Applications of computational science for understanding enzymatic deconstruction of cellulose

Understanding the molecular-level mechanisms that enzymes employ to deconstruct plant cell walls is a fundamental scientific challenge with significant ramifications for renewable fuel production from biomass. In nature, bacteria and fungi use enzyme cocktails that include processive and non-processive cellulases and hemicellulases to convert cellulose and hemicellulose to soluble sugars. Catalyzed by an accelerated biofuels R&D portfolio, there is now a wealth of new structural and experimental insights related to cellulases and the structure of plant cell walls. From this background, computational approaches commonly used in other fields are now poised to offer insights complementary to experiments designed to probe mechanisms of plant cell wall deconstruction. Here we outline the current status of computational approaches for a collection of critical problems in cellulose deconstruction. We discuss path sampling methods to measure rates of elementary steps of enzyme action, coarse-grained modeling for understanding macromolecular, cellulosomal complexes, methods to screen for enzyme improvements, and studies of cellulose at the molecular level. Overall, simulation is a complementary tool to understand carbohydrate-active enzymes and plant cell walls, which will enable industrial processes for the production of advanced, renewable fuels.     

The physiology of the normal human breast: an exploratory study

The physiology of the nonlactating human breast likely plays a key role in factors that contribute to the etiology of breast cancer and other breast conditions. Although there has been extensive research into the physiology of lactation, few reports explore the physiology of the resting mammary gland, including mechanisms by which compounds such as hormones, drugs, and potential carcinogens enter the breast ducts. The purpose of this study was to explore transport of exogenous drugs into ductal fluid in nonlactating women and determine if their concentrations in the fluid are similar to those observed in the breast milk of lactating women. We selected two compounds that have been well characterized during lactation, caffeine and cimetidine. Caffeine passively diffuses into breast milk, but cimetidine is actively transported and concentrated in breast milk. After ingestion of caffeine and cimetidine, 14 nonlactating subjects had blood drawn and underwent ductal lavage at five time points over 12 h to measure drug levels in the fluid and blood. The concentrations of both caffeine and cimetidine in lavage fluid were substantially less than those observed in breast milk. Our results support recent evidence that the cimetidine transporter is not expressed in the nonlactating mammary gland, and highlight intriguing differences in the physiology and molecular transport of the lactating and nonlactating breast. The findings of this exploratory study warrant further exploration into the physiology of the nonlactating mammary gland to elucidate factors involved in disease initiation and progression.     

Improved ethanol yield and reduced minimum ethanol selling price (MESP) by modifying low severity dilute acid pretreatment with deacetylation and mechanical refining: 2) Techno-economic analysis

ABSTRACT: BACKGROUND: Our companion paper discussed the yield benefits achieved by integrating deacetylation, mechanical refining, and washing with low acid and low temperature pretreatment. To evaluate the impact of the modified process on the economic feasibility, a techno-economic analysis (TEA) was performed based on the experimental data presented in the companion paper. RESULTS: The cost benefits of dilute acid pretreatment technology combined with the process alternatives of deacetylation, mechanical refining, and pretreated solids washing were evaluated using cost benefit analysis within a conceptual modeling framework. Control cases were pretreated at much lower acid loadings and temperatures than used those in the NREL 2011 design case, resulting in much lower annual ethanol production. Therefore, the minimum ethanol selling prices (MESP) of the control cases were $0.41-$0.77 higher than the $2.15/gallon MESP of the design case. This increment is highly dependent on the carbohydrate content in the corn stover. However, if pretreatment was employed with either deacetylation or mechanical refining, the MESPs were reduced by $0.23-$0.30/gallon. Combing both steps could lower the MESP further by $0.44 ~ $0.54. Washing of the pretreated solids could also greatly improve the final ethanol yields. However, the large capital cost of the solid--liquid separation unit negatively influences the process economics. Finally, sensitivity analysis was performed to study the effect of the cost of the pretreatment reactor and the energy input for mechanical refining. A 50% cost reduction in the pretreatment reactor cost reduced the MESP of the entire conversion process by $0.11-$0.14/gallon, while a 10-fold increase in energy input for mechanical refining will increase the MESP by $0.07/gallon. CONCLUSION: Deacetylation and mechanical refining process options combined with low acid, low severity pretreatments show improvements in ethanol yields and calculated MESP for cellulosic ethanol production.     

Paradigmatic status of an endo- and exoglucanase and its effect on crystalline cellulose degradation

Background

Presently, different studies are conducted related to the topic of biomass potential to generate through anaerobic fermentation process alternative fuels supposed to support the existing fossil fuel resources, which are more and more needed, in quantity, but also in quality of so called green energy. The present study focuses on depicting an optional way of capitalizing agricultural biomass residues using anaerobic fermentation in order to obtain biogas with satisfactory characteristics.. The research is based on wheat bran and a mix of damaged ground grains substrates for biogas production.

Results

The information and conclusions delivered offer results covering the general characteristics of biomass used , the process parameters with direct impact over the biogas production (temperature regime, pH values) and the daily biogas production for each batch relative to the used material.

Conclusions

All conclusions are based on processing of monitoring process results , with accent on temperature and pH influence on the daily biogas production for the two batches. The main conclusion underlines the fact that the mixture batch produces a larger quantity of biogas, using approximately the same process conditions and input, in comparison to alone analyzed probes, indicating thus a higher potential for the biogas production than the wheat bran substrate. Adrian Eugen Cioabla, Ioana Ionel, Gabriela-Alina Dumitrel and Francisc Popescu contributed equally to this work     

Do communication training programs improve students' communication skills? - a follow-up study

ABSTRACT: BACKGROUND: Although it is taken for granted that history-taking and communication skills are learnable, this learning process should be confirmed by rigorous studies, such as randomized pre- and post-comparisons. The purpose of this paper is to analyse whether a communication course measurably improves the communicative competence of third-year medical students at a German medical school and whether technical or emotional aspects of communication changed differently. METHOD: A sample of 32 randomly selected students performed an interview with a simulated patient before the communication course (pre-intervention) and a second interview after the course (post-intervention), using the Calgary-Cambridge Observation Guide (CCOG) to assess history taking ability. RESULTS: On average, the students improved in all of the 28 items of the CCOG. The 6 more technically-orientated communication items improved on average from 3.4 for the first interview to 2.6 in the second interview (p < 0.0001), the 6 emotional items from 2.7 to 2.3 (p = 0.023). The overall score for women improved from 3.2 to 2.5 (p = 0.0019); male students improved from 3.0 to 2.7 (n.s.). The mean interview time significantly increased from the first to the second interview, but the increase in the interview duration and the change of the overall score for the students' communication skills were not correlated (Pearson's r = 0.03; n.s.). CONCLUSIONS: Our communication course measurably improved communication skills, especially for female students. These improvements did not depend predominantly on an extension of the interview time. Obviously, "technical" aspects of communication can be taught better than "emotional" communication skills.     

Microvascular endothelial cells from human omentum lack an inward rectifier K+ current

In most macrovascular endothelial cell (EC) preparations, resting membrane potential is determined by the inwardly rectifying K+ current (I(K1)), whereas in microvascular EC the presence of I(K1) varies markedly. Cultured microvascular EC from small vessels of human omentum were examined by means of the voltage-clamp technique to elucidate the putative role of I(K1) in maintaining resting membrane potential. Macrovascular EC from human iliac artery and bovine aorta served as reference. Human omentum EC showed an outwardly rectifying current-voltage relation. Inward current was hardly sensitive to variations of extracellular [K+] and Ba2+ block suggesting lack of I(K1). However, substitution of extracellular [Na+] and/or [Cl-] affected the current-voltage relation indicating that Na+ and Cl- contribute to basal current. Furthermore, outward current was reduced by tetraethylammonium (10 mM), and cell-attached recordings suggested the presence of a Ca2+-activated K+ current. In contrast to human omentum EC, EC from human iliac artery and bovine aorta possessed inwardly rectifying currents which were sensitive to variations of extracellular [K+] and blocked by Ba2+. Thus, the lack of I(K1) in human omentum EC suggests that resting membrane potential is determined by Na+ and Cl- currents in addition to K+ outward currents.     

Dimerisation of myomesin: implications for the structure of the sarcomeric M-band

The sarcomeric M-band is thought to provide a link between the thick and the elastic filament systems. So far, relatively little is known about its structural components and their three-dimensional organisation. Myomesin seems to be an essential component of the M-band, since it is expressed in all types of vertebrate striated muscle fibres investigated and can be found in its mature localisation pattern as soon as the first myofibrils are assembled. Previous work has shown that the N-terminal and central part of myomesin harbour binding sites for myosin, titin and muscle creatine kinase. Intrigued by the highly conserved domain layout of the C-terminal half, we screened for new interaction partners by yeast two-hybrid analysis. This revealed a strong interaction of myomesin with itself. This finding was confirmed by several biochemical assays. Our data suggest that myomesin can form antiparallel dimers via a binding site residing in its C-terminal domain 13. We suggest that, similar to alpha-actinin in the Z-disc, the myomesin dimers cross-link the contractile filaments in the M-band. The new and the already previously identified myomesin interaction sites are integrated into the first three-dimensional model of the sarcomeric M-band on a molecular basis.     

Beta-D-glucosidase reaction kinetics from isothermal titration microcalorimetry

The cellobiase activities of nine thermal stable mutants of Thermobifida fusca BglC were assayed by isothermal titration microcalorimetry (ITC). The mutations were previously generated using random mutagenesis and identified by high-temperature screening as imparting improved thermal stability to the beta-D-glucosidase enzyme. Analysis of the substrate-saturation curves obtained by ITC for the wild-type enzyme and the nine thermally stabilized mutants revealed that the wild type and all the mutants were subject to binding of a second substrate molecule. Furthermore, the "inhibited" enzyme-substrate complexes were shown to retain catalytic activity. In the case of three of the BglC mutants (N178I, N317Y/L444F, and N317Y/L444F/A433V), binding of a second substrate molecule resulted in improved cellobiose turnover rates at lower substrate concentrations. No correlation between denaturation temperatures of the mutants and activity on cellobiose at 25 degrees C was evident. However, one particular mutant, BglC S319C, was significantly improved in both thermal tolerance and cellobiase activity with respect to those of the wild-type BglC. The triple mutant, N317Y/L444F/A433V, had a 5 degrees C increase in denaturation temperature while maintaining activity levels similar to that of the wild type at higher substrate concentrations. ITC provided a highly sensitive and nondestructive means to continuously monitor the reaction of BglC with cellobiose, resulting in abundant data sets that could be rigorously analyzed by fitting to known enzyme kinetics models. One distinct advantage of using data from the ITC was the empirical validation of the pseudo steady state assumption, a necessary condition for obtaining solutions to the proposed mechanisms.