Here, we provide a detailed protocol for the single protein production (SPP) system, which is designed to produce only a single protein of interest in living Escherichia coli cells. Induction of MazF, an mRNA interferase that cleaves RNA at ACA nucleotide sequences, results in complete cell growth arrest. However, if mRNA encoding a protein of interest is engineered to be devoid of ACA base triplets and is induced at 15 1C using pCold vectors in MazF-expressing cells, only the protein from this mRNA is produced at a yield of 20-30% of total cellular protein; other cellular protein synthesis is almost completely absent. In theory, any protein can be produced by the SPP system. Protein yields are typically unaffected even if the culture is condensed up to 40-fold, reducing the cost of protein production by up to 97.5%. The SPP system has a number of key features important for protein production, including highyield and prolonged production of isotope-labeled protein at a very high signal-to-noise ratio. The procedure can be completed in 7 d after cloning of an ACA-less target gene into the expression system.

The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease.IMPORTANCE Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus drug development. We identified and characterized an inhibitor of pUL89 endonuclease activity that also inhibits human cytomegalovirus replication in cell culture. pUL89 endonuclease, therefore, should be explored as a potential target for antiviral development against human cytomegalovirus.KEYWORDS human cytomegalovirus, terminase, pUL89 inhibitor, hydroxypyridonecarboxylic acid, metal chelation H uman cytomegalovirus (HCMV) infection is a significant cause of morbidity and mortality in immunocompromised patients, including transplant recipients and AIDS patients (reviewed in reference 1). In addition, HCMV infects 1% of all newborns and is a leading cause of brain damage and hearing loss (2). Currently, most of the U.S. Food and Drug Administration (FDA)-approved anti-HCMV drugs target the viral polymerase, including ganciclovir (GCV), valganciclovir, cidofovir, and foscarnet (reviewed in

The potential toxicity of existing chemical dispersants on the marine environment has motivated the search for environmentally friendly dispersants with excellent dispersion ability. Here, an effective Pickering emulsifier is developed based on the synergy of natural biopolymer, Xanthan Gum (XG), and silica nanoparticles. The oil−in−seawater emulsion stabilized by a combination of XG and silica demonstrates great stability and smaller droplet size, which is favorable for the following natural degradation of oil. The synergistic emulsification mechanism has been investigated systematically. The presence of XG favors the adsorption of silica nanoparticles at the oil−seawater interface and also is considerably effective in enhancing the viscosity of continuous phase. These contributions of XG slow down the droplet coalescence and creaming significantly. Confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) images of emulsions indicate a thick layer of aggregated XG/silica particles at the oil− water interface. This thick layer provides an effective steric barrier. In this study, the synergy between XG and silica not only enhances the dispersion effectiveness, but also reduces the amount of nanoparticles dramatically. This finding opens up a new path for the development of a novel, high efficiency, ecologically acceptable, and cheaper dispersant for emulsifying crude oil following a spill.

Insertion of phenyl isocyanate into the Ln−N σ bond of (MeC5H4)2Ln(i-Pr)2(THF) (Ln = Y (1), Er (2), Yb (3)), which were synthesized by reactions of (MeC5H4)2LnCl(THF) with LiN(i-Pr)2, led to the isolation of {(MeC5H4)2Ln(THF)[O CN(i-Pr)2 NPh]} (Ln = Y (4), Er (5), Yb (6)). The latter is the active species for phenyl isocyanate polymerizations. The X-ray structure analysis of 4 shows that Y is coordinated to two methylcyclopentadienyl groups and one bidentate anion [O CN(i-Pr)2 NPh]- via a partial single bond, partial donor bond interaction with N1 and O1 atoms.

4 H 8 ) 2 Eu (4), via tandem silylamine elimination/homolysis of the Eu-N bond reactions. The formation pathway for complex 3 was proposed. All the compounds were fully characterized by spectroscopic methods and elemental analyses, and the structures of complexes 3 and 4 were additionally determined by single-crystal X-ray diffraction analyses. It was found that complexes 3 and 4 can function as single-component MMA polymerization initiators, which represent the first examples of europium(II) complexes as single-component MMA polymerization catalysts. The solvents and temperature effects' on the activities of the catalysts were also discussed.

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One remediation technique of oil spills is the application of dispersants to oil slicks, which is essentially a process of emulsification. Tetradecane and crude oil-inseawater emulsions formed with silica nanoparticles modified in situ with rhamnolipid produced a longer stability and smaller droplet size. The interactions of silica particles with rhamnolipid were characterized by contact angle, interfacial tension, TEM, and SEM measurements. The images of confocal fluorescence microscopy and SEM showed the oil droplet microstructure and the morphology of nanoparticles at the oil droplet−water interface. The average emulsion droplet size and emulsion index were investigated. These results indicated a synergistic stabilization upon rhamnolipid addition. The synergy was even more efficient in the case of seawater with a high salinity. Here, because of the strong flocculation caused by high salinity, silica nanoparticles alone were not an effective emulsifier in seawater. The modification of silica nanoparticles by rhamnolipid changed the contact angle and promoted their adsorption at the oil−seawater interface, which provided an efficient barrier to droplet coalescence. The emulsification of rhamnolipid-modified silica nanoparticles worked well in crude oil−seawater system. So, this could be a new method to deal with the issue of the marine oil spill by environmentally benign silica particles and rhamnolipid.

We investigated the effect of carbon nanotubes (CNTs) location on the property and performance of prepared membranes. Four different types of membranes were prepared, including (1) thin film composite (TFC, polyamide active layer on polysulfone substrate), (2) nanocomposite-supported thin film composite (nTFC, polyamide active layer on CNT-embedded polysulfone substrate), (3) thin film nanocomposite (TFN, CNT-incorporated polyamide active layer on polysulfone substrate), and (4) nanocomposite-supported thin film nanocomposite (nTFN, CNT-incorporated polyamide active layer on CNT-embedded polysulfone substrate). The water permeability followed the sequence of nTFN > TFN > nTFC > TFC. However, the rejection and salt permeability exhibited the opposite trend. The incompatibility between CNTs and polymers provided nanocorridors, through which both water and solutes could pass. The nTFN membrane exhibited the highest porosity and lowest structural parameter. Moreover, the nTFN membrane possessed the best antifouling capacity by preventing foulants to attach to the surface and clog the substrate pores. This work offered some systematic knowledge to design novel membranes with improved performance for desalination and water purification applications.