Inter-Bragg crystallographic phase retrieval from shape transforms, stacking faults and substitutional disorder.

One of the brilliant ideas of John Spence when he saw the first diffraction patterns from the Linac Coherent Light Source was that one could solve the crystallographic phase problem by utilising the intensities between Bragg peaks. Because these intensities are due to the Fourier transform of the shape of the crystal, the approach came to be known as "shape-transform phasing." Shape-transform phasing was developed over the next ten years and formed the basis for many other interesting ideas and pursuits. Here we describe the current best implementation of the original idea using a lattice occupancy formalism and show that certain types of crystal defects can also be modelled via this approach, allowing the molecular structure to be recovered from the additional information offered by the inter-Bragg intensities from these crystal defects.

Expected resolution limits of x-ray free-electron laser single-particle imaging for realistic source and detector properties.

The unprecedented intensity of x-ray free-electron laser sources has enabled single-particle x-ray diffraction imaging (SPI) of various biological specimens in both two-dimensional projection and three dimensions (3D). The potential of studying protein dynamics in their native conditions, without crystallization or chemical staining, has encouraged researchers to aim for increasingly higher resolutions with this technique. The currently achievable resolution of SPI is limited to the sub-10 nanometer range, mainly due to background effects, such as instrumental noise and parasitic scattering from the carrier gas used for sample delivery. Recent theoretical studies have quantified the effects of x-ray pulse parameters, as well as the required number of diffraction patterns to achieve a certain resolution, in a 3D reconstruction, although the effects of detector noise and the random particle orientation in each diffraction snapshot were not taken into account. In this work, we show these shortcomings and address limitations on achievable image resolution imposed by the adaptive gain integrating pixel detector noise.

On the characterization of a 1 m long, ultra-precise KB-focusing mirror pair for European XFEL by means of slope measuring deflectometry.

Recently, the European X-Ray Free Electron Laser (XFEL) has successfully produced its first X-ray photon pulse trains. This unique photon source will provide up to 27 000 photon pulses per second for experiments in different fields of science. In order to accomplish this, ultra-precise mirrors of dedicated shape are used to guide and focus these photons along beamlines of up to 930 m in length from the source in the undulator section to the desired focal point at an experimental station. We will report on a Kirkpatrick-Baez-mirror pair designed to focus hard-X-rays in the energy range from 3 to 16 keV to a 100 nm scale at the SPB/SFX instrument of the European XFEL. Both mirrors are elliptical cylinder-like shaped. The figure error of these 1 m long mirrors was specified to be better than 2 nm pv in terms of the height domain; this corresponds to a slope error of about 50 nrad rms (at least a best effort finishing is requested). This is essential to provide optimal experimental conditions including preservation of brilliance and wavefront. Such large and precise optics represents a challenge for the required deterministic surface polishing technology, elastic emission machining in this case, as well as for the metrology mandatory to enable a precise characterization of the topography on the mirror aperture. Besides the slope errors, the ellipse parameters are also of particular interest. The mirrors were under inspection by means of slope measuring deflectometry at the BESSY-NOM slope measuring profiler at the Helmholtz Zentrum Berlin. The NOM measurements have shown a slope error of 100 nrad rms on a aperture length of 950 mm corresponding to a residual figure deviation ≤20 nm pv for both mirrors. Additionally we found a strong impact of the mirror support conditions on the mirror shape finally measured. We will report on the measurement concept to characterize such mirrors as well as to discuss the achieved results.

Clustering.

Clustering techniques are used to arrange genes in some natural way, that is, to organize genes into groups or clusters with similar behavior across relevant tissue samples (or cell lines). These techniques can also be applied to tissues rather than genes. Methods such as hierarchical agglomerative clustering, k-means clustering, the self-organizing map, and model-based methods have been used. Here we focus on mixtures of normals to provide a model-based clustering of tissue samples (gene signatures) and of gene profiles, including time-course gene expression data.

Electronic damage in S atoms in a native protein crystal induced by an intense X-ray free-electron laser pulse.

Current hard X-ray free-electron laser (XFEL) sources can deliver doses to biological macromolecules well exceeding 1 GGy, in timescales of a few tens of femtoseconds. During the pulse, photoionization can reach the point of saturation in which certain atomic species in the sample lose most of their electrons. This electronic radiation damage causes the atomic scattering factors to change, affecting, in particular, the heavy atoms, due to their higher photoabsorption cross sections. Here, it is shown that experimental serial femtosecond crystallography data collected with an extremely bright XFEL source exhibit a reduction of the effective scattering power of the sulfur atoms in a native protein. Quantitative methods are developed to retrieve information on the effective ionization of the damaged atomic species from experimental data, and the implications of utilizing new phasing methods which can take advantage of this localized radiation damage are discussed.

Simple convergent-nozzle aerosol injector for single-particle diffractive imaging with X-ray free-electron lasers.

A major challenge in high-resolution x-ray free-electron laser-based coherent diffractive imaging is the development of aerosol injectors that can efficiently deliver particles to the peak intensity of the focused X-ray beam. Here, we consider the use of a simple convergent-orifice nozzle for producing tightly focused beams of particles. Through optical imaging we show that 0.5 µm particles can be focused to a full-width at half maximum diameter of 4.2 µm, and we demonstrate the use of such a nozzle for injecting viruses into a micro-focused soft-X-ray FEL beam.

Profile of suicidal behaviour in Guyana. A retrospective study from 2010 -2012

OBJECTIVE: To assess cases of suicidal behaviour in Guyana to characterize the suicidal profile of Guyanese at present. DESIGN AND METHODS: A nationally represented sample of 899 cases involved in suicidal behaviour, 555 who committed suicide and another 344 attempted suicides from 2010-2012, were surveyed using an epidemiologic study. Risk factors and other associations were identified by professionals through the use of a statistical review and the administration of questionnaires; the information was processed. RESULTS: Suicide was one of the biggest problems in Guyana as evidenced by the country being listed as having the highest estimated rate of suicide worldwide (44.3/100,000) with an mean of 200 lives being lost each year due to suicide. Data on respondents (patient or parents) were used for analysis. The most affected age group is 20 to 49 years (50%) Males committed suicide more frequently, with a ratio of almost 4:1 and the most commonly used methods in suicide cases were poisoning (pesticide / herbicide) with more than 65 % of cases, followed by hanging (>20%). East Indians accounted for >80 %; most of the cases were geographically concentrated in Regions 6,5,4,3 and 2 similar to the estimated cases of suicidal attempts. CONCLUSIONS: The profile of suicide had been well identified in Guyana, and it permits the development of a comprehensive multisectoral suicide prevention strategy to reduce the magnitude of suicidal behavior in Guyana.

X-ray imaging and multiferroic coupling of cycloidal magnetic domains in ferroelectric monodomain BiFeO3.

Magnetic domains at the surface of a ferroelectric monodomain BiFeO(3) single crystal have been imaged by hard x-ray magnetic scattering. Magnetic domains up to several hundred microns in size have been observed, corresponding to cycloidal modulations of the magnetization along the wave vector k=(δ,δ,0) and symmetry equivalent directions. The rotation direction of the magnetization in all magnetic domains, determined by diffraction of circularly polarized light, was found to be unique and in agreement with predictions of a combined approach based on a spin-model complemented by relativistic density-functional simulations. Imaging of the surface shows that the largest adjacent domains display a 120° vortex structure.

Genetic relatedness of Streptococcus pneumoniae isolates from paired blood and respiratory specimens.

To assess the relatedness of Streptococcus pneumoniae isolates recovered concurrently from blood and respiratory tract specimens from patients with pneumonia, we analyzed 24 paired isolates by pulsed-field gel electrophoresis (PFGE), serotyping, and antimicrobial susceptibility testing. PFGE, serotype, and/or susceptibility patterns were identical for 22 of 24 pairs. Susceptibility results for blood isolates should guide therapy.

Application of gene shaving and mixture models to cluster microarray gene expression data.

Researchers are frequently faced with the analysis of microarray data of a relatively large number of genes using a small number of tissue samples. We examine the application of two statistical methods for clustering such microarray expression data: EMMIX-GENE and GeneClust. EMMIX-GENE is a mixture-model based clustering approach, designed primarily to cluster tissue samples on the basis of the genes. GeneClust is an implementation of the gene shaving methodology, motivated by research to identify distinct sets of genes for which variation in expression could be related to a biological property of the tissue samples. We illustrate the use of these two methods in the analysis of Affymetrix oligonucleotide arrays of well-known data sets from colon tissue samples with and without tumors, and of tumor tissue samples from patients with leukemia. Although the two approaches have been developed from different perspectives, the results demonstrate a clear correspondence between gene clusters produced by GeneClust and EMMIX-GENE for the colon tissue data. It is demonstrated, for the case of ribosomal proteins and smooth muscle genes in the colon data set, that both methods can classify genes into co-regulated families. It is further demonstrated that tissue types (tumor and normal) can be separated on the basis of subtle distributed patterns of genes. Application to the leukemia tissue data produces a division of tissues corresponding closely to the external classification, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), for both methods. In addition, we also identify genes specific for the subgroup of ALL-Tcell samples. Overall, we find that the gene shaving method produces gene clusters at great speed; allows variable cluster sizes and can incorporate partial or full supervision; and finds clusters of genes in which the gene expression varies greatly over the tissue samples while maintaining a high level of coherence between the gene expression profiles. The intent of the EMMIX-GENE method is to cluster the tissue samples. It performs a filtering step that results in a subset of relevant genes, followed by gene clustering, and then tissue clustering, and is favorable in its accuracy of ranking the clusters produced.

A simple implementation of a normal mixture approach to differential gene expression in multiclass microarrays.

MOTIVATION: An important problem in microarray experiments is the detection of genes that are differentially expressed in a given number of classes. We provide a straightforward and easily implemented method for estimating the posterior probability that an individual gene is null. The problem can be expressed in a two-component mixture framework, using an empirical Bayes approach. Current methods of implementing this approach either have some limitations due to the minimal assumptions made or with more specific assumptions are computationally intensive. RESULTS: By converting to a z-score the value of the test statistic used to test the significance of each gene, we propose a simple two-component normal mixture that models adequately the distribution of this score. The usefulness of our approach is demonstrated on three real datasets.

A mixture model-based approach to the clustering of microarray expression data.

MOTIVATION: This paper introduces the software EMMIX-GENE that has been developed for the specific purpose of a model-based approach to the clustering of microarray expression data, in particular, of tissue samples on a very large number of genes. The latter is a nonstandard problem in parametric cluster analysis because the dimension of the feature space (the number of genes) is typically much greater than the number of tissues. A feasible approach is provided by first selecting a subset of the genes relevant for the clustering of the tissue samples by fitting mixtures of t distributions to rank the genes in order of increasing size of the likelihood ratio statistic for the test of one versus two components in the mixture model. The imposition of a threshold on the likelihood ratio statistic used in conjunction with a threshold on the size of a cluster allows the selection of a relevant set of genes. However, even this reduced set of genes will usually be too large for a normal mixture model to be fitted directly to the tissues, and so the use of mixtures of factor analyzers is exploited to reduce effectively the dimension of the feature space of genes. RESULTS: The usefulness of the EMMIX-GENE approach for the clustering of tissue samples is demonstrated on two well-known data sets on colon and leukaemia tissues. For both data sets, relevant subsets of the genes are able to be selected that reveal interesting clusterings of the tissues that are either consistent with the external classification of the tissues or with background and biological knowledge of these sets. AVAILABILITY: EMMIX-GENE is available at http://www.maths.uq.edu.au/~gjm/emmix-gene/

cyp7b1 catalyses the 7alpha-hydroxylation of dehydroepiandrosterone and 25-hydroxycholesterol in rat prostate.

Dehydroepiandrosterone (DHEA) is the most prominent circulating steroid in humans, and it is a precursor for sex-steroid synthesis in peripheral tissues, including the prostate. Recently, enzyme-mediated pre-receptor metabolism has been recognized as a key step in determining steroid action in vivo. Hydroxylation of 3beta-steroids at the 7alpha-position has been reported in rat and human prostate to be a major inhibitory pathway to sex-steroid synthesis/action. However, the molecular identity of the enzyme responsible is so far unknown. We recently described a novel cytochrome P450 enzyme, cyp7b1, strongly expressed in the hippocampus of rodent brain, which catalyses the metabolism of DHEA, pregnenolone and 25-hydroxycholesterol to 7alpha-hydroxy products. In the light of this new enzyme, we have examined its possible role in 7alpha-hydroxylation conversion in rat prostate. NADPH-dependent 7alpha-hydroxylation was confirmed for 3beta-hydroxysteroids including DHEA and androstenediol, as well as 25-hydroxycholesterol. Kinetic analysis yielded an apparent K(m) of 14+/-1 microM for 7alpha-hydroxylation of DHEA in the prostate gland, a value similar to that recorded for recombinant cyp7b1 enzyme [13.6 microM; Rose, Stapleton, Dott, Kieny, Best, Schwarz, Russell, Bjoorkheim, Seckl and Lathe (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4925-4930]. The V(max) value of the prostate was 46+/-2 pmol/min per mg, and this activity was inhibited by clotrimazole, a P450-enzyme blocker. Moreover, RNA analysis (reverse-transcription PCR, Northern blotting and in situ hybridization) revealed a high expression of cyp7b1 mRNA in the rat prostate, restricted to the epithelium, suggesting that cyp7b1 catalyses oxysterol 7alpha-hydroxylation in the prostate gland.

Ontogeny of the neurosteroid enzyme Cyp7b in the mouse.

The function of the major adrenal steroid dehydroepiandrosterone (DHEA) is not known. It has been reported to improve learning and memory in mice and can exert neuroprotective and trophic effects, particularly in the hippocampus. We recently described a cytochrome P450 (Cyp7b), that catalyses the 7alpha-hydroxylation of DHEA and related steroids and sterols. In this paper, we have used mRNA in situ hybridisation to map the ontogeny of cyp7b in the foetal and adult mouse. Cyp7b mRNA is highly expressed throughout from embryonal (E) day 12.5 (the earliest day studied). There is also expression throughout the body, including the spine, thymus, developing kidneys, lungs and urogenital region. Widespread expression becomes more restricted towards birth: in newborn mice expression is largely limited to the hippocampus, with some expression being detected in kidney. The overall decline in mRNA, and increasing restriction to the hippocampus, is reflected in the DHEA hydroxylation activity of brain homogenates. This pattern of cyp7b mRNA expression in specific organs could be consistent with a protective role in foetal development, with highest expression seen when the foetus is most vulnerable to steroid excess (i.e.) early gestation.

Developing culturally competent marriage and family therapists: guidelines for working with Hispanic families.

As the Hispanic population of the United States continues to grow, so will the need for therapists who have been trained to work with Hispanic families. This content analysis of the available treatment literature generated several specific guidelines that can be used in training and evaluating culturally competent therapists. Guidelines included: Use family therapy, act as advocate for the family, assess immigration experience, assess acculturation, respect father, interview family subsystems separately, do not force changes, provide concrete suggestions, and warmly engage the family. Empirical and conceptual support for each guideline is discussed and several conclusions are made regarding culturally competent therapy with Hispanic families.

Statistical analysis of environmental monitoring data: does a worst case time for monitoring clean rooms exist?

To determine the relationship between the sampling time of the environmental monitoring, i.e., viable counts, in aseptic filling areas and the microbial count and frequency of alerts for air, surface and personnel microbial monitoring, statistical analyses were conducted on 1) the frequency of alerts versus the time of day for routine environmental sampling conducted in calendar year 1994, and 2) environmental monitoring data collected at 30-minute intervals during routine aseptic filling operations over two separate days in four different clean rooms with multiple shifts and equipment set-ups at a parenteral manufacturing facility. Statistical analyses showed, except for one floor location that had significantly higher number of counts but no alert or action level samplings in the first two hours of operation, there was no relationship between the number of counts and the time of sampling. Further studies over a 30-day period at the floor location showed no relationship between time of sampling and microbial counts. The conclusion reached in the study was that there is no worst case time for environmental monitoring at that facility and that sampling any time during the aseptic filling operation will give a satisfactory measure of the microbial cleanliness in the clean room during the set-up and aseptic filling operation.

Altering records: discrediting your best witness.

There is a proper way to make a correction to a medical record. If the correction is entered properly, contemporaneously, has bearing on current patient care, does not embellish or amplify your actions as being correct, and is consistent with other entries in the record it should be sound. Entries that are not properly made can destroy a professional liability defense. Plaintiff's attorneys will look for a reason to allege altered records. It is a plaintiff's attorney's strategy to use such an allegation to make the case less defensible, force a settlement or increase the value of a case through the threat of punitive damages. Do not give them the opportunity.