A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin

During light-driven proton transport bacteriorhodopsin shuttles between two protein conformations. A large-scale structural change similar to that in the photochemical cycle is produced in the D85N mutant upon raising the pH, even without illumination. We report here that (i) the pKa values for the change in crystallographic parameters and for deprotonation of the retinal Schiff base are the same, (ii) the retinal isomeric configuration is nearly unaffected by the protein conformation, and (iii) preventing rotation of the C13—C14 double bond by replacing the retinal with an all-trans locked analogue makes little difference to the Schiff base pKa. We conclude that the direct cause of the conformational shift is destabilization of the structure upon loss of interaction of the positively charged Schiff base with anionic residues that form its counter-ion.

Crystal structure of the Sec18p N-terminal domain

Yeast Sec18p and its mammalian orthologue N-ethylmaleimide-sensitive fusion protein (NSF) are hexameric ATPases with a central role in vesicle trafficking. Aided by soluble adapter factors (SNAPs), Sec18p/NSF induces ATP-dependent disassembly of a complex of integral membrane proteins from the vesicle and target membranes (SNAP receptors). During the ATP hydrolysis cycle, the Sec18p/NSF homohexamer undergoes a large-scale conformational change involving repositioning of the most N terminal of the three domains of each protomer, a domain that is required for SNAP-mediated interaction with SNAP receptors. Whether an internal conformational change in the N-terminal domains accompanies their reorientation with respect to the rest of the hexamer remains to be addressed. We have determined the structure of the N-terminal domain from Sec18p by x-ray crystallography. The Sec18p N-terminal domain consists of two β-sheet-rich subdomains connected by a short linker. A conserved basic cleft opposite the linker may constitute a SNAP-binding site. Despite structural variability in the linker region and in an adjacent loop, all three independent molecules in the crystal asymmetric unit have the identical subdomain interface, supporting the notion that this interface is a preferred packing arrangement. However, the linker flexibility allows for the possibility that other subdomain orientations may be sampled.

Biodiversity of Costa Rican salamanders: Implications of high levels of genetic differentiation and phylogeographic structure for species formation

Although salamanders are characteristic amphibians in Holarctic temperate habitats, in tropical regions they have diversified evolutionarily only in tropical America. An adaptive radiation centered in Middle America occurred late in the history of a single clade, the supergenus Bolitoglossa (Plethodontidae), and large numbers of species now occur in diverse habitats. Sublineages within this clade decrease in number from the northern to southern parts of Middle America, and in Costa Rica, there are but three. Despite this phylogenetic constraint, Costa Rica has many species; the number of salamander species on one local elevational transect in the Cordillera de Talamanca may be the largest for any such transect in the world. Extraordinary variation in sequences of the mitochondrial gene cytochrome b within a clade of the genus Bolitoglossa in Costa Rica reveals strong phylogeographic structure within a single species, Bolitoglossa pesrubra. Allozymic variation in 19 proteins reveals a pattern largely concordant with the mitochondrial DNA phylogeography. More species exist than are currently recognized. Diversification occurs in restricted geographic areas and involves sharp geographic and elevational differentiation and zonation. In their degree of genetic differentiation at a local scale, these species of the deep tropics exceed the known variation of extratropical salamanders, which also differ in being less restricted in elevational range. Salamanders display “tropicality” in that although speciose, they are usually local in distribution and rare. They display strong ecological and physiological differentiation that may contribute importantly to morphological divergence and species formation.

Structure-based assignment of the biochemical function of a hypothetical protein: A test case of structural genomics

Many small bacterial, archaebacterial, and eukaryotic genomes have been sequenced, and the larger eukaryotic genomes are predicted to be completely sequenced within the next decade. In all genomes sequenced to date, a large portion of these organisms’ predicted protein coding regions encode polypeptides of unknown biochemical, biophysical, and/or cellular functions. Three-dimensional structures of these proteins may suggest biochemical or biophysical functions. Here we report the crystal structure of one such protein, MJ0577, from a hyperthermophile, Methanococcus jannaschii, at 1.7-Å resolution. The structure contains a bound ATP, suggesting MJ0577 is an ATPase or an ATP-mediated molecular switch, which we confirm by biochemical experiments. Furthermore, the structure reveals different ATP binding motifs that are shared among many homologous hypothetical proteins in this family. This result indicates that structure-based assignment of molecular function is a viable approach for the large-scale biochemical assignment of proteins and for discovering new motifs, a basic premise of structural genomics.

Mineral surfaces and bioavailability of heavy metals: A molecular-scale perspective

There is a continual influx of heavy metal contaminants and pollutants into the biosphere from both natural and anthropogenic sources. A complex variety of abiotic and biotic processes affects their speciation and distribution, including adsorption onto and desorption from mineral surfaces, incorporation in precipitates or coprecipitates, release through the dissolution of minerals, and interactions with plants and microbes. Some of these processes can effectively isolate heavy metals from the biosphere, whereas others cause their release or transformation to different species that may be more (or less) bioavailable and/or toxic to organisms. Here we focus on abiotic adsorption and precipitation or coprecipitation processes involving the common heavy metal contaminant lead and the metalloids arsenic and selenium in mine tailings and contaminated soils. We have used extremely intense x-rays from synchrotron sources and a structure-sensitive method known as x-ray absorption fine structure (XAFS) spectroscopy to determine the molecular-level speciation of these elements at concentrations of 50 to several thousand ppm in the contaminated environmental samples as well as in synthetic sorption samples. Our XAFS studies of As and Pb in the mine tailings show that up to 50% of these contaminants in the samples studied may be present as adsorbed species on mineral surfaces, which makes them potentially more bioavailable than when present in sparingly soluble solid phases. Our XAFS studies of Se(VI) sorption on Fe2+-containing sulfates show that this element undergoes redox reactions that transform it into less bioavailable and less toxic species. This type of information on molecular-level speciation of heavy metal and metalloid contaminants in various environmental settings is needed to prioritize remediation efforts and to assess their potential hazard to humans and other organisms.

Determining RNA solution structure by segmental isotopic labeling and NMR: application to Caenorhabditis elegans spliced leader RNA 1.

Recent developments in multidimensional heteronuclear NMR spectroscopy and large-scale synthesis of uniformly 13C- and 15N-labeled oligonucleotides have greatly improved the prospects for determination of the solution structure of RNA. However, there are circumstances in which it may be advantageous to label only a segment of the entire RNA chain. For example, in a larger RNA molecule the structural question of interest may reside in a localized domain. Labeling only the corresponding nucleotides simplifies the spectrum and resonance assignments because one can filter proton spectra for coupling to 13C and 15N. Another example is in resolving alternative secondary structure models that are indistinguishable in imino proton connectivities. Here we report a general method for enzymatic synthesis of quantities of segmentally labeled RNA molecules required for NMR spectroscopy. We use the method to distinguish definitively two competing secondary structure models for the 5' half of Caenorhabditis elegans spliced leader RNA by comparison of the two-dimensional [15N] 1H heteronuclear multiple quantum correlation spectrum of the uniformly labeled sample with that of a segmentally labeled sample. The method requires relatively small samples; solutions in the 200-300 microM concentration range, with a total of 30 nmol or approximately 40 micrograms of RNA in approximately 150 microliters, give strong NMR signals in a short accumulation time. The method can be adapted to label an internal segment of a larger RNA chain for study of localized structural problems. This definitive approach provides an alternative to the more common enzymatic and chemical footprinting methods for determination of RNA secondary structure.

Very-long-baseline radio interferometry surveys of the compact structure in active galactic nuclei.

Very-long-baseline radio interferometry (VLBI) imaging surveys have been undertaken since the late 1970s. The sample sizes were initially limited to a few tens of objects but the snapshot technique has now allowed samples containing almost 200 sources to be studied. The overwhelming majority of powerful compact sources are asymmetric corejects of one form or another, most of which exhibit apparent superluminal motion. However 5-10% of powerful flat-spectrum sources are 100-parsec (pc)-scale compact symmetric objects; these appear to form a continuum with the 1-kpc-scale double-lobed compact steep-spectrum sources, which make up 15-20% of lower frequency samples. It is likely that these sub-galactic-size symmetric sources are the precursors to the large-scale classical double sources. There is a surprising peak around 90 degrees in the histogram of misalignments between the dominant source axes on parsec and kiloparsec scales; this seems to be associated with sources exhibiting a high degree of relativistic beaming. VLBI snapshot surveys have great cosmological potential via measurements of both proper motion and angular size vs. redshift as well as searches for gravitational "millilensing."

The parsec-scale jet in M87.

We briefly review the observed structure and evolution of the M87 jet on scales less, similar1 parsec (pc; 1 pc = 3.09 x 10(16) m). Filamentary features, limb-brightening, and side-to-side oscillation are common characteristics of the pc-scale, and kpc-scale jets. The most prominent emission features on both the pc and subpc scales appear stationary (v/c < 0.1). Nonetheless, based on the jet's flux evolution, the presence of kpc-scale superluminal motion, and the absence of a visible counter-jet, we argue for the presence of an underlying relativistic flow, consistent with unified models. The initial jet collimation appears to occur on scales <0.1 pc, thus favoring electromagnetic processes associated with a black hole and accretion disk.

On the possibility of coherently stimulated recombination and cosmological structure generation: recombination instability.

Possible instabilities during cosmological recombination may produce an epoch of nonlinear density growth and fractal-like structural patterns out to the horizon scale at that epoch (approximately 200 Mpc today). With this motivation, we examine the consequences of the change in effective radiative recombination reaction rate coefficients produced by intense stimulated emission. The proton-electron recombination is considered as a natural laser, leading to the formation of spatially nonuniform distributions of neutral matter earlier than the recombination epoch.

The 12-Item General Health Questionnaire (GHQ-12): Reliability,external validity and factor structure in the Spanish population

The purpose of this study was to analyze the internal consistency and the external and structure validity of the 12-Item General Health Questionnaire (GHQ-12) in the Spanish general population. A stratified sample of 1001 subjects, ages between 25 and 65 years, taken from the general Spanish population was employed. The GHQ-12 and the Inventory of Situations and Responses of Anxiety-ISRA were administered. A Cronbach’s alpha of .76 (Standardized Alpha: .78) and a 3-factor structure (with oblique rotation and maximum likelihood procedure) were obtained. External validity of Factor I (Successful Coping) with the ISRA is very robust (.82; Factor II, .70; Factor III, .75). The GHQ-12 shows adequate reliability and validity in the Spanish population. Therefore, the GHQ-12 can be used with efficacy to assess people’s overall psychological well-being and to detect non-psychotic psychiatric problems. Additionally, our results confirm that the GHQ-12 can best be thought of as a multidimensional scale that assesses several distinct aspects of distress, rather than just a unitary screening measure.

Mechanical annealing of metallic electrodes at the atomic scale

The process of creating an atomically defined and robust metallic tip is described and quantified using measurements of contact conductance between gold electrodes and numerical simulations. Our experiments show how the same conductance behavior can be obtained for hundreds of cycles of formation and rupture of the nanocontact by limiting the indentation depth between the two electrodes up to a conductance value of approximately 5G0 in the case of gold. This phenomenon is rationalized using molecular dynamics simulations together with density functional theory transport calculations which show how, after repeated indentations (mechanical annealing), the two metallic electrodes are shaped into tips of reproducible structure. These results provide a crucial insight into fundamental aspects relevant to nanotribology or scanning probe microscopies.

The Spanish version of the Prenatal Breast-feeding Self-efficacy Scale: Reliability and validity assessment

Background: Only a minority of infants are exclusively breastfed for the recommended 6 months postpartum. Breast-feeding self-efficacy is a mother's confidence in her ability to breastfeed and is predictive of breastfeeding behaviors. The Prenatal Breast-feeding Self-efficacy Scale (PBSES) was developed among English-speaking mothers to measure breastfeeding self-efficacy before delivery. Objectives: To translate the PBSES into Spanish and assess its psychometric properties. Design: Reliability and validity assessment. Setting: A public hospital in Yecla, Spain. Participants: A convenience sample of 234 pregnant women in their third trimester of pregnancy. Methods: The PBSES was translated into Spanish using forward and back translation. A battery of self-administered questionnaires was completed by participants, including a questionnaire on sociodemographic variables, breastfeeding experience and intention, as well as the Spanish version of the PBSES. Also, data on exclusive breastfeeding at discharge were collected from hospital database. Dimensional structure, internal consistency and construct validity of the Spanish version of PBSES were assessed. Results: Confirmatory factor analysis suggested the presence of one construct, self-efficacy, with four dimensions or latent variables. Cronbach's alpha coefficient for internal consistency was 0.91. Response patterns based on decision to breastfeed during pregnancy provided evidence of construct validity. In addition, the scores of the Spanish version of the PBSES significantly predicted exclusive breastfeeding at discharge. Conclusions: The Spanish version of PBSES shows evidences of reliability, and contrasting group and predictive validity. Confirmatory factor analysis indicated marginal fit and further studies are needed to provide new evidence on the structure of the scale. The Spanish version of the PBSES can be considered a reliable measure and shows validity evidences.

Development and psychometric testing of the clinical learning environment, supervision and nurse teacher evaluation scale (CLES+T): The Spanish version

Background: The Clinical Learning Environment, Supervision and Nurse Teacher scale is a reliable and valid instrument to evaluate the quality of the clinical learning process in international nursing education contexts. Objectives: This paper reports the development and psychometric testing of the Spanish version of the Clinical Learning Environment, Supervision and Nurse Teacher scale. Design: Cross-sectional validation study of the scale. Setting: 10 public and private hospitals in the Alicante area, and the Faculty of Health Sciences (University of Alicante, Spain). Participants: 370 student nurses on clinical placement (January 2011–March 2012). Methods: The Clinical Learning Environment, Supervision and Nurse Teacher scale was translated using the modified direct translation method. Statistical analyses were performed using PASW Statistics 18 and AMOS 18.0.0 software. A multivariate analysis was conducted in order to assess construct validity. Cronbach’s alpha coefficient was used to evaluate instrument reliability. Results: An exploratory factorial analysis identified the five dimensions from the original version, and explained 66.4% of the variance. Confirmatory factor analysis supported the factor structure of the Spanish version of the instrument. Cronbach’s alpha coefficient for the scale was .95, ranging from .80 to .97 for the subscales. Conclusion: This version of the Clinical Learning Environment, Supervision and Nurse Teacher scale instrument showed acceptable psychometric properties for use as an assessment scale in Spanish-speaking countries.

Probing large-scale wind structures in Vela X–1 using off-states with INTEGRAL

Vela X–1 is the prototype of the class of wind-fed accreting pulsars in high-mass X-ray binaries hosting a supergiant donor. We have analysed in a systematic way 10 years of INTEGRAL data of Vela X–1 (22–50 keV) and we found that when outside the X-ray eclipse, the source undergoes several luminosity drops where the hard X-rays luminosity goes below ∼3 × 1035 erg s−1, becoming undetected by INTEGRAL. These drops in the X-ray flux are usually referred to as ‘off-states’ in the literature. We have investigated the distribution of these off-states along the Vela X–1 ∼ 8.9 d orbit, finding that their orbital occurrence displays an asymmetric distribution, with a higher probability to observe an off-state near the pre-eclipse than during the post-eclipse. This asymmetry can be explained by scattering of hard X-rays in a region of ionized wind, able to reduce the source hard X-ray brightness preferentially near eclipse ingress. We associate this ionized large-scale wind structure with the photoionization wake produced by the interaction of the supergiant wind with the X-ray emission from the neutron star. We emphasize that this observational result could be obtained thanks to the accumulation of a decade of INTEGRAL data, with observations covering the whole orbit several times, allowing us to detect an asymmetric pattern in the orbital distribution of off-states in Vela X–1.

Towards a professional army. Changes to the structure of the officer cadre and the manning system of the Russian armed forces. OSW Commentary No. 73, 2012-03-28

The structure of the Russian army’s personnel has undergone a major transformation in recent years. The Armed Forces are no longer a downsized continuation of the Soviet-era mass army, but are gradually becoming a de facto professional army in which conscription, now employed on a diminishing scale, will primarily constitute a first step towards a continued professional military career. The cornerstone for the process of professionalisation has been laid by a personnel reform which cut the number of officer posts by nearly half and considerably restricted the recruitment of new officers, thus restoring traditional proportions to the structure of the officer corps. The plans to ultimately implement a manning system based predominantly on contract service are a natural consequence of these changes. The ongoing professionalisation of the Russian Armed Forces should be treated as a conscious effort which is mainly necessitated by global trends: despite the demographic changes taking place, Russia could still maintain an army with a declared strength of one million soldiers, most of them conscripts.