All-codon scanning identifies p53 cancer rescue mutations

In vitro scanning mutagenesis strategies are valuable tools to identify critical residues in proteins and to generate proteins with modified properties. We describe the fast and simple All-Codon Scanning (ACS) strategy that creates a defined gene library wherein each individual codon within a specific target region is changed into all possible codons with only a single codon change per mutagenesis product. ACS is based on a multiplexed overlapping mutagenesis primer design that saturates only the targeted gene region with single codon changes. We have used ACS to produce single amino-acid changes in small and large regions of the human tumor suppressor protein p53 to identify single amino-acid substitutions that can restore activity to inactive p53 found in human cancers. Single-tube reactions were used to saturate defined 30-nt regions with all possible codon changes. The same technique was used in 20 parallel reactions to scan the 600-bp fragment encoding the entire p53 core domain. Identification of several novel p53 cancer rescue mutations demonstrated the utility of the ACS approach. ACS is a fast, simple and versatile method, which is useful for protein structure–function analyses and protein design or evolution problems

Predicating from an early age: edusemiotics and the potential of children’s preconceptions

This paper aims to explain how semiotics and constructivism can collaborate in an educational epistemology by developing a joint approach to prescientific conceptions. Empirical data and findings of constructivist research are interpreted in the light of Peirce’s semiotics. Peirce’s semiotics is an anti-psychologistic logic (CP 2.252; CP 4.551; W 8:15; Pietarinen in Signs of logic, Springer, Dordrecht, 2006; Stjernfelt in Diagrammatology. An investigation on the borderlines of phenomenology, ontology and semiotics, Springer, Dordrecht, 2007) and relational logic. Constructivism was traditionally developed within psychology and sociology and, therefore, some incompatibilities can be expected between these two schools. While acknowledging the differences, we explain that constructivism and semiotics share the assumption of realism that knowledge can only be developed upon knowledge and, therefore, an epistemological collaboration is possible. The semiotic analysis performed confirms the constructivist results and provides a further insight into the teacher-student relation. Like the constructivist approach, Peirce’s doctrine of agapism infers that the personal dimension of teaching must not be ignored. Thus, we argue for the importance of genuine sympathy in teaching attitudes. More broadly, the article also contributes to the development of postmodern humanities. At the end of the modern age, the humanities are passing through a critical period of transformation. There is a growing interest in semiotics and semiotic philosophy in many areas of the humanities. Such a case, on which we draw, is the development of a theoretical semiotic approach to education, namely edusemiotics (Stables and Semetsky, Pedagogy and edusemiotics: theoretical challenge/practical opportunities, Sense Publishers, Rotterdam, 2015)

Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo

Meeting Abstracts: Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo Clearwater Beach, FL, USA. 9-11 June 201

Observing Shocks

Macroeconomists have observed business cycle fluctuations over time by constructing and manipulating models in which shocks have increasingly played a greater role. Shock is a term of art that pervades modern economics appearing in nearly one-quarter of all journal articles in economics and in nearly half in macroeconomics. Surprisingly, its rise as an essential element in the vocabulary of economists can be dated only to the early 1970s. We trace the history of shocks in macroeconomics from Ragnar Frisch and Eugen Slutsky in the 1920s and 1930s through real business cycle and DSGE models and to the use of shocks as generators of impulse-response functions, which are in turn used as data in matching estimators. The history is organized around the observability of shocks. As well as documenting a critical conceptual development in economics, the history of shocks shows that James Bogen and James Woodward’s distinction between data and phenomena must be substantially relativized if it is to be at all plausible

Membrane Stresses Induced by Overproduction of Free Fatty Acids in Escherichia coli▿†

Microbially produced fatty acids are potential precursors to high-energy-density biofuels, including alkanes and alkyl ethyl esters, by either catalytic conversion of free fatty acids (FFAs) or enzymatic conversion of acyl-acyl carrier protein or acyl-coenzyme A intermediates. Metabolic engineering efforts aimed at overproducing FFAs in Escherichia coli have achieved less than 30% of the maximum theoretical yield on the supplied carbon source. In this work, the viability, morphology, transcript levels, and protein levels of a strain of E. coli that overproduces medium-chain-length FFAs was compared to an engineered control strain. By early stationary phase, an 85% reduction in viable cell counts and exacerbated loss of inner membrane integrity were observed in the FFA-overproducing strain. These effects were enhanced in strains endogenously producing FFAs compared to strains exposed to exogenously fed FFAs. Under two sets of cultivation conditions, long-chain unsaturated fatty acid content greatly increased, and the expression of genes and proteins required for unsaturated fatty acid biosynthesis were significantly decreased. Membrane stresses were further implicated by increased expression of genes and proteins of the phage shock response, the MarA/Rob/SoxS regulon, and the nuo and cyo operons of aerobic respiration. Gene deletion studies confirmed the importance of the phage shock proteins and Rob for maintaining cell viability; however, little to no change in FFA titer was observed after 24 h of cultivation. The results of this study serve as a baseline for future targeted attempts to improve FFA yields and titers in E. coli