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Scanning probe microscopy makes it possible to image and spectroscopically characterize nanoscale objects, and to manipulate.sup.1-3 and excite.sup.4-8 them; even time-resolved experiments are now routinely achieved.sup.9,10. This combination of capabilities has enabled proof-of-principle demonstrations of nanoscale devices, including logic operations based on molecular cascades.sup.11, a single-atom transistor.sup.12, a single-atom magnetic memory cell.sup.13 and a kilobyte atomic memory.sup.14. However, a key challenge is fabricating device structures that can overcome their attraction to the underlying surface and thus protrude from the two-dimensional flatlands of the surface. Here we demonstrate the fabrication of such a structure: we use the tip of a scanning probe microscope to lift a large planar aromatic molecule (3,4,9,10-perylenetetracarboxylic-dianhydride) into an upright, standing geometry on a pedestal of two metal (silver) adatoms. This atypical and surprisingly stable upright orientation of the single molecule, which under all known circumstances adsorbs flat on metals.sup.15,16, enables the system to function as a coherent single-electron field emitter. We anticipate that other metastable adsorbate configurations might also be accessible, thereby opening up the third dimension for the design of functional nanostructures on surfaces. Precision control over matter at the atomic scale enables a planar dye molecule to be lifted up and placed on its edge--a configuration that is surprisingly stable., Author(s): Taner Esat [sup.1] [sup.2] , Niklas Friedrich [sup.1] [sup.2] , F. Stefan Tautz [sup.1] [sup.2] , Ruslan Temirov [sup.1] [sup.2] Author Affiliations: (1) Peter Grünberg Institute (PGI-3), Forschungszentrum Jülich, [...]

A new breed of protein engineers is finding that the best way to create a molecule is to build it from scratch. A new breed of protein engineers is finding that the best way to create a molecule is to build it from scratch., Author(s): Jeffrey M. Perkel Author Affiliations: The computational protein designers By designing a protein from the ground up, researchers can create molecules with forms and functions not found in nature. [...]

Anharmonic and harmonic rate constants for the unimolecular dissociation of C[H.sub.3]OOH and C[D.sub.3]OOD were calculated using the Rice-Ramsperger-Kassel-Marcus (RRKM) theory at the MP2/6-311++G(3df,3pd) level of theory. The anharmonic effect of the reactions was investigated. Comparison of results for the decompositions of C[H.sub.3]OOH and C[D.sub.3]OOD shows that the direct bond dissociation channel, C[H.sub.3] ([D.sub.3]) O + OH (D), is the most dominant reaction. The anharmonic effect plays an important role in the unimolecular dissociation of both C[H.sub.3]OOH and C[D.sub.3]OOD. For channels C[H.sub.3] ([D.sub.3]) O + OH (D) and C[H.sub.3] ([D.sub.3]) + H (D) [O.sub.2], the anharmonic effect of the unimolecular dissociation of C[D.sub.3]OOD is more pronounced than that of the unimolecular dissociation of C[H.sub.3]OOH. For channel [H.sub.2] (D2) CO + [H.sub.2] (D2) O, the anharmonic effect of the unimolecular dissociation of C[H.sub.3]OOH is more pronounced than that of the unimolecular dissociation of C[D.sub.3]OOD. The isotope effect is more distinct in the anharmonic oscillator model. Key words: anharmonic effect, unimolecular reaction, rate constant, Rice-Ramsperger-Kassel-Marcus (RRKM) theory. Au moyen de la theorie de Rice-Ramsperger-Kassel-Marcus (RRKM), nous avons calcule les constantes de vitesses harmonique et anharmonique de la dissociation unimoleculaire de l'acide acetique (C[H.sub.3]OOH) et de son homologue deutere (C[D.sub.3]OOD) au niveau de calcul MP2/6-311++G(3df,3pd). Nous avons etudie l'effet anharmonique des reactions. En comparant les resultats pour la decomposition du C[H.sub.3]OOH et du C[D.sub.3]OOD, nous avons observe que la dissociation directe de la liaison en C[H.sub.3]([D.sub.3])O + OH(D) constitue la principale voie de reaction. L'effet anharmonique joue un role important dans la dissociation unimoleculaire des deux composes, soit le C[H.sub.3]OOH et le C[D.sub.3]OOD. Pour les voies C[H.sub.3]([D.sub.3])O + OH(D) et C[H.sub.3]([D.sub.3]) + H(D)[O.sub.2], l'effet anharmonique de la dissociation unimoleculaire du C[D.sub.3]OOD est plus marque que celui de la dissociation unimoleculaire du C[H.sub.3]OOH. A l'inverse, pour ce qui est de la voie [H.sub.2](D2)CO + [H.sub.2](D2)O, c'est l'effet anharmonique de la dissociation unimoleculaire du C[H.sub.3]OOH qui est plus marque que celui de la dissociation unimoleculaire du C[D.sub.3]OOD. L'effet isotopique est plus perceptible dans le modele de l'oscillateur anharmonique que dans le modele harmonique. [Traduit par la Redaction] Mots-cles: effet anharmonique, reaction unimoleculaire, constante de vitesse, theorie de Rice-Ramsperger-Kassel-Marcus (RRKM)., Introduction Hydrogen peroxide and organic peroxides play important roles in atmospheric processes. They act as indicators of the oxidative capacity of the atmosphere through their involvement in gas phase chemistry [...]

Phospholamban (PLN) is an essential regulator of cardiac muscle contractility. The homopentameric assembly of PLN is the reservoir for active monomers that, upon deoligomerization form 1:1 complexes with the sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA), thus modulating the rate of calcium uptake. In lipid bilayers and micelles, monomeric PLN exists in equilibrium between a bent (or resting) T state and a more dynamic (or active) R state. Here, we report the high-resolution structure and topology of the T state of a monomeric PLN mutant in lipid bilayers, using a hybrid of solution and solid-state NMR restraints together with molecular dynamics simulations in explicit lipid environments. Unlike the previous structural ensemble determined in micelles, this approach gives a complete picture of the PLN monomer structure in a lipid bilayer. This hybrid ensemble exemplifies the tilt, rotation, and depth of membrane insertion, revealing the interaction with the lipids for all protein domains. The N-terminal amphipathic helical domain la (residues 1-16) rests on the surface of the lipid membrane with the hydrophobic face of domain la embedded in the membrane bilayer interior. The helix comprised of domain lb (residues 23-30) and transmembrane domain II (residues 31-52) traverses the bilayer with a tilt angle of [approximately equal to] 24[degrees]. The specific interactions between PLN and lipid membranes may represent an additional regulatory element of its inhibitory function. We propose this hybrid method for the simultaneous determination of structure and topology for membrane proteins with compact folds or proteins whose spatial arrangement is dictated by their specific interactions with lipid bilayers. hybrid method | membrane proteins | oriented solid-state NMR | molecular modeling | PISEMA

The aim of the study was to assess the impact of mitral regurgitation (MR) on left ventricular (LV) anatomic and molecular remodeling and function and to determine whether early LV remodeling and function predict long-term outcome in experimental organic MR. A new rodent model of chronic MR was created. Twenty-eight rats had surgically induced MR, twelve rats had a sham operation, and twelve rats had no operation. LV diameters, volume, and mass and LV ejection fraction (LVEF) and LV fractional shortening (LVFS) were assessed using echocardiography in the early stage of MR (6 and 12 wk after induction of MR). LV hemodynamics was assessed invasively. Cardiac [alpha]-and [beta]-myosin heavy chains and sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase 2 (SERCA2) were measured to assess molecular remodeling and contractility. Cox's proportional hazard ratios (HR) were used to identify outcome predictors. Early LV dilation was demonstrated in rats with MR when LVEF and LVFS were still normal. LV remodeling was associated with an increase in LV end-diastolic pressure and decrease in maximal change in pressure over time. Shifting of a- to [beta]-myosin and reduced SERCA2 were observed in rats with MR. Cox's proportional hazard analysis showed that LV end-diastolic diameters (HR, 1.2-2.4; P = 0.007) and LV end-diastolic volume (HR, 1.1-1.4; P = 0.005) at 6 wk and LV mass index (HR, 1.1-2.0; P = 0.004) at 12 wk after induction of MR were significantly associated with 1-yr mortality. However, LVEF (HR, 0.7-6.8 for the 6 wk, P > 0.05; and HR, 0.4-3.2 for the 12 wk, P > 0.05) and LVFS (HR, 0.4-1.4 for the 6 wk; and 0.4-3.1 for the 12 wk, P > 0.05) did not predict late death. Chronic MR leads to LV anatomic and cellular remodeling and impaired contractility. The time course of LV remodeling and function changes in the rat model of MR is similar to humans. Prediction of outcome may be achieved by assessments of early LV remodeling. molecular biology; animal model

Recent improvements in ELSD instrumentation and a better understanding of the limitations and advantages of this detection method are increasing its popularity, especially for the analysis of molecules lacking chromophores [...]

Various studies provide insights into developmental processes and molecular mechanisms controlling the formation and remodelling of the lymphatic vessels. The current understanding of the mechanisms of lymphatic development and contribution of lymphatic vessels to pathological conditions are reviewed and the essential and active role of lymphatic vessels in various pathological conditions is demonstrated.

The ever-improving time and space resolution and molecular detection sensitivity of fluorescence microscopy offer unique opportunities to deepen our insights into the function of chemical and biological catalysts. Because single-molecule microscopy allows for counting the turnover events one by one, one can map the distribution of the catalytic activities of different sites in solid heterogeneous catalysts, or one can study time-dependent activity fluctuations of individual sites in enzymes or chemical catalysts. By experimentally monitoring individuals rather than populations, the origin of complex behavior, e.g., in kinetics or in deactivation processes, can be successfully elucidated. Recent progress of temporal and spatial resolution in single-molecule fluorescence microscopy is discussed in light of its impact on catalytic assays. Key concepts are illustrated regarding the use of fluorescent reporters in catalytic reactions. Future challenges comprising the integration of other techniques, such as diffraction, scanning probe, or vibrational methods in single-molecule fluorescence spectroscopy are suggested.

Single chlorophyll-a molecules, a vital resource for the sustenance of life on Earth, have been investigated by using scanning tunneling microscope manipulation and spectroscopy on a gold substrate at 4.6 K. Chlorophyll-a binds on Au(111) via its porphyrin unit while the phytyl-chain is elevated from the surface by the support of four [CH.sub.3] groups. By injecting tunneling electrons from the scanning tunneling microscope tip, we are able to bend the phytyl-chain, which enables the switching of four molecular conformations in a controlled manner. Statistical analyses and structural calculations reveal that all reversible switching mechanisms are initiated by a single tunneling-electron energy-transfer process, which induces bond rotation within the phytyl-chain.

Biological molecular motors have a number of unique advantages over artificial motors, including efficient conversion of chemical energy into mechanical work and the potential for self-assembly into larger structures, as is seen in muscle sarcomeres and bacterial and eukaryotic flagella. The development of an appropriate interface between such biological materials and synthetic devices should enable us to realize useful hybrid micromachines. Here we describe a microrotary motor composed of a 20-[micro]m-diameter silicon dioxide rotor driven on a silicon track by the gliding bacterium Mycoplasma mobile. This motor is fueled by glucose and inherits some of the properties normally attributed to living systems. glucose | micro actuator | motor protein | nanobiotechnology | Mycoplasma gliding

We investigate the phase behavior of surface-functionalized silica colloids at both the molecular and macroscopic levels. This investigation allows us to relate collective properties such as aggregation, gelation, and aging directly to molecular interfacial behavior. By using surface-specific vibrational spectroscopy, we reveal dramatic changes in the conformation of alkyl chains terminating submicrometer silica particles. In fluid suspension at high temperatures, the interfacial molecules are in a liquid-like state of conformational disorder. As the temperature is lowered, the onset of gelation is identified by macroscopic phenomena, including changes in turbidity, heat release, and diverging viscosity. At the molecular level, the onset of this transition coincides with straightening of the carbon-carbon backbones of the interfacial molecules. In later stages, their intermolecular crystalline packing improves. It is the increased density of this ordered boundary layer that increases the van der Waals attraction between particles, causing the colloidal gas to aggregate. The approach presented here can provide insights into phase transitions that occur through surface modifications in a variety of colloidal systems. sum frequency generation | surface spectroscopy | transition | nonlinear optical scattering | calorimetry

We report a previously undescribed spectroscopic probe that makes use of electrons rescattered during the process of high-order harmonic generation. We excite coherent vibrations in S[F.sub.6] using impulsive stimulated Raman scattering with a short laser pulse. A second, more intense laser pulse generates high-order harmonics of the fundamental laser, at wavelengths of [approximately equal to] 20-50 nm. The high-order harmonic yield is observed to oscillate, at frequencies corresponding to all of the Raman-active modes of S[F.sub.6], with an asymmetric mode most visible. The data also show evidence of relaxation dynamics after impulsive excitation of the molecule. Theoretical modeling indicates that the high harmonic yield should be modulated by both Raman and infrared-active vibrational modes. Our results indicate that high harmonic generation is a very sensitive probe of vibrational dynamics and may yield more information simultaneously than conventional ultrafast spectroscopic techniques. Because the de Broglie wavelength of the recolliding electron is on the order of interatomic distances, i.e., [approximately equal to] 1.5 [Angstrom], small changes in the shape of the molecule lead to large changes in the high harmonic yield. This work therefore demonstrates a previously undescribed spectroscopic technique for probing ultrafast internal dynamics in molecules and, in particular, on the chemically important ground-state potential surface. spectroscopy | ultrafast | x-rays

Annelida, one of the most successful animal phyla, exhibits an amazing variety of morphological forms. Disparity between some of the forms is so great that until molecular tools were used, some annelid lineages (for example, echiurids and pogonophorans) were not commonly recognized as belonging to the group. Although it is easy to assign annelids to a given family, understanding the deeper relationships within the group has been difficult. The main working hypothesis for annelid phylogeny is based on morphological cladistic analysis. However, the recent work using molecular tools has caused a revision of our view of annelid evolutionary history. For example, Scolecida and Palpata appear not to be natural groups, and the phylogenetic positions of some aberrant taxa (for example, Siboglinidae, Poeobius, Pisione) have been determined. Herein, we discuss some of the main molecular markers that have been used to elucidate annelid phylogeny and the contribution that such work is making to our understanding. A table highlighting the molecular literature and the genes used is included.

The signal from many single-molecule experiments monitoring molecular processes, such as enzyme turnover by means of fluorescence and opening and closing of ion channel through the flux of ions, consists of a time series of stochastic 'on' and 'off' (or open and closed) periods, termed a two-state trajectory. This signal reflects the dynamics in the underlying multisubstate on--off kinetic scheme (KS) of the process. The determination of the underlying KS is difficult and sometimes even impossible because of the loss of information in the mapping of the multidimensional KS onto two dimensions. Here we introduce a previously undescribed procedure that efficiently and optimally relates the signal to all equivalent underlying KS. This procedure partitions the space of KS into canonical (unique) forms that can handle any KS and obtains the topology and other details of the canonical form from the data without the need for fitting. Also established are relationships between the data and the topology of the canonical form to the on-off connectivity of a KS. The suggested canonical forms constitute a powerful tool in discriminating between KS. Based on our approach, the upper bound on the information content in two-state trajectories is determined. canonical mechanisms | data analysis | fitting free relationships | mapping | on-off kinetic schemes

The actin cytoskeleton represents a key regulator of multiple essential cellular functions in both eukaryotes and prokaryotes. In eukaryotes, these functions depend on the orchestrated dynamics of actin filament assembly and disassembly. However, the dynamics of the bacterial actin homolog MreB have yet to be examined in vivo. In this study, we observed the motion of single fluorescent MreB-yellow fluorescent protein fusions in living Caulobacter cells in a background of unlabeled MreB. With time-lapse imaging, polymerized MreB [filamentous MreB (fMreB)] and unpolymerized MreB [globular MreB (gMreB)] monomers could be distinguished: gMreB showed fast motion that was characteristic of Brownian diffusion, whereas the labeled molecules in fMreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer provides an indication that, like actin, MreB monomers treadmill through MreB filaments by preferential polymerization at one filament end and depolymerization at the other filament end. From these data, we extract several characteristics of single MreB filaments, including that they are, on average, much shorter than the cell length and that the direction of their polarized assembly seems to be independent of the overall cellular polarity. Thus, MreB, like actin, exhibits treadmilling behavior in vivo, and the long MreB structures that have been visualized in multiple bacterial species seem to represent bundles of short filaments that lack a uniform global polarity. bacteria | cytoskeleton | single-molecule fluorescence

The Einstein, Podolski, and Rosen (EPR) experiment in the case of the breakup of polyatomic molecule into two mutually entangled fragments is analyzed. The experiments have shown that each separated fragment can retain coherences induced in its parent molecule by a broad band laser pulse, regardless of whether a measurement is performed on its entangled partner.

We report here the results of a chemical genetic screen using small molecules with known pharmacologies coupled with a cortical brain slice-based model for ischemic stroke. We identified a small-molecule compound not previously appreciated to have neuroprotective action in ischemic stroke, the cardiac glycoside neriifolin, and demonstrated that its properties in the brain slice assay included delayed therapeutic potential exceeding 6 h. Neriifolin is structurally related to the digitalis class of cardiac glycosides, and its putative target is the [Na.sup.+]/[K.sup.+]-ATPase. Other cardiac glycoside compounds tested also showed neuroprotective activity, although with lower apparent potencies. In subsequent whole-animal studies, we found that neriifolin provided significant neuroprotection in a neonatal model of hypoxia/ischemia and in a middle cerebral artery occlusion model of transient focal ischemia. The neuroprotective potential of [Na.sup.+/[K.sup.+]-ATPase is of particular interest because of its known 'druggability'; indeed, Food and Drug Administration-approved, small-molecule compounds such as digitoxin and digoxin have been in clinical usage for congestive heart failure and arrhythmias for several decades. Thus, an existing cardiac glycoside or closely related compound could provide an accelerated path toward clinical trial testing for ischemic stroke. Our findings underscore the important role that hypothesis-neutral, high-content, tissue-based screens can play in the identification of new candidate drugs and drug targets for the treatment of diseases for which validated therapeutic pathways are not currently available. biotechnology | drug discovery | high-content screening | translational medicine

The nuclear lamina is a network of structural filaments, the A and B type lamins, located at the nuclear envelope and throughout the nucleus. Lamin filaments provide the nucleus with mechanical stability and support many basic activities, including gene regulation. Mutations in LMNA, the gene encoding A type lamins, cause numerous human diseases, including the segmental premature aging disease Hutchinson--Gilford progeria syndrome (HGPS). Here we show that structural and mechanical properties of the lamina are altered in HGPS cells. We demonstrate by live-cell imaging and biochemical analysis that lamins A and C become trapped at the nuclear periphery in HGPS patient cells. Using micropipette aspiration, we show that the lamina in HGPS cells has a significantly reduced ability to rearrange under mechanical stress. Based on polarization microscopy results, we suggest that the lamins are disordered in the healthy nuclei, whereas the lamins in HGPS nuclei form orientationally ordered microdomains. The reduced deformability of the HGPS nuclear lamina possibly could be due to the inability of these orientationally ordered microdomains to dissipate mechanical stress. Surprisingly, intact HGPS cells exhibited a degree of resistance to acute mechanical stress similar to that of cells from healthy individuals. Thus, in contrast to the nuclear fragility seen in Imna null cells, the lamina network in HGPS cells has unique mechanical properties that might contribute to disease phenotypes by affecting responses to mechanical force and misregulation of mechanosensitive gene expression. laminopathy | mechanics | nucleus | photobleaching | micropipette aspiration

Microtubules are hollow cylindrical structures that constitute one of the three major classes of cytoskeletal filaments. On the mesoscopic length scale of a cell, their material properties are characterized by a single stiffness parameter, the persistence length [l.sub.p]. Its value, in general, depends on the microscopic interactions between the constituent tubulin dimers and the architecture of the microtubule. Here, we use single-particle tracking methods combined with a fluctuation analysis to systematically study the dependence of [l.sub.p] on the total filament length L. Microtubules are grafted to a substrate with one end free to fluctuate in three dimensions. A fluorescent bead is attached proximally to the free tip and is used to record the thermal fluctuations of the microtubule's end. The position distribution functions obtained with this assay allow the precise measurement of [l.sub.p] for microtubules of different contour length L. Upon varying L between 2.6 and 47.5 [micro]m, we find a systematic increase of [l.sub.p] from 110 to 5,035 [micro]m. At the same time we verify that, for a given filament length, the persistence length is constant over the filament within the experimental accuracy. We interpret this length dependence as a consequence of a nonnegligible shear deflection determined by subnanometer relative displacement of adjacent protofilaments. Our results may shine new light on the function of microtubules as sophisticated nanometer-sized molecular machines and give a unified explanation of seemingly uncorrelated spreading of microtubules' stiffness previously reported in literature. nanomechanics | protofilaments | single-particle tracking | thermal fluctuation analysis

Self-assembly of Fe(ll) and the ditopic ligand 1,4-bis(2,2':6',2'-terpyridine-4'-yl) benzene results in equilibrium structures in solutions, so-called metallosupramolecular coordination polyelectrolytes (MEPEs). It is exceedingly difficult to characterize such macromolecular assemblies, because of the dynamic nature. Therefore, hardly any structural information is available for this type of material. Here, we show that from dilute solutions, where small aggregates predominate, it is possible to grow nanoscopic crystals at an interface. A near atomic resolution structure of MEPE is obtained by investigating the nanoscopic crystals with electron diffraction in combination with molecular modeling. The analysis reveals a primitive monoclinic unit cell ([P2.sub.1]/c space group, a = 10.4 [Angstrom], b = 10.7 [Angstrom], c = 34.0 [Angstrom] [alpha] = [gamma] = 90[degrees], [beta] = 95[degrees], [rho] = 1.26 g/[cm.sup.3], and Z = 4). The MEPE forms linear rods, which are organized into sheets. Four sheets intersect the unit cell, while adjacent sheets are rotated by 90[degrees] with respect to each other. The pseudooctahedral coordination geometry of the Fe(ll) centers is confirmed by Mossbauer spectroscopy. The combination of diffraction and molecular modeling presented here may be of general utility to address problems in structural materials science. electron diffraction | Mossbauer spectroscopy | molecular modeling | supramolecular chemistry

GSH is the most abundant redox molecule in cells and thus the most important determinant of cellular redox status. Thiols in proteins can undergo a wide range of reversible redox modifications (e.g., Sglutathionylation, S-nitrosylation, and disulfide formation) during times of increased exposure to reactive oxygen and nitrogen species, which can affect protein activity. These reversible thiol modifications regulated by GSH may be nanoswitches to turn on and off proteins, similar to phosphorylation, in cells. In the cytoplasm, an altered redox state can activate (e.g., MAPKs and NF-E2-related factor-2) and inhibit (e.g., phosphatases and caspases) proteins, whereas in the nucleus, redox alterations can inhibit DNA binding of transcription factors (e.g., NF-[kappa]B and activator protein-1). The consequences include the promotion of expression of antioxidant genes and alterations of hepatocyte survival as well as the balance between necrotic versus apoptotic cell death. Therefore, the understanding of the redox regulation of proteins may have important clinical ramifications in understanding the pathogenesis of liver diseases. thiol; disulfide; S-glutathionylation

Natively unfolded phenylalanine-glycine (FG)-repeat domains are alleged to form the physical constituents of the selective barrier-gate in nuclear pore complexes during nucleocytoplasmic transport. Presently, the biophysical mechanism behind the selective gate remains speculative because of a lack of information regarding the nanomechanical properties of the FG domains. In this work, we have applied the atomic force microscope to measure the mechanical response of individual and clusters of FG molecules. Single-molecule force spectroscopy reveals that FG molecules are unfolded and highly flexible. To provide insight into the selective gating mechanism, an experimental platform has been constructed to study the collective behavior of surface-tethered FG molecules at the nanoscale. Measurements indicate that the collective behavior of such FG molecules gives rise to an exponentially decaying long-range steric repulsive force. This finding indicates that the molecules are thermally mobile in an extended polymer brush-like conformation. This assertion is confirmed by observing that the brush-like conformation undergoes a reversible collapse transition in less polar solvent conditions. These findings reveal how FG-repeat domains may simultaneously function as an entropic barrier and a selective trap in the near-field interaction zone of nuclear pore complexes; i.e., selective gate. force spectroscopy | nanomechanics | natively unfolded proteins | nuclear pore complex | selective gating

PKC[epsilon] has been shown to play a key role in the effect of the Gram-negative bacterial product LPS; however, the target for PKC[epsilon] in LPS signaling is unknown. LPS signaling is mediated by Toll-like receptor 4, which uses four adapter proteins, MyD88, MyD88 adapter-like (Mal), Toll/IL-1R domain-containing adapter inducing IFN-[beta] (Trif), and Trif-related adapter molecule (TRAM). Here we show that TRAM is transiently phosphorylated by PKC[epsilon] on serine-16 in an LPS-dependent manner. Activation of IFN regulatory factor 3 and induction of the chemokine RANTES, which are both TRAM-dependent, were attenuated in PKC[epsilon]-deficient cells. TRAMS16A is inactive when overexpressed and is attenuated in its ability to reconstitute signaling in TRAM-deficient cells. We have therefore uncovered a key process in Toll-like receptor 4 signaling, identifying TRAM as the target for PKC[epsilon]. innate immunity | LPS | myristoylation

The drive toward more sensitive single-molecule manipulation techniques has led to the recent development of optical tweezers capable of resolving the motions of biological systems at the subnanometer level, approaching the fundamental limit set by Brownian fluctuations. One successful approach has been the dual-trap optical tweezers, in which the system of study is held at both ends by microspheres in two separate optical traps. We present here a theoretical description of the Brownian limit on the spatial resolution of such systems and verify these predictions by direct measurement in a Brownian noise-limited dual-trap optical tweezers. We find that by detecting the positions of both trapped microspheres, correlations in their motions can be exploited to maximize the resolving power of the instrument. Remarkably, we show that the spatial resolution of dual optical traps with dual-trap detection is always superior to that of more traditional, single-trap designs, despite the added Brownian noise of the second trapped microsphere. single molecule | subnanometer resolution | signal-to-noise ratio

The enormous complexity of biological networks has led to the suggestion that networks are built of modules that perform particular functions and are 'reused' in evolution in a manner similar to reusable domains in protein structures or modules of electronic circuits. Analysis of known biological networks has revealed several modules, many of which have transparent biological functions. However, it remains to be shown that identified structural modules constitute evolutionary building blocks, independent and easily interchangeable units. An alternative possibility is that evolutionary modules do not match structural modules. To investigate the structure of evolutionary modules and their relationship to functional ones, we integrated a metabolic network with evolutionary associations between genes inferred from comparative genomics. The resulting metabolic-genomic network places metabolic pathways into evolutionary and genomic context, thereby revealing previously unknown components and modules. We analyzed the integrated metabolic-genomic network on three levels: macro-, meso-, and microscale. The macroscale level demonstrates strong associations between neighboring enzymes and between enzymes that are distant on the network but belong to the same linear pathway. At the mesoscale level, we identified evolutionary metabolic modules and compared them with traditional metabolic pathways. Although, in some cases, there is almost exact correspondence, some pathways are split into independent modules. On the microscale level, we observed high association of enzyme subunits and weak association of isoenzymes independently catalyzing the same reaction. This study shows that evolutionary modules, rather than pathways, may be thought of as regulatory and functional units in bacterial genomes. clustering | evolution | modules

Myosin-V is a linear molecular motor that hydrolyzes ATP to move processively toward the plus end of actin filaments. Motion of this motor under low forces has been studied recently in various single-molecule assays. In this paper we show that myosin-V reacts to high forces as a mechanical ratchet. High backward loads can induce rapid and processive backward steps along the actin filament. This motion is completely independent of ATP binding and hydrolysis, in contrast, forward forces cannot induce ATP-independent forward steps. We can explain this pronounced mechanical asymmetry by a model in which the strength of actin binding of a motor head is modulated by the lever arm conformation. Knowledge of the complete force-velocity dependence of molecular motors is important to understand their function in the cellular environment. backward movement | molecular motor r optical tweezers | asymmetry | kinesin

By applying atomic force microscope (AFM)-based force spectroscopy together with computational modeling in the form of molecular force-field simulations, we have determined quantitatively the actuation energetics of a synthetic motor-molecule. This multidisciplinary approach was performed on specifically designed, bistable, redox-controllable [2]rotaxanes to probe the steric and electrostatic interactions that dictate their mechanical switching at the single-molecule level. The fusion of experimental force spectroscopy and theoretical computational modeling has revealed that the repulsive electrostatic interaction, which is responsible for the molecular actuation, is as high as 65 kcal*[mol.sup.-1], a result that is supported by ab initio calculations. computational modeling | force spectroscopy | molecular motors | switchable rotaxanes

Although the endothelial expression of various adhesion molecules substantially differs between pulmonary microvessels, their importance for neutrophil and lymphocyte sequestration in ventilator-induced lung injury (VILI) has not been systematically analyzed. We investigated the kinetics of polymorphonuclear cells (PMN) and mononuclear cells (MN) in the acinar microcirculation of the isolated rat lung with VILI by real-time confocal laser fluorescence microscopy, with or without inhibition of ICAM-1, VCAM-1, or P-selectin by monoclonal antibodies (MAb). Adhesion molecules in each microvessel were estimated by intravital fluorescence microscopy or immunohistochemical staining. In high tidal volume-ventilated lungs, 1) ICAM-1, VCAM-1, and P-selectin were differently upregulated in venules, arterioles, and capillaries; 2) venular PMN rolling was improved by inhibition of ICAM-1, VCAM-1, or P-selectin, whereas arteriolar PMN rolling was improved by ICAM-1 or VCAM-1 inhibition; 3) capillary PMN entrapment was ameliorated only by anti-ICAM-1 MAb; and 4) MN rolling in venules and arterioles and MN entrapment in capillaries were improved by ICAM-1 and VCAM-1 inhibition. In conclusion, the contribution of endothelial adhesion molecules to abnormal leukocyte behavior in VILI-injured microcirculation is microvessel and leukocyte specific. ICAM-1- and VCAM-1-dependent, but P-selectin-independent, arteriolar PMN rolling, which is expected to reflect the initial stage of tissue injury, should be taken as a phenomenon unique to ventilator-associated lung injury. neutrophil; lymphocyte; rolling; adhesion

The folding mechanisms of proteins are increasingly being probed through single-molecule experiments in which the protein is immobilized on a surface. Nevertheless, a clear understanding of how the surface might affect folding, and whether or not it changes folding from its bulk behavior, is lacking. In this work, we use molecular dynamics simulations of a model [beta]-barrel protein tethered to a surface to systematically investigate how the surface impacts folding. In the bulk, this protein folds in a three-state manner through a compact intermediate state, and its transition state (TS) has a well formed hydrophobic core. Upon tethering, we find that folding rates and stability are impacted differently by the surface, with dependencies on both the length and location of the tether. Significant changes in folding times are observed for tether points that do not alter the folding temperature. Tethering also locally enhances the formation of structure for residues proximal to the tether point. We find that neither the folding mechanism nor the TS of this protein are altered if the tether is in a fully structured or completely unstructured region of the TS, By contrast, tethering in a partially structured region of the TS leads to dramatic changes. For one such tether point, the intermediate present in bulk folding is eliminated, leading to a two-state folding process with a heterogeneous, highly unstructured TS ensemble. These results have implications for both the design of single-molecule experiments and biotechnological applications of tethered proteins. molecular dynamics simulations | protein surface interactions | single-molecule spectroscopy

Torque is generated in the rotary motor at the base of the bacterial flagellum by ion translocating stator units anchored to the peptidoglycan cell wall. Stator units are composed of the proteins MotA and MotB in proton-driven motors, and they are composed of PomA and PomB in sodium-driven motors. Strains of Escherichia coli lacking functional stator proteins produce flagella that do not rotate, and induced expression of the missing proteins leads to restoration of motor rotation in discrete speed increments, a process known as 'resurrection.' Early work suggested a maximum of eight units. More recent indications that WT motors may contain more than eight units, based on recovery of disrupted motors, are inconclusive. Here we demonstrate conclusively that the maximum number of units in a motor is at least 11. Using back-focal-plane interferometry of 1-[micro]m polystyrene beads attached to flagella, we observed at least 11 distinct speed increments during resurrection with three different combinations of stator proteins in E. coli. The average torques generated by a single unit and a fully induced motor were lower than previous estimates. Speed increments at high numbers of units are smaller than those at low numbers, indicating that not all units in a fully induced motor are equivalent. molecular motors | single molecules | MotA | PomA | resurrection