Computationally-guided thermostabilization of the primary endoglucanase from Hypocrea jecorina for cellulosic biofuel production

The creation of thermostable enzymes has wide-ranging applications in industrial, scientific, and pharmaceutical settings. As various stabilization techniques exist, it is often unclear how to best proceed. To this end, we have redesigned Cel5A (HjCel5A) from Hypocrea jecorina (anamorph Trichoderma reesei) to comparatively evaluate several significantly divergent stabilization methods: 1) consensus design, 2) core repacking, 3) helix dipole stabilization, 4) FoldX ΔΔG approximations, 5) Triad ΔΔG approximations, and 6) entropy reduction through backbone stabilization. As several of these techniques require structural data, we initially solved the first crystal structure of HjCel5A to 2.05 Å. Results from the stabilization experiments demonstrate that consensus design works best at accurately predicting highly stabilizing and active mutations. FoldX and helix dipole stabilization, however, also performed well. Both methods rely on structural data and can reveal non-conserved, structure-dependent mutations with high fidelity. HjCel5A is a prime target for stabilization. Capable of cleaving cellulose strands from agricultural waste into fermentable sugars, this protein functions as the primary endoglucanase in an organism commonly used in the sustainable biofuels industry. Creating a long-lived, highly active thermostable HjCel5A would allow cellulose hydrolysis to proceed more efficiently, lowering production expenses. We employed information gleaned during the survey of stabilization techniques to generate HjCel5A variants demonstrating a 12-15 °C increase in the temperature at which 50% of the total activity persists, an 11-14 °C increase in optimal operating temperature, and a 60% increase over the maximal amount of hydrolysis achievable using the wild type enzyme. We anticipate that our comparative analysis of stabilization methods will prove useful in future thermostabilization experiments.

Ecosystem structure in disturbed and restored subtropical seagrass meadows

Shallow seagrass ecosystems frequently experience physical disturbance from vessel groundings. Specific restoration methods that modify physical, chemical, and biological aspects of disturbances are used to accelerate recovery. This study evaluated loss and recovery of ecosystem structure in disturbed seagrass meadows through plant and soil properties used as proxies for primary and secondary production, habitat quality, benthic metabolism, remineralization, and nutrient storage and exchange. The efficacy of common seagrass restoration techniques in accelerating recovery was also assessed. Beyond removal of macrophyte biomass, disturbance to seagrass sediments resulted in loss of organic matter and stored nutrients, and altered microbial and infaunal communities. Evidence of the effectiveness of restoration actions was variable. Fill placement prevented additional erosion, but the resulting sediment matrix had different physical properties, low organic matter content and nutrient pools, reduced benthic metabolism, and less primary and secondary production relative to the undisturbed ecosystem. Fertilization was effective in increasing nitrogen and phosphorus availability in the sediments, but concurrent enhancement of seagrass production was not detected. Seagrass herbivores removed substantial seagrass biomass via direct grazing, suggesting that leaf loss to seagrass herbivores is a spatially variable but critically important determinant of seagrass transplanting success. Convergence of plant and sediment response variables with levels in undisturbed seagrass meadows was not detected via natural recovery of disturbed sites, or through filling and fertilizing restoration sites. However, several indicators of ecosystem development related to primary production and nutrient accumulation suggest that early stages of ecosystem development have begun at these sites. This research suggests that vessel grounding disturbances in seagrass ecosystems create more complex and persistent resource losses than previously understood by resource managers. While the mechanics of implementing common seagrass restoration actions have been successfully developed by the restoration community, expectations of consistent or rapid recovery trajectories following restoration remain elusive.

A structure design method for compliant parallel manipulators with actuation isolation

Since precise linear actuators of a compliant parallel manipulator suffer from their inability to tolerate the transverse motion/load in the multi-axis motion, actuation isolation should be considered in the compliant manipulator design to eliminate the transverse motion at the point of actuation. This paper presents an effective design method for constructing compliant parallel manipulators with actuation isolation, by adding the same number of actuation legs as the number of the DOF (degree of freedom) of the original mechanism. The method is demonstrated by two design case studies, one of which is quantitatively studied by analytical modelling. The modelling results confirm possible inherent issues of the proposed structure design method such as increased primary stiffness, introduced extra parasitic motions and cross-axis coupling motions.

The impact of personal background and school contextual factors on academic competence and mental health functioning across the primary-secondary school transition

Students negotiate the transition to secondary school in different ways. While some thrive on the opportunity, others are challenged. A prospective longitudinal design was used to determine the contribution of personal background and school contextual factors on academic competence (AC) and mental health functioning (MHF) of 266 students, 6-months before and after the transition to secondary school. Data from 197 typically developing students and 69 students with a disability were analysed using hierarchical linear regression modelling. Both in primary and secondary school, students with a disability and from socially disadvantaged backgrounds gained poorer scores for AC and MHF than their typically developing and more affluent counterparts. Students who attended independent and mid-range sized primary schools had the highest concurrent AC. Those from independent primary schools had the lowest MHF. The primary school organisational model significantly influenced post-transition AC scores; with students from Kindergarten--Year 7 schools reporting the lowest scores, while those from the Kindergarten--Year 12 structure without middle school having the highest scores. Attending a school which used the Kindergarten--Year 12 with middle school structure was associated with a reduction in AC scores across the transition. Personal background factors accounted for the majority of the variability in post-transition AC and MHF. The contribution of school contextual factors was relatively minor. There is a potential opportunity for schools to provide support to disadvantaged students before the transition to secondary school, as they continue to be at a disadvantage after the transition.

Sensible use of primary energy in organic greenhouse production.

This document addresses the direct and indirect use of energy in European organic greenhouse horticulture (OGH) with the aim of reviewing available means for making it more environmental friendly and identifying knowledge gaps that should be addressed to attain this aim. The first observation is that there is no common regulation for energy use in OGH, which is not unexpected, since the need for climatisation is not uniformly distributed in the EU (and outside). Accordingly, the EU directive on organic agriculture does not set limitations on the use of energy, but rather promotes the responsible use of energy and of natural resources. The restrictions and rules of most private standards are slightly more stringent. Some standards have specific restrictions on the amount and sources of energy and/or on the seasonal use of energy for heating. Some standards also address processes that may affect (in)direct energy use, such as cultivation methods, mulching, lighting and growing media or substrates. However, most private standards have no or little restrictions or regulations on energy use. Accordingly, it should not surprise that very little quantitative information is available about energy use in OGH. In the present document we have filled the gaps with data with estimates drawn on energy use in conventional greenhouses. With respect to ongoing research, whereas many of the present research results about energy use and saving in conventional greenhouses are relevant (and also applied) in OGH, little research is devoted to address the energy use that is peculiar to OGH, particularly energy use for humidity control. In short, there are still a lot of knowledge gaps to improve quality and to lower energy use in organic greenhouses. The purpose of this document is a summary of present relevant knowledge about energy use and energy saving and of the perspective for improvement. In particular, the goal is to make an overview on the methods and technologies which can be used to reduce the energy use in OGH. We start from the assumption that methods and technologies that are used for reducing direct and indirect energy in conventional greenhouses can also be applied in organic greenhouses. Research on reducing energy use in conventional greenhouses is also more widely available because the area of conventional greenhouse horticulture is much larger than the area of OGH. When implementing these methods and techniques we should take into account the specific characteristics of organic agriculture like soil-based cultivation, use of organic fertilizers and the limited use of crop protection products. This document is organised as follows: first we report the results of a survey about energy use and relevant standards in the countries participating to the COST action (chapter 1); then we review the energy use for climatisation: heating (chapter 2) and humidity (chapter 3). In chapter 4 we review the available design and management means that would either reduce energy use and/or increase energy use efficiency by increasing productivity of OGH. In chapter 5 we present a short summary of existing information on indirect energy use, that is the energy required to manufacture production means (greenhouse structure and cover, fertilisers, equipment etc.) and for crop protection, particularly steaming, and briefly discuss possible savings. Finally (chapter 6) we review briefly the potential for application of renewable energy sources in OGH.

New dimeric supramolecular structure of mixed (phthalocyaninato)(porphyrinato)-europium(III) sandwiches : preparation and spectroscopic characteristics

The mixed double-decker Eu\[Pc(15C5)4](TPP) (1) was obtained by base-catalysed tetramerisation of 4,5-dicyanobenzo-15-crown-5 using the half-sandwich complex Eu(TPP)(acac) (acac = acetylacetonate), generated in situ, as the template. For comparative studies, the mixed triple-decker complexes Eu2\[Pc(15C5)4](TPP)2 (2) and Eu2\[Pc(15C5)4]2(TPP) (3) were also synthesised by the raise-by-one-story method. These mixed ring sandwich complexes were characterised by various spectroscopic methods. Up to four one-electron oxidations and two one-electron reductions were revealed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). As shown by electronic absorption and infrared spectroscopy, supramolecular dimers (SM1 and SM3) were formed from the corresponding double-decker 1 and triple-decker 3 in the presence of potassium ions in MeOH/CHCl3.

Influence of host plant structure and microclimate on the abundance and behaviour of Tephritid fly

Microclimate and host plant architecture significantly influence the abundance and behavior of insects. However, most research in this field has focused at the invertebrate assemblage level, with few studies at the single-species level. Using wild Solanum mauritianum plants, we evaluated the influence of plant structure (number of leaves and branches and height of plant) and microclimate (temperature, relative humidity, and light intensity) on the abundance and behavior of a single insect species, the monophagous tephritid fly Bactrocera cacuminata (Hering). Abundance and oviposition behavior were signficantly influenced by the host structure (density of foliage) and associated microclimate. Resting behavior of both sexes was influenced positively by foliage density, while temperature positively influenced the numbers of resting females. The number of ovipositing females was positively influenced by temperature and negatively by relative humidity. Feeding behavior was rare on the host plant, as was mating. The relatively low explanatory power of the measured variables suggests that, in addition to host plant architecture and associated microclimate, other cues (e.g., olfactory or visual) could affect visitation and use of the larval host plant by adult fruit flies. For 12 plants observed at dusk (the time of fly mating), mating pairs were observed on only one tree. Principal component analyses of the plant and microclimate factors associated with these plants revealed that the plant on which mating was observed had specific characteristics (intermediate light intensity, greater height, and greater quantity of fruit) that may have influenced its selection as a mating site.