Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.     

Dynamic mechanical properties of some polyurethane-acrylic copolymer interpenetrating polymer networks

Dynamic mechanical properties have been investigated for interpenetrating-network systems based on polyol-cured polyurethanes (PU) and 2 to 1 n-butyl acrylate-n-butyl methacrylate (Ac) networks. The systems were formed simultaneously (SIN) from all of the precursors and reactants for both networks in the same vessel, and sequentially (SIPN) by swelling a precured PU with the reactants that will form the Ac network. If the Ac network is formed after gelation of the PU, the IPNs are transparent and appear to have single T (tan δ_max) between those of the homonetworks; visible-phase separation occurs if the Ac is intentionally polymerized prior to PU gelation. Damping curves were lower and broader and the T (tan δ_max) and rubber moduli were higher for the SIN than for the SIPN systems. Up to 65 percent Ac, the T (tan δ_max) data for both SIN and SIPN fit the Gordon-Taylor equation if a T (tan δ_max) for the Ac homonetwork 7°C higher than observed is used, suggesting a higher crosslink density for the Ac network under these conditions. The differences in properties of the SIN and SIPN are assumed to be dependent on sample homogeneity and upon the presence of a tin catalyst in the SIN preparation. This can result in limited Ac-network formation and consequent phase separation before PU gelation has occurred, and the catalyst may also increase the extent of interaction, such as grafting or hydrogen-bond formation between the networks.     

R. K. Merton — Life time of influence

In this article, “Life time of influence” refers to Robert K. Merton's impact broadly, and emblematically, to his influence upon my work. The article discusses 1) the scope and influence of Merton's ideas about social structure and explanations of social processes; 2) his vast scholarship establishing the study of science as a social institution, with implications for theory and research; and 3) his fostering of the social study of science through immense published work, and through impact upon an inter-generational network of scholars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Capture of Airborne Particulate Using Surface Applied Emulsions: Potential for Postdetonation Dirty Bomb Cleanup

Recent research has proposed the use of asphalt and tall-oil-pitch emulsions for stabilizing radioactive contamination deposited on surfaces in urban areas. The objective of this project was to investigate whether surface applied emulsions could capture airborne radioactive particulate. Laboratory experiments included wind-blown particulate capture studies using an acrylic column and particulate retainment experiments using a wind box capable of producing wind speeds of 96?km/h. A probe methodology was developed to relate particulate retainment to a tack force on the emulsion surface. Experiments were also performed to determine the potential for such emulsions to absorb particulate matter into their emulsion matrix. Tall-oil-pitch emulsions outperformed asphalt emulsions in terms of particulate retention, tack force, and the ability to absorb magnesium silicate. Both tall-oil-pitch and asphalt emulsions were capable of extracting 22–24?g?m?2 of powder from particulate-laden airflow. Tall-oil-pitch emulsions were capable of retaining as much as 5–10% of magnesium silicate powder applied (i.e., retainment densities of 10–20?g?m?2) even after seven?days of curing and after applying 96.5?km/h (60?mph) wind. Tall-oil-pitch emulsions were able to absorb surface-applied magnesium silicate (approximately 0.1–0.2?g of magnesium silicate per 1.0?g of emulsion within three?days) into their emulsion matrix, preventing the magnesium silicate from being exposed to the external environment. Initial results with these five different emulsion formulations suggested particulate capture was feasible. Future emulsion formulations (i.e., longer curing times with greater acid concentrations) should be tested to optimize this postdetonation response strategy.     

General acid/base catalysis in the active site of Escherichia coli thioredoxin

Enzymic catalysts of thiol:disulfide oxidoreduction contain two cysteine residues in their active sites. Another common residue is an aspartate (or glutamate), the role of which has been unclear. Escherichia coli thioredoxin (Trx) is the best characterized thiol:disulfide oxidoreductase, and in Trx these three active-site residues are Cys32, Cys35, and Asp26. Structural analyses had indicated that the carboxylate of Asp26 is positioned properly for the deprotonation of the thiol of Cys35, which would facilitate its attack on Cys32 in enzyme-substrate mixed disulfides. Here, Asp26 of Trx was replaced with isologous asparagine and leucine residues. D26N Trx and D26L Trx are reduced and oxidized more slowly than is wild-type Trx during catalysis by E.coli thioredoxin reductase. Stopped-flow spectroscopy demonstrated that the cleavage of the mixed disulfide between Trx and a substrate is slower in the D26N and D26L enzymes. Buffers increase the rate of mixed disulfide cleavage in these variants but not in wild-type Trx. These results indicate that Asp26 serves as an acid/base in the oxidation/reduction reactions catalyzed by Trx. Specifically, Asp26 protonates (during substrate oxidation) or deprotonates (during substrate reduction) the thiol of Cys35. A similar role is likely filled by the analogous aspartate (or glutamate) residue in protein disulfide isomerase, DsbA, and other thiol:disulfide oxidoreductases. Moreover, these results provide the first evidence for general acid/base catalysis in a thiol:disulfide interchange reaction.     

Fueguian cranial morphology: the adaptation to a cold, harsh environment

Craniometric data from the three extinct tribes that inhabited Tierra del Fuego (Selk'nam, Yámana, and Kawéskar) were gathered following Howell's measurement technique. We studied 180 skulls preserved at thirteen different institutions. Analysis of variance (ANOVA) between groups showed that morphological similarities among Fueguian groups are far more important than some differences between marine (Yámana and Kawéskar) and terrestrial (Selk'nam) groups. A principal component analysis (PCA) generated from the correlation matrix shows that Fueguians fall as outliers with respect to the typical Mongoloid morphology. In addition, a UPGMA tree generated from a squared Euclidean distance matrix indicates that Fueguian groups have a morphological pattern that is very distinct from that of other present-day Amerindian groups, with the exception of the Eskimos. One of the variables that contributes substantially to the differentiation of Eskimos and Fueguians is the nasal height. This suggests that nasal morphology in both groups could be a response to adaptive pressures related to the cold environment. However, other morphological particularities of Fueguian skulls, such as craniofacial robustness and variables of craniofacial width, can be attributed to a large masticatory stress. As a whole, the morphological features of Fueguian groups can be regarded as a general adaptive response to a very harsh environment, along with the retention of some plesiomorphic features. Assuming that the initial entry in Tierra de Fuego took place around 10,000 years BP, before the disappearance of the last land bridges in the Magellan Straits, then this adaptation might have arisen in a relatively short period, hastened by the extreme environmental conditions.