Austin v. Norfolk S Corp

USDC for the Western District of Pennsylvani

A condition-specific codon optimization approach for improved heterologous gene expression in Saccharomyces cerevisiae

All authors are with the Department of Chemical Engineering, The University of Texas at Austin, 200 E Dean Keeton St. Stop C0400, Austin, TX 78712, USA -- Hal S. Alper is with the Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, TX 78712, USA -- Amanda M. Lanza Current Address: Bristol-Myers Squibb, Biologics Development, 35 South Street, Hopkinton, MA 01748, USABackground: Heterologous gene expression is an important tool for synthetic biology that enables metabolic engineering and the production of non-natural biologics in a variety of host organisms. The translational efficiency of heterologous genes can often be improved by optimizing synonymous codon usage to better match the host organism. However, traditional approaches for optimization neglect to take into account many factors known to influence synonymous codon distributions. Results: Here we define an alternative approach for codon optimization that utilizes systems level information and codon context for the condition under which heterologous genes are being expressed. Furthermore, we utilize a probabilistic algorithm to generate multiple variants of a given gene. We demonstrate improved translational efficiency using this condition-specific codon optimization approach with two heterologous genes, the fluorescent protein-encoding eGFP and the catechol 1,2-dioxygenase gene CatA, expressed in S. cerevisiae. For the latter case, optimization for stationary phase production resulted in nearly 2.9-fold improvements over commercial gene optimization algorithms. Conclusions: Codon optimization is now often a standard tool for protein expression, and while a variety of tools and approaches have been developed, they do not guarantee improved performance for all hosts of applications. Here, we suggest an alternative method for condition-specific codon optimization and demonstrate its utility in Saccharomyces cerevisiae as a proof of concept. However, this technique should be applicable to any organism for which gene expression data can be generated and is thus of potential interest for a variety of applications in metabolic and cellular engineering.Chemical EngineeringInstitute for Cellular and Molecular [email protected]

Three Applications of an Austin/Wittgenstein Ontological Insight

On the first page of How to do Things with Words, Austin claims that `making a statement" is primary, and `statement" derivative – a `logical construction", as he calls it, out of the makings of statements. Wittgenstein, in similar vein, takes `explaining the meaning" to be primary with `meaning" a derivative notion. He says that `[m]eaning is what an explanation of meaning explains (Wittgenstein 1974, 68). Part of Wittgenstein"s point is that giving explanations of meaning is, like the making of statements, a perfectly common, everyday occurrence, but asking what meaning is is a perverse question of the sort that gives philosophy a bad name – Austin makes the same point in his paper `The Meaning of a Word" (Austin 1961, 23-43). Wittgenstein"s diagnosis of why philosophers are misled is very simple: the mistake lies in supposing that, for every noun there is an object named (unum nomen, unum nominatum) and so coming to believe that there is something – some thing – named by the noun `meaning". He says that he wants to cure us of the temptation to look about us for some object which you might call `the meaning" (Wittgenstein 1958, 1). This is hardly a new insight. Kant famously argued, in the Transcendental Aesthetic, that the noun `time" does not name a thing and one consequence of this conclusion is that talk of the Big Bang as marking the beginning of time is nonsensical. Are there some comparably important conclusions that can be drawn from the thesis that the nouns `meaning" and `statement" do not name objects? The answer, as I hope to demonstrate, is `Yes"

The case for new academic workspaces

Executive summary: This report draws upon the combined efforts of a number of estates professionals, architects, academics, designers, and senior managers involved in the planning of new university buildings for the 21st century. Across these perspectives, all would agree – although perhaps for different reasons - that this planning is difficult and that a number of particular considerations apply in the design of academic workspaces. Despite these difficulties, they will also agree that when this planning goes well, ‘good’ buildings are truly transformational – for both the university as a whole and the people who work and study in them. The value of well-designed buildings goes far beyond their material costs, and endures long after those costs have been forgotten ..

The Quest for the Causal Joint

This study is an examination of three proposals for a \"causal joint\" model of God\'s action in the world. Adapting the thought of Austin Farrer and David Burrell, the author seeks to show how these hypotheses are theologically flawed. The flaws stem from an overemphasis on the doctrine of creatio continua. Without an affirmation of both creatio ex nihilo and creatio continua, the latter is mistakenly removed from its theological context and adds unnecessary incoherence to the doctrine of creation

Alternative Computational Protocols for Supercharging Protein Surfaces for Reversible Unfolding and Retention of Stability

Bryan S. Der, Ron Jacak, Brian Kuhlman, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of AmericaChristien Kluwe, Aleksandr E. Miklos, Andrew D. Ellington , Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas, United States of AmericaChristien Kluwe, Aleksandr E. Miklos, George Georgiou, Andrew D. Ellington, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, United States of AmericaAleksandr E. Miklos, Andrew D. Ellington , Applied Research Laboratories, University of Texas at Austin, Austin, Texas, United States of AmericaSergey Lyskov, Jeffrey J. Gray, Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States of AmericaBrian Kuhlman, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of AmericaReengineering protein surfaces to exhibit high net charge, referred to as “supercharging”, can improve reversibility of unfolding by preventing aggregation of partially unfolded states. Incorporation of charged side chains should be optimized while considering structural and energetic consequences, as numerous mutations and accumulation of like-charges can also destabilize the native state. A previously demonstrated approach deterministically mutates flexible polar residues (amino acids DERKNQ) with the fewest average neighboring atoms per side chain atom (AvNAPSA). Our approach uses Rosetta-based energy calculations to choose the surface mutations. Both protocols are available for use through the ROSIE web server. The automated Rosetta and AvNAPSA approaches for supercharging choose dissimilar mutations, raising an interesting division in surface charging strategy. Rosetta-supercharged variants of GFP (RscG) ranging from −11 to −61 and +7 to +58 were experimentally tested, and for comparison, we re-tested the previously developed AvNAPSA-supercharged variants of GFP (AscG) with +36 and −30 net charge. Mid-charge variants demonstrated ~3-fold improvement in refolding with retention of stability. However, as we pushed to higher net charges, expression and soluble yield decreased, indicating that net charge or mutational load may be limiting factors. Interestingly, the two different approaches resulted in GFP variants with similar refolding properties. Our results show that there are multiple sets of residues that can be mutated to successfully supercharge a protein, and combining alternative supercharge protocols with experimental testing can be an effective approach for charge-based improvement to refolding.This work was supported by the Defense Advanced Research Projects Agency (HR-0011-10-1-0052 to A.E.) and the Welch Foundation (F-1654 to A.E.), the National Institutes of Health grants GM073960 (B.K.) and R01-GM073151 (J.G. and S.L.), the Rosetta Commons (S.L.), the National Science Foundation graduate research fellowship (2009070950 to B.D.), the UNC Royster Society Pogue fellowship (B.D.), and National Institutes of Health grant T32GM008570 for the UNC Program in Molecular and Cellular Biophysics. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Center for Systems and Synthetic BiologyCellular and Molecular BiologyApplied Research LaboratoriesEmail: [email protected]

Caddo Ceramic Vessels from the S. E. Watson (41RR8) and Hook’s Ferry (41RR9) Sites, Red River County, Texas

There are 15 ancestral Caddo ceramic vessels from the S. E. Watson (n=13) and Hook’s Ferry (n=2) sites in the collections of the Texas Archeological Research Laboratory at The University of Texas at Austin. The S. E. Watson site is a Caddo mound center, village, and cemetery on Pecan Bayou near its confluence with the Red River. Another Caddo mound was reported at nearby 41RR67, on the Chapman Plantation, although it may have been destroyed by Red River flooding. The Hook’s Ferry site (41RR9) is situated in the Red River floodplain just east of the Jonesborough site (41RR15), north of Salt Well Slough, and ca. 3 km upstream from the large village and mound center at the Sam Kaufman site (41RR16)