Engineering of the Escherichia coli Im7 immunity protein as a loop display scaffold

Protein scaffolds derived from non-immunoglobulin sources are increasingly being adapted and engineered to provide unique binding molecules with a diverse range of targeting specificities. The ColE7 immunity protein (Im7) from Escherichia coli is potentially one such molecule, as it combines the advantages of (i) small size, (ii) stability conferred by a conserved four anti-parallel alpha-helical framework and (iii) availability of variable surface loops evolved to inactivate members of the DNase family of bacterial toxins, forming one of the tightest known protein-protein interactions. Here we describe initial cloning and protein expression of Im7 and its cognate partner the 15 kDa DNase domain of the colicin E7. Both proteins were produced efficiently in E.coli, and their in vitro binding interactions were validated using ELISA and biosensor. In order to assess the capacity of the Im7 protein to accommodate extensive loop region modifications, we performed extensive molecular modelling and constructed a series of loop graft variants, based on transfer of the extended CDR3 loop from the IgG1b12 antibody, which targets the gp120 antigen from HIV-1. Loop grafting in various configurations resulted in chimeric proteins exhibiting retention of the underlying framework conformation, as measured using far-UV circular dichroism spectroscopy. Importantly, there was low but measurable transfer of antigen-specific affinity. Finally, to validate Im7 as a selectable scaffold for the generation of molecular libraries, we displayed Im7 as a gene 3 fusion protein on the surface of fd bacteriophages, the most common library display format. The fusion was successfully detected using an anti-Im7 rabbit polyclonal antibody, and the recombinant phage specifically recognized the immobilized DNase. Thus, Im7 scaffold is an ideal protein display scaffold for the future generation and for the selection of libraries of novel binding proteins.     

SAR by oxime-containing peptide libraries: application to Tsg101 ligand optimization

HIV-1 viral assembly requires a direct interaction between a Pro-Thr-Ala-Pro ("PTAP") motif in the viral protein Gag-p6 and the cellular endosomal sorting factor Tsg101. In an effort to develop competitive inhibitors of this interaction, an SAR study was conducted based on the application of post solid-phase oxime formation involving the sequential insertion of aminooxy-containing residues within a nonamer parent peptide followed by reaction with libraries of aldehydes. Approximately 15-20-fold enhancement in binding affinity was achieved by this approach.     

Increased Activity of Cell Surface Peptidases in HeLa Cells Undergoing UV-Induced Apoptosis Is Not Mediated by Caspase 3

We have previously shown that in HeLa cells treated with a variety of agents there is an increase in cell surface peptidase (CSP) activity in those cells undergoing apoptosis. The increase in CSP activity observed in UVB-irradiated cells undergoing apoptosis was unaffected when the cultures were treated with the aminopeptidase inhibitor bestatin, and matrix metalloprotease inhibitor BB3103, but greatly enhanced when treated with the caspase 3 inhibitor-DEVD, and reduced in the presence of the poly(ADP-ribose) polymerase (PARP) inhibitor-3-aminobenzamide (3AB). Neither 3AB nor DEVD had an effect on the gross morphology of the apoptotic cells observed under electron microscopy, nor did they have an effect on phosphatidylserine eversion on the cell membrane, or that of PARP cleavage. All the agents except for DEVD had no effect on the level of caspase 3 activity in the cells. The results suggest that other caspases may cleave PARP in these cells. Both 3AB and DEVD treatment reduced the level of actin cleavage seen in the apoptotic cells. The increase in CSP activity observed in cells undergoing UVB-induced apoptosis appears to involve PARP but not caspase 3.     

Feasibility of a perfluorocarbon tracer based network to support monitoring, verification, and accounting of sequestered CO₂

Carbon capture and sequestration (CCS) will act as a bridging technology necessary to facilitate a transition from fossil fuels to a sustainable energy based economy. The Department of Energy (DOE) target leak rate for sequestration reservoirs is 1% of total sequestered CO(2) over the lifetime of the reservoir. This is 0.001% per year for a 1000 year lifetime of a storage reservoir. Effective detection of CO(2) leaks at the surface may require incorporation of a tracer tag into the sequestered CO(2). We applied a simple Gaussian Plume model to predict dispersion of a direct leak into the atmosphere and used the results to examine the requirements for designing a perfluorocarbon (PFT) monitoring network and tracer tagging strategy. Careful consideration must be given to the climate implications of using these compounds. The quantity of PFTs needed for tagging sequestered CO(2) is too large to be practical for routine monitoring. Tagging at a level that will result in 1.5 times background at a sampler 1 km from a leak of 0.01% per year will require 625 kg per year of PFT. This is a leak rate 10 times greater than the 1000 year DOE requirement and will require 19 tons of injected PFT over the 30 year lifetime of a 1000 mega watt coal fired plant. The utility of PFTs or any other tracer will be lost if the background levels are allowed to rise indiscriminately. A better use of PFTs is as a tool in sequestration research. Instead, geological surveys of sequestration sites will be necessary to locate potential direct pathways and develop targeted monitoring strategies. A global agreement on the use of tracers for monitoring CCS projects should be developed.     

The Ferroelectric Field Effect within an Integrated Core/Shell Nanowire

The synthesis of cylindrical silicon‐core and ferroelectric oxide perovskite‐shell nanowires and their response characteristics as individual three‐terminal nanoscale electronic devices is reported. The co‐axial nanowire geometry facilitates large ferroelectric field‐effect modulation (>10⁴) of nanowire conductivity following sequential application and removal of an applied dc field. Source‐drain current–voltage traces collected during sweeps of ferroelectric gate potential and switching of the component of shell outward and inward polarization provide direct evidence of ferroelectric coupling on nanowire channel conductance. Despite a very small (1:20) ferroelectric‐to‐semiconductor channel thickness ratio, an unexpectedly strong electrostatic coupling of ferroelectric polarization to channel conductance is observed because of the co‐axial gate geometry and curvature‐induced strain enhancement of ferroelectric polarization.     

Polymer-graphene nanocomposites as ultrafast-charge and -discharge cathodes for rechargeable lithium batteries

Electroactive polymers are a new generation of "green" cathode materials for rechargeable lithium batteries. We have developed nanocomposites combining graphene with two promising polymer cathode materials, poly(anthraquinonyl sulfide) and polyimide, to improve their high-rate performance. The polymer-graphene nanocomposites were synthesized through a simple in situ polymerization in the presence of graphene sheets. The highly dispersed graphene sheets in the nanocomposite drastically enhanced the electronic conductivity and allowed the electrochemical activity of the polymer cathode to be efficiently utilized. This allows for ultrafast charging and discharging; the composite can deliver more than 100 mAh/g within just a few seconds.     

A study of non-operational dynamic responses of disk in 3.5 in. hard disk drive to impact load

Hard disk drives have to be designed to sustain operational and non-operational shock. There are many analytical models and numerical schemes proposed and many experiments conducted for analyzing the transient impact responses of hard disk drives. The existing researches have been focused on the slider-suspension responses at the head-disk interface in which the linear models have been used and the effects of spindle motor have been ignored. In this study, the complex vibrations of disk of 3.5 in. hard disk drive (HDD) under shock are experimentally investigated. The hammer impact test and linear drop test are conducted for the HDD to study the effect of shock on the disk responses. The results show that the nonlinear rock modes substantially contribute to the vibrations of disk when HDD is under shock impact. The nonlinear properties of the disk responses and the mode damping ratio are evaluated by using empirical mode decomposition approach.     

Labor‐Management Cooperation: Operation MOST

Unionized contractors and construction unions are in difficulty today because of their apparent increasing lack of competitiveness with the non‐union sector of the industry. Probiems contributing to this are examined, as are efforts by labor, management, and others to remedy the problems. In Columbus, Ohio, labor and management established Operation MOST as a cooperative program to regain the share of the construction market lost to non‐union firms. This program encompasses a variety of activities and has been very successful. It is now being copied in various locations throughout the country. Several recommendations are made as to activities that should be undertaken by the parties to continue their cooperative spirit and to allow the unionized contractors to improve their cost competitiveness.