Functional evolution of IGF2:IGF2R domain 11 binding generates novel structural interactions and a specific IGF2 antagonist

Among the 15 extracellular domains of the mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R), domain 11 has evolved a binding site for IGF2 to negatively regulate ligand bioavailability and mammalian growth. Despite the highly evolved structural loops of the IGF2:domain 11 binding site, affinity-enhancing AB loop mutations suggest that binding is modifiable. Here we examine the extent to which IGF2:domain 11 affinity, and its specificity over IGF1, can be enhanced, and we examine the structural basis of the mechanistic and functional consequences. Domain 11 binding loop mutants were selected by yeast surface display combined with high-resolution structure-based predictions, and validated by surface plasmon resonance. We discovered previously unidentified mutations in the ligand-interacting surface binding loops (AB, CD, FG, and HI). Five combined mutations increased rigidity of the AB loop, as confirmed by NMR. When added to three independently identified CD and FG loop mutations that reduced the koff value by twofold, these mutations resulted in an overall selective 100-fold improvement in affinity. The structural basis of the evolved affinity was improved shape complementarity established by interloop (AB-CD) and intraloop (FG-FG) side chain interactions. The high affinity of the combinatorial domain 11 Fc fusion proteins functioned as ligand-soluble antagonists or traps that depleted pathological IGF2 isoforms from serum and abrogated IGF2-dependent signaling in vivo. An evolved and reengineered high-specificity M6P/IGF2R domain 11 binding site for IGF2 may improve therapeutic targeting of the frequent IGF2 gain of function observed in human cancer

The 1981 NASA/ASEE Summer Faculty Fellowship Program: Research reports

Research reports related to spacecraft industry technological advances, requirements, and applications were considered. Some of the topic areas addressed were: (1) Fabrication, evaluation, and use of high performance composites and ceramics, (2) antenna designs, (3) electronics and microcomputer applications and mathematical modeling and programming techniques, (4) design, fabrication, and failure detection methods for structural materials, components, and total systems, and (5) chemical studies of bindary organic mixtures and polymer synthesis. Space environment parameters were also discussed

The performance of high speed reciprocating polymer seals in water

Bibliography: pages 93-94.The elastohydrodynamic lubrication of polymeric piston seals in a water powered rockdrill has been modelled in this thesis. Current seal designs, consist of an Ultra High Molecular Weight Polyethylene (UHMWPE) ring, energised to remain in contact with the reciprocating piston, using a nitrile rubber o-ring. The design of the seals is such that they do not facilitate the formation of a lubricating film, and the high contact stresses at the seal face due to the seal pressure cause excessive wear of the seals. The elastohydrodynamic model is based on a numerical algorithm in which the elasticity equations for the seal deflection and the one dimensional lubrication equation have been solved simultaneously. The polymeric ring has been simplified due to its axisymmetry, and modelled in cross section as an elastic beam resting on a spring base, while the deformation of the o-ring has been considered independently since the moduli of the two materials differs by nearly two orders of magnitude. The o-ring was modelled using the non-linear Mooney-Rivlin constitutive equation implemented in the finite element code ABAQUS. With this model, the contact stresses existing between the o-ring and the back of the polymer seal have been determined for a range of o-ring squeezes and rubber hardnesses. The resultant lubrication and o-ring forces acting on the rectangular polymer seal have been used in a finite difference formulation of the seal (or beam on a spring base) to determine the seal deflection above the piston

Electronic properties of well-oriented graphite at low temperatures

Imperial Users onl

Multi-Functional Polymer Vesicles: Applications in Chemotherapy and Photodynamic Therapy

The field of drug delivery is rapidly expanding to bridge the gap between novel drugs that are created and their effective entry into diseased tissue. In one growing area of research, synthetic polymers are being utilized to meet these needs. The precise control over their chemistry allows polymers to be tuned to the drug delivery application and make them attractive candidates for research. The focus of this dissertation is to engineer responsive polymersomes for drug delivery and understand their ability to reduce drug toxicity, increase absorption in diseased cells and tissue, and control the release of drug in vitro and in vivo. Gemcitabine, a nucleoside analog, was encapsulated in the aqueous core of nano-polymersomes composed of the biodegradable and biocompatible polymer PEO-PCL, and the in vitro toxicity of this novel drug delivery construct was tested against Panc-1 cells. The polymersome formulation performed at par with the free drug with one-log cell killing at 1 ìM of gemcitabine. The polymersome was also able to control the release of gemcitabine, and this release was modulated by the degradation kinetics of the ester linkages in the membrane. Photodynamic therapy was performed against OVCAR-5 (ovarian cancer) cells. A hydrophobic photosensitizer, benzoporphrin derivative monoacid A (BPD-MA) was encapsulated the in the membrane of polymersomes composed of PEO14-PBD22 (OB14.5) polymer, and its phototoxicity was compared to an existing photosensitizer formulation called verteporfin that is currently used in the clinic for age-related macular degeneration (AMD). The polymersome formulation outperformed verteporfin both at the in vitro and in vivo level. Additionally, we investigated the photorupture of giant polymersomes encapsulating a near IR fluorophore (porphyrin dimer, PZn2) in the hydrophobic membrane and dextran in the aqueous lumen. Polymersomes synthesized from softer polymers released more of a reporter dye than stiffer polymersomes when illuminated with 690 nm of light. Finally, we investigated the fractionation of giant polymersomes in a deterministic lateral displacement device and developed a hydrodynamic model to predict this fractionation based on the attractive and repulsive forces experienced by the polymersome

Parameter elimination in particle Gibbs sampling

Bayesian inference in state-space models is challenging due to high-dimensional state trajectories. A viable approach is particle Markov chain Monte Carlo, combining MCMC and sequential Monte Carlo to form "exact approximations" to otherwise intractable MCMC methods. The performance of the approximation is limited to that of the exact method. We focus on particle Gibbs and particle Gibbs with ancestor sampling, improving their performance beyond that of the underlying Gibbs sampler (which they approximate) by marginalizing out one or more parameters. This is possible when the parameter prior is conjugate to the complete data likelihood. Marginalization yields a non-Markovian model for inference, but we show that, in contrast to the general case, this method still scales linearly in time. While marginalization can be cumbersome to implement, recent advances in probabilistic programming have enabled its automation. We demonstrate how the marginalized methods are viable as efficient inference backends in probabilistic programming, and demonstrate with examples in ecology and epidemiology

Leukocyte HMGB1 Guides Neoangiogenesis in Regenerating Skeletal Muscle

HMGB1 is ubiquitously present in the cell nucleus and highly conserved among species. It is released by necrotic cells and actively secreted by immune cells in the extracellular environment, where it behaves as a DAMP (Damage-Associate Molecular Pattern). Up to now all studies addressing the role of HMGB1 in wound healing have been carried out either in vitro or using pharmacological inhibitors. To assess the relative contribution of leukocyte HMGB1 and of passively released HMGB1 we have set up an ad hoc animal model. In this system, haematopoietic cells selectively lack HMGB1 by transplanting Hmgb1+/+ or Hmgb1-/- foetal livers into WT recipients. The role of leukocyte HMGB1 in tissue repair has been analyzed after skeletal muscle damage. I have used histological and magnetic resonance imaging (MRI) to non-invasively evaluate muscle inflammation and tissue architecture. In the absence of Hmgbl, there is a lower number of regenerating fibres at day7 after injury and an aberrant repair with smaller fibres at day 15. In vitro no differential effects of Hmgb1-/- and Hmgb1+/+ macrophages are detected on satellite cells differentiation/fusion, suggesting that events taking place in vivo are independent of the direct macrophage/satellite cell cross talk. No obvious differences are observed in the extent of leukocyte infiltration, as assessed both by cytofluorimetric and MRI analysis, suggesting that leukocyte Hmgb1 is not required for the attraction of inflammatory cells. qPCR unveils a role for leukocyte Hmgb1 in the production of angiogenic stimuli such as Ang2 and TNFα, probably in a hypoxia-dependent manner. Accordingly, the number of CD31 cells is dramatically reduced in the absence of leukocyte Hmgb1. Collectively our data suggest that HMGB1 secreted by leukocytes guides muscle regeneration through a paracrine role on vascular bed reconstitution

Development of mechanical reliability testing techniques with application to thin films and piezo MEMS components

This work focuses on the development of a method for probing the mechani- cal response of thin film materials based on miniature tensile testing. A number of mechanisms that may compromise the performance and potentially limit the operational lifetime of MEMS devices which incorporate functional ferroelectric ceramics were also identified and investigated. Reliability of piezo MEMS com- ponents was studied at a wafer and at a device level through the development of appropriate techniques based on miniature tensile testing, time- resolved mi- cro RAMAN spectroscopy and laser Doppler vibrometry. Micro tensile testing was further used for the extraction of the elastic properties of various thin film materials. A miniature tensile stage was developed in common with DEBEN UK for the mechanical characterization of functional thin film materials like PZT and ZnO ceramics, which are commonly used in MEMS fabrication. The stage is of- fered with a piezo electric motor which can be fitted with interchangeable heads. These can be combined with di.erent types of mounting jaws, enabling both con- ventional tensile testing and compression testing to be performed. Strains and displacements were measured in- situ using an optical, non destructive method based on CCD imaging. The elastic constants of polymer (LCP), LCP-Au bi- layers and electroplated Ni were defined in good agreement with the literature. However yield of successfully released ceramic samples was rather poor so a col- laboration with IMTEK at Germany was established. Using their facilities batch processing of a large number of wafers was possible. Cont/d

Improved micro-contact resistance model that considers material deformation, electron transport and thin film characteristics

This paper reports on an improved analytic model forpredicting micro-contact resistance needed for designing microelectro-mechanical systems (MEMS) switches. The originalmodel had two primary considerations: 1) contact materialdeformation (i.e. elastic, plastic, or elastic-plastic) and 2) effectivecontact area radius. The model also assumed that individual aspotswere close together and that their interactions weredependent on each other which led to using the single effective aspotcontact area model. This single effective area model wasused to determine specific electron transport regions (i.e. ballistic,quasi-ballistic, or diffusive) by comparing the effective radius andthe mean free path of an electron. Using this model required thatmicro-switch contact materials be deposited, during devicefabrication, with processes ensuring low surface roughness values(i.e. sputtered films). Sputtered thin film electric contacts,however, do not behave like bulk materials and the effects of thinfilm contacts and spreading resistance must be considered. Theimproved micro-contact resistance model accounts for the twoprimary considerations above, as well as, using thin film,sputtered, electric contact