Targeted Mutagenesis of the Oligopeptide Repeat Domain of the Yeast Prion Sup35

The formation of prions in the baker’s yeast Saccharomyces cerevisiae is determined by amino acid composition rather than the primary sequence of amino acids. The infectious amyloid proteins known as prions undergo nucleation and propagation, two distinct activities critical for prion formation. The ability for prions to be transferred from cell to cell, or propagate, is of interest not only in yeast prions but also in prion diseases such as the mammalian spongiform encephalopathies. Prion formation has been widely studied in yeast prions, however, the fundamental mechanisms behind the specific process of propagation of prions from cell to cell are not yet understood. In the most well-studied yeast prion, the prion form [PSI+] of Sup35, a domain of 5 ½ degenerate oligopeptide repeats called the oligopeptide repeat domain (ORD) has been shown to be important for prion propagation and to have a distinct amino acid composition as compared to the nucleation domain region. A library mutagenesis experiment has identified amino acids that favor or disfavor prion propagation in yeast cells. To confirm the results of the random library mutagenesis experiment, we generated several clones in which a portion of the ORD (the fourth oligopeptide repeat) was replaced with defined sequences expected to propagate or fail to propagate

Traffic Flow: An Approach towards Modeling the Right Lane Rule

We attempted to model and analyze the effect of the right hand rule for the 2014 COMAP Math Modeling Competition. In order to analyze the right hand rule we started with Greenshield’s macroscopic approach and modified it to simulate the effects of the right hand rule. By analyzing the resulting changes in the flow and density of the system we determined the performance of the rule in varying traffic densities. Next we looked at the performance by modeling traffic flow when the rule is strictly adhered to, as compared to an intermediate, where the rule is followed until the critical density is reached. In the intermediate model we show how traffic flow can be maximized if people no longer follow the right hand rule after the critical density

Mapping the Beta-Sheet Structure of the Yeast Prion Sup35 through Creation of Targeted Mutant Forms

Proteins with an aggregated form rich in beta-sheet structure are known as amyloids, of which a subset are infectious. These infectious proteins are known as prions and cause diseases including bovine spongiform encephalopathy (“Mad Cow” disease). Several prions have been identified in the baker’s yeast, Saccharomyces cerevisiae. One of the most well-studied yeast prions is the protein Sup35. To understand the fine protein structure of Sup35 better, we used PCR-based mutagenesis to introduce a lysine residue (a charged amino acid) at five defined places in the protein sequence of Sup35. We describe our process for creating these mutant versions and the results of DNA sequencing of each mutant version. The next step will be to assess prion formation and stability of clones with the correct sequences. Understanding the behavior of yeast prions has proven helpful in understanding human amyloid diseases and further studies on these yeast prions, including Sup35, will expand our knowledge further

Primary herpetic gingivo-stomatitis: clinical studies from general practice

Primary herpetic gingivo-stomatitis, a well -defined clinical entity, appears to present a peculiarly difficult diagnostic challenge to the family doctor. Possible reasons for this have been examined in a series of studies over a period of three years by one general practitioner in a large group practice. Analysis of the early clinical manifestations of the disease, studied in a prospective survey, showed upper respiratory catarrh to be a prominent feature. The difficulty of spotting this and other early features of the disease against the background of similar presentations of different - and more common - illnesses was demonstrated in a one -year prospective survey of all febrile children seen by this family doctor. Laboratory support for the main clinical survey led to antibody studies of affected children, of a control group, and of mothers of both groups. The findings supported the clinical diagnosis, confirmed the high prevalence of infection in the mothers, and provided information on some changes in antibody response in relation to the length of the follow-up period. Recurrent clinical infections and possible sequelae were already evident, even within the relatively short duration of the survey. These phenomena, examined against the control group employed in the antibody studies, included herpes facialis, various infections, and recurrent mouth ulceration. The findings were compared with results of a retrospective study of cases of probable herpetic stomatitis and of control children, selected from the group practice records. Similar results were obtained in this survey. Comparison of households containing affected children against others from the practice showed the disease occurred more frequently in a setting of poor hygiene and larger sized families, factors which also hinder the family doctor in diagnosis. From the pattern of admission diagnoses of cases treated in the Edinburgh City Hospital during the three years of the studies, it was clear that other general practitioners in the area shared difficulties in diagnosis. The results of all these surveys have been examined in the light of published reports, with particular reference to diagnosis in general practice. The differential diagnosis has been discussed and illustrated by examples drawn from the day-to-day work of the practice. It is concluded that greater attention should be paid to the disease - which is as common as rubella, mumps or the more severe cases of chickenpox - in undergraduate teaching and in post-graduate research. The inadequacy of drug therapy highlights the importance of prophylaxis, and a greater public awareness of the potentially infectious nature of the common "cold sore" needs to be promoted

An Analysis of Putative Roles for the CCR4-NOT Deadenylase-Complex Subunit Regena (NOT2) in microRNA-Mediated Gene Silencing in \u3cem\u3eDrosophila Melanogaster\u3c/em\u3e

microRNAs (miRNAs) are one class of small non-coding ribonucleic acid (RNA) molecules essential to development and homeostasis in plants and animals. miRNAs silence gene expression through complementary base pairing with target gene messenger RNAs and association with the miRNA-induced silencing complex (miRISC). The identification and characterization of cellular factors required for miRNA-mediated gene silencing is incomplete. A forward genetic screen was carried out in Drosophila melanogaster to generate flies defective for gene silencing. Silencing was assayed by expression of a Green Fluorescent Protein (GFP) reporter fused to the Brd gene 3’ UTR, which is regulated by miRNAs. Genetic analysis revealed that the CCR4-NOT deadenylase-complex subunit Regena (NOT2) is required for miRNA-mediated silencing of the reporter. In addition, perturbation of Regena function altered Drosophila eye development and resulting adult eye morphology. miRNAs are thought to silence target gene expression through a combination of translational repression and target mRNA degradation, though the detailed mechanism of this process is a matter of controversy. Novel genetic reagents to explore miRNA function in vivo have been generated and characterized. Ongoing efforts aim to explore whether Regena is required to silence other miRNA targets in vivo, and whether Regena is required for miRNA-mediated gene silencing at different stages of the Drosophila life cycle. Elucidation of the lesion in the Regena (NOT2) gene and the molecular nature of GFP reporter silencing will contribute to an understanding of the mechanism of miRNA-mediated gene silencing in vivo

New Methods for the Adsorption of Carbon Dioxide and Water Vapor from Manned Spacecraft Atmospheres: Application and Modeling

This viewgraph presentation describes new methodologies for carbon dioxide and humidity removal from manned spacecraft atmospheres. The mathematical modeling of various adsorbents is also presented

IMPACT OF A MORE INTENSIVE INSECT PEST INFESTATION LEVEL ON COTTON PRODUCTION: TEXAS HIGH PLAINS

This study evaluated implications of increased bollworm problems in a 20-county area of the Texas High Plains relative to cotton yields and economic impact. Results did not indicate a serious effect of bollworms upon lint yield when insecticides were used for control. However, estimated annual reduction in farmer profit due to the bollworm for 1979-81 was over $30 million. Yields were estimated to decline about 300,000 bales without insecticide use and about 30,000 bales with insecticide use. This decline suggests potentially serious implications for the comparative economic position of cotton in this region if insecticide resistance were to develop among insect pests.Crop Production/Industries,

Aquatic biosurvey of the Lovell River on UNH land

We assessed the physical, chemical and biological conditions at two sites along the Lovell River on University of New Hampshire (UNH) -owned conservation land. The discharge was 4.4 m3 s-1 at Site 1 and 5.7 m3 s -1 downstream at Site 2. Canopy coverage ranged from 8-25%. Canopy was dominated by Eastern Hemlock (79-84%). Much of the stream was strewn with large boulders and the substrate consisted of rocks of highly variable sizes ( 3-549 cm dia.). Specific conductivity (22.1-23.3 µS), pH (6.4) and temperature (7.9-8.3 °C) varied little between sites. Macro-invertebrate bio-indices indicated either excellent water quality with no apparent organic pollution (3.0/10) or good water quality with possible slight organic pollution (4.4/10)

Simulation of Unique Pressure Changing Steps and Situations in Psa Processes

A more rigorous cyclic adsorption process simulator is being developed for use in the development and understanding of new and existing PSA processes. Unique features of this new version of the simulator that Ritter and co-workers have been developing for the past decade or so include: multiple absorbent layers in each bed, pressure drop in the column, valves for entering and exiting flows and predicting real-time pressurization and depressurization rates, ability to account for choked flow conditions, ability to pressurize and depressurize simultaneously from both ends of the columns, ability to equalize between multiple pairs of columns, ability to equalize simultaneously from both ends of pairs of columns, and ability to handle very large pressure ratios and hence velocities associated with deep vacuum systems. These changes to the simulator now provide for unique opportunities to study the effects of novel pressure changing steps and extreme process conditions on the performance of virtually any commercial or developmental PSA process. This presentation will provide an overview of the cyclic adsorption process simulator equations and algorithms used in the new adaptation. It will focus primarily on the novel pressure changing steps and their effects on the performance of a PSA system that epitomizes the extremes of PSA process design and operation. This PSA process is a sorbent-based atmosphere revitalization (SBAR) system that NASA is developing for new manned exploration vehicles. This SBAR system consists of a 2-bed 3-step 3-layer system that operates between atmospheric pressure and the vacuum of space, evacuates from both ends of the column simultaneously, experiences choked flow conditions during pressure changing steps, and experiences a continuously changing feed composition, as it removes metabolic CO2 and H20 from a closed and fixed volume, i.e., the spacecraft cabin. Important process performance indicators of this SBAR system are size, and the corresponding CO2 and H20 removal efficiencies, and N2 and O2 loss rates. Results of the fundamental behavior of this PSA process during extreme operating conditions will be presented and discussed

CO2 Capacity Sorbent Analysis using Volumetric Measurement Approach

In support of air revitalization system sorbent selection for future space missions, Ames Research Center (ARC) has performed CO2 capacity tests on various sorbents to complement structural strength tests from Marshall Space Flight Center (MSFC). The materials of interest are: Grace Davison Grade 544 13x, Honeywell UOP APG III, VSA-10, BASF 13x, and Grace Davison Grade 522 5A. Each sorbents CO2 capacity was measured using a Micromeritics ASAP 2020 Physisorption Volumetric Analysis machine to produce 0C, 10C, 25C, 50C, and 75C isotherms. These datasets were then extrapolated using Langmuir 3-Site and Toth isotherm models to compare with previously measured capacity data from MSFC using a thermogravimetric analysis approach. The modeling and extrapolation from ARC data correlated well with data measured at MSFC