Site-specific attachment to recombinant antibodies via introduced surface cysteine residues

Many diagnostic and therapeutic applications of monoclonal antibodiesrequire the covalent linking of effector or reporter moleculesto the immunoglobulin polypeptides. Existing methods generallyinvolve the non-selective modification of amino acid side chains,producing one or more randomly distributed attachment sites.This results in heterogeneous labelling of the antibody moleculesand often to a decrease in antigen-binding due to the modificationof residues close to the antigen-binding site. We report a novelstrategy for site-specifically labelling antibodies throughsurface cysteine residues. Examination of molecular structureswas used to identify amino acids of the C_H1 domain of the IgGheavy chain that were accessible to solvent but not to largermolecules. Site-directed mutagenesis was used to substitutecysteine residues at these positions in the heavy chain of amouse/human chimaeric version of the tumour-binding monoclonalantibody, B72.3. Expression of the modified antibody genes inmammalian cells yielded correctly assembled proteins that hadthiol groups in pre-determined positions and showed no lossof antigen-binding activity. One of the mutants was used todemonstrate the site-specific attachment of a radio-iodinatedligand to the chimaeric B72.3 antibody.     

HEAT TRANSFER FROM A HORIZONTAL BUBBLING SURFACE TO AN OVERLYING WATER POOL†

An experimenl was performed to study the effects of bubbling from a circular, horizontal, fiat plate on the heat transfer to an overlying water pool. The plate had drilled orifices through which nitrogen was injected into an overlying pool of water at atmospheric pressure. For “deep” pools, the heat transfer coefficient was found to increase only about 20% over a range of superficial gas velocities from 0.6 to 8.5cm/s, A turbulent heat transfer model developed by Konsetov was found to agree well with the data from this experiment. This model and the experimental data suggest that under certain conditions the heat transfer coefficient is similar for both horizontal and vertical surfaces. These conditions are, that the bubbles only contribute to the stirring action in the pool and only when the bubbling pool is considered “deep”. When the pool height fell below 60% of its diameter, the heat transfer coefficient decreased almost linearly with pool height. This suggests that there occurs a reduction in the turbulent fluctuations or in the characteristic length scale based on the average turbulent eddy size as the pool height decreases.     

Rapid characterization of the ultraviolet induced fiber Bragg grating complex coupling coefficient as a function of irradiance and exposure time

We report the application of optical frequency domain reflectometry and a discrete-layer-peeling inverse scattering algorithm to the spatial characterization of the UV induced complex coupling coefficient during fiber Bragg grating growth. The fiber grating is rapidly characterized using this technique to give irradiance dependent growth as a function of exposure time, thereby providing the complete characterization of the coupling coefficient in the form of a "growth surface," which is related to the fiber's photosensitivity. We compare measurements of fiber Bragg grating growth in SMF-28 when exposed to continuous wave 244 nm irradiation from 0 to 90 W cm(-2) for exposure times up to 3230 s with a selection of other fibers including high germanium concentration fiber and erbium doped fiber.     

Silicon Photovoltaics Using Conducting Photonic Crystal Back‐Reflectors

Currently, research is being directed towards thinning conventional 200–300µm thick silicon photovoltaic cells by an order of magnitude or more. The benefits of reducing the cell thickness include decreased material costs, enhanced cell flexibility, and reduced effects of light‐induced degradation. However, one of the major challenges associated with reducing the active region to this extent is the corresponding reduction of light absorption. To mitigate this effect it has been proposed that the cell should incorporate enhanced light‐trapping strategies. One potential approach to enhance light trapping in thin photovoltaic cells is to structure the back‐reflector in the form of a photonic crystal (PC). It has recently been shown that two fundamental attributes of PC back‐reflectors optically coupled to thin semiconductor films contribute to enhanced absorption in the semiconductor: (i) the PC back‐reflector behaves as a perfect mirror, exhibiting complete reflection over stop‐gap frequencies; and (ii) the PC–semiconductor film interface couples incident light into resonant states that propagate along the plane of the film, thereby further enhancing the absorption. Although the ability of PC back‐reflectors to enhance absorption is encouraging, significant challenges arise when attempting to incorporate this light trapping technique in photovoltaic devices. Herein, we describe the underlying physical mechanisms that give rise to absorption enhancements in thin Si wafers featuring PC back‐reflectors, and describe hurdles that will have to be surmounted in order to reduce‐to‐practice a PC back‐reflector into an actual PV device.     

Multiple path analysis of reflectance from turbid media

A novel method to calculate the reflectance of light from a turbid medium is presented. The method takes an approach similar to that of the Beer-Lambert law, where the intensity of light is attenuated by an exponential factor involving the path length and the absorption coefficient. Due to scatter, however, there are many path lengths; in the present method all possible path lengths are weighted by their probabilities and summed over. A path length probability density is derived by considering a photon random walk through the medium. The result is a simple expression for the reflectance based on the physical properties of the medium.     

Retrieval of stratospheric aerosol size and composition information from solar infrared transmission spectra

Infrared transmission spectra were recorded by the Jet Propulsion Laboratory MkIV interferometer during flights aboard the NASA DC-8 aircraft as part of the Airborne Arctic Stratospheric Expedition II (AASE II) mission in the early months of 1992. In our research, we infer the properties of the stratospheric aerosols from these spectra. The instrument employs two different detectors, a HgCdTe photoconductor for 650-1850 cm(-1) and an InSb photodiode for 1850-5650 cm(-1), to simultaneously record the solar intensity throughout the mid-infrared. These spectra have been used to retrieve the concentrations of a large number of gases, including chlorofluorocarbons, NOy species, O3, and ozone-depleting gases. We demonstrate how the residual continua spectra, obtained after accounting for the absorbing gases, can be used to obtain information about the stratospheric aerosols. Infrared extinction spectra are calculated for a range of modeled aerosol size distributions and compositions with Mie theory and fitted to the measured residual spectra. By varying the size distribution parameters and sulfate weight percent, we obtain the microphysical properties of the aerosols that best fit the observations. The effective radius of the aerosols is found to be between 0.4 and 0.6 microm, consistent with that derived from a large number of instruments in this post-Pinatubo period. We demonstrate how different parts of the spectral range can be used to constrain the range of possible values of this size parameter and show how the broad spectral bandpass of the MkIV instrument presents a great advantage for retrieval ofboth aerosol size a nd composition over instruments with a more limited spectral range. The aerosol composition that provides the best fit to the measured spectra is a 70-75% sulfuric acid solution, in good agreement with that obtained from thermodynamic considerations.     

Oriented Nanofibrous Polymer Scaffolds Containing Protein‐Loaded Porous Silicon Generated by Spray Nebulization

Oriented composite nanofibers consisting of porous silicon nanoparticles (pSiNPs) embedded in a polycaprolactone or poly(lactide‐co‐glycolide) matrix are prepared by spray nebulization from chloroform solutions using an airbrush. The nanofibers can be oriented by an appropriate positioning of the airbrush nozzle, and they can direct growth of neurites from rat dorsal root ganglion neurons. When loaded with the model protein lysozyme, the pSiNPs allow the generation of nanofiber scaffolds that carry and deliver the protein under physiologic conditions (phosphate‐buffered saline (PBS), at 37 °C) for up to 60 d, retaining 75% of the enzymatic activity over this time period. The mass loading of protein in the pSiNPs is 36%, and in the resulting polymer/pSiNP scaffolds it is 3.6%. The use of pSiNPs that display intrinsic photoluminescence (from the quantum‐confined Si nanostructure) allows the polymer/pSiNP composites to be definitively identified and tracked by time‐gated photoluminescence imaging. The remarkable ability of the pSiNPs to protect the protein payload from denaturation, both during processing and for the duration of the long‐term aqueous release study, establishes a model for the generation of biodegradable nanofiber scaffolds that can load and deliver sensitive biologics.     

Designer reagents for mass spectrometry-based proteomics: clickable cross-linkers for elucidation of protein structures and interactions

We present novel homobifunctional amine-reactive clickable cross-linkers (CXLs) for investigation of three-dimensional protein structures and protein-protein interactions (PPIs). CXLs afford consolidated advantages not previously available in a simple cross-linker, including (1) their small size and cationic nature at physiological pH, resulting in good water solubility and cell-permeability, (2) an alkyne group for bio-orthogonal conjugation to affinity tags via the click reaction for enrichment of cross-linked peptides, (3) a nucleophilic displacement reaction involving the 1,2,3-triazole ring formed in the click reaction, yielding a lock-mass reporter ion for only clicked peptides, and (4) higher charge states of cross-linked peptides in the gas-phase for augmented electron transfer dissociation (ETD) yields. Ubiquitin, a lysine-abundant protein, is used as a model system to demonstrate structural studies using CXLs. To validate the sensitivity of our approach, biotin-azide labeling and subsequent enrichment of cross-linked peptides are performed for cross-linked ubiquitin digests mixed with yeast cell lysates. Cross-linked peptides are detected and identified by collision induced dissociation (CID) and ETD with linear quadrupole ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) and LTQ-Orbitrap mass spectrometers. The application of CXLs to more complex systems (e.g., in vivo cross-linking) is illustrated by Western blot detection of Cul1 complexes including known binders, Cand1 and Skp2, in HEK 293 cells, confirming good water solubility and cell-permeability.     

Process optimization using sequential design of experiment: A case study

In practice, engineers seek to find reasonable solutions for complex and unstructured problems, which are common in many areas. The workable solutions for these problems are never a one-shot experiment and data analysis procedure. Rather, the proper solution for these problems requires an inductive-deductive process which involves a series of experiments. To teach engineers the sequential learning strategy in solving complex problems, this article presents a case study on the startup of an ethanol–water distillation column that illustrates the scientific process of response surface methodology. The goal of this experiment is generally to find a good, robust solution that produces high grade concentration of ethanol with maximum profit. This case illustrates the sequential application of response surface methodology and consists of an initial fractional factorial design, a steepest ascent design, a full factorial design, and a central composite face-centered cube design. The analysis of the data in the previous steps gives engineers a guidance about the design of experiment in the next step. This study uses the desirability function approach to obtain a compromise optimization between the concentration of ethanol and the profit, which gives a robust solution to the complex problem. Finally, we conduct appropriate confirmation experiments to verify the optimization results. The case study emphasizes the importance of sequential nature and provides a useful guidance for engineers to solve complex problems.