Flexible Attachment Research Articles

RGD constructs with physical anchor groups as polymer co‐electrospinnable cell adhesives

The tissue integration of synthetic polymers can be promoted by displaying RGD peptides at the biointerface with the objective of enhancing colonization of the material by endogenous cells. A firm but flexible attachment of the peptide to the polymer matrix, still allowing interaction with receptors, is therefore of interest. Here, the covalent coupling of flexible physical anchor groups, allowing for temporary immobilization on polymeric surfaces via hydrophobic or dipole–dipole interactions, to a RGD peptide was investigated. For this purpose, a stearate or an oligo(ethylene glycol) (OEG) was attached to GRGDS in 51–69% yield. The obtained RGD linker constructs were characterized by NMR, IR and MALDI‐ToF mass spectrometry, revealing that the commercially available OEG and stearate linkers are in fact mixtures of similar compounds. The RGD linker constructs were co‐electrospun with poly(p‐dioxanone) (PPDO). After electrospinning, nitrogen could be detected on the surface of the PPDO fibers by X‐ray photoelectron spectroscopy. The nitrogen content exceeded the calculated value for the homogeneous material mixture suggesting a pronounced presentation of the peptide on the fiber surface. Increasing amounts of RGD linker constructs in the electrospinning solution did not lead to a detection of an increased amount of peptide on the scaffold surface, suggesting inhomogeneous distribution of the peptide on the PPDO fiber surface. Human adipose‐derived stem cells cultured on the patches showed similar viability as when cultured on PPDO containing pristine RGD. The fully characterized RGD linker constructs could serve as valuable tools for the further development of tissue‐integrating polymeric scaffolds. Copyright © 2016 John Wiley & Sons, Ltd.

Flexible Attachment Designs for Rapid Tooling: A Contribution to Greater Design Freedom within Pin-type Moulding, Spatially Curved CFRP Panels

Form flexible moulding by pin-type tooling enables the mass-customised production of spatially curved panels. As the design freedom of such panels is limited to the arrangement density of pins, more compactly arranged pins come to mind. However, increasing this density is limited since the increase in the number of pins and thereby also the mould costs is disproportionately large. To manufacture panels with filigree geometries while using lower pin arrangement densities, attaching rapid tooling moulds is useful. Since such moulds are to be attached on uneven interpolation surfaces, novel flexible and non-destructive (dis-)attachment designs of rapid tooling attachments were developed and investigated. The results enable greater design freedom within pin-type moulding, spatially curved panels.

A versatile approach to grafting biofouling resistant coatings from polymeric membrane surfaces using an adhesive macroinitiator

The use of a polydopamine-based macroinitiator provides a flexible attachment method that is virtually independent of membrane substrate. The subsequent ARGET-ATRP controllably grafts the stable biofouling resistant polyzwitterion coating.

Identification and Synthesis of Macrolide Pheromones of the Grain Beetle Oryzaephilus Surinamensis and the Frog Spinomantis Aglavei

Macrolide lactones, the so called cucujolides derived from unsaturated fatty acids, are aggregation pheromones of cucujid grain beetles. Thirty years ago, Oehlschlarger et al. showed that (3Z,6Z)-dodeca-3,6-dien-11-olide (4) and the respective 12-olide (7) attract the sawtoothed grain beetle Oryzaephilus surinamensis, whereas (5Z,8Z,13R)-tetradeca-5,8-dien-13-olide (5) increases the response synergistically. The frass of this beetle is attractive for its parasitoid Cephalonomia tarsalis, which potentially can be used for pest control. A GC/MS analysis of attractive frass showed the presence of 5, together with an unknown isomer. Cucujolide V was tentatively identified also in the femoral glands, pheromone-releasing structures, of the Madagascan mantelline frog Spinomantis aglavei. Therefore, a new route to synthesize doubly unsaturated macrolides allowing the flexible attachment of the side chain was developed. A straightforward method to obtain Z configured macrolides involves ring-closing alkyne metathesis (RCAM) followed by Lindlar-catalyzed hydrogenation. This methodology was extended to homoconjugated diene macrolides by using RCAM after introduction of one Z configured double bond in the precursor by Wittig reaction. A tungsten benzylidyne complex was used as the catalyst in the RCAM reaction, which afforded the products in high yield at room temperature. With the synthetic material at hand, the unknown isomer was identified as the new natural product (5Z,8Z,12R)-tetradeca-5,8-dien-12-olide, cucujolide X (8). Furthermore, the route also allowed the synthesis of cucujolide V in good yield. The natural products were identified by the synthesis of enantiomerically pure or enriched material and gas chromatography on chiral phases. The new macrolide (R)-8 proved to be biologically active, attracting female O. surinamensis, but no males. The synthetic material allowed the identification of (R)-5 in both the beetle and the frog.

Solution structure of CCP modules 10-12 illuminates functional architecture of the complement regulator, factor H.

The 155-kDa plasma glycoprotein factor H (FH), which consists of 20 complement control protein (CCP) modules, protects self-tissue but not foreign organisms from damage by the complement cascade. Protection is achieved by selective engagement of FH, via CCPs 1–4, CCPs 6–8 and CCPs 19–20, with polyanion-rich host surfaces that bear covalently attached, activation-specific, fragments of complement component C3. The role of intervening CCPs 9–18 in this process is obscured by lack of structural knowledge. We have concatenated new high-resolution solution structures of overlapping recombinant CCP pairs, 10–11 and 11–12, to form a three-dimensional structure of CCPs 10–12 and validated it by small-angle X-ray scattering of the recombinant triple‐module fragment. Superimposing CCP 12 of this 10–12 structure with CCP 12 from the previously solved CCP 12–13 structure yielded an S-shaped structure for CCPs 10–13 in which modules are tilted by 80–110° with respect to immediate neighbors, but the bend between CCPs 10 and 11 is counter to the arc traced by CCPs 11–13. Including this four-CCP structure in interpretation of scattering data for the longer recombinant segments, CCPs 10–15 and 8–15, implied flexible attachment of CCPs 8 and 9 to CCP 10 but compact and intimate arrangements of CCP 14 with CCPs 12, 13 and 15. Taken together with difficulties in recombinant production of module pairs 13–14 and 14–15, the aberrant structure of CCP 13 and the variability of 13–14 linker sequences among orthologues, a structural dependency of CCP 14 on its neighbors is suggested; this has implications for the FH mechanism.

Dynamic Interaction of the Escherichia coli Cell Division ZipA and FtsZ Proteins Evidenced in Nanodiscs

The full-length ZipA protein from Escherichia coli, one of the essential components of the division proto-ring that provides membrane tethering to the septation FtsZ protein, has been incorporated in single copy into nanodiscs formed by a membrane scaffold protein encircling an E. coli phospholipid mixture. This is an acellular system that reproduces the assembly of part of the cell division components. ZipA contained in nanodiscs (Nd-ZipA) retains the ability to interact with FtsZ oligomers and with FtsZ polymers. Interactions with FtsZ occur at similar strengths as those involved in the binding of the soluble form of ZipA, lacking the transmembrane region, suggesting that the transmembrane region of ZipA has little influence on the formation of the ZipA·FtsZ complex. Peptides containing partial sequences of the C terminus of FtsZ compete with FtsZ polymers for binding to Nd-ZipA. The affinity of Nd-ZipA for the FtsZ polymer formed with GTP or GMPCPP (a slowly hydrolyzable analog of GTP) is moderate (micromolar range) and of similar magnitude as for FtsZ-GDP oligomers. Polymerization does not stabilize the binding of FtsZ to ZipA. This supports the role of ZipA as a passive anchoring device for the proto-ring with little implication, if any, in the regulation of its assembly. Furthermore, it indicates that the tethering of FtsZ to the membrane shows sufficient plasticity to allow for its release from noncentral regions of the cytoplasmic membrane and its subsequent relocation to midcell when demanded by the assembly of a division ring.

Mimicking cell membrane-like structures on alkylated silicon surfaces by peptide amphiphiles

We present a new strategy for flexible attachment of peptide amphiphiles on functionalized silicon surfaces. This method involves the production of an alkylated surface on which a lipidated peptide can then be attached through hydrophobic interaction. We applied this to two derivatives of amphiphilic peptide molecules with the same amino acid sequence (A-A-A-A-G-G-G-E-R-G-D) but different in alkyl chain lengths (palmitic acid, undecanoic acid). The basis of this work was to develop substrates which are more biocompatible and bioactive. The ultra-thin peptide amphiphile films were characterized using electrical impedance spectroscopy (EIS), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (ATR-FTIR) spectroscopy. The results demonstrated that the length of the alkyl chain in the peptide amphiphile affects the packing and coverage of the peptides on the silicon surface.

Das aktive Mittelohrimplantat in der Rehabilitation von sensorineuralen, kombinierten und Schallleitungs-Hörstörungen

Active middle ear implants, such as the Vibrant Soundbridge, are used as an important part in the rehabilitation of sensorineural, conductive hearing, or mixed hearing loss. The attachment of the Vibrant Soundbridge at the round window and the usage of the Vibroplasty couplers strongly expanded the application of the Vibrant Soundbridge.The Vibrant Soundbridge is developed for patients who have an intolerance to hearing aids and a moderate to profound sensorineural hearing loss. The VSB also provides an optimal solution for patients with failed middle ear reconstructions or patients with atresia. To capture the improvement with the VSB Implant with different hearing losses a literature analysis was conducted. The functional gain was analyzed for 107 patients with conductive hearing loss and for 214 patients with sensorineural hearing loss out of 14 studies.Patients with conductive and mixed hearing loss resulted in a functional gain from 30 to 58 dB with the VSB. Patients with a pure sensorineural hearing loss showed a functional gain of 23-30 dB. The VSB bone conduction threshold shift was analyzed for all studies conducted in the years between 2000 and 2009. In 11 of the 16 studies there was no significant (p=0.05) change found. In 5 studies, the pre- to post-surgical bone conduction threshold shift was less than 10 dB. None of these studies measured a threshold shift of more than 10 dB.The flexible attachment at either the long process of the incus with sensorineural hearing loss, with an conductive hearing loss at the round window or the use of Vibroplasty couplers at the oval window, head of the stapes or round window makes the VSB an extremely versatile instrument. If patients can't wear conventional hearing aids, had failed middle ear reconstructions or atresia the VSB presents, due to the significant hearing improvement in any type of hearing loss, an ideal solution.

Structural Characterization of Intramolecular Hg2+ Transfer between Flexibly Linked Domains of Mercuric Ion Reductase

The enzyme mercuric ion reductase MerA is the central component of bacterial mercury resistance encoded by the mer operon. Many MerA proteins possess metallochaperone-like N-terminal domains (NmerA) that can transfer Hg2+ to the catalytic core domain (Core) for reduction to Hg0. These domains are tethered to the homodimeric Core by ∼30-residue linkers that are susceptible to proteolysis, the latter of which has prevented characterization of the interactions of NmerA and the Core in the full-length protein. Here, we report purification of homogeneous full-length MerA from the Tn21 mer operon using a fusion protein construct and combine small-angle X-ray scattering and small-angle neutron scattering with molecular dynamics simulation to characterize the structures of full-length wild-type and mutant MerA proteins that mimic the system before and during handoff of Hg2+ from NmerA to the Core. The radii of gyration, distance distribution functions, and Kratky plots derived from the small-angle X-ray scattering data are consistent with full-length MerA adopting elongated conformations as a result of flexibility in the linkers to the NmerA domains. The scattering profiles are best reproduced using an ensemble of linker conformations. This flexible attachment of NmerA may facilitate fast and efficient removal of Hg2+ from diverse protein substrates. Using a specific mutant of MerA allowed the formation of a metal-mediated interaction between NmerA and the Core and the determination of the position and relative orientation of NmerA to the Core during Hg2+ handoff.

Numerical Simulation and Testing of Water Impact of Structural Attachment Elements of a Reusable Thermal Protection System

Multiple mission reuse capability has become extremely important, towards reducing costs of space transportation. Carbon / Carbon (C/C) composites are well proven, functionally, for repeated use in re-entry missions. A re-entry capsule with sphere-cone-flare external shape, currently under realisation, will fly with C/C Thermal Protection System (TPS) in its peak heating region. The biggest challenge in design of such a reusable hot structure TPS is the management of thermo-structural loads. Differential Coefficient of Thermal Expansion (CTE) is the main cause of stress on the structural assembly elements. A set of flexible super alloy attachment brackets have been configured to take care of this differential thermal expansion of various TPS elements. The brackets also have to survive the impact transient load, on splashdown. This load was estimated using explicit non linear Finite Element method by considering the whole structure a rigid body. A separate FE model with actual stiffness of the structural attachments and the hot structure was generated, to predict the stresses caused by the load. In order to demonstrate the margins and survivability of the assembly, as a whole, a water impact test with actual qualification model of the assembly was carried out in a 10 m deep shock tank. The test also helped to validate the prediction. Considering factors such as cost, time and process constraints involved in realising C/C TPS for the test, it was decided to replace the same with an equivalent structure that satisfied all design and functional requirements. The test article was dropped vertically to simulate an impact velocity of 12 m/s and was adequately instrumented with accelerometers and strain gauges. The test results correlate reasonably well with the prediction.

Trimeric structure and flexibility of the L1ORF1 protein in human L1 retrotransposition

The LINE-1 (L1) retrotransposon emerges as a major source of human interindividual genetic variation, with important implications for evolution and disease. L1 retrotransposition is poorly understood at the molecular level, and the mechanistic details and evolutionary origin of the L1-encoded L1ORF1 protein (L1ORF1p) are particularly obscure. Here three crystal structures of trimeric L1ORF1p and NMR solution structures of individual domains reveal a sophisticated and highly structured, yet remarkably flexible, RNA-packaging protein. It trimerizes via an N-terminal, ion-containing coiled coil that serves as scaffold for the flexible attachment of the central RRM and the C-terminal CTD domains. The structures explain the specificity for single-stranded RNA substrates, and a mutational analysis indicates that the precise control of domain flexibility is critical for retrotransposition. Although the evolutionary origin of L1ORF1p remains unclear, our data reveal previously undetected structural and functional parallels to viral proteins.

ITER Port Plug Engineering Trainee Program: Design, manufacturing and integration of structural components (analysis of the attachment)

The structural connection of helium cooled plasma facing components in ITER to the water cooled structural port plug shield requires an attachment system, which is able to cope with two main contradicting requirements: The attachment system has to be rigid in order to withstand mechanical loads, e.g. due to the deadweight or static and transient electro-magnetic loads. On the other hand, the attachment system has to be flexible in order to compensate the different thermal strains in between the plasma facing test devices (300–550 °C) and the port plug structure (∼120 °C). The paper presents the latest developments of an attachment system consisting of flexible attachment blocks with lamellae. The optimization steps as well as the connection to the shield are described. The results of the thermo-mechanical analyses under a defined worst-case scenario confirm the feasibility of the lamella design. This work has been performed in the frame of the EFDA goal orientated trainee program on port plug engineering.

Application Analysis of Form-Finding on the Large Building Construction Tower Crane Flexible Attachments

A modeling method of tower crane flexible attachments based on ANSYS is researched for more practical crane. Form-finding is used to model and analyze flexible attachments and the analytic equations for typical example are derived. Comparing the analytic solutions with ANSYS solutions, the biggest relative error is 0.45% which is the foot moment relative error while cantilever beam top transverse force is 5×105N. The results show that the form-finding method accords with engineering practice, and is reliable and suitable for structural analysis of flexible attachment tower crane.

Fiber-Reinforced Polymer Decks for Movable Bridges

Fiber-reinforced polymer (FRP) bridge decks are ideal for re-decking movable bridges. They can match the weight of existing steel grid decking to minimize bridge balance adjustment. They are bicycle- and motorcycle-friendly and quieter than steel grids. They protect the structure from precipitation and are corrosion resistant. In 2002, the Oregon Department of Transportation (ODOT) installed FRP decks on two movable bridges near Astoria. Cementitious grout pads supported the deck on the steel stringers, and the deck was secured to the stringers by welded steel studs in cementitious grout pockets. The wearing surface was to have asphalt concrete, but was changed to epoxy polymer concrete after the asphalt slid off a bascule leaf during a prolonged test lift. The grout details had failed on one of these bridges, resulting in deck replacement, and both bridges had experienced distress in the wearing surface. The attachment details and wearing surfaces used on these bridges did not accommodate the higher deflections associated with FRP decks, and were regarded as unacceptable. In 2005, the ODOT installed a FRP deck on a movable bridge in Florence. Neoprene sheets supported the deck on the steel stringers, and the deck was secured to the stringers with large structural blind fasteners installed from below. The wearing surface was urethane polymer concrete, placed by the broom and seed method. This installation showed none of the distress experienced on the two bridges at Astoria. The strong but flexible attachment details and wearing surface used on this bridge accommodate deflections well and were recommended for future installations.

Membrane Alignment of the Pore-Forming Component TatAd of the Twin-Arginine Translocase from Bacillus subtilis Resolved by Solid-State NMR Spectroscopy

The twin-arginine translocase (Tat) provides protein export in bacteria and plant chloroplasts and is capable of transporting fully folded proteins across the membrane. We resolved the conformation and membrane alignment of the pore-forming subunit TatA(d) from Bacillus subtilis using solid-state NMR spectroscopy. The relevant structured part of the protein, TatA(2-45), contains a transmembrane segment (TMS) and an amphiphilic helix (APH). It was reconstituted in planar bicelles, which represent the lipid environment of a bacterial membrane. The SAMMY solid-state NMR experiment was used to correlate (15)N chemical shifts and (1)H-(15)N dipolar couplings in the backbone and side chains of the (15)N-labeled protein. The observed wheel-like patterns ("PISA wheels") in the resulting 2-dimensional spectra confirm the α-helical character of the two segments and reveal their alignment in the lipid bilayer. Helix tilt angles (τ(TMS) = 13°, τ(APH) = 64°) were obtained from uniformly labeled protein, and azimuthal rotations (ρ(Val15) = 235°, ρ(Ile29) = 25°) were obtained from selective labels. These constraints define two distinct families of allowed structures for TatA in the membrane-bound state. The manifold of solutions could be narrowed down to a unique structure by using input from a liquid-state NMR study of TatA in detergent micelles, as recently described [Hu, Y.; Zhao, E.; Li, H.; Xia, B.; Jin, C. J. Am. Chem. Soc. 2010, DOI: 10.1021/ja1053785]. Interestingly, the APH showed an unexpectedly slanted alignment in the protein, different from that of the isolated APH peptide. This finding implies that the amphiphilic region of TatA is not just a flexible attachment to the transmembrane anchor but might be able to form intra- or even intermolecular salt-bridges, which could play a key role in pore assembly.

Finite Element Analysis on Flexible Attachments of Luffing Jib Tower Crane

According to the mechanical characteristics of a certain luffing jib tower crane structure, finite element calculation model of the whole machine considering suspended cable under complicated working conditions is built. Nonlinear large displacements static structural analysis of the crane flexible attachment in the on-linear flexible scope is conducted with software ANSYS. The influence of variational attachment parameter on crane structure and anchoring points performance and the flexible attachment rule of crane are researched. Curves of influence of distance between two layers and tower overhanging height on crane structure and anchoring points performance and the reasonable layers and distance of flexible attachment are obtained and attachment rule of flexible attachment tower crane is derived.

Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

The structure and dynamics of proteins underlies the workings of virtually every biological process. Existing biophysical methods are inadequate to measure protein structure at atomic resolution, on a rapid time scale, with limited amounts of protein, and in the context of a cell or membrane. FRET can measure distances between two probes, but depends on the orientation of the probes and typically works only over long distances comparable with the size of many proteins. Also, common probes used for FRET can be large and have long, flexible attachment linkers that position dyes far from the protein backbone. Here, we improve and extend a fluorescence method called transition metal ion FRET that uses energy transfer to transition metal ions as a reporter of short-range distances in proteins with little orientation dependence. This method uses a very small cysteine-reactive dye monobromobimane, with virtually no linker, and various transition metal ions bound close to the peptide backbone as the acceptor. We show that, unlike larger fluorophores and longer linkers, this donor-acceptor pair accurately reports short-range distances and changes in backbone distances. We further extend the method by using cysteine-reactive metal chelators, which allow the technique to be used in protein regions of unknown secondary structure or when native metal ion binding sites are present. This improved method overcomes several of the key limitations of classical FRET for intramolecular distance measurements.

Mechanism of DNA substrate recognition by the mammalian DNA repair enzyme, Polynucleotide Kinase.

Mammalian polynucleotide kinase (mPNK) is a critical DNA repair enzyme whose 5′-kinase and 3′-phoshatase activities function with poorly understood but striking specificity to restore 5′-phosphate/3′-hydroxyl termini at sites of DNA damage. Here we integrated site-directed mutagenesis and small-angle X-ray scattering (SAXS) combined with advanced computational approaches to characterize the conformational variability and DNA-binding properties of mPNK. The flexible attachment of the FHA domain to the catalytic segment, elucidated by SAXS, enables the interactions of mPNK with diverse DNA substrates and protein partners required for effective orchestration of DNA end repair. Point mutations surrounding the kinase active site identified two substrate recognition surfaces positioned to contact distinct regions on either side of the phosphorylated 5′-hydroxyl. DNA substrates bind across the kinase active site cleft to position the double-stranded portion upstream of the 5′-hydroxyl on one side, and the 3′-overhang on the opposite side. The bipartite DNA-binding surface of the mPNK kinase domain explains its preference for recessed 5′-termini, structures that would be encountered in the course of DNA strand break repair.

Multimeric Options for the Auto-Activation of the Saccharomyces cerevisiae FAS Type I Megasynthase

The fungal type I fatty acid synthase (FAS) is a 2.6 MDa multienzyme complex, catalyzing all necessary steps for the synthesis of long acyl chains. To be catalytically competent, the FAS must be activated by a posttranslational modification of the central acyl carrier domain (ACP) by an intrinsic phosphopantetheine transferase (PPT). However, recent X-ray structures of the fungal FAS revealed a barrel-shaped architecture, with PPT located at the outside of the barrel wall, spatially separated from the ACP caged in the inner volume. This separation indicated that the activation has to proceed before the assembly to the mature complex, in a conformation where the ACP and PPT domains can meet. To gain insight into the auto-activation reaction and also into the fungal FAS assembly pathway, we structurally and functionally characterized the Saccharomyces cerevisiae FAS type I PPT as part of the multienzyme protein and as an isolated domain.

Double hearing protection device

A double hearing protection system (120) comprising an earplug portion (122) of the system contains a tether attachment mechanism (123) that may be selectively attached to the interior of a circumaural hearing protector (125) designed with the mating attachment (154). The tether (123) retracts into the earcup (125) when both the earplug (122) and circumaural hearing protector (125) are worn, ensuring that the earseal (126) against the head is not broken by the retractable tether (123). The attachment point (154) inside the earcup (125) mates to the end of the tether (123) in a way that prevents foreign object damage in dangerous work environments, and limits the vibratory path from the earcup (125) to the earplug (122) through a flexible attachment mechanism (154).