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]

Microsystem strategies for sample preparation in biological detection.

The objective of this LDRD was to develop microdevice strategies for dealing with samples to be examined in biological detection systems. This includes three sub-components: namely, microdevice fabrication, sample delivery to the microdevice, and sample processing within the microdevice. The first component of this work focused on utilizing Sandia's surface micromachining technology to fabricate small volume (nanoliter) fluidic systems for processing small quantities of biological samples. The next component was to develop interfaces for the surface-micromachined silicon devices. We partnered with Micronics, a commercial company, to produce fluidic manifolds for sample delivery to our silicon devices. Pressure testing was completed to examine the strength of the bond between the pressure-sensitive adhesive layer and the silicon chip. We are also pursuing several other methods, both in house and external, to develop polymer-based fluidic manifolds for packaging silicon-based microfluidic devices. The second component, sample processing, is divided into two sub-tasks: cell collection and cell lysis. Cell collection was achieved using dielectrophoresis, which employs AC fields to collect cells at energized microelectrodes, while rejecting non-cellular particles. Both live and dead Staph. aureus bacteria have been collected using RF frequency dielectrophoresis. Bacteria have been separated from polystyrene microspheres using frequency-shifting dielectrophoresis. Computational modeling was performed to optimize device separation performance, and to predict particle response to the dielectrophoretic traps. Cell lysis is continuing to be pursued using microactuators to mechanically disrupt cell membranes. Novel thermal actuators, which can generate larger forces than previously tested electrostatic actuators, have been incorporated with and tested with cell lysis devices. Significant cell membrane distortion has been observed, but more experiments need to be conducted to determine the effects of the observed distortion on membrane integrity and cell viability. Finally, we are using a commercial PCR DNA amplification system to determine the limits of detectable sample size, and to examine the amplification of DNA bound to microspheres. Our objective is to use microspheres as capture-and-carry chaperones for small molecules such as DNA and proteins, enabling the capture and concentration of the small molecules using dielectrophoresis. Current tests demonstrated amplification of DNA bound to micron-sized polystyrene microspheres using 20-50 microliter volume size reactions

Care seeking for fatal illness episodes in Neonates: a population-based study in rural Bangladesh

<p>Abstract</p> <p>Background</p> <p>Poor neonatal health is a major contributor to under-five mortality in developing countries. A major constraint to effective neonatal survival programme has been the lack of population level data in developing countries. This study investigated the consultation patterns of caregivers during neonatal fatal illness episodes in the rural Matlab sub-district of eastern Bangladesh.</p> <p>Methods</p> <p>Neonatal deaths were identified through a population-based demographic surveillance system in Matlab ICDDR,B maternal and child health (MCH) project area and an adjoining government service area. Trained project staff administered a structured questionnaire on care seeking to mothers at home who had experienced a neonatal death. Univariate, bivariate and binary multivariate logistic regressions were performed to describe care seeking during the fatal illness episode.</p> <p>Results</p> <p>Of the 365 deaths recorded during 2003 and 2004, 84% died in the early (0-7 days) neonatal period, with the remaining deaths occurring over the subsequent 8 to 28 days. The first resort of care by parents was a qualified doctor or paramedic in 37% of cases, followed by traditional and unqualified health care providers in 25%, while 38% sought no care. Thus, almost two thirds (63%) of neonates who died received only traditional and unqualified care or no care at all during their final illness episode. About 22% sought care from more than one provider, including 6% from 3 or more providers. Such plurality in care seeking was more likely among male infants, in the late neonatal period, and in the MCH project area.</p> <p>Conclusions</p> <p>The high proportion of neonatal deaths that had received traditional care or no medical care in a rural area of Bangladesh highlights the need to develop community awareness about prompt medical care seeking for neonatal illnesses and to improve access to effective health care. Integration of traditional care providers into mainstream health programs should also be considered.</p

University of Maryland Memorabilia - Accession 1232

This University of Maryland Memorabilia collection includes a 1914 yearbook titled, Terra Mariae from the University of Maryland at Baltimore. There is a handwritten note on the first page, Daddy - Freshman - p. 202-203 . This collection also includes a Christmas greeting card with an illustration of Mackenzie Hall and a signature in the right hand bottom corner by Sara Lee. The origin or provenance of this collection is entirely unknown.https://digitalcommons.winthrop.edu/manuscriptcollection_findingaids/1993/thumbnail.jp

Neurosurgical Manuscripts of Neuro-Ophthalmological Significance

There is an increasing shift from open craniotomy to endoscopic or keyhole surgical approaches in patients with suprasellar lesions because of shorter operating times, shorter hospital stays and improved quality of life

Neurosurgical Manuscripts of Neuro-Ophthalmological Significance

There is an increasing shift from open craniotomy to endoscopic or keyhole surgical approaches in patients with suprasellar lesions because of shorter operating times, shorter hospital stays and improved quality of life

American Jews and the Holocaust: History, memory and identity

Formation of American Jewish identity in the 1930s and 1940s was a multi-dimensional process. The Jewish American experience in the United States is unique because they faced a moral dilemma of loyalties and ties. They felt an obligation and responsibility to save their European brethren but were prevented from presenting a cohesive voice to protest the annihilation of Europe's Jews in the Holocaust. Examining American Jews' struggles with anti-Semitism, assimilation and ultimately the Holocaust, makes evident the complex process of immigrant identity formation in the United States and illuminates the specific ways in which the Jewish immigrant experience is unique.This thesis project will interpret oral history interviews from Jewish Americans who dealt with conflicting identities as Jews and as Americans. By creating a website that integrates oral history interviews with photographs and themes drawn from relevant scholarship, this project will be an opportunity for high school history teachers to begin a dialogue with students discussing issues around the Holocaust and the moral dilemma Jewish Americans faced being Americans yet wanting to aid their European religious brethren. The collection of these oral histories is crucial because they can help students discuss more openly the contradictions and conflicts underlying American Jewish identity.The website will ask thought provoking questions to lead students to a better understanding of the historical limitations that shaped American Jewish identity during the interwar years. The questions will be based on Jewish identity, anti-Semitism and Holocaust awareness. Creating a website to begin this dialogue will encourage a broader range of viewers to access the information. The website is a pilot, designed to encourage educators and other institutions to add additional and relevant information that can further the discussion. More broadly, this overall project can provide a first step in a larger dialogue to foster understanding of how global and domestic misconceptions can deter a population from aiding victims of a religious genocide.Thesis (M.A.)--University of Maryland, Baltimore County, 2009.School code: 0434