atomic force microscope study under aqueous fluid, such as physiological buffer or bacterial growth medium, presents challenges as cells will often desorb from the supporting surface or be dislodged by the atomic force microscope tip during imaging. An established method of immobilizing coccoid bacteria is to trap cells in polycarbonate track etched filter pores. We have significantly improved this method by modifying the pore diameter of commercially available filters to correspond to the diameter of the target strain, enabling high-resolution imaging of stationary organisms under buffer and dividing organisms under growth media.

RepD-mediated recruitment of PcrA helicase at the Staphylococcus aureus pC221 plasmid replication origin, oriD.

Plasmid encoded replication initiation (Rep) proteins recruit host helicases to plasmid replication origins. Previously, we showed that RepD recruits directionally the PcrA helicase to the pC221 oriD, remains associated with it, and increases its processivity during plasmid unwinding. Here we show that RepD forms a complex extending upstream and downstream of the core oriD. Binding of RepD causes remodelling of a region upstream from the core oriD forming a 'landing pad' for the PcrA. PcrA is recruited by this extended RepD-DNA complex via an interaction with RepD at this upstream site. PcrA appears to have weak affinity for this region even in the absence of RepD. Upon binding of ADPNP (non-hydrolysable analogue of ATP), by PcrA, a conformational rearrangement of the RepD-PcrA-ATP initiation complex confines it strictly within the boundaries of the core oriD. We conclude that RepD-mediated recruitment of PcrA at oriD is a three step process. First, an extended RepD-oriD complex includes a region upstream from the core oriD; second, the PcrA is recruited to this upstream region and thirdly upon ATP-binding PcrA relocates within the core oriD.

Imaging the substructure of antibodies with tapping-mode AFM in air: the importance of a water layer on mica.

Monoclonal antibodies (immunoglobulin G; IgG) against the N-terminal domain of the A subunit of DNA gyrase have been imaged using tapping-mode atomic force microscopy under ambient conditions on hydrophilic mica surfaces. The familiar tri-nodal submolecular resolution of IgG (i.e. 50-kDa resolution) has been achieved when operating the microscope with the tip predominantly in the attractive force regime. Under common laboratory conditions of about 40% relative humidity, the resolution of this substructure was not achieved owing to motion of the antibodies on the surface and/or image distortion from tip-sample instabilities. Reproducible imaging of the tri-nodal antibody substructure was achieved by desiccating the samples for extended periods of time (1 week or more) before imaging. This effect is attributed to the presence of a humidity-dependent thin water layer (a few molecules or nanometres thick), which has been observed previously using the surface force apparatus and scanning polarization force microscopy. Desiccation of the mica surfaces allowed enough water to be removed from the mica surface to prevent this effect. Degradation in the image quality over the imaging period of an hour or two was observed, owing to re-adsorption of water under the ambient laboratory conditions.

Beta(2)-microglobulin and its deamidated variant, N17D form amyloid fibrils with a range of morphologies in vitro.

Amyloid fibrils formed by incubation of recombinant wild-type human beta(2)-microglobulin (beta(2)M) ab initio in vitro at low pH and high ionic strength are short and highly curved. By contrast, fibrils extracted from patients suffering from haemodialysis-related amyloidosis and those formed by seeding growth of the wild-type protein in vitro with fibrils ex vivo are longer and straighter than those previously produced ab initio in vitro. Here we explore the effect of growth conditions on morphology of beta(2)M fibrils formed ab initio in vitro from the wild-type protein, as well as a variant form of beta(2)M in which Asn17 is deamidated to Asp (N17D). We show that deamidation results in significant destabilisation of beta(2)M at neutral pH. Despite this, acidification is still necessary to form amyloid from the mutant protein in vitro. Interestingly, at low pH and low ionic strength long, straight fibrils of recombinant beta(2)M are formed in vitro. The fibrils comprise three distinct morphological types when examined using electron microscopy (EM) and atomic force microscopy (AFM) that vary in periodicity and the number of constituent protofibrils. Using kinetic experiments we suggest that the immature fibrils observed previously do not represent intermediates in the assembly of fully mature amyloid, at least under the conditions studied here.

Micromechanical and structural properties of a pennate diatom investigated by atomic force microscopy.

The mechanisms behind natural nanofabrication of highly structured silicas are increasingly being investigated. We have explored the use of a standard Nanoscope III Multimode atomic force microscope (AFM) to study the silica shell of diatoms. The delicate structures of the shell surface of the diatom Navicula pelliculosa (Bréb.) Hilse were imaged and the shell's micromechanical properties were measured semi-quantitatively with a resolution down to approximately 10 nm. The technique to measure elasticity and hardness with the AFM was demonstrated to be useable even on these hard glass-like surfaces. Different experimental configurations and evaluation methods were tested. They gave a consistent result of the shell micromechanical properties. The first results showed that the diatom shell's overall hardness and elasticity was similar to that of known silicas. However, regions with different mechanical properties were distinguished. The elastic modulus varied from 7 to 20 GPa, from 20 to 100 GPa and from 30 to hundreds of GPa depending on the location. In general, the hardness measurements showed similar spatial differences. The hardness values ranged from 1 to 12 GPa but one specific part of the shell was even harder. Hence, certain localized regions of the shell were significantly harder or more elastic. These regions coincide with known characteristic features and mechanisms appearing at the different stages of the shell's growth. These results show that this method serves as a complementary tool in the study of silica biomineralization, and can detect eventual crystalline phases.

Direct observation of one-dimensional diffusion and transcription by Escherichia coli RNA polymerase.

The dynamics of nonspecific and specific Escherichia coli RNA polymerase (RNAP)-DNA complexes have been directly observed using scanning force microscopy operating in buffer. To this end, imaging conditions had to be found in which DNA molecules were adsorbed onto mica strongly enough to be imaged, but loosely enough to be able to diffuse on the surface. In sequential images of nonspecific complexes, RNAP was seen to slide along DNA, performing a one-dimensional random walk. Heparin, a substance known to disrupt nonspecific RNAP-DNA interactions, prevented sliding. These observations suggest that diffusion of RNAP along DNA constitutes a mechanism for accelerated promoter location. Sequential images of single, transcribing RNAP molecules were also investigated. Upon addition of 5 microM nucleoside triphosphates to stalled elongation complexes in the liquid chamber, RNAP molecules were seen to processively thread their template at rates of 1.5 nucleotide/s in a direction consistent with the promoter orientation. Transcription assays, performed with radiolabeled, mica-bound transcription complexes, confirmed this rate, which was about three times smaller than the rate of complexes in solution. This assay also showed that the pattern of pause sites and the termination site were affected by the surface. By using the Einstein-Sutherland friction-diffusion relation the loading force experienced by RNAP due to DNA-surface friction is estimated and discussed.

Small angle X-ray scattering of wheat seed-storage proteins: alpha-, gamma- and omega-gliadins and the high molecular weight (HMW) subunits of glutenin.

Small angle X-ray scattering in solution was performed on seed-storage proteins from wheat. Three different groups of gliadins (alpha-, gamma- and omega-) and a high molecular weight (HMW) subunit of glutenin (1Bx20) were studied to determine molecular size parameters. All the gliadins could be modelled as prolate ellipsoids with extended conformations. The HMW subunit existed as a highly extended rod-like particle in solution with a length of about 69 nm and a diameter of about 6.4 nm. Specific aggregation effects were observed which may reflect mechanisms of self-assembly that contribute to the unique viscoelastic properties of wheat dough.

Oriented, active Escherichia coli RNA polymerase: an atomic force microscope study.

Combining a system for binding proteins to surfaces (Sigal, G. B., C. Bamdad, A. Barberis, J. Strominger, and G. M. Whitesides. 1996. Anal. Chem. 68:490-497) with a method for making ultraflat gold surfaces (Hegner, M., P. Wagner, and G. Semenza. 1993. Surface Sci. 291:39-46 1993) has enabled single, oriented, active Escherichia coli RNA polymerase (RNAP) molecules to be imaged under aqueous buffer using tapping-mode atomic force microscopy (AFM). Recombinant RNAP molecules containing histidine tags (hisRNAP) on the C-terminus were specifically immobilized on ultraflat gold via a mixed monolayer of two different omega-functionalized alkanethiols. One alkanethiol was terminated in an ethylene-glycol (EG) group, which resists protein adsorption, and the other was terminated in an N-nitrilotriacetic acid (NTA) group, which binds the histidine tag through two coordination sites with a nickel ion. AFM images showed that these two alkanethiols phase-segregate. Specific binding of the hisRNAP molecules was followed in situ by injecting proteins directly into the AFM fluid cell. The activity of the hisRNAP bound to the NTA groups was confirmed with a 42-base circular single-stranded DNA template (rolling circle), which the RNAP uses to produce huge RNA transcripts. These transcripts were imaged in air after the samples were rinsed and dried, since RNA also has low affinity for the EG-thiol and cannot be imaged under the buffers we used.

Phase imaging of moving DNA molecules and DNA molecules replicated in the atomic force microscope.

Phase imaging with a tapping mode atomic force microscope (AFM) has many advantages for imaging moving DNA and DNA-enzyme complexes in aqueous buffers at molecular resolution. In phase images molecules can be resolved at higher scan rates and lower forces than in height images from the AFM. Higher scan rates make it possible to image faster processes. At lower forces the molecules are imaged more gently. Moving DNA molecules are also resolved more clearly in phase images than in height images. Phase images in tapping mode AFM show the phase difference between oscillation of the piezoelectric crystal that drives the cantilever and oscillation of the cantilever as it interacts with the sample surface. Phase images presented here show moving DNA molecules that have been replicated with Sequenase in the AFM and DNA molecules tethered in complexes with Escherichia coli RNA polymerase.

Escherichia coli RNA polymerase activity observed using atomic force microscopy.

Fluid tapping-mode atomic force microscopy (AFM) was used to observe Escherichia coli RNA polymerase (RNAP) transcribing two different linear double-stranded (ds) DNA templates. The transcription process was detected by observing the translocation of the DNA template by RNAP on addition of ribonucleoside 5'-triphosphates (NTPs) in sequential AFM images. Stalled ternary complexes of RNAP, dsDNA and nascent RNA were adsorbed onto a mica surface and imaged under continuously flowing buffer. On introduction of all four NTPs, we observed some DNA molecules being pulled through the RNAP, some dissociating from the RNAP and others which did not move relative to the RNAP. The transcription rates were observed to be approximately 0.5-2 bases/s at our NTP concentrations, approximately 5 microM. The RNA transcripts were not unambiguously imaged in fluid. However, in experiments using a small single-stranded (ss) circular DNA template, known as a rolling circle, transcripts up to 1 or 2 microns long could be observed with tapping mode AFM once the samples were dried and imaged in air. This confirmed our observations of the transcriptional activity of RNAP adsorbed onto mica. This work illustrates that the development of tapping-mode in fluid has made it possible to use AFM to follow biological processes at the molecular level and get new insights about the variability of activity of individual molecules bound to a surface.

Protein tracking and detection of protein motion using atomic force microscopy.

Height fluctuations over three different proteins, immunoglobulin G, urease, and microtubules, have been measured using an atomic force microscope (AFM) operating in fluid tapping mode. This was achieved by using a protein-tracking system, where the AFM tip was periodically repositioned above a single protein molecule (or structure) as thermal drifting occurred. Height (z-piezo signal) data were taken in 1 - or 2-s time slices with the tip over the molecule and compared to data taken on the support. The measured fluctuations were consistently higher when the tip was positioned over the protein, as opposed to the support the protein was adsorbed on. Similar measurements over patches of an amphiphile, where the noise was identical to that on the support, suggest that the noise increase is due to some intrinsic property of proteins and is not a result of different tip-sample interactions over soft samples. The orientation of the adsorbed proteins in these preliminary studies was not known; thus it was not possible to make correlations between the observed motion and specific protein structure or protein function beyond noting that the observed height fluctuations were greater for an antibody (anti-bovine IgG) and an enzyme (urease) than for microtubules.

Molecular images of cereal proteins by STM.

Scanning tunnelling microscopy (STM) has been used to study a seed storage protein of wheat known as gamma-gliadin. The protein was deposited onto highly oriented pyrolytic graphite (HOPG) from solutions of trifluoroethanol (TFE) and 1% acetic acid. Samples were dried down and then scanned in air. Transmission electron microscopy (TEM) was also used to visualise the distribution of protein on the substrate. Small-angle X-ray scattering (SAXS) was used to compare the molecular size and shape obtained with those from the STM images.