New developments in X-ray optics for macromolecular crystallography using laboratory X-ray sources.

A variety of different devices has been described recently for conditioning the X-ray beam incident on the sample for structural studies on proteins and other macromolecular crystals.     

Population genetic structure of Plasmopara viticola after 125 years of colonization in European vineyards

To examine the within- and among-population genetic structure of Plamopara viticola oosporic populations in Europe, 8991 lesions from 32 vineyard plots were collected and analysed. Four multi-allelic microsatellite markers were used to genotype the pathogen. All populations had high levels of gene and genotypic diversity. Most populations were in Hardy–Weinberg equilibrium and thus randomly mating. Among P. viticola populations, significant low to moderate genetic differentiation was observed, even between geographically close populations. This genetic differentiation was also evident in the neighbour-joining phylogenetic genetic distance tree, showing clear substructure and distinguishing mainly five clusters based on geographical origin. Significant isolation by distance was found in central European P. viticola populations, suggesting a step-wise migration model. No significant isolation by distance was found within Greek populations, most probably owing to natural geographical barriers such as the sea and mountains, as well as the frequent population bottlenecks occurring in these populations, preventing natural migration among populations. The high variability of P. viticola provides explanation for its successful infestation of the heterogeneous European vineyards in the last 125 years after its introduction.     

Nutrient transport systems in brain: 40 years of progress

In the last 40 years, especially with the application of new neurochemical and molecular biologic techniques, there has been an explosive progress in understanding how nutrients are transported across the blood–brain barrier and choroid plexus into brain and CSF, and how nutrient homeostasis in brain is achieved. In most cases, there are separate transporters, or in a few cases, systems that transport related substances (e.g., biotin, lipoic, and pantothenic acids). This review focuses on three crucial nutrients (glucose, ascorbic acid, and folates) for which there is substantial new information including ‘knock down’ and ‘knockout’ models in mice and/or humans. The overall objective is to show that this new knowledge leads not just to a more thorough understanding (e.g., of ‘why’ questions like: why do neurons require up to 10 mM ascorbic acid intracellulary?); but in some cases leads to clinically important predictions that allow treatment of heretofore devastating neurologic disorders.     

Learning-by-doing: experience from 20 years of teaching LCA to future engineers

Purpose

In support of the sustainable development of our societies, future engineers should have elementary knowledge in sustainability assessment and use of life cycle assessment. Publications on pedagogical experience with teaching life cycle assessment (LCA) in high-level education are however scarce. Here, we describe and discuss 20 years of experience in teaching LCA at MSc level in an engineering university with the ambition to share our insights and inspire teaching of LCA as part of a university curriculum.

Methods

We detail the design of an LCA course taught at the Technical University of Denmark since 1997. The course structure relies on (i) a structured combination of theoretical teaching, practical assignments and hands-on practice on LCA case studies, and (ii) the conduct of real-life LCA case studies in collaboration with companies or other organisations. Through the semester-long duration of the course, students from different engineering backgrounds perform full-fledged LCA studies in groups, passing through two iterations—a screening LCA supporting a more targeted LCA.

Results and discussion

The course design, which relies on a learning-by-doing principle, is transparently described to inspire LCA teachers among the readers. Historical evolution and statistics about the course, including its 192 case studies run in collaboration with 105 companies and institutions, are analysed and serve as basis to discuss the benefits and challenges of its different components, such as the theory acquisition, the assignment work, the LCA software learning, the conduct of case studies, the merits of industrial collaborations and grading approaches.

Conclusions

We demonstrate the win-win situation created by the setting of the course, in which the students are actively engaged and learn efficiently how to perform an LCA while the collaborating companies often get useful insights into their analysed case studies. The course can also be an eye opener for companies unfamiliar with LCA, who get introduced to life cycle thinking and the potential benefits of LCA. We have no hesitation in recommending industries and LCA teachers to engage into such collaborations even in the fundamental teaching of LCA techniques.

     

20 years of human mtDNA pathologic point mutations: Carefully reading the pathogenicity criteria

Despite the strong purifying selection that occurs during embryonic development, the particular location and features of mitochondrial DNA make it especially susceptible to accumulating point mutations, giving rise to a large number of mitochondrial DNA variants. Many of these will have moderate or no phenotypic effects but others will be the cause of very dramatic diseases, usually known as mitochondriopathies. Because of the abundance of different mitochondrial DNA variants, it is not easy to determine whether a new mutation is pathogenic. To facilitate this task, different criteria have been proposed, but they are often either too severely or too loosely applied. Citing examples from the literature, in this paper we discuss some critical aspects of these criteria.     

X-ray Crystal Structure of the Rotavirus Inner Capsid Particle at 3.8 Å Resolution

The rotavirus inner capsid particle, known as the “double-layered particle” (DLP), is the “payload” delivered into a cell in the process of viral infection. Its inner and outer protein layers, composed of viral protein (VP) 2 and VP6, respectively, package the 11 segments of the double-stranded RNA (dsRNA) of the viral genome, as well as about the same number of polymerase molecules (VP1) and capping-enzyme molecules (VP3). We have determined the crystal structure of the bovine rotavirus DLP. There is one full particle (outer diameter ∼ 700 Å) in the asymmetric unit of the P2₁2₁2₁ unit cell of dimensions a = 740 Å, b = 1198 Å, and c = 1345 Å. A three-dimensional reconstruction from electron cryomicroscopy was used as a molecular replacement model for initial phase determination to about 18.5 Å resolution, and the 60-fold redundancy of icosahedral particle symmetry allowed phases to be extended stepwise to the limiting resolution of the data (3.8 Å). The structure of a VP6 trimer (determined previously by others) fits the outer layer density with very little adjustment. The T = 13 triangulation number of that layer implies that there are four and one-third VP6 trimers per icosahedral asymmetric unit. The inner layer has 120 copies of VP2 and thus 2 copies per icosahedral asymmetric unit, designated VP2A and VP2B. Residues 101-880 fold into a relatively thin principal domain, comma-like in outline, shaped such that only rather modest distortions (concentrated at two “subdomain” boundaries) allow VP2A and VP2B to form a uniform layer with essentially no gaps at the subunit boundaries, except for a modest pore along the 5-fold axis. The VP2 principal domain resembles those of the corresponding shells and homologous proteins in other dsRNA viruses: λ1 in orthoreoviruses and VP3 in orbiviruses. Residues 1-80 of VP2A and VP2B fold together with four other such pairs into a “5-fold hub” that projects into the DLP interior along the 5-fold axis; residues 81-100 link the 10 polypeptide chains emerging from a 5-fold hub to the N-termini of their corresponding principal domains, clustered into a decameric assembly unit. The 5-fold hub appears to have several distinct functions. One function is to recruit a copy of VP1 (or of a VP1-VP3 complex), potentially along with a segment of plus-strand RNA, as a decamer of VP2 assembles. The second function is to serve as a shaft around which can coil a segment of dsRNA. The third function is to guide nascent mRNA, synthesized in the DLP interior by VP1 and 5′-capped by the action of VP3, out through a 5-fold exit channel. We propose a model for rotavirus particle assembly, based on known requirements for virion formation, together with the structure of the DLP and that of VP1, determined earlier.     

The C : N : P stoichiometry of autotrophs – theory and observations

Evolution has set biochemical constraints on the chemical composition of living organisms. These constraints seem to lead to increases in N : C and P : C ratios with increasing relative growth rate for all types of organisms. The N : P ratio also seems to decrease with relative growth rate for heterotrophs whereas autotrophs may show a more complex behaviour. Here I will show that, from biochemical considerations, N : C should increase linearly and P : C quadratically with relative growth rate in autotrophs with the consequence that N : P increases at low relative growth rates, passes a maximum and then decreases at high relative growth rates. These predictions are verified against observations for a freshwater alga (Selenastrum minutum) and a tree seedling (Betula pendula). Changes in temperature, light or other factors that affect the growth rate of autotrophs interact with nutrient supply in such a way that there are no simple rules for as to how N : P will change.     

For a few years more: reductions in plant diversity 70 years after the last fire in Mediterranean forests

Plant Ecology - Changes in community diversity and dynamics after fires in Mediterranean ecosystems are rarely investigated more than a few years after the fire even though pronounced changes can...     

Gene synthesis technology: recent developments and future prospects

Gene synthesis is a potentially powerful tool in molecular biology that has not yet reached widespread use because of the relatively high cost and labor-intensive nature of the process. This paper reviews some recent technological developments and current research activities of this laboratory which promise to greatly reduce the cost of gene synthesis and to increase the speed and efficiency of the process. We recently developed an improved device for "segmented" synthesis of oligonucleotides, which utilizes porous Teflon wafers containing derivatized controlled pore glass supports to simultaneously synthesize up to 100 different DNA sequences. The stepwise coupling efficiency with the "wafer synthesis device" is as high as that attained with current automated "gene machines" producing 1-4 oligonucleotides at a time, whereas the reagent usage is only 20-50% that of the current DNA synthesizers. At present, we are optimizing the conditions for rapid, efficient assembly of genes on a solid-phase support, wherein ordered, stepwise annealing/washing is performed to segmentally elongate a "starting" oligonucleotide attached to a solid-phase support. We expect that the wafer synthesis device (operated at reduced scale of synthesis), together with solid-phase gene assembly, will permit the synthesis and assembly of an average size gene (1 kb) in one week at a cost of less than $1000. These developments should make gene synthesis a routine and powerful tool in molecular biology.     

A phylogenetic perspective on 160 years of troubled taxonomy of Niphargus (Crustacea: Amphipoda)

Niphargus is the largest genus of freshwater amphipods. Its systematics from the species to the family level has always been problematic. This study is the first comprehensive phylogenetic treatment of the chiefly subterranean group in 160 years of its taxonomic history. It includes 103 niphargid species plus outgroups, representing about one‐third of all nominal species. The samples originated mainly from type localities or adjacent sites and covered most of the morphological variability of the genus. Character sampling included nuclear 28S and mitochondrial 12S rDNA sequences, and 122 morphological characters. Quantitative morphological traits were coded using two alternative methods. The first one searches for gaps in the variability range of each character, while the second one uses absolute differences between the standardized raw data as weights. Different data sets yielded alternative topologies. All data support the monophyly of Niphargidae, while Niphargopsis— another niphargid genus — was consistently nested within Niphargus, loosing justification for its separate status. We predict a similar fate for all or most of the remaining six small niphargid genera, which were not yet scrutinized phylogenetically. Different topologies agreed in species composition of five large, well‐supported clades, although the hierarchic relationships between them remain unresolved. These clades reject all previously proposed taxonomic subdivisions of Niphargus, implying a high degree of morphological homoplasy that renders any morphology‐based groups questionable. The clade members are distributed within well‐established zoogeographical regions that do not exceed 1300 km across the longest diagonal. These results provide a framework for future studies on niphargid systematics, the evolution of endemism and cryptic diversity in subterranean environments, the mechanisms leading to exceptional morphological heterogeneity, historical biogeography, and applied ecological issues.     

Managed realignment in the UK – the first 5 years of colonization by birds

With current losses of saltmarsh running at > 100 ha per year in the UK, creation of new intertidal habitats through managed realignment is likely to be increasingly used. Potentially, this has biodiversity as well as engineering benefits. However, assessing the conservation value of many of the current UK schemes is difficult as the biological monitoring has been generally poor, with a few notable exceptions. At the Tollesbury and Orplands realignment sites, Essex, bird communities were dominated by terrestrial species during the first year of inundation and waterbird communities rapidly developed during the second and third years. Five years after the initial breach in the sea wall, communities were similar to surrounding mudflats but with some notable exceptions. Dunlin Calidris alpina and Common Redshank Tringa totanus that prey on the early colonizing Nereis and Hydrobia used the sites in the first 2 years. Eurasian Oystercatcher Haematopus ostralegus did not occur on the realignment site as there were no large bivalves, whereas Red Knot Calidris canutus used the site after 4–5 years coincidentally with the appearance of Macoma balthica . The differences in the bird communities occurred because UK sites are often small, enclosed and poorly drained. If at a suitable height in the tidal frame, UK managed realignment sites are successful in that they have developed saltmarsh and biologically active mudflats but they may lack the full range of biodiversity found in surrounding natural intertidal habitats, even decades after inundation.     

20,000 years of interactions between climate,vegetation and land use in Northern Greece

Vegetation History and Archaeobotany - Detailed knowledge about the history of vegetation, fire and land use is scarce in Northern Greece. We analysed lake sediments from Limni Zazari (Northern...     

Present and future developments of stereotactic technology

Incorporation of a surgical computer system into stereotactic methodology provides the facility for efficient utilization of the multiple data bases at the disposal of the modern stereotactician. Computed tomography, magnetic resonance imaging, and digital fluoroscopy data gathered in stereotactic conditions are digitized into a stereotactic surgical matrix for surgical planning and interactive surgical procedures. The advantages of this system are illustrated in stereotactic biopsy, interstitial irradiation, and laser resections of intracranial tumors.