Towards Efficient Nonenzymatic DNA Ligation: Comparing Key Parameters for Maximizing Ligation Rates and Yields with Carbodiimide Activation**

Chemical ligation is an important tool for the generation of synthetic DNA structures, which are used for a wide range of applications. Surprisingly, reported chemical ligation yields can range from 30 to 95 % for the same chemical activating agent and comparable DNA structures. We report a systematic study of DNA ligation by using a well-defined bimolecular test system and a water-soluble carbodiimide (EDC) as a phosphate-activating agent. Our results emphasize the interplay between template-substrate complex stability and the rates of the chemical steps of ligation, with 3′ phosphate substrates providing yields near 100 % after 24 hours for particularly favorable reaction conditions. Ligation rates are also shown to be sensitive to the identity of the base pairs flanking a nick site, with as much as threefold variation. Finally, the observation that DNA substrates are modified by EDC at rates that can be comparable with ligation rates emphasizes the importance of considering side reactions when designing protocols to maximize ligation yields.     

NMR spectroscopy for determination of cationic polymer concentrations

Organic polyelectrolytes are utilized extensively in wastewater treatment, but their fate after use is poorly understood. Analytical methods used for polymer determination in less complex systems appear to fail in application to wastewater systems, contributing to the lack of knowledge. Thus, the development of 1H NMR spectroscopy is reported here for specifically quantitating certain cationic flocculant polymers in environmental samples. Proton observe frequencies of 250 or 400 MHz proton were used. A copolymer of acrylamide and acryloyloxyethyltrimethylammonium chloride was used, representative of cationic flocculant polymers possessing quaternary ammonium groups with terminal methyls that provide a sharp singlet at a chemical shift of approximately 3.06 ppm. A strong linear relationship was demonstrated between polymer concentration and either height or area of this peak. Recoveries were up to 96% at higher concentrations (250 mg/L), and were greater than when using viscosity or charge titration methods for polymer determination. Lesser recoveries at lower concentrations (70% at 5 mg/L) were attributed to adsorptive losses. The detection limit of this method was determined to be <0.5 mg/L. Use of the method was exemplified by analysis of anaerobically digested sludges for residual polymer following a range of dosages, showing the resulting isotherm.     

Effects of oil dispersants and oil emulsions on marine animals

The toxicities to marine animals of nine oil dispersants, three oil emulsions with Corexit and of a dispersion of Oman crude oil, have been studied in continuous flow aquarium systems at 96-h exposures followed by a recovery period in clean sea-water. New types of dispersants were found to be less toxic than older types and oil emulsions more toxic than dispersants alone or crude oil alone. Fishes and bivalves were found most sensitive. Crustaceans were the most resistant to dispersants but very susceptible to oil emulsions. The tolerance of different species was found to be related to their mode of life, more active species being more susceptible. Delayed mortality of bivalves increased their susceptibility if the recovery period was included. Effects on locomotory behaviour of fishes and crustaceans, breathing rate of fish, valve-closure of bivalves and byssal thread formation of common mussels have been demonstrated for both dispersants and oil emulsions. The general sequence of such effects was: increased activity; successively impaired activity; immobilization; and death. Recovery is good for fish and crustaceans but poor for bivalves due to the delayed effects. Ecological consequences of dispersants and oil pollution in the marine environment are discussed.     

波哥大城市公共空间的改革

波哥大是哥伦比亚的首都,城市位于国家中部海拔2600m的高原上。自1999年以来,首都经历了一场重大的改革,这项改革利用公共空间规划与可持续城市交通作为主要手段以营造更人性化的城市环境。介绍重要事件、项目、实践以及帮助推进波哥大城市形成的人物,重点论述公共空间改革过程与园林设计师的职责。著名的波哥大公共空间改革主要分为以下几个具体内容：步行区、道路基础设施(特别是自行车专用道的实施)、公园与绿道的改造恢复、城市绿化以及“千年捷运”巴士快速公交系统。1999-2004年之间,城市经历了高速投资,并完成了一系列重要基础设施项目。尽管波哥大的变化在空间上看起来最为明显,实际上改革已经影响到城市生活的方方面面。     

Characterization and Technological Properties of Lactic Acid Bacteria Isolated from Traditional Argentinean Goat’s Milk Products

A total of 286 lactic acid bacteria isolated from goat’s dairy products in northwest of Argentina were characterized. Lactobacilli (38%) and cocci (62%) were identified according to morphological, physiological, and relevant technological properties. L. plantarum (14%) and Enterococcus (34%) were the predominant species. S. thermophilus, Pediococcus and L. plantarum were the highest acid producers. Eight strains of L. fermentum produced bacteriocins or metabolites similar to bacteriocins. The API-ZYM test was applied to 39 isolates. Eight strains were selected from their both technological properties and enzymatic activities for use as starter or adjunct culture in the manufacture of artisanal goat cheeses.     

Aktuelle Aktivitäten zu den europäischen Rohstoff-Initiativen

The European Initiatives manifested in the Communications of the European Commission on Raw Materials, in the Strategic Implementation Plan of the European Innovation Partnership on Raw Materials and in the 2014 Call of the European Institute for Innovation & Technology (EIT) for a Knowledge and Innovation Community (KIC) on Raw Materials lead to manifold Austrian activities on Government level and on level of the Montanuniversitaet Leoben. The ESEE Region, thus East- and Southeast Europe has a special role.     

Cover Image,Volume 99, Issue 13

The cover image is based on the Research Article Study of the physical and chemical changes during the maturation of three cocoa clones, EET8, CCN51, and ICS60 by Andrés F Cubillos Bojacá et al., DOI: 10.1002/jsfa.9882 . Cover image ©AGROSAVIA Images.

     

α-Alumina and spinel react into single-phase high-alumina spinel in <3 seconds during flash sintering

In situ X-ray diffraction measurements at the Advanced Photon Source show that α-Al₂O₃ and MgAl₂O₄ react nearly instantaneously and completely, and nearly completely to form single-phase high-alumina spinel during voltage-to-current type of flash sintering experiments. The initial sample was constituted from powders of α-Al₂O₃, MgAl₂O₄ spinel, and cubic 8 mol% Y₂O₃-stabilized ZrO₂ (8YSZ) mixed in equal volume fractions, the spinel to alumina molar ratio being 1:1.5. Specimen temperature was measured by thermal expansion of the platinum standard. These measurements correlated well with a black-body radiation model, using appropriate values for the emissivity of the constituents. Temperatures of 1600-1736°C were reached during the flash, which promoted the formation of alumina-rich spinel. In a second set of experiments, the flash was induced in a current-rate method where the current flowing through the specimen is controlled and increased at a constant rate. In these experiments, we observed the formation of two different compositions of spinel, MgO•3Al₂O₃ and MgO•1.5Al₂O₃, which evolved into a single composition of MgO•2.5Al₂O₃ as the current continued to increase. In summary, flash sintering is an expedient way to create single-phase, alumina-rich spinel.     

An Alcohol Dehydrogenase from the Short-Chain Dehydrogenase/Reductase Family of Enzymes for the Lactonization of Hexane-1,6-diol

Biocatalytic production of lactones, and in particular ϵ-caprolactone (CL), have gained increasing interest as a greener route to polymer building blocks, especially through the use of Baeyer–Villiger monooxygenases (BVMOs). Despite several advances in the field, BVMOs, however, still suffer several practical limitations. Alcohol dehydrogenase (ADH)-mediated lactonization of diols in turn has received far less attention and very few enzymes have been identified for the conversion of diols to lactones, with horse-liver ADH (HLADH) remaining the catalyst of choice. Screening of a diverse panel of ADHs, AaSDR-1, a member of the short-chain dehydrogenase/reductase family, was found to produce ϵ-caprolactone from hexane-1,6-diol. Moreover, cofactor regeneration by an NADH oxidase eliminated the requirement of co-substrates, yielding water as the sole by-product. Despite lower turnover frequencies as compared to HLADH, higher selectivity was found for the production of CL, with HLADH forming significant amounts of 6-hydroxyhexanoic acid and adipic acid through aldehyde dehydrogenation/oxidation of the gem-diol intermediates. Also, CL yield were shown to be dependent on buffer choice, as structural elucidation of a Tris adduct confirmed the buffer amine to react with aliphatic aldehydes forming a Schiff-base intermediate which through further ADH oxidation, forms a tricyclic acetal product.