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There is growing interest in therapeutic intervention that targets disease-relevant RNAs using small molecules. While there have been some successes in RNA-targeted small-molecule discovery, a deeper understanding of structure–activity relationships in pursuing these targets has remained elusive. One of the best-studied tertiary-structured RNAs is the theophylline aptamer, which binds theophylline with high affinity and selectivity. Although not a drug target, this aptamer has had many applications, especially pertaining to genetic control circuits. Heretofore, no compound has been shown to bind the theophylline aptamer with greater affinity than theophylline itself. However, by carrying out a high-throughput screen of low-molecular-weight compounds, several unique hits were identified that are chemically distinct from theophylline and bind with up to 340-fold greater affinity. Multiple atomic-resolution X-ray crystal structures were determined to investigate the binding mode of theophylline and four of the best hits. These structures reveal both the rigidity of the theophylline aptamer binding pocket and the opportunity for other ligands to bind more tightly in this pocket by forming additional hydrogen-bonding interactions. These results give encouragement that the same approaches to drug discovery that have been applied so successfully to proteins can also be applied to RNAs.

Liquefied petroleum gas (LPG) is a promising diesel fuel alternative for heavy-duty vehicle applications due to its CO2 reduction potential, high knock resistance, easy liquefication capability, and lower fuel cost. Direct injection (DI) of liquid-phase LPG has emerged as a promising technology in spark-ignited (SI) engines due to the benefits from the in-cylinder charge cooling effect in comparison to external mixture formation systems. But this DI LPG technology requires a fuel delivery system that can supply the desired amount of LPG fuel in the liquid state at high pressure. This work first describes the design and component integration of an LPG fuel system that delivers fuel from the tank to the injector in the liquid state at around 172 bar, mainly focusing on thermal management aspects to avoid multi-phase behavior within the system. A detailed description of the injector developmental work, from reverse engineering of stock injectors to manufacturing a prototype LPG direct injector, is also presented, including nozzle modifications to accommodate a high LPG flow rate for heavy-duty applications. A one-dimensional (1D) flow model of the fuel delivery system is developed using the MATLAB/Simulink software platform to guide the selection and sizing of components and characterize the prototype injector. Bench testing of the fuel delivery system is performed with the unmodified stock and prototype LPG injectors using a Viscor calibration fluid and LPG to study the effect of fuel pressure and current profiles on the injected fuel quantity. The simulation models are shown to be capable of predicting the experimental results. Durability tests are also performed to understand the failure modes of different components in the fuel delivery system.

group (C geoffroyi, C jacchus, C kuhlii, and C penicillata), this relatively young primate radiation is endemic to the Brazilian Cerrado, Caatinga, and Atlantic Forest biomes. Significant impacts on Callithrix within these biomes resulting from anthropogenic activity include (1) population declines, particularly for the aurita group; (2) widespread geographic displacement, biological invasions, and range expansions of C jacchus and C penicillata; (3) anthropogenic hybridization; and (4) epizootic Yellow Fever and Zika viral outbreaks. A number of Brazilian legal and conservation initiatives are now in place to protect the threatened aurita group and increase research about them. Due to their small size and rapid life history, marmosets are prized biomedical models. As a result, there are increasingly sophisticated genomic Callithrix resources We provide here a current overview of marmoset (Callithrix) evolution, hybridization, species biology, basic/biomedical research, and conservation initiatives. Composed of 2 subgroups, the aurita group (C aurita and C flaviceps) and the jacchus available and burgeoning marmoset functional, immuno-, and epigenomic research. In both the laboratory and the wild, marmosets have given us insight into cognition, social group dynamics, human disease, and pregnancy. Callithrix jacchus and C penicillata are emerging neotropical primate models for arbovirus disease, including Dengue and Zika.Wild marmoset populations are helping us understand sylvatic transmission and human spillover of Zika and Yellow Fever viruses. All of these factors are positioning marmosets as preeminent models to facilitate understanding of facets of evolution, hybridization, conservation, human disease, and emerging infectious diseases.

Coal-fired Direct Injection Carbon Engine – Gas Turbine (DICE-GT) Compound-Reheat Combined Cycle (CRCC), is a combined cycle power plant comprising a multiplicity of coal-fired reciprocating internal combustion engines (RICE), which is commonly referred to by the acronym DICE (Direct Injection Carbon Engine); a natural gas-fired gas turbine (turbine and combustor); a heat recovery steam generator (HRSG); a steam turbine generator; and an integrally geared and intercooled centrifugal air compressor. The concept uses coal-water slurry/fluid in the DICE and natural gas (or hydrogen) in the gas turbine for unmatched efficiency (well above 50% net LHV) and modularity (block sizes of 120, 240 and 360 MW) with easy adaptability to carbon capture and sequestration with minimal additional cost, complexity and performance hit.

Conceived of as a response to the 2007 MLA Report on the state of foreign language teaching and learning in the United States, this article takes up some of the report's recommendations and puts them into practice. The first part of the article offers a response to current debates concerning foreign language programs and lays the conceptual foundation for a unit intended to help bridge the gap between second and third year courses in German Studies. This is followed by a pedagogical unit developed around Gregor Schnitzler's (2002) comedy thriller, Was tun, wenn's brennt? The film provides material for students of German to explore the idea of Vergangenheitsbewaltigung (‘coming to terms with the past’) beyond the context of the Nazi period, focusing instead on post-war periods of German history.

Using a comprehensive set of discovery and optimization tools, antibodies were produced with the ability to neutralize SARS coronavirus (SARS-CoV) infection in Vero E6 cells and in animal models. These anti-SARS antibodies were discovered using a novel DNA display method, which can identify new antibodies within days. Once neutralizing antibodies were identified, a comprehensive and effective means of converting the mouse sequences to human frameworks was accomplished using HuFR™ (human framework reassembly) technology. The best variant (61G4) from this screen showed a 3.5–4-fold improvement in neutralization of SARS-CoV infection in vitro. Finally, using a complete site-saturation mutagenesis methodology focused on the CDR (complementarity determining regions), a single point mutation (51E7) was identified that improved the 80% plaque reduction neutralization of the virus by greater than 8-fold. These discovery and evolution strategies can be applied to any emerging pathogen or toxin where a causative agent is known.

In an attempt to enhance the overall assembly, yield and half-life of recombinant antibody proteins, we have cloned and expressed several IgG1 C(H)3 domains and examined their folding/refolding characteristics. We utilized a cytoplasmic bacterial expression system with a thioredoxin reductase knock-out strain of BL21(DE3) to produce bovine, murine and human C(H)3. Under identical conditions, expression of bovine C(H)3 resulted consistently in the highest yields of properly folded/oxidized protein. Circular dichroism and fluorescence experiments demonstrate that oxidized bovine and murine C(H)3 have surprisingly similar structures and stabilities, considering the marginal sequence conservation between the two molecules. Residue frequency analysis using a limited data set of 36 unique Fc sequences originating from 19 different mammalian species targeted five specific sites for optimization within bovine C(H)3. Combination of three of these mutants increased the thermal stability of the molecule to 86 degrees C. Comparison of this approach to similar studies using larger sequence databases and/or different selection criteria suggests sequence database design can increase the success rate for identifying residue sites worth optimizing. This optimized C(H)3 domain can be used as a particularly stable platform for functional design and can be grafted into full-length antibody sequences to enhance their thermodynamic parameters and shelf-life.

The tuberactinomycins are a group of cyclic peptide antibiotics, which are potent inhibitors of prokaryotic protein synthesis. We report the inhibitory effect of viomycin, di-β-lysylcapreomycin IIA and tuberactinomycin A on group I intron self-splicing. They compete with the guanosine cofactor for the G-binding site located in the conserved core of the intron. They are 100-fold more active than all other competitive inhibitors described so far (dGTP, arginine or streptomycin), inhibiting splicing at concentrations between 10 and 50 μM. Mutation of the G-binding site leads to partial resistance, and the inhibitory effect of these drugs is dependent on Mg 2+ concentration. This suggests that the tuberactinomycins have more than one contact site with the intron RNA: via the G-binding site and via additional contacts with the RNA backbone. Positioning the tuberactinomycins in the three-dimensional model of the td intron core suggests that the charged lysyl side-chain (RI) is in contact with the backbone of the P1 helix. Structure/function analyses with various tuberactinomycin analogues with different activities confirm the involvement of this side-chain in inhibition of group I self-splicing. The demonstration of a new class of splicing inhibitors, the peptide antibiotics, illustrates how antibiotics may interact with catalytic RNA.

Designing and developing highly engineered products requires direct (and more dynamic) associations between customers’ specifications and product characteristics (or its behaviors). In order to meet the specified customer performance, cost, and integrity goals, a multitude of specialized analyses, heuristics, shortcuts, look-up tables, equations, algorithms, finite elements, and material substitution at multiple levels (system, subsystems, components and parts) are ought to be performed. The product geometries of such engineered product are often complex and many parts are designed interactively from scratch using a 3D commercial computer-aided design (CAD) — lately often referred as Product Life-cycle Management (PLM) system. Today, this very “PLM-based” engineered product-design process is often “static”, very “feature or geometry-dependent,” “knowledge-intensive,” and therefore, engineers often takes considerable time (months) to complete this manual process. Today, more and more companies want to quickly reengineer a product from a multitude of family solutions (corresponding to various design trade-off studies). They are interested in some dynamic form of a decision-based system that could automatically filter through a multitude of historical product solutions and quickly reconfigure one that meets the customer requirements with the least cost, weight, and time investment. Such decision-based product automation is not an easy task by any means. Product definitions without knowing specific geometry are hard to conceive, capture generically, and reuse widely (via any generative tool). A typical product development process —by its nature—is highly dynamic, nonlinear, discrete, feature-dependent, and part-dependent. The solution is not easy, since problem formulation is time-bound, has numerous discrete inputs, topologies, and several mathematical discontinuities. This paper discusses the system architecture of the Knowledge-driven Automation (KDA) program — established at Parker in 2002. It addresses many of the above product development issues and problems. In particular, authors describe a Knowledge-based System Engineering Process for Obtaining Engineering Design Solutions in a Commercial PLM Setting. The architecture and solutions use a number of innovative knowledge-based engineering (KBE) concepts and procedures. Through strategic use of generative modeling, spreadsheet tables, part and assembly templates, system engineering concepts, and our proprietary “smart part concepts,” authors were able to engineer-to-configure a family of hydraulic actuators automatically from their customer specifications using a set of PLM (CATIA V5 and its underlying knowledgeware) tools.Copyright © 2005 by ASME

The RNA-world hypothesis proposes that, before the advent of DNA and protein, life was based on RNA, with RNA serving as both the repository of genetic information and the chief agent of catalytic function1. An argument against an RNA world is that the components of RNA lack the chemical diversity necessary to sustain life. Unlike proteins, which contain 20 different amino-acid subunits, nucleic acids are composed of only four subunits which have very similar chemical properties. Yet RNA is capable of a broad range of catalytic functions2,3,4,5,6,7. Here we show that even three nucleic-acid subunits are sufficient to provide a substantial increase in the catalytic rate. Starting from a molecule that contained roughly equal proportions of all four nucleosides, we used in vitro evolution to obtain an RNA ligase ribozyme that lacks cytidine. This ribozyme folds into a defined structure and has a catalytic rate that is about 105-fold faster than the uncatalysed rate of template-directed RNA ligation.

Human hepatitis delta virus (HDV) poses a health threat in populations where chronic hepatitis B is endemic. It is a single-stranded RNA virus of 1700 nucleotides and both genomic and antigenomic sequences contain ribozymes which are important for viral replication. Using ribozyme constructs we show that several classes of antibiotics inhibit the self-cleavage reaction of the HDV ribozyme. Antibiotics of the aminoglycoside, peptide and tetracycline classes all inhibit HDV cleavage in vitro at micromolar concentrations. Neomycin (an aminoglycoside) inhibits HDV self-cleavage with a Ki value of 28 (+/- 10) microM. Neomycin inhibition can be reversed by increasing magnesium ion concentration in a competitive manner. Lead acetate cleaves positions G76, A42 and G28, which surround the ribozyme cleavage site. Both Mg2+ and neomycin prevent lead cleavage. Footprinting experiments using base-specific chemical probes revealed enhanced modifications of a set of bases by neomycin, overlapping with the above mentioned lead cleavages. These observations may indicate that neomycin directly displaces divalent metal ions essential for catalysis.

Lysinomicin, a naturally-occurring pseudodisaccharide, inhibits translation in prokaryotes. We report that lysinomicin (and three related compounds) are able to inhibit the self-splicing of group I introns, thus identifying pseudodisaccharides as a novel class of group I intron splicing inhibitors. Lysinomicin inhibited the self-splicing of the sunY intron of phage T4 with a Ki of 8.5 microM (+/- 5 microM) and was active against other group I introns. Inhibition was found to be competitive with the substrate guanosine, unlike aminoglycoside antibiotics, which act non-competitively to inhibit the splicing of group I introns. Competitive inhibitors of group I intron splicing known to date all contain a guanidino group that was thought to be required for inhibition; lysinomicin lacks a guanidino group.

Ubiquitination is a post-translational modification wherein a small (8 kDa) and highly abundant protein called ubiquitin is covalently attached to specific protein substrates in cells. This “glinkage” plays an important role in the regulation of many cellular functions including division, differentiation, protein trafficking, response to DNA damage, and gene transcription, among others. A lysine 48-linked chain of four or more ubiquitins targets a substrate for degradation by the 26S proteasome. A wealth of experimental evidence supports the notion that derangement of ubiquitin-mediated proteolysis contributes to various human malignancies including breast cancer. During metastasis, breast tumor cells undergo a process that is similar to the epithelial-mesenchymal-transition (EMT). This process is characterized by multiple steps ultimately enabling breast tumor cells to leave their place of origin and to colonize new organs. Little is known about the intrinsic ubiquitination activity during this process and how the ubiquitination pattern differs in metastatic cells compared to their non-malignant counterparts. Furthermore, there are no adequate experimental tools to identify the ubiquitination of protein substrates on a proteome-wide scale. The ubiquitin-proteasome system has been recognized as a valid target for the treatment of some malignancies (e.g. Bortezomib), thus deciphering the change of tumor cells' intrinsic enzymatic activity could warrant for the development of enhanced diagnostic tools and effective treatment of breast cancer patients. To overcome this shortcoming, we have developed an innovative approach that combines protein microarrays (where >8,300 human recombinant proteins are spotted onto glass microscope slides) with an ubiquitination reaction to determine novel ubiquitinated substrates during EMT. We obtained breast tumor material using the 4T1 mouse model of breast cancer, which consists of five isogenic murine mammary carcinoma cells lines (67NR, 168FARN, 4TO7, 4T1, and 66cl4) that spontaneously arose from a single mammary tumor in BALB/c mice and exhibit differing metastatic abilities resembling the multi-step process of EMT. Lysed tumor tissue was subsequently analyzed for a change of the ubiquitination signature. More than 230 alterations in specific target protein ubiquitination were detected in the cell lines used, indicating a possible regulatory role of protein uniquitination during the process of breast cancer progression. The identification of the biological consequences of some of these protein modifications will aid in gaining more insight into how the cells' intrinsic enzymatic activity has become distorted and how this could drive the metastatic process of breast cancer. Our novel identification of target protein ubiquitination could ultimately serve as a diagnostic and prognostic tool and refine the regimens for breast cancer therapies. (This work is supported by California Breast Cancer Research Program grant CBCRP-14FB-0114 to S.G.) Citation Information: Cancer Res 2009;69(23 Suppl):C21.