Combination of computational prescreening and experimental library construction can accelerate enzyme optimization by directed evolution

Chiral compounds can be produced efficiently by using biocatalysts. However, wild-type enzymes often do not meet the requirements of a production process, making optimization by rational design or directed evolution necessary. Here, we studied the lipase-catalyzed hydrolysis of the model substrate 1-(2-naphthyl)ethyl acetate both theoretically and experimentally. We found that a computational equivalent of alanine scanning mutagenesis based on QM/MM methodology can be applied to identify amino acid positions important for the activity of the enzyme. The theoretical results are consistent with concomitant experimental work using complete saturation mutagenesis and high-throughput screening of the target biocatalyst, a lipase from Bacillus subtilis. Both QM/MM-based calculations and molecular biology experiments identify histidine 76 as a residue that strongly affects the catalytic activity. The experiments demonstrate its important influence on enantioselectivity.     

Justifications,Strategies, and Critical Success Factors in Successful ITIL Implementations in U.S. and Australian Companies: An Exploratory Study

Abstract

A growing number of organizations are implementing the ITIL (IT Infrastructure Library) “best practice” framework in an attempt to improve their IT service management processes. However, not all ITIL implementations are successful and some companies have been disappointed with the outcomes. This exploratory research reports on four case studies of “successful” implementations of IT service management using the process-based ITIL V2 framework. Two companies are located in the U.S. and two in Australia. The cases demonstrate a mix of implementation justifications and strategies. Critical success factors (CSFs) suggested in the literature are compared against those attributed to these successful ITIL implementations. Some CSFs, including executive management support, interdepartmental communication and collaboration, use of consultants, training and careful software selection are confirmed. Three new CSFs are identified: creating an ITIL-friendly culture, process as a priority, and customer-focused metrics. Practitioner guidelines, to assist IT managers, who are contemplating adopting ITIL for process improvement and organisational transformation, are also provided together with some challenges encountered and their associated resolutions.     

Publishers’ relationships on the web: An examination of how publishers’ websites are causing changes in relations in the book industry

This research, funded by the British National Bibliography Research Fund, examined how publishers’ websites are causing changes in relations in the book industry in order to gain further understanding of the implications of the impact of the Internet on the publishing chain. The paper is set within the context of the development of electronic commerce and how business to consumer commerce is now being overtaken by the activity in business to business trading. Publishers have followed the main business trends in using their websites to develop relationships directly with the consumer, but have been rather slower in developing their business to business activity through the Internet. This study investigated what changes were taking place as a result of current publisher activity on the Web and how these changes were affecting the traditional lines of communication in the book industry. An analysis of a range of consumer publishers’ websites was carried out to see what facilities they were offering both to the general public and to business partners within the industry. Questionnaires and interviews were then conducted to establish how publishers, booksellers and wholesalers were using publishers’ websites and whether these sites were beneficial to the industry as a whole. Facilities found on the websites were examined and analysed in order to determine their usefulness and how they might be developed to aid business to business commerce. Additionally, several issues relating to online sales, changes in sales patterns and changes in working methods were discussed. Although an amount of quantitative data is included in the report, many of the issues raised relied on the perceptions and opinions of practitioners in the book trade.     

Calculation of the stability ratio of suspensions using Emulsion Stability Simulations

According to Emulsion Stability Simulations (ESS), the flocculation of two non-deformable drops in the primary minimum of the interaction potential, necessarily leads to their coalescence. This property is used here for the evaluation of the stability ratio (W) of solid particles, interacting with the same inter-particle potential as the one of non-deformable droplets. Two different methodologies are used. The first one consists on the repeated evaluation of the coalescence time between two particles. The second one consists on the estimation of the time required for a decrease in the number of aggregates of the dispersion equal to n₀/2 (where n₀ is the initial number of aggregates). The results of the simulations are contrasted with the stability ratio of an anionic latex suspension subject to several ionic strengths (400-1000 mM). The first methodology is far more efficient for the evaluation of W although it misses the development of the aggregates and their growth. Absolute coagulation rates (kf) can also be obtained using one N-particle simulation for the calculation of the fast flocculation rate , and several two-particle simulations for the evaluation of W. This combined procedure is also more efficient than the N-particle evaluation of .     

The in vitro and in vivo investigation of inhaled migraine therapies using a novel aerosol delivery system consisting of an air pressurized capsule device (APCD) in combination with a pMDI spacer for endotracheal dosing into beagle dogs

Abstract

Context: Aerosol delivery to animals in preclinical settings has historically been very challenging, requiring the use of techniques, such as intratracheal instillation and dry powder insufflation, that are somewhat invasive, inefficient and not representative of clinical inhalation.

Objective: The objective of this work is to develop a system to deliver dry powder to dogs in an efficient and effective manner for the study of new anti-migraine compounds in development.

Materials and methods: The new device uses a metered aliquot of a dry gas to force dry powder drug from a pre-filled HPMC capsule into an AeroChamber® spacer for subsequent inhalation by the animal.

Results: The delivery of two invesigational migraine drugs via the new device was assessed in vitro using abbreviated Andersen cascade impaction and showed the device is capable of generating a reproducible delivered dose of up to ～68% with more than 50% of the dose in the respirable range. In vivo studies have also been performed showing that this device effectively delivered the migraine drugs to spontaneously breathing dogs using a proprietary validated dog inhalation model.

Discussion: Results confirmed that the air pressurized capsule device (APCD) was effective in delivering the APIs to lungs of the animals. The in vivo data verified the advantages of inhaled delivery over oral delivery for this class of drugs and were used to establish the cardiopulmonary and respiratory side effect liability profile for these compounds.

Conclusions: This work has demonstrated the utility of this device for quick and accurate screening of prospective drug candidates, representing a significant improvement in ease of use and reprodicibility over current delivery methods.     

The effect of fertilizer placement on nitrogen uptake and yield of wheat and maize in Chinese loess soils

Field trials were carried out to study the fate of¹⁵N-labelled urea applied to summer maize and winter wheat in loess soils in Shaanxi Province, north-west China. In the maize experiment, nitrogen was applied at rates of 0 or 210 kg N ha^–1, either as a surface application, mixed uniformly with the top 0.15 m of soil, or placed in holes 0.1 m deep adjacent to each plant and then covered with soil. In the wheat experiment, nitrogen was applied at rates of 0, 75 or 150 kg N ha^–1, either to the surface, or incorporated by mixing with the top 0.15 m, or placed in a band at 0.15 m depth. Measurements were made of crop N uptake, residual fertilizer N and soil mineral N. The total above-ground dry matter yield of maize varied between 7.6 and 11.9 t ha^–1. The crop recovery of fertilizer N following point placement was 25% of that applied, which was higher than that from the surface application (18%) or incorporation by mixing (18%). The total grain yield of wheat varied between 4.3 and 4.7 t ha^–1. In the surface applications, the recovery of fertilizer-derived nitrogen (25%) was considerably lower than that from the mixing treatments and banded placements (33 and 36%). The fertilizer N application rate had a significant effect on grain and total dry matter yield, as well as on total N uptake and grain N contents. The main mechanism for loss of N appeared to be by ammonia volatilization, rather than leaching. High mineral N concentrations remained in the soil at harvest, following both crops, demonstrating a potential for significant reductions in N application rates without associated loss in yield.     

Diode-laser-controlled millimetre-wave propagation in a silicon waveguide

We report the dynamic behaviour of the phase shift and attenuation of millimetre waves in a silicon waveguide when it is illuminated by a diode laser. The effect of diffusion of the induced plasma on the phase shift and attenuation is also described.     

Selenium levels in cows fed pasture and concentrates or a total mixed ration and supplemented with selenized yeast to produce milk with supra-nutritional selenium concentrations

Seventy multiparous Holstein-Friesian cows were fed different amounts of pasture and concentrates, or a total mixed ration (TMR), for 42 d in mid-lactation to test the hypothesis that the concentration of Se in milk would depend on the amount of Se consumed, when the Se is primarily organic in nature, regardless of the diet of the cows. Of the 70 cows, 60 grazed irrigated perennial pasture at daily allowances of either 20 or 40 kg of dry matter (DM)/cow. These cows received 1 of 3 amounts of concentrates, either 1, 3, or 6 kg of DM/cow per day of pellets, and at each level of concentrate feeding, the pellets were formulated to provide 1 of 2 quantities of Se from Se yeast, either about 16 or 32 mg of Se/d. The other 10 cows were included in 2 additional treatments where a TMR diet was supplemented with 1 kg of DM/d of pellets formulated to include 1 of the 2 quantities of supplemental Se. Total Se intakes ranged from 14.5 to 35.9 mg/d, and of this, the Se-enriched pellets provided 93, 91, and 72% of the Se for cows allocated 20 and 40 kg of pasture DM/d or the TMR, respectively. No effects of the amount of Se consumed on any milk production variable, or on somatic cell count, body weight, and body condition score, for either the pasture-fed or TMR-fed cows were found. Milk Se concentrations responded quickly to the commencement of Se supplementation, reaching 89% of steady state levels at d 5. When milk Se concentrations were at steady state (d 12 to 40), each 1 mg of Se eaten increased the Se concentration of milk by 5.0 μg/kg (R² = 0.97), and this response did not seem to be affected by the diet of the cows or their milk production. The concentration of Se in whole blood was more variable than that in milk, and took much longer to respond to change in Se status, but it was not affected by diet at any time either. For the on-farm production of Se-enriched milk, it is important to be able to predict milk Se concentration from Se input. In our study, type of diet did not affect this relationship.     

The importance of the lumpectomy surgical margin status in long-term results of breast conservation

BACKGROUND: The impact of the surgical margin status on long-term local control rates for breast cancer in women treated with lumpectomy and radiation therapy is unclear. METHODS: The records of 289 women with 303 invasive breast cancers who were treated with lumpectomy and radiation therapy from 1972 to 1992 were reviewed. The surgical margin was classified as positive (transecting the inked margin), close (less than or equal to 2 mm from the margin), negative, or indeterminate, based on the initial biopsy findings and reexcision specimens, as appropriate. Various clinical and pathologic factors were analyzed as potential prognostic factors for local recurrence in addition to the margin status, including T classification, N classification, age, histologic features, and use of adjuvant therapy. The mean follow-up was 6.25 years. RESULTS: The actuarial probability of freedom from local recurrence for the entire group of patients at 5 and 10 years was 94% and 87%, respectively. The actuarial probability of local control at 10 years was 98% for those patients with negative surgical margins versus 82% for all others (P = 0.007). The local control rate at 10 years was 97% for patients who underwent reexcision and 84% for those who did not. Reexcision appears to convey a local control benefit for those patients with close, indeterminate, or positive initial margins, when negative final margins are attained (P = 0.0001). Final margin status was the most significant determinant of local recurrence rates in univariate analysis. By multivariate analysis, the final margin status and use of adjuvant chemotherapy were significant prognostic factors. CONCLUSIONS: The attainment of negative surgical margins, initially or at the time of reexcision, is the most significant predictor of local control after breast-conserving treatment with lumpectomy and radiation therapy.