Femtosecond X-ray protein nanocrystallography

X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (～200?nm to 2?μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.     

A conditional knockout resource for the genome-wide study of mouse gene function

Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.     

Oxysterols direct B-cell migration through EBI2

EBI2 (also called GPR183) is an orphan G-protein-coupled receptor that is highly expressed in spleen and upregulated upon Epstein-Barr-virus infection. Recent studies indicated that this receptor controls follicular B-cell migration and T-cell-dependent antibody production. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol metabolism. The biological effects of oxysterols have largely been credited to the activation of nuclear hormone receptors. Here we isolate oxysterols from porcine spleen extracts and show that they are endogenous ligands for EBI2. The most potent ligand and activator is 7α,25-dihydroxycholesterol (OHC), with a dissociation constant of 450?pM for EBI2. In vitro, 7α,25-OHC stimulated the migration of EBI2-expressing mouse B and T cells with half-maximum effective concentration values around 500 pM, but had no effect on EBI2-deficient cells. In vivo, EBI2-deficient B cells or normal B cells desensitized by 7α,25-OHC pre-treatment showed reduced homing to follicular areas of the spleen. Blocking the synthesis of 7α,25-OHC in vivo with clotrimazole, a CYP7B1 inhibitor, reduced the content of 7α,25-OHC in the mouse spleen and promoted the migration of adoptively transferred pre-activated B cells to the T/B boundary (the boundary between the T-zone and B-zone in the spleen follicle), mimicking the phenotype of pre-activated B cells from EBI2-deficient mice. Our results show an unexpected causal link between EBI2, an orphan G-protein-coupled receptor controlling B-cell migration, and the known immunological effects of certain oxysterols, thus uncovering a previously unknown role for this class of molecules.     

From Bureaucracy to Learning Organization: Critical Minimum Specification Design as Space for Sensemaking

What does it take of a municipal organization to be a significant participant in the dynamic, learning oriented interaction within a triple helix constellation of partners aimed at regional development? The short answer is that it needs to develop the receptivity (Morgan, 1996) and the flexibility to act in novel ways, which are the traits of a learning organization. However, how these abilities are enhanced in organizations that are basically organized as bureaucracies aimed at maintaining a stable and standardized function of its operations, is an under-researched phenomenon within the triple helix discussions. In this paper, I investigate this question through an analysis of a change and development process within a municipal organization that aimed to enhance the capacity of the organization to interact receptively and dynamically with their environment, by changing their organizational structure from a bureaucratic to a post-bureaucratic type. The main finding is that rather than the new formal structure as such; it is the process of sensemaking following the introduction of this new formal structure that creates the capacity for learning and development. However, this outcome is dependent on the sensemaking process being turned over from a defensive mode into a learning oriented mode. The paper explores how this turn may be facilitated.     

Low retinal noise in animals with low body temperature allows high visual sensitivity

The weakest pulse of light a human can detect sends about 100 photons through the pupil and produces 10-20 rhodopsin isomerizations in a small retinal area. It has been postulated that we cannot see single photons because of a retinal noise arising from randomly occurring thermal isomerizations. Direct recordings have since demonstrated the existence of electrical 'dark' rod events indistinguishable from photoisomerization signals. Their mean rate of occurrence is roughly consistent with the 'dark light' in psychophysical threshold experiments, and their thermal parameters justify an identification with thermal isomerizations. In the retina of amphibians, a small proportion of sensitive ganglion cells have a performance-limiting noise that is low enough to be well accounted for by these events. Here we study the performance of dark-adapted toads and frogs and show that the performance limit of visually guided behaviour is also set by thermal isomerizations. As visual sensitivity limited by thermal events should rise when the temperature falls, poikilothermous vertebrates living at low temperatures should then reach light sensitivities unattainable by mammals and birds with optical factors equal. Comparison of different species at different temperatures shows a correlation between absolute threshold intensities and estimated thermal isomerization rates in the retina.     

A Strategic Study of Establishing the Industrial Needs in Pattern Design and Technology in Mainland China

Pattern design and, technology play a very important role in the garment industry. In order to improve the level of pattern making and design of the garment industry, a survey was conducted to investigate the industrial needs in pattern design and technology in mainland China. The data were collected from the employers and employees from the garment industry and students in the major of fashion and clothing studies. It indicated that there was a gap between the employer and employee, especially the requirements of the industrial needs and the course contents covered by the tertiary schools. The employers expected to recruit more experienced pattern designers, at the same time, they were not reluctant to hire fresh graduates and spent more resources on the training of employees. The students knew little about their employment situation of the garment industry, spent too little time on the course study and learned too little practical skills in pattern design. They could not make use of the knowledge which prevented them from being employed by the garment industry. Efforts should be taken by both the tertiary schools and the garment industry. The students should be aspirated towards the profession of pattern cutters and the syllabuses of pattern making should be more practical and industrial orientated. The solution might benefit the garment industry a lot in a long run.     

A comparative evaluation of objective and subjective weather forecasts in the united states

This paper is concerned primarily with the evaluation and comparison of objective and subjective weather forecasts. Operational forecasts of three weather elements are considered: (1) probability forecasts of precipitation occurrence, (2) categorical (i.e. non-probabilistic) forecasts of maximum and minimum temperatures and (3) categorical forecasts of cloud amount. The objective forecasts are prepared by numerical-statistical procedures, whereas the subjective forecasts are based on the judgements of individual forecasters. In formulating the latter, the forecasters consult information from a variety of sources, including the objective forecasts themselves. The precipitation probability forecasts are found to be both reliable and skilful, and evaluation of the temperature/cloud amount forecasts reveals that they are quite accurate/skilful. Comparison of the objective and subjective forecasts of precipitation occurrence indicates that the latter are generally more skilful than the former for shorter lead times (e.g. 12–24 hours), whereas the two types of forecasts are of approximately equal skill for longer lead times (e.g. 36–48 hours). Similar results are obtained for the maximum and minimum temperature forecasts. Objective cloud amount forecasts are more skilful than subjective cloud amount forecasts for all lead times. Examination of trends in performance over the last decade reveals that both types of forecasts for all three elements increased in skill (or accuracy) over the period, with improvements in objective forecasts equalling or exceeding improvements in subjective forecasts. The role and impact of the objective forecasts in the subjective weather forecasting process are discussed in some detail. The need to conduct controlled experiments and other studies of this process, with particular reference to the assimilation of information from different sources, is emphasized. Important characteristics of the forecasting system in meteorology are identified, and they are used to describe similarities and differences between weather forecasting and forecasting in other fields. Acquisition of some of these characteristics may be beneficial to other forecasting systems.     

Quality/value relationships for imperfect weather forecasts in a prototype multistage decision-making model

Some theoretical results concerning the nature of the relationship between the scientific quality and economic value of imperfect weather forecasts are obtained. A prototype multistage decision-making model is considered, involving only two possible actions and two possible states of weather. This particular form of model is motivated by a real-world application known as the fruit-frost problem. For an infinite-horizon, discounted version of this model it is shown that economic value remains zero below a forecast quality threshold and then rises monotonically but nonlinearly above this threshold. In particular, the relative sensitivity of economic value to changes in the quality of forecasts increases as perfect information is approached. This curve is compared with quality/value relationships that have been obtained for other versions of the model; namely, a single-stage model and a multistage, finite-horizon model.