Label-free live-cell imaging with confocal Raman microscopy.

Confocal Raman spectroscopy is a noninvasive alternative to established cell imaging methods because it does not require chemical fixation, the use of fluorescent markers, or genetic engineering. In particular, single live-cell, high-resolution imaging by confocal Raman microscopy is desirable because it allows further experiments concerning the individually investigated cells. However, to derive meaningful images from the spectroscopic data, one must identify cell components within the dataset. Using immunofluorescence images as a reference, we derive Raman spectral signatures by means of information measures to identify cell components such as the nucleus, the endoplasmic reticulum, the Golgi apparatus, and mitochondria. The extracted signatures allow us to generate representations equivalent to conventional (immuno)fluorescence images with more than three cell components at a time, exploiting the Raman spectral information alone.

A leucine-rich repeat assembly approach for homology modeling of the human TLR5-10 and mouse TLR11-13 ectodomains.

So far, 13 groups of mammalian Toll-like receptors (TLRs) have been identified. Most TLRs have been shown to recognize pathogen-associated molecular patterns from a wide range of invading agents and initiate both innate and adaptive immune responses. The TLR ectodomains are composed of varying numbers and types of leucine-rich repeats (LRRs). As the crystal structures are currently missing for most TLR ligand-binding ectodomains, homology modeling enables first predictions of their three-dimensional structures on the basis of the determined crystal structures of TLR ectodomains. However, the quality of the predicted models that are generated from full-length templates can be limited due to low sequence identity between the target and templates. To obtain better templates for modeling, we have developed an LRR template assembly approach. Individual LRR templates that are locally optimal for the target sequence are assembled into multiple templates. This method was validated through the comparison of a predicted model with the crystal structure of mouse TLR3. With this method, we also constructed ectodomain models of human TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 and mouse TLR11, TLR12, and TLR13 that can be used as first passes for a computational simulation of ligand docking or to design mutation experiments. This template assembly approach can be extended to other repetitive proteins.

Intermittency in amplitude modulated dynamic atomic force microscopy.

From a mathematical point of view, the atomic force microscope (AFM) belongs to a special class of continuous time dynamical systems with intermittent impact collisions. Discontinuities of the velocity result from the collisions of the tip with the surface. Transition to chaos in non-linear systems can occur via the following four routes: bifurcation cascade, crisis, quasi-periodicity, and intermittency. For the AFM period doubling and period-adding cascades are well established. Other routes into chaos, however, also may play an important role. Time series data of a dynamic AFM experiment indicates a chaotic mode that is related to the intermittency route into chaos. The observed intermittency is characterized as a type III intermittency. Understanding the dynamics of the system will help improve the overall system performance by keeping the operation parameters of dynamic AFM in a range, where chaos can be avoided or at least controlled.

TollML: a database of toll-like receptor structural motifs.

Toll-like receptors (TLRs) play a key role in the innate immune system. TLRs recognize pathogen-associated molecular patterns and initiate an intracellular kinase cascade to induce an immediate defensive response. During recent years TLRs have become the focus of tremendous research interest. A central repository for the growing amount of relevant TLR sequence information has been created. Nevertheless, structural motifs of most sequenced TLR proteins, such as leucine-rich repeats (LRRs), are poorly annotated in the established databases. A database that organizes the structural motifs of TLRs could be useful for developing pattern recognition programs, structural modeling and understanding functional mechanisms of TLRs. We describe TollML, a database that integrates all of the TLR sequencing data from the NCBI protein database. Entries were first divided into TLR families (TLR1-23) and then semi-automatically subdivided into three levels of structural motif categories: (1) signal peptide (SP), ectodomain (ECD), transmembrane domain (TD) and Toll/IL-1 receptor (TIR) domain of each TLR; (2) LRRs of each ECD; (3) highly conserved segment (HCS), variable segment (VS) and insertions of each LRR. These categories can be searched quickly using an easy-to-use web interface and dynamically displayed by graphics. Additionally, all entries have hyperlinks to various sources including NCBI, Swiss-Prot, PDB, LRRML and PubMed in order to provide broad external information for users. The TollML database is available at http://tollml.lrz.de.

Inhibition of Toll-like receptors TLR4 and 7 signaling pathways by SIGIRR: a computational approach.

Toll-like receptors (TLRs) belong to the Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) superfamily which is defined by a common cytoplasmic Toll/interleukin-1 receptor (TIR) domain. TLRs recognize pathogen-associated molecular patterns and initiate an intracellular kinase cascade to trigger an immediate defensive response. SIGIRR (single immunoglobulin interleukin-1 receptor-related molecule), another member of the TLR/IL-1R superfamily, acts as a negative regulator of MyD88-dependent TLR signaling. It attenuates the recruitment of MyD88 adaptors to the receptors with its intracellular TIR domain. Thus, SIGIRR is a highly important molecule for the therapy of autoimmune diseases caused by TLRs. So far, the structural mechanism of interactions between SIGIRR, TLRs and adaptor molecules is unclear. To develop a working hypothesis for this interaction, we constructed three-dimensional models for the TIR domains of TLR4, TLR7, MyD88 and SIGIRR based on computational modeling. Through protein-protein docking analysis, we developed models of essential complexes involved in the TLR4 and 7 signaling and the SIGIRR inhibiting processes. We suggest that SIGIRR may exert its inhibitory effect through blocking the molecular interface of TLR4, TLR7 and the MyD88 adaptor mainly via its BB-loop region.

Homology modeling of human Toll-like receptors TLR7, 8, and 9 ligand-binding domains.

Toll-like receptors (TLRs) play a key role in the innate immune system. The TLR7, 8, and 9 compose a family of intracellularly localized TLRs that signal in response to pathogen-derived nucleic acids. So far, there are no crystallographic structures for TLR7, 8, and 9. For this reason, their ligand-binding mechanisms are poorly understood. To enable first predictions of the receptor-ligand interaction sites, we developed three-dimensional structures for the leucine-rich repeat ectodomains of human TLR7, 8, and 9 based on homology modeling. To achieve a high sequence similarity between targets and templates, structural segments from all known TLR ectodomain structures (human TLR1/2/3/4 and mouse TLR3/4) were used as candidate templates for the modeling. The resulting models support previously reported essential ligand-binding residues. They also provide a basis to identify three potential receptor dimerization mechanisms. Additionally, potential ligand-binding residues are identified using combined procedures. We suggest further investigations of these residues through mutation experiments. Our modeling approach can be extended to other members of the TLR family or other repetitive proteins.

Characterizing synchronization in time series using information measures extracted from symbolic representations.

We present a methodology to characterize synchronization in time series based on symbolic representations. Each time series is mapped onto a sequence of p -dimensional delay vectors that are subsequently transformed into symbols by means of a rank-ordering of their values. Based on these representations, we propose a transcription scheme between symbols of the respective time series to study synchronization properties. Group-theoretical considerations and the use of information measures allow us to classify regimes of synchronization and to assess its strength. We apply our method to a prototype nonlinear system, which reveals a rich variety of coupled dynamics. We investigate in detail the robustness of the derived synchronization measure against noise and compare its value with that of the established measures.

LRRML: a conformational database and an XML description of leucine-rich repeats (LRRs).

BACKGROUND: Leucine-rich repeats (LRRs) are present in more than 6000 proteins. They are found in organisms ranging from viruses to eukaryotes and play an important role in protein-ligand interactions. To date, more than one hundred crystal structures of LRR containing proteins have been determined. This knowledge has increased our ability to use the crystal structures as templates to model LRR proteins with unknown structures. Since the individual three-dimensional LRR structures are not directly available from the established databases and since there are only a few detailed annotations for them, a conformational LRR database useful for homology modeling of LRR proteins is desirable. DESCRIPTION: We developed LRRML, a conformational database and an extensible markup language (XML) description of LRRs. The release 0.2 contains 1261 individual LRR structures, which were identified from 112 PDB structures and annotated manually. An XML structure was defined to exchange and store the LRRs. LRRML provides a source for homology modeling and structural analysis of LRR proteins. In order to demonstrate the capabilities of the database we modeled the mouse Toll-like receptor 3 (TLR3) by multiple templates homology modeling and compared the result with the crystal structure. CONCLUSION: LRRML is an information source for investigators involved in both theoretical and applied research on LRR proteins. It is available at http://zeus.krist.geo.uni-muenchen.de/~lrrml.

Supramolecular self-assembly initiated by solid-solid wetting.

We present a preparation method for self-assembled supra-molecular monolayers of unsubstituted organic semiconductors and pigments on a solid substrate, applicable under ambient conditions. The deposition is based on a solid-solid wetting phenomenon, whereas the subsequent layer growth proceeds according to standard models. Molecular adsorption results from direct contact of the compound in a nanocrystalline state with the solid surface. Based on complementary force field calculations, we propose that molecules disintegrate from the crystalline state and adsorb on the surface because of a gain in binding energy. The preparation method is exemplified by means of a linear hydrogen-bonded system, namely quinacridone (QAC) on graphite. In addition, the chosen system allows us to actively guide the self-assembly after deliberate removal of molecules from a predefined area.

Dynamics of grain boundaries in two-dimensional hydrogen-bonded molecular networks.

The temporal evolution of domain boundaries of hydrogen-bonded molecular monolayers at the liquid-solid interface is evaluated by recording series of subsequent scanning tunneling microscopy (STM) images. Comparison of dissimilar benzene carboxylic acids reveals a clear distinction between one- and two-dimensional H-bonded network structures. Trimesic acid forms a two-dimensionally H-bonded networked structure, whereas terephthalic acid organizes in a dense packing of H-bonded linear chains on a graphite surface. In addition, TMA forms a sixfold lattice on a threefold graphite substrate, whereas TPA exhibits only a twofold lattice, causing a high grain-boundary line energy for the latter. In the case of TMA the nanostructure was mostly stable during the observation time. For TPA, Ostwald ripening-that is, the growth of larger islands at the expense of smaller islands-was observed. To explain the various experimentally observed timescales of the dynamics occurring at grain boundaries, molecular mechanics simulations were applied to calculate the binding energy of edge molecules, that is, the line energy, of finite islands of both trimesic and terephthalic acid on a graphite substrate.

Incorporation and manipulation of coronene in an organic template structure.

A two-dimensional molecular template structure of 1,3,5-benzenetricarboxylic acid (trimesic acid, TMA) was formed on a highly oriented pyrolytic graphite surface (HOPG) by self-assembly at the liquid-solid interface. Scanning tunneling microscopy (STM) investigations show high-resolution images of the porous structure on the surface. After the host structure was created, coronene molecules were inserted as guest molecules into the pores. STM results indicate that some of the guest molecules rotate inside their molecular bearing. Further investigations show that single coronene molecules can be directly kicked out of their pores by means of STM.

Discrimination of aqueous and aeolian paleoenvironments by atomic force microscopy-- a database for the characterization of martian sediments.

In the search for aqueous habitats on Mars direct proof of (ancient) flowing water is still lacking, although remote sensing has provided indications of young fluvial systems. To demonstrate that such proof can be given, we examined surface marks on recent terrestrial sand grains by atomic force microscopy (AFM) and applied a quantitative three-dimensional analysis that can numerically distinguish between aeolian and aquatic transport mechanisms in sedimentary deposits on Earth. The surfaces of natural quartz grains as well as olivine, feldspar pyroxene, and monazite sands of known origin were imaged, each image yielding a three-dimensional map of the mineral surface. A fully automated analysis of distribution patterns of the structural elements that constitute the grain surfaces shows that wind-transported quartz grains have short linear elements irregularly distributed on the surface. Linear elements on water-transported grains, however, are longer with orientations that reflect the mineral symmetry. Because the surface patterns found on aqueous grains are due to preferential etching, they can be used as diagnostic fingerprints for the existence of past or present aqueous transport systems. We used a cluster analysis of the cross-correlation distance of distribution patterns in the structures of aeolian and aquatic sand grains to build a phenogram that provides a map for the relationship of the various sediments found on earth. The analysis shows that the method is highly significant and that water and wind transport can clearly be differentiated. In particular, feldspar and olivine sands contributed even more to the discrimination than quartz grains, which indicated that the method is promising for its application on future missions to Mars. Assuming that martian aqueous sand grains exhibit similar erosional patterns to mineral grains on Earth, simple AFM experiments on a Mars lander would be capable of proving the activity of flowing water in modern runoff systems and of analyzing the paleoenvironments of Mars.