Structures, kinetics, and modeling of glycoside hydrolase family 44 endoglucanases from Clostridium acetobutylicum and Ruminococcus flavefaciens FD-1

Glycoside hydrolases (GH) are enzymes that hydrolyze glycosidic bonds between carbohydrates. Family 44 contains endo- and xylo- glucanases capable of hydrolyzing cellulose and hemicelluose. Two members produced by Clostridium acetobutylicum and Ruminococcus flavefaciens FD-1 are the subjects of characterization work in this dissertation. The EG from Clostridium acetobutylicum was characterized both structurally and kinetically. Crystallization and x-ray diffraction to 2.2-y resolution revealed a TIM barrel-like structure with additional ψ-loop and β-sandwich folds. The EG hydrolyzes cellotetraose and larger cellooligosaccharides, as well as carboxymethylcellulose and xylan at approximately the same rates. It converts lichenan to oligosaccharides of intermediate size and attacks Avicel to a limited extent. The enzyme has an optimal temperature in a 10-min assay of 55yC and an optimal pH of 5.0. The EG from Ruminococcus flavefaciens FD-1 enzyme\u27s catalytic domain, with and without the CBM, hydrolyzes cellotetraose, cellopentaose, cellohexaose, carboxymethylcellulose (CMC), birchwood and larchwood xylan, lichenan, and Avicel, but not cellobiose, cellotriose, mannan, or pullulan. The presence of the CBM increases catalytic efficiencies on both CMC and birchwood xylan. The products of cellooligosaccharide hydrolysis show both asymmetric cleavage and disproportionate product distributions, indicating the reactions taking place are more complicated than hydrolysis alone. A reaction model, including hydrolysis and transglycosylation, was designed that describes disproportionation of hydrolysis products of cellopentaose and cellohexaose by the two GH44 EGs produced by C. acetobutylicum and R. flavefaciens FD-1. The model fit the C. acetobutylicum EG data well for the digestions of cellotetraose, cellopentaose, and cellohexaose, while agreeing with the absence of measured transglycosylation products in capillary electrophoresis chromatographs due to cellooligosaccharide insolubility or concentrations near or below the limit of detection. Characterization of binding constants and thermodynamic properties of the novel CBM associated with the R. flavefaciens catalytic domain and its synergistic effect when the two were linked is described. All constructs bind cellotetraose, cellopentaose, and cellohexaose with increasing affinity. No binding of xylotetraose, xylopentaose or xylohexaose was observed. The CBM and fusion protein have larger association constants for CMC than for larchwood xylan. A synergistic effect was observed with the fusion protein on cellopentaose and cellohexaose. All binding experiments were energetically favorable and enthalpy-driven

A comparative verification of forecasts from two operational solar wind models

The solar wind (SW) and interplanetary magnetic field (IMF) have a significant influence on the near‐Earth space environment. In this study we evaluate and compare forecasts from two models that predict SW and IMF conditions: the Hakamada‐Akasofu‐Fry (HAF) version 2, operational at the Air Force Weather Agency, and Wang‐Sheeley‐Arge (WSA) version 1.6, executed routinely at the Space Weather Prediction Center. SW speed (Vsw) and IMF polarity (Bpol) forecasts at L1 were compared with Wind and Advanced Composition Explorer satellite observations. Verification statistics were computed by study year and forecast day. Results revealed that both models’ mean Vsw are slower than observed. The HAF slow bias increases with forecast duration. WSA had lower Vsw forecastobservation difference (F‐O) absolute means and standard deviations than HAF. HAF and WSA Vsw forecast standard deviations were less than observed. Vsw F‐O mean square skill rarely exceeds that of recurrence forecasts. Bpol is correctly predicted 65%–85% of the time in both models. Recurrence beats the models in Bpol skill in nearly every year forecast day category. Verification by “event” (flare events ≤5 days before forecast start) and “nonevent” (no flares) forecasts showed that most HAF Vsw bias growth, F‐O standard deviation decrease, and forecast standard deviation decrease were due to the event forecasts. Analysis of single time step Vsw increases of ≥20% in the nonevent forecasts indicated that both models predicted too many occurrences and missed many observed incidences. Neither model had skill above a random guess in predicting Vsw increase arrival time at L1

EC253 Nutrients, Feeds and Example Rations for Swine

Extension Circular 253 discusses nutrients, feeds and example rations for swine

Fixing Hollingsworth: Standing in Initiative Cases

In Hollingsworth v. Perry, the Supreme Court dismissed an appeal filed by the “Official Proponents” of California’s Proposition 8, which banned same-sex marriage in California. Chief Justice Roberts’ majority opinion held that initiative sponsors lack Article III standing to defend their ballot measures even when state officials refuse to defend against constitutional challenges. As a result, Hollingsworth provides state officers with the ability to overrule laws that were intended to bypass the government establishment—in effect, an “executive veto” of popularly-enacted initiatives. The Article examines this new “executive veto” in depth. It places Hollingsworth in context, discussing the initiative process in California, and the history of the federal lawsuit challenging Proposition 8. An in-depth discussion of Hollingsworth follows. The particular issue presented by the appellants, their claim to standing based on their status as representatives of the People of California, and the Court’s treatment of that issue, is scrutinized. This includes the Court’s rejection of California law on the legal status of initiative proponents, and its adoption of the Restatement of Agency as the basis for Article III standing. After concluding that Hollingsworth establishes an “executive veto” over the initiative process, the Article proceeds to examine the potential effect of this in California and the thirty-six other “direct democracy” states. Finally, the authors present a series of “fixes” to Hollingsworth’s executive veto. These could assure defense of initiatives in the future, protecting them from the fate that Proposition 8 suffered in Hollingsworth

Nucleate Boiling Heat Transfer Of Liquid Nitrogen From Plasma Deposited Polymer Coated Surfaces

In certain cases, nucleate boiling heat transfer from polymerized tetrafluoroethylene coated surfaces was more than five times the rate of heat transfer found for uncoated surfaces at a given temperature difference. It was also found that for the polymer coated surfaces the transition from nucleate to film boiling was much slower than for uncoated surfaces. © 1978

Comparison of Training Methods to Develop Maximum Fitness for Bicycle Roller Racing

Health, Physical Education, and Recreatio