Molecular kinetics and cooperative effects in friction and adhesion of fast reversible bonds.

Molecular mechanisms of adhesion and friction include the rupture of single and multiple bonds. The strength of adhesion and friction thus depends on the molecular kinetics and cooperative effects in the lifetime of bonds under stress. We measured the rate dependence of friction and adhesion mediated by supramolecular guest-host bonds using atomic force microscopy (AFM). The tip of the AFM and the surface were functionalized with cyclodextrin hosts. The influence of molecular kinetics on adhesion and friction was studied using three different ditopic guest molecules that connected the AFM tip and the surface. Adamantane, ferrocene, and azobenzene were the guest end groups of the connector molecules that formed inclusion complexes with the cyclodextrin hosts. The results confirm the importance of the molecular off-rate and of cooperative effects for the strength of adhesion and friction. Positive cooperativity also shapes the dependence of friction on the concentration of connector molecules, which follows the Hill-Langmuir model. Based on the Hill coefficient of 3.6, reflecting a characteristic rupture of at least 3-4 parallel bonds, a rescaling of the pulling rate is suggested that shifts the rate dependence of adhesion and friction for the three different molecules towards one master curve.

Discrimination of proteins through interaction with pyrene-labelled polymer aggregates.

A pyrene-labelled PDMAEMA (2-(dimethylamino)ethyl methacrylate) polymer was synthesized through a controlled radical RAFT polymerisation approach. An average pyrene content of 3.65% was determined by UV/Vis and 1H NMR measurements. DLS measurements reveal the formation of polymer aggregates with an average size of 172 nm in aqueous phosphate buffer indicating the presence of hydrophobic interactions between pyrene and/or DMAEMA moieties of adjacent polymer chains. Furthermore, this aggregation results in the appearance of two characteristic emission bands at 394 and 488 nm analyzed by fluorescence measurements. Based on spectral changes of the so-called monomer and excimer emission intensity, the specific discrimination of various non-metallo- and metallo proteins was realized using an optical fingerprint approach. DLS and fluorescence measurements show a significant dependence of the structural characteristics of the analytes on the presence of different binding modes between the hydrophilic DMAEMA side groups of the polymer and the proteins, resulting in a molecular disassembly of the aggregates and/or fluorescence quenching. Furthermore, pH, temperature and ionic strength dependence of the sensor polymer with BSA was investigated to optimise the external parameters. Based on these results, the most specific discrimination of the analyzed proteins was obtained using a sodium chloride concentration of 50 mM and a pH of 7.0. This study gives fundamental insights into the sensing performance of a novel one-component pyrene-based polymer system and its application as a protein sensor.

Real-Time Ultrasound Doppler Enhances Precision in Image-Guided Approaches to the Cerebellopontine Angle.

OBJECTIVE: To evaluate efficacy and reliability of intraoperative Doppler sonography in localizing the transverse and sigmoid sinuses during lateral suboccipital craniotomy. METHODS: A 16-Mhz intraoperative micro-Doppler ultrasound (16Mhz, Multi-Dop pro, Compumedics, Singen, Germany) was applied to detect the medial border of the sigmoid sinus and the inferior border of the transverse sinus in 25 patients. Micro-Doppler measurements were compared with magnetic resonance- and computed tomography-based image guidance (Kolibri, Brainlab, Munich, Germany). Visual detectability of the sinuses with the operating microscope was also documented. RESULTS: Inadvertent incision of the transverse or sigmoid sinuses did not occur in any patient when the 2 localizing methods have been used in combination. The mean mismatch of image-guided system and micro-Doppler was 2.64 mm (range, 0-6 mm; standard deviation, 1.55 mm). With the microscope the transverse sinus was invisible in 7 patients, the sigmoid sinus was visually undetectable in 1 case. The micro-Doppler indicated blood flow outside the visible borders of the sinuses in 5 patients. CONCLUSIONS: A combination of image-guidance and micro-Doppler enhances the accuracy in localizing the margins of the transverse and sigmoid sinuses using the retrosigmoid approach, thus preventing inadvertent injury. The method could potentially be applied during other craniotomies involving the exposure of a venous sinus.

Interactions between shape-persistent macromolecules as probed by AFM.

Water-soluble shape-persistent cyclodextrin (CD) polymers with amino-functionalized end groups were prepared starting from diacetylene-modified cyclodextrin monomers by a combined Glaser coupling/click chemistry approach. Structural perfection of the neutral CD polymers and inclusion complex formation with ditopic and monotopic guest molecules were proven by MALDI-TOF and UV-vis measurements. Small-angle neutron and X-ray (SANS/SAXS) scattering experiments confirm the stiffness of the polymer chains with an apparent contour length of about 130 Å. Surface modification of planar silicon wafers as well as AFM tips was realized by covalent bound formation between the terminal amino groups of the CD polymer and a reactive isothiocyanate-silane monolayer. Atomic force measurements of CD polymer decorated surfaces show enhanced supramolecular interaction energies which can be attributed to multiple inclusion complexes based on the rigidity of the polymer backbone and the regular configuration of the CD moieties. Depending on the geometrical configuration of attachment anisotropic adhesion characteristics of the polymer system can be distinguished between a peeling and a shearing mechanism.

Single-molecule force spectroscopy of fast reversible bonds.

In single-molecule force spectroscopy, the unbinding force is often used to quantify the interaction strength of single molecular bonds. We analyze force spectroscopy of fast reversible bonds probed in thermodynamic equilibrium by considering the dynamics of force probe and molecular linker. The effect of cantilever and linker dynamics is systematically addressed by measuring the unbinding force of single cyclodextrin inclusion complexes by atomic force spectroscopy for a variety of molecular linkers and varying force probe stiffness. The unbinding force of individual bonds probed in thermodynamic equilibrium is not unique for the molecular system but scales with , the square root of the force probe stiffness, and is largely independent of the molecular linker stiffness. The observations are explained by an effective potential resulting from fast linker fluctuations and fast rebinding kinetics which is probed by an AFM cantilever. The slow cantilever dynamics in the kHz range act as mechanical low pass filter, allowing for fast rebinding kinetics of the molecular complex in the order of 106 kHz. The binding energy of the complex can be estimated from the unbinding force as a function of cantilever stiffness, however with some uncertainty arising from lack of a model in three dimensions.

Site-specific conjugation of 8-ethynyl-BODIPY to a protein by [2 + 3] cycloaddition.

We report a straightforward synthesis of 8-ethynyl-BODIPY derivatives and their potential as fluorescent labeling compounds using an alkyne-azide click chemistry approach. The ethynyl substituted BODIPY dyes at the meso-position were reacted under Cu(+) catalysis and mild physiological conditions in organic and biological model systems using benzyl azide and a Barstar protein which was selectively modified by a single amino acid substituted methionine at the N-terminus (Met1) → azidohomoalanine (Aha). Conjugation with the protein and the model azide was indicated by a significant blue shift upon formation of the triazole moiety system, which allowed easy distinction between free and coupled dyes. This blue shift was rationalized by the perpendicular orientation of the triazole relative to the chromophore using time dependent density functional theory (TDDFT) calculations. A full spectroscopic and thermodynamic characterization of the protein revealed that a fluorophore was incorporated without the cross influence of protein stability and functional integrity. Furthermore, model reactions of 8-ethynyl-BODIPY derivatives with benzyl azide under copper-free conditions indicate second order kinetics with high rate constants comparable with those found for the strain-promoted azide-alkyne cycloaddition (SPAAC). In this way, we establish a unique and highly efficient method to introduce alkyne-BODIPY into a protein scaffold potentially useful for diverse applications in areas ranging from fundamental protein dynamics studies to biotechnology or cell biology.

Dynamic effects in friction and adhesion through cooperative rupture and formation of supramolecular bonds.

We introduce a molecular toolkit for studying the dynamics in friction and adhesion from the single molecule level to effects of multivalency. As experimental model system we use supramolecular bonds established by the inclusion of ditopic adamantane connector molecules into two surface-bound cyclodextrin molecules, attached to a tip of an atomic force microscope (AFM) and to a flat silicon surface. The rupture force of a single bond does not depend on the pulling rate, indicating that the fast complexation kinetics of adamantane and cyclodextrin are probed in thermal equilibrium. In contrast, the pull-off force for a group of supramolecular bonds depends on the unloading rate revealing a non-equilibrium situation, an effect discussed as the combined action of multivalency and cantilever inertia effects. Friction forces exhibit a stick-slip characteristic which is explained by the cooperative rupture of groups of host-guest bonds and their rebinding. No dependence of friction on the sliding velocity has been observed in the accessible range of velocities due to fast rebinding and the negligible delay of cantilever response in AFM lateral force measurements.

Switching adhesion and friction by light using photosensitive guest-host interactions.

Friction and adhesion between two ß-cyclodextrin functionalized surfaces can be switched reversibly by external light stimuli. The interaction between the cyclodextrin molecules attached to the tip of an atomic force microscope and a silicon wafer surface is mediated by complexation of ditopic azobenzene guest molecules. At the single molecule level, the rupture force of an individual complex is 61 ± 10 pN.

Red/near-infrared luminescence tuning of group-14 element complexes of dipyrrins based on a central atom.

A dipyrrin complex has been one of the most utilized fluorescent dyes, and a variety of dipyrrin complexes show intriguing functions based on the various coordination structures of the central element. We now report the synthesis, structure, and photophysical properties of germanium and stannane complexes of the N2O2-type tetradentate dipyrrin, L·Ge and L·Sn, which are heavier analogues of the previously reported dipyrrin silicon complex, L·Si. The central group-14 atoms of the monomeric complexes have geometries close to trigonal bipyramidal (TBP), in which the contribution of the square-pyramidal (SP) character becomes higher as the central atom is heavier. Interestingly, L·Sn formed a dimeric structure in the crystal. All complexes L·Si, L·Ge, and L·Sn showed a fluorescence in the red/NIR region. Fluorescence quantum yields of L·Ge and L·Sn are higher than that of L·Si. These results indicated that the central atom on the dipyrrin complexes contributes not only to the geometry difference but also to tuning the fluorescence properties.

Reasons for ordering computed tomography scans of the head in patients with minor brain injury.

BACKGROUND: Minor brain injury is a frequent condition. Validated clinical decision rules can help in deciding whether a computed tomogram (CT) of the head is required. We hypothesized that institutional guidelines are not frequently used, and that psychological factors are a common reason for ordering an unnecessary CT. METHODS: Physicians at the emergency department of a tertiary care hospital completed an anonymous questionnaire before ordering a CT of the head for a patient presenting with a GCS of 13-15 after a head trauma. RESULTS: Over a period of 10 months, 1018 CTs of the head were performed in patients presenting with a GCS of 13-15 after a head trauma; 168 (16.5%) questionnaires were completed. The most four common reasons for ordering a CT were "to confirm/rule out traumatic intracranial lesion" (in 94% of all questionnaires), "to expedite diagnosis" (63%) "guidelines" (58%) and "fear of missing a traumatic intracranial lesion" (50%). A positive answer for "fear of being sued" was declared in 21%, and "pressure from the patient or his relatives" in 8% of all questionnaires. Of 71 questionnaires without "guidelines" as a positive answer, there were 40 (56%) positive answers of "fear of missing a traumatic cerebral lesion". CONCLUSION: Besides guidelines, fear of missing a traumatic intracranial lesion played a role in ordering head CTs. Although the physicians had been instructed in the use of guidelines, including validated clinical decision rules, this did not prevent them from ordering unnecessary CTs.

Blue fluorescent amino acids as in vivo building blocks for proteins.

In vivo expression of colored proteins without post-translational modification or chemical functionalization is highly desired for protein studies and cell biology. Cell-permeable tryptophan analogues, such as azatryptophans, have proved to be almost ideal isosteric substitutes for natural tryptophan in cellular proteins. Their unique spectral features, such as markedly red-shifted fluorescence, are transmitted into protein structures upon incorporation. Among the azaindoles under study (2-, 4-, 5-, 6-, and 7-azaindole) 4-azaindole has exhibited the largest Stokes shift (approximately 130 nm) in steady-state fluorescence measurements. It is also highly biocompatible and as 4-azatryptophan it can be translated into target protein sequences. However, its quantum yield and fluorescence intensity are still significantly lower when compared with natural indole/tryptophan. Since azatryptophans are hydrophilic, their presence in the hydrophobic core of proteins could be harmful. In order to overcome these limitations we have performed nitrogen methylation of azaindoles and generated mono- and dimethylated azaindoles. Some of these methyl derivatives retain the pronounced red shift present in the parent 4-azaindole, but with much higher fluorescence intensity (reaching the level of indole/tryptophan). Therefore, the blue fluorescence of azaindole-containing proteins could be further enhanced by the use of methylated analogues. Further substitution of any azaindole ring with either endo- or exocyclic nitrogen will not yield a spectral fluorescence maximum shift beyond 450 nm under steady-state conditions in the physiological milieu. However, green fluorescence is a special feature of tautomeric species of azaindoles in various nonaqueous solvents. Thus, the design or evolution of the protein interior combined with the incorporation of these azaindoles might lead to the generation of specific chromophore microenvironments that facilitate tautomeric or protonated/deprotoned states associated with green fluorescence.

Redox active donor-substituted punicin derivatives.

The redox active plant material punicin from Punica granatum, 2-hydroxy-1-(pyridinium-1-yl)-5-olate, and some derivatives were modified by substitution with 2-oxochromen-4-olate moieties to give donor-substituted molecules which form distinct types of atropisomeric mesomeric betaines and tetrapolar substances. Oxidation to new negatively-charged solvatochromic quinones was achieved on treatment of these betaines with CAN, taking advantage of the stabilizing properties of the electron-donating 2-oxochromen-4-olate partial structure. The quinones can be employed in model redox reactions. Cyclovoltametric studies have been performed.

Imidazol-2-and-4-ylidene by decarboxylation. Studies on the cross-conjugated mesomeric betaine-alkaloid norzooanemonine and its pseudo-cross-conjugated isomer.

1,3-Dimethylimidazolium-2-carboxylate and -4-carboxylate (norzooanemonine), which belong to two distinct classes of heterocyclic mesomeric betaines, undergo thermal decarboxylations to the N-heterocyclic carbenes imidazol-2-ylidene and imidazol-4-ylidene, respectively. These carbenes can be detected by ESI mass spectrometry and can be trapped by isocyanates to imidazolium-amidates, the structure of which was proved by independent syntheses. We performed calculations to characterize the different types of conjugation in the imidazolium-carboxylates.