Three-dimensional micro-channel fabrication in polydimethylsiloxane(PDMS) elastomer

This paper describes a fabrication technique for building three-dimensional (3-D) micro-channels in polydimethylsiloxane (PDMS) elastomer. The process allows for the stacking of many thin (less than 100-μm thick) patterned PDMS layers to realize complex 3-D channel paths. The master for each layer is formed on a silicon wafer using an epoxy-based photoresist (SU 8). PDMS is cast against the master producing molded layers containing channels and openings. To realize thin layers with openings, a sandwich molding configuration was developed that allows precise control of the PDMS thickness. The master wafer is clamped within a sandwich that includes flat aluminum plates, a flexible polyester film layer, a rigid Pyrex wafer, and a rubber sheet. A parametric study is performed on PDMS surface activation in a reactive-ion-etching system and the subsequent methanol treatment for bonding and aligning very thin individual components to a substrate. Low RF power and short treatment times are better than high RF power and long treatment times, respectively, for instant bonding. Layer-to-layer alignment of less then 15 μm is achieved with manual alignment techniques that utilize surface tension driven self-alignment methods. A coring procedure is used to realize off-chip fluidic connections via the bottom PDMS layer, allowing the top layer to remain smooth and flat for complete optical access     

Phaseolin diversity as a possible strategy to improve the nutritional value of common beans (Phaseolus vulgaris)

This article proposes a new way to improve the protein quality of the common bean (Phaseolus vulgaris). It is based on the natural variability found in the different types of phaseolin, its main storage protein (40–50% of the total protein). Despite the fact that it is deficient in methionine content, phaseolin still represents the main source of that amino acid in the seed. More than 40 genetic variants, differing in subunit number (2–6) and molecular weight (40–54 kDa) have been analyzed. The similarity of the amino acid composition among phaseolins, suggests that a nutritional improvement cannot be expected from that side. Conversely, important variation in phaseolin susceptibility to proteolysis (ranging from 57% to 96% after cooking) has been observed, increasing the theoretical availability of methionine by up to 37%. Therefore, breeding programs based on highly-digestible phaseolin types could lead to the production of beans with higher protein quality.     

Integrating microfabricated fluidic systems and NMR spectroscopy

The philosophy of miniature total analysis systems (mu-TAS) hinges on the integration of multiple chemical processing steps and the means of analyzing their results on the same miniaturized system. We have constructed chip-based capillary electrophoresis (CE) devices equipped with an integrated planar radio-frequency detector coil used for nuclear magnetic resonance spectroscopy (NMR). Separations were accomplished in the devices, but satisfactory NMR spectra could only be obtained from samples of high concentration. The relative sensitivity is explained and the scaling law dichotomy of CE and NMR explored.     

Identification of a neurosteroid binding site contained within the GluR2-S1S2 domain

Glutamate receptors play a major role in neural cell plasticity, growth, and maturation. The degree to which ionotropic glutamate receptors (iGluR) conduct current is dependent on binding of extracellular ligands, of which glutamate is the native agonist. Although the glutamate binding site of the GluR2 class of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) iGluR has been structurally characterized, the allosteric sites attributed to neurosteroid binding have yet to be localized. Here, using intrinsic tryptophan fluorescence spectroscopy, we show that the extracellular glutamate binding core of the GluR2 class of AMPA receptors also binds to two neurosteroids, pregnenolone sulfate (PS) and 3α-hydroxy-5β-pregnan-20-one sulfate, both of which negatively modulate its activity. Interest in these sulfated neurosteroids stems from their differential modulation of other members of the iGluR family and their potential use as endogeneous agents for stroke therapy. In particular, whereas PS inhibits AMPA and other non-N-methyl-d-aspartate (NMDA) family members, it activates the NMDA receptor. In addition to providing evidence for binding of these neurosteroids to the glutamate binding core of the GluR2 class of AMPA receptors, our data suggests that both neurosteroids bind in a similar manner, consistent with their modulation of activity of this class of iGluR. Interestingly, the conformational change induced upon binding of these neurosteroids is distinct from that induced upon glutamate binding.     

Fabrication of Biofunctionalized Quasi‐Three‐Dimensional Microstructures of a Nonfouling Comb Polymer Using Soft Lithography

This paper describes a simple set of patterning methods that are applicable to diverse substrates and allow the routine and rapid fabrication of protein patterns embedded within a background that consists of quasi‐three‐dimensional microstructures of a cell‐resistant polymer. The ensemble of methods reported here utilizes three components to create topographically nonfouling polymeric structures that present cell‐adhesive protein patterns in the regions between the microstructures: the first component is an amphiphilic comb polymer that is comprised of a methyl methacrylate backbone and pendant oligo(ethylene glycol) moieties along the side chain, physically deposited films of which are protein‐ and cell‐resistant. The second component of the fabrication methodology involves the use of different variants of soft lithography, such as microcontact printing to create nonfouling topographical features of the comb polymer that demarcate cell‐adhesive regions of the third component: a cell‐adhesive extracellular protein or peptide. The ensemble of methods reported in this paper was used to fabricate quasi‐three‐dimensional patterns that present topographical and biochemical cues on a variety of substrates, and was shown to successfully maintain cellular patterns for up to two months in serum‐containing medium. We believe that this, and other such methods under development that allow independent and systematic control of chemistry, topography and substrate compliance will provide versatile “test‐beds” for fundamental studies in cell biology as well as allow the discovery of rational design principles for the development of biomaterials and tissue‐engineering scaffolds.     

Inhibition of HSP90 with Pochoximes: SAR and Structure‐Based Insights

The pochoximes, based on the radicicol pharmacophore, are potent inhibitors of heat shock protein 90 (HSP90) that retain their activity in vivo. Herein we report an extended library that broadly explores the structure–activity relationship (SAR) of the pochoximes with four points of diversity. Several modifications were identified that afford improved cellular efficacy, new opportunities for conjugation, and further diversifications. Cocrystal structures of pochoximes A and B with HSP90 show that pochoximes bind to a different conformation of HSP90 than radicicol and provide a rationale for the enhanced affinity of the pochoximes relative to radicicol and the pochonins.     

Potential for increasing the amounts of bioavailable zinc in dry beans (Phaseolus vulgaris L) through plant breeding

A whole‐body radioassay procedure was used to assess the bioavailability to rats of zinc (Zn) in seeds of 18 genotypes of beans (Phaseolus vulgaris L) that were grown hydroponically. Dry beans that were labelled intrinsically with ⁶⁵Zn were added to test meals fed to rats that were marginally Zn‐deficient. The amount of Zn in the seeds varied between genotypes and ranged from 26.7 to 62.4 µg g^?1 (from 0.41 to 0.95 µmol g^?1) dry weight (DW). Similarly, the amount of iron (Fe) in the beans varied nearly twofold (from 52.3 to 96.3 µg g^?1 DW), and Zn and Fe concentrations were positively correlated. Concentrations of myo‐inositolhexaphosphate (IP6) plus myo‐inositolpentaphosphate (IP5) varied from 18.1 to 27.3 µmol g^?1 DW. Cultivars with white‐coloured seeds contained relatively small amounts of tannins varying from 0.12 to 0.16 mg g^?1 DW (determined as catechin equivalents) compared to those with coloured seed coats (up to 2.58 mg g^?1 DW). All rats readily ate the test meals so that Zn intake varied directly with seed‐Zn concentration. As indicated by ⁶⁵Zn absorption, the bioavailability to rats of Zn in the seeds varied between genotypes and ranged from about 78 to 95% of the total Zn in the seeds. The bioavailability of Zn to marginally Zn‐deficient rats was not affected markedly by either IP5 + IP6 or tannin in the dry beans. These results demonstrate that the concentration of Zn in dry beans can be increased through traditional plant‐breeding techniques and that this may result in significant increases in the amount of bioavailable Zn in the beans. Increasing the amount of Zn in beans may contribute significantly to improving the Zn status of individuals dependent on beans as a staple food. © 2002 Society of Chemical Industry