Molecular cooperativity of drebrin1-300 binding and structural remodeling of F-actin.

Drebrin A, an actin-binding protein, is a key regulatory element in synaptic plasticity of neuronal dendrites. Understanding how drebrin binds and remodels F-actin is important for a functional analysis of their interactions. Conventionally, molecular models for protein-protein interactions use binding parameters derived from bulk solution measurements with limited spatial resolution, and the inherent assumption of homogeneous binding sites. In the case of actin filaments, their structural and dynamic states-as well as local changes in those states-may influence their binding parameters and interaction cooperativity. Here, we probed the structural remodeling of single actin filaments and the binding cooperativity of DrebrinA(1-300) -F-actin using AFM imaging. We show direct evidence of DrebrinA(1-300)-induced cooperative changes in the helical structure of F-actin and observe the binding cooperativity of drebrin to F-actin with nanometer resolution. The data confirm at the in vitro molecular level that variations in the F-actin helical structure can be modulated by cooperative binding of actin-binding proteins.

Image analysis and length estimation of biomolecules using AFM.

There are many examples of problems in pattern analysis for which it is often possible to obtain systematic characterizations, if in addition a small number of useful features or parameters of the image are known a priori or can be estimated reasonably well. Often the relevant features of a particular pattern analysis problem are easy to enumerate, as when statistical structures of the patterns are well understood from the knowledge of the domain. We study a problem from molecular image analysis, where such a domain-dependent understanding may be lacking to some degree and the features must be inferred via machine-learning techniques. In this paper, we propose a rigorous, fully-automated technique for this problem. We are motivated by an application of atomic force microscopy (AFM) image processing needed to solve a central problem in molecular biology, aimed at obtaining the complete transcription profile of a single cell, a snapshot that shows which genes are being expressed and to what degree. Reed et al (Single molecule transcription profiling with AFM, Nanotechnology, 18:4, 2007) showed the transcription profiling problem reduces to making high-precision measurements of biomolecule backbone lengths, correct to within 20-25 bp (6-7.5 nm). Here we present an image processing and length estimation pipeline using AFM that comes close to achieving these measurement tolerances. In particular, we develop a biased length estimator on trained coefficients of a simple linear regression model, biweighted by a Beaton-Tukey function, whose feature universe is constrained by James-Stein shrinkage to avoid overfitting. In terms of extensibility and addressing the model selection problem, this formulation subsumes the models we studied.

Identifying individual DNA species in a complex mixture by precisely measuring the spacing between nicking restriction enzymes with atomic force microscope.

We discuss a novel atomic force microscope-based method for identifying individual short DNA molecules (<5000 bp) within a complex mixture by measuring the intra-molecular spacing of a few sequence-specific topographical labels in each molecule. Using this method, we accurately determined the relative abundance of individual DNA species in a 15-species mixture, with fewer than 100 copies per species sampled. To assess the scalability of our approach, we conducted a computer simulation, with realistic parameters, of the hypothetical problem of detecting abundance changes in individual gene transcripts between two single-cell human messenger RNA samples, each containing roughly 9000 species. We found that this approach can distinguish transcript species abundance changes accurately in most cases, including transcript isoforms which would be challenging to quantitate with traditional methods. Given its sensitivity and procedural simplicity, our approach could be used to identify transcript-derived complementary DNAs, where it would have substantial technical and practical advantages versus established techniques in situations where sample material is scarce.

Localized nanoscopic surface measurements of nickel-modified mica for single-molecule DNA sequence sampling.

Cleaved, cation-derivatized Muscovite mica is utilized extensively in atomic force microscopy (AFM) imaging because of its flatness over large areas (millimeter cleavage planes with local root-mean-square roughness < 0.3 nm), ease of preparation, and ability to adsorb charged biomolecules such as DNA (work by Hansma and Laney, Guthold et al., and McMaster et al.). In particular, NiCl(2) treatment has become a common method for controlling DNA adsorption on mica substrates while retaining the mica's ultraflat surface (work by Pietrement et al.). While several studies have modeled the mica/metal ion/DNA system using macroscopic colloidal theory (DLVO, etc.; Pietrement et al., Sushko et al., Pastre et al., and Cheng et al.), nickel/mica's physicochemical properties have not been well characterized on the nanoscale. Efforts to manipulate and engineer DNA nanostructures would benefit greatly from a better understanding of the surface chemistry of nickel/mica. Here we present in situ nanometer- and attogram-scale measurements and thermodynamic simulation results that show that the surface chemistry of nickel-treated mica is more complex than generally appreciated by AFM practitioners because of metal-ion speciation effects present at neutral pH. We also show that, under certain preparations, nickel/mica allows in situ nanoscopic nucleotide sequence mapping within individual surface-adsorbed DNA molecules by permitting localized, controlled desorption of the double helix by soluble DNA binding enzymes. These results should aid efforts to precisely control the DNA/mica binding affinity, particularly at the physiological pH ranges required by enzymatic biochemistry (pH 7.0-8.5), and facilitate the development of more complex and useful biochemical manipulations of adsorbed DNA, such as single-molecule sequencing.

Nanocharacterization in dentistry.

About 80% of US adults have some form of dental disease. There are a variety of new dental products available, ranging from implants to oral hygiene products that rely on nanoscale properties. Here, the application of AFM (Atomic Force Microscopy) and optical interferometry to a range of dentistry issues, including characterization of dental enamel, oral bacteria, biofilms and the role of surface proteins in biochemical and nanomechanical properties of bacterial adhesins, is reviewed. We also include studies of new products blocking dentine tubules to alleviate hypersensitivity; antimicrobial effects of mouthwash and characterizing nanoparticle coated dental implants. An outlook on future "nanodentistry" developments such as saliva exosomes based diagnostics, designing biocompatible, antimicrobial dental implants and personalized dental healthcare is presented.

A breakthrough therapy for dentin hypersensitivity: how dental products containing 8% arginine and calcium carbonate work to deliver effective relief of sensitive teeth.

OBJECTIVE: These studies have utilized a range of state-of-the-art surface techniques to gain insight into the mechanism of action of a new technology for dentin hypersensitivity relief based upon arginine and calcium carbonate and, in particular, to address important questions regarding the nature and extent of dentin tubule occlusion. METHODS: Confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) have been used to assess tubule occlusion. Energy dispersive x-ray (EDX) and electron spectroscopy for chemical analysis (ESCA) have been used to identify the composition of the dentin plug. CLSM has also been used to compare the mechanism of action of the toothpaste and the desensitizing prophylaxis paste, to address whether both the arginine and the calcium carbonate components are essential to occlusion, to identify the location of the arginine within the occluded dentin, and to demonstrate resistance of the occlusion to acid challenge. Hydraulic conductance has been used to assess the effectiveness of the arginine-calcium carbonate technology in arresting dentin fluid movement, to evaluate the effects of pulpal pressure on the robustness of the occlusion, and to confirm the resistance of the occlusion to an acid challenge. RESULTS: The CLSM, SEM, and AFM studies demonstrate that the arginine-calcium carbonate technology is highly effective in rapidly and completely occluding dentin tubules. The EDX and ESCA studies show that the dentin surface deposit and occluded tubule plug contain high levels of calcium and phosphate, as well as carbonate. CLSM has confirmed that the toothpaste and the desensitizing prophylaxis paste have the same mechanism of action, that the arginine and calcium carbonate components are both essential to the effectiveness of these products, and that the arginine becomes incorporated into the dentin plug. The hydraulic conductance studies demonstrate that the occlusion provided by the arginine-calcium carbonate technology results in highly significant reductions in dentin fluid flow, and that the tubule plug is resistant to normal pulpal pressure and acid challenge. CONCLUSION: A breakthrough technology based upon arginine and calcium carbonate provides clinically proven benefits with respect to rapid and lasting relief of dentin hypersensitivity. It is unique in that two of its key components, arginine and calcium, are found naturally in saliva, and that the arginine and calcium carbonate work together to accelerate the natural mechanisms of occlusion to deposit a dentin-like mineral, containing calcium and phosphate, within the dentin tubules and in a protective layer on the dentin surface.

Atomic force microscope observation of branching in single transcript molecules derived from human cardiac muscle.

We have used an atomic force microscope to examine a clinically derived sample of single-molecule gene transcripts, in the form of double-stranded cDNA, (c: complementary) obtained from human cardiac muscle without the use of polymerase chain reaction (PCR) amplification. We observed a log-normal distribution of transcript sizes, with most molecules being in the range of 0.4-7.0 kilobase pairs (kb) or 130-2300 nm in contour length, in accordance with the expected distribution of mRNA (m: messenger) sizes in mammalian cells. We observed novel branching structures not previously known to exist in cDNA, and which could have profound negative effects on traditional analysis of cDNA samples through cloning, PCR and DNA sequencing.