Nanopore Biosensors: Monitoring of an ATP‐Binding Aptamer and its Conformational Changes Using an α‐Hemolysin Nanopore (Small 1/2011)

An aptamer is a specific oligonucleotide sequence that spontaneously forms a secondary structure capable of selectively binding an analyte. An aptamer’s conformation is the key to specific binding of a target molecule, even in the case of very closely related targets. Nanopores are a sensitive tool for the single‐molecule analysis of DNA, peptides, and proteins transporting through the pore. Herein, a single α‐hemolysin natural nanopore is utilized to sense the conformational changes of an adenosine 5’‐triphosphate (ATP)‐binding aptamer (ABA). The known DNA sequence of the ABA is used as a model to develop real‐time monitoring of molecular conformational changes that occur by binding targets. The native, folded ABA structure has a nanopore unfolding time of 4.17 ms, compared with 0.29 ms for the ABA:ATP complex. A complementary 14‐mer strand, which binds the ABA sequence in the key nucleic acids responsible for folding, forms linear duplex DNA, resulting in a nanopore transit time of 0.50 ms and a higher capture probability than that of the folded ABA oligomer. Competition assays between the ABA:ATP and ABA:reporter complexes are carried out, and the results suggest that the ABA:ATP complex is formed preferentially. The nanopore allows for the detection of an ABA in its folded, ATP‐bound, and linear conformations.     

Adhesion Studies of Fluoropolymers

A comparative study of the treatment of polytetrafluoroethylene (PTFE) and poly(vinyl fluoride) (PVF) with “Tetra-Etch” has been carried out. The treatment of PTFE resulted in extensive changes in surface chemistry and topography, whereas with PVF there was no significant change in topography and the chemical changes were much less marked. However, treatment of both polymers resulted in large increases in bond strength.

Multiple bonding experiments in which samples are repeatedly fractured and re-bonded were carried out with untreated PTFE and PVF. These resulted in moderate increases in bond strength with PTFE and large increases with PVF. The results indicate that weak boundary layer (WBL) removal is a key element in adhesion improvement by “Tetra-Etch” on PVF. With PTFE, WBL removal also improves adhesion, but the chemical and/or topographical changes introduced by the “Tetra-Etch” are required for optimum performance.     

Hydrogen refueling station compression and storage optimization with tube-trailer deliveries

Hydrogen refueling stations require high capital investment, with compression and storage comprising more than half of the installed cost of refueling equipment. Refueling station configurations and operation strategies can reduce capital investment while improving equipment utilization. Argonne National Laboratory developed a refueling model to evaluate the impact of various refueling compression and storage configurations and tube trailer operating strategies on the cost of hydrogen refueling. The modeling results revealed that a number of strategies can be employed to reduce fueling costs. Proper sizing of the high-pressure buffer storage reduces the compression requirement considerably, thus reducing refueling costs. Employing a tube trailer to initially fill the vehicle's tank also reduces the compression and storage requirements, further reducing refueling costs. Reducing the cut-off pressure of the tube trailer for initial vehicle fills can also significantly reduce the refueling costs. Finally, increasing the trailer's return pressure can cut refueling costs, especially for delivery distances less than 100 km, and in early markets, when refueling stations will be grossly underutilized.     

The recognition of the nonclassical major histocompatibility complex (MHC) class I molecule, T10, by the gammadelta T cell, G8

Recent studies have shown that many nonclassical major histocompatibility complex (MHC) (class 1b) molecules have distinct antigen-binding capabilities, including the binding of nonpeptide moieties and the binding of peptides that are different from those bound to classical MHC molecules. Here, we show that one of the H-2T region-encoded molecules, T10, when produced in Escherichia coli, can be folded in vitro with beta2-microglobulin (beta2m) to form a stable heterodimer in the absence of peptide or nonpeptide moieties. This heterodimer can be recognized by specific antibodies and is stimulatory to the gammadelta T cell clone, G8. Circular dichroism analysis indicates that T10/beta2m has structural features distinct from those of classical MHC class I molecules. These results suggest a new way for MHC-like molecules to adopt a peptide-free structure and to function in the immune system.     

Increased oxidant resistance of alveolar macrophages isolated from rats repeatedly exposed to cadmium aerosols

This study investigated potential mechanisms of oxidant resistance in alveolar macrophages (AM) isolated from Lewis rats exposed repeatedly to cadmium aerosols. Macrophages from Cd-adapted animals significantly greater resistance to oxidant-induced cytotoxicity than control cells when challenged with hydrogen peroxide in vitro. Elevations in glutathione peroxidase and glutathione reductase activities were associated with increased oxidant tolerance but catalase activity was unchanged. Metallothionein (MT) expression (protein and mRNA) was dramatically up-regulated in response to in vivo Cd exposure. A study using immunocytochemistry and in situ hybridization techniques revealed significantly heterogeneity in the expression of metallothionein by AMs. The percentage of AMs positive for MT (protein and mRNA) and the degree of MT expression within individual cells increased in response to additional Cd exposures. A putative state of activation was suggested by differences in size and number of inclusion bodies in macrophages from Cd-adapted animals and by secretion of a cytokine with interleukin-1-like characteristics. In summary, AMs from Cd-adapted animals are distinguished from control cells with respect to: (1) increased oxidant resistance, (2) secretion of cytokines, (3) elevations in enzymes associated with glutathione metabolism, and (4) up-regulation in metallothionein expression.     

Relation between maternal diet and subsequent primitive neuroectodermal brain tumors in young children

BACKGROUND: It has been hypothesized that a high dietary intake of nitrosamines and their precursors, nitrites and nitrates, is a risk factor for brain tumors. Vitamins C and E inhibit the formation of nitrosamines and thus may be protective. METHODS: We conducted a case-control study of maternal diet and the risk of primitive neuroectodermal tumors of the brain in children. The case patients were under the age of six years at diagnosis in 1986 to 1989. The controls were selected by random-digit telephone dialing and were matched for age and race to 166 case patients. Telephone interviews with the mothers included questions on the frequency of consumption of alcohol, vitamin and mineral supplements, and 53 foods during pregnancy. RESULTS: Significant protective trends were observed for vegetables (odds ratio for the highest quartile group for intake relative to the lowest, 0.37; P for trend = 0.005), fruits and fruit juices (odds ratio, 0.28; P = 0.003), vitamin A (odds ratio, 0.59; P = 0.03), vitamin C (odds ratio, 0.42; P = 0.009), nitrate (odds ratio, 0.44; P = 0.002), and folate (odds ratio, 0.38; P = 0.005). A nonsignificant trend of increasing risk was observed for nitrosamine (odds ratio, 1.65; P = 0.15). The use of iron (odds ratio, 0.43; P = 0.004), calcium (odds ratio, 0.42; P = 0.05), and vitamin C (odds ratio, 0.35; P = 0.04) supplements at any time during the pregnancy and the use of multivitamins during the first six weeks (odds ratio, 0.56; P = 0.02) were associated with decreased risk. In multivariate analyses, folate, early multivitamin use, and iron supplements generally remained protective. CONCLUSIONS: These results do not support the hypothesis that nitrosamines have a role in the development of primitive neuroectodermal tumors in young children, but they do suggest that certain other aspects of maternal diet can influence the risk.     

Protein kinase C is activated in platelets subjected to pathological shear stress

High levels of fluid shear stress at the blood vessel wall directly stimulate von Willebrand factor (vWF)-mediated platelet adhesion and aggregation and thereby contribute to the pathogenesis of arterial thrombosis. We have found that a pathological level of arterial wall shear stress (90 dynes/cm2) induces platelet aggregation that is associated with the phosphorylation of pleckstrin, a M(r) 47,000 protein kinase C substrate (p47). Shear-induced p47 phosphorylation depends entirely on vWF binding to platelet glycoprotein (Gp) Ib and GpIIb-IIIa, and the specific inhibition of protein kinase C with the staurosporine analogue Ro 31-7549 inhibits the full aggregation response to shear. Shear stress-induced platelet p47 phosphorylation occurs independent of any measurable change in diacylglycerol mass or hydrolysis of phosphatidylinositol 4,5-bisphosphate. These results indicate that mechanical shear stress induces vWF to bind to platelet GpIb and GpIIb-IIIa, stimulating a diacylglycerol-independent pathway of protein kinase C activation that contributes to platelet aggregation in response to shear.