Atomic mutagenesis of the ribosomal Sarcin-Ricin-Loop to study EF-G GTPase activation

Translocation factor EF-G, possesses a low basal GTPase activity, which is stimulated by the ribosome. One potential region of the ribosome that triggers GTPase activity of EF-G is the Sarcin-Ricin-Loop (SRL) (helix 95) in domain VI of the 23S rRNA. Structural data showed that the tip of the SRL closely approaches GTP in the active center of EF-G, structural probing data confirmed that EF-G interacts with nucleotides G2655, A2660, G2661 and A2662.1-3 The exocyclic group of adenine at A2660 is required for stimulation of EF-G GTPase activity by the ribosome as demonstrated using atomic mutagenesis.4 Recent crystal structures of EF-G on the ribosome, gave more insights into the molecular mechanism of EF-G GTPase activity.5 Based on the structure of EF-Tu on the ribosome1, the following mechanism of GTPase activation was proposed: upon binding of EF-G to the ribosome, the conserved His92 (E.coli) changes its position, pointing to the γ-phosphate of GTP. In this activated state, the phosphate of residue A2662 of the SRL positions the catalytic His in its active conformation. It was further proposed that the phosphate oxygen of A2662 is involved in a charge-relay system, enabling GTP hydrolysis. In order to test this mechanism, we use the atomic mutagenesis approach, which allows introducing non-natural modifications in the SRL, in the context of the complete 70S ribosome. Therefore, we replaced one of the non-bridging oxygens of A2662 by a methyl group. A methylphosphonat is not able to position or activate a histidine, as it has no free electrons and therefore no proton acceptor function. These modified ribosomes were then tested for stimulation of EF-G GTPase activity. First experiments show that one of the two stereoisomers incorporated into ribosomes does not stimulate GTPase activity of EF-G, whereas the other is active. From this we conclude that indeed the non-bridging phosphate oxygen of A2662 is involved in EF-G GTPase activation by the ribosome. Ongoing experiments aim at revealing the contribution of this non-bridging oxygen at A2662 to the mechanism of EF-G GTPase activation at the atomic level.

Atomic mutagenesis of the ribosomal Sarcin-Ricin-Loop to study EF-G GTPase activation

Translocation factor EF-G, possesses a low basal GTPase activity, which is stimulated by the ribosome. One potential region of the ribosome that triggers GTPase activity of EF-G is the Sarcin-Ricin-Loop (SRL) (helix 95) in domain VI of the 23S rRNA. Structural data showed that the tip of the SRL closely approaches GTP in the active center of EF-G, structural probing data confirmed that EF-G interacts with nucleotides G2655, A2660, G2661 and A2662.1-3 The exocyclic group of adenine at A2660 is required for stimulation of EF-G GTPase activity by the ribosome as demonstrated using atomic mutagenesis.4 Recent crystal structures of EF-G on the ribosome, gave more insights into the molecular mechanism of EF-G GTPase activity.5 Based on the structure of EF-Tu on the ribosome1, the following mechanism of GTPase activation was proposed: upon binding of EF-G to the ribosome, the conserved His92 (E.coli) changes its position, pointing to the γ-phosphate of GTP. In this activated state, the phosphate of residue A2662 of the SRL positions the catalytic His in its active conformation. It was further proposed that the phosphate oxygen of A2662 is involved in a charge-relay system, enabling GTP hydrolysis. In order to test this mechanism, we use the atomic mutagenesis approach, which allows introducing non-natural modifications in the SRL, in the context of the complete 70S ribosome. Therefore, we replaced one of the non-bridging oxygens of A2662 by a methyl group. A methylphosphonat is not able to position or activate a histidine, as it has no free electrons and therefore no proton acceptor function. These modified ribosomes were then tested for stimulation of EF-G GTPase activity. First experiments show that one of the two stereoisomers incorporated into ribosomes does not stimulate GTPase activity of EF-G, whereas the other is active. From this we conclude that indeed the non-bridging phosphate oxygen of A2662 is involved in EF-G GTPase activation by the ribosome. Ongoing experiments aim at revealing the contribution of this non-bridging oxygen at A2662 to the mechanism of EF-G GTPase activation at the atomic level.

Lake surface water temperatures of European Alpine lakes (1989–2013) based on the Advanced Very High Resolution Radiometer (AVHRR) 1 km data set

Lake water temperature (LWT) is an important driver of lake ecosystems and it has been identified as an indicator of climate change. Consequently, the Global Climate Observing System (GCOS) lists LWT as an essential climate variable. Although for some European lakes long in situ time series of LWT do exist, many lakes are not observed or only on a non-regular basis making these observations insufficient for climate monitoring. Satellite data can provide the information needed. However, only few satellite sensors offer the possibility to analyse time series which cover 25 years or more. The Advanced Very High Resolution Radiometer (AVHRR) is among these and has been flown as a heritage instrument for almost 35 years. It will be carried on for at least ten more years, offering a unique opportunity for satellite-based climate studies. Herein we present a satellite-based lake surface water temperature (LSWT) data set for European water bodies in or near the Alps based on the extensive AVHRR 1 km data record (1989–2013) of the Remote Sensing Research Group at the University of Bern. It has been compiled out of AVHRR/2 (NOAA-07, -09, -11, -14) and AVHRR/3 (NOAA-16, -17, -18, -19 and MetOp-A) data. The high accuracy needed for climate related studies requires careful pre-processing and consideration of the atmospheric state. The LSWT retrieval is based on a simulation-based scheme making use of the Radiative Transfer for TOVS (RTTOV) Version 10 together with ERA-interim reanalysis data from the European Centre for Medium-range Weather Forecasts. The resulting LSWTs were extensively compared with in situ measurements from lakes with various sizes between 14 and 580 km2 and the resulting biases and RMSEs were found to be within the range of −0.5 to 0.6 K and 1.0 to 1.6 K, respectively. The upper limits of the reported errors could be rather attributed to uncertainties in the data comparison between in situ and satellite observations than inaccuracies of the satellite retrieval. An inter-comparison with the standard Moderate-resolution Imaging Spectroradiometer (MODIS) Land Surface Temperature product exhibits RMSEs and biases in the range of 0.6 to 0.9 and −0.5 to 0.2 K, respectively. The cross-platform consistency of the retrieval was found to be within ~ 0.3 K. For one lake, the satellite-derived trend was compared with the trend of in situ measurements and both were found to be similar. Thus, orbital drift is not causing artificial temperature trends in the data set. A comparison with LSWT derived through global sea surface temperature (SST) algorithms shows lower RMSEs and biases for the simulation-based approach. A running project will apply the developed method to retrieve LSWT for all of Europe to derive the climate signal of the last 30 years. The data are available at doi:10.1594/PANGAEA.831007.

Invited Abstract: Issues with State-based Energy Consumption Modelling

Energy consumption modelling by state based approaches often assume constant energy consumption values in each state. However, it happens in certain situations that during state transitions or even during a state the energy consumption is not constant and does fluctuate. This paper discusses those issues by presenting some examples from wireless sensor and wireless local area networks for such cases and possible solutions.

The mammalian central nervous synaptic cleft contains a high density of periodically organized complexes.

Cryo-electron microscopy of vitreous section makes it possible to observe cells and tissues at high resolution in a close-to-native state. The specimen remains hydrated; chemical fixation and staining are fully avoided. There is minimal molecular aggregation and the density observed in the image corresponds to the density in the object. Accordingly, organotypic hippocampal rat slices were vitrified under high pressure and controlled cryoprotection conditions, cryosectioned at a final thickness of approximately 70 nm and observed below -170 degrees C in a transmission electron microscope. The general aspect of the tissue compares with previous electron microscopy observations. The detailed analysis of the synapse reveals that the density of material in the synaptic cleft is high, even higher than in the cytoplasm, and that it is organized in 8.2-nm periodic transcleft complexes. Previously undescribed structures of presynaptic and postsynaptic elements are also described.

Intersystem crossing rates of S1 state keto-amino cytosine at low excess energy

The amino-keto tautomer of supersonic jet-cooled cytosine undergoes intersystem crossing (ISC) from the v = 0 and low-lying vibronic levels of its S1(¹ππ*) state. We investigate these ISC rates experimentally and theoretically as a function of S1 state vibrational excess energy Eexc. The S1 vibronic levels are pumped with a ~5 ns UV laser, the S1 and triplet state ion signals are separated by prompt or delayed ionization with a second UV laser pulse. After correcting the raw ISC yields for the relative S1 and T1ionization cross sections, we obtain energy dependent ISC quantum yields Q corr ISC =1% –5%. These are combined with previously measured vibronic state-specific decay rates, giving ISC rates kISC = 0.4–1.5 ⋅ 10⁹ s⁻¹, the corresponding S1⇝S0internal conversion (IC) rates are 30–100 times larger. Theoretical ISC rates are computed using SCS-CC2 methods, which predict rapid ISC from the S1; v = 0 state with kISC = 3 ⋅ 10⁹ s⁻¹ to the T1(³ππ*) triplet state. The surprisingly high rate of this El Sayed-forbidden transition is caused by a substantial admixture of ¹nOπ* character into the S1(¹ππ*) wave function at its non-planar minimum geometry. The combination of experiment and theory implies that (1) below Eexc = 550 cm⁻¹ in the S1 state, S1⇝S0internal conversion dominates the nonradiative decay with kIC ≥ 2 ⋅ 10¹⁰ s⁻¹, (2) the calculated S1⇝T1 (¹ππ*⇝³ππ*) ISC rate is in good agreement with experiment, (3) being El-Sayed forbidden, the S1⇝T1 ISC is moderately fast (kISC = 3 ⋅ 10⁹ s⁻¹), and not ultrafast, as claimed by other calculations, and (4) at Eexc ~ 550 cm⁻¹ the IC rate increases by ~50 times, probably by accessing the lowest conical intersection (the C5-twist CI) and thereby effectively switching off the ISC decay channels.

Mycoplasma-induced minimally conscious state

Mycoplasma pneumoniae (M. pneumoniae) frequently causes community-acquired respiratory tract infection and often presents as atypical pneumonia. Following airborne infection and a long incubation period, affected patients mostly suffer from mild or even asymptomatic and self-limiting disease. In particular in school-aged children, M. pneumoniae is associated with a wide range of extrapulmonary manifestations including central nervous system (CNS) disease. In contrast to children, severe CNS manifestations are rarely observed in adults. We report a case of a 37 year-old previously healthy immunocompetent adult with fulminant M. pneumoniae-induced progressive encephalomyelitis who was initially able to walk to the emergency department. A few hours later, she required controlled mechanical ventilation for ascending transverse spinal cord syndrome, including complete lower extremity paraplegia. Severe M. pneumoniae-induced encephalomyelitis was postulated, and antimicrobial, anti-inflammatory and immunosuppressive therapy was applied on the intensive care unit. Despite early and targeted therapy using four different immunosuppressive strategies, clinical success was limited. In our patient, locked-in syndrome developed followed by persistent minimally conscious state. The neurological status was unchanged until day 230 of follow-up. Our case underlines that severe M. pneumoniae- related encephalomyelitis must not only be considered in children, but also in adults. Moreover, it can be fulminant and fatal in adults. Our case enhances the debate for an optimal antimicrobial agent with activity beyond the blood-brain barrier. Furthermore, it may underline the difficulty in clinical decision making regarding early antimicrobial treatment in M. pneumoniae disease, which is commonly self-limited.

Cost analysis of opt-in consent procedures for the Texas statewide immunization information system, ImmTrac

Background. Childhood immunization programs have dramatically reduced the morbidity and mortality associated with vaccine-preventable diseases. Proper documentation of immunizations that have been administered is essential to prevent duplicate immunization of children. To help improve documentation, immunization information systems (IISs) have been developed. IISs are comprehensive repositories of immunization information for children residing within a geographic region. The two models for participation in an IIS are voluntary inclusion, or "opt-in," and voluntary exclusion, or "opt-out." In an opt-in system, consent must be obtained for each participant, conversely, in an opt-out IIS, all children are included unless procedures to exclude the child are completed. Consent requirements for participation vary by state; the Texas IIS, ImmTrac, is an opt-in system.^ Objectives. The specific objectives are to: (1) Evaluate the variance among the time and costs associated with collecting ImmTrac consent at public and private birthing hospitals in the Greater Houston area; (2) Estimate the total costs associated with collecting ImmTrac consent at selected public and private birthing hospitals in the Greater Houston area; (3) Describe the alternative opt-out process for collecting ImmTrac consent at birth and discuss the associated cost savings relative to an opt-in system.^ Methods. Existing time-motion studies (n=281) conducted between October, 2006 and August, 2007 at 8 birthing hospitals in the Greater Houston area were used to assess the time and costs associated with obtaining ImmTrac consent at birth. All data analyzed are deidentified and contain no personal information. Variations in time and costs at each location were assessed and total costs per child and costs per year were estimated. The cost of an alternative opt-out system was also calculated.^ Results. The median time required by birth registrars to complete consent procedures varied from 72-285 seconds per child. The annual costs associated with obtaining consent for 388,285 newborns in ImmTrac's opt-in consent process were estimated at $702,000. The corresponding costs of the proposed opt-out system were estimated to total $194,000 per year. ^ Conclusions. Substantial variation in the time and costs associated with completion of ImmTrac consent procedures were observed. Changing to an opt-out system for participation could represent significant cost savings. ^

Expression study of the atherosclerosis mouse aorta reveals significant disturbance of calcium signaling pathway

Atherosclerosis is a complex disease resulting from interactions of genetic and environmental risk factors leading to heart failure and stroke. Using an atherosclerotic mouse model (ldlr-/-, apobec1-/- designated as LDb), we performed microarray analysis to identify candidate genes and pathways, which are most perturbed in changes in the following risk factors: genetics (control C57BL/6 vs. LDb mice), shearstress (lesion-prone vs. lesion-resistant regions in LDb mice), diet (chow vs. high fat fed LDb mice) and age (2-month-old vs. 8-month old LDb mice). ^ Atherosclerotic lesion quantification and lipid profile studies were performed to assess the disease phenotype. A microarray study was performed on lesion-prone and lesion-resistant regions of each aorta. Briefly, 32 male C57BL/6 and LDb mice (n =16/each) were fed on either chow or high fat diet, sacrificed at 2- and 8-months old, and RNA isolated from the aortic lesion-prone and aortic lesion-resistant segments. Using 64 Affymetrix Murine 430 2.0 chips, we profiled differentially expressed genes with the cut off value of FDR ≤ 0.15 for t-test, and q <0.0001 for the ANOVA. The data were normalized using two normalization methods---invariant probe sets (Loess) and Quantile normalization, the statistical analysis was performed using t-tests and ANOVA, and pathway characterization was done using Pathway Express (Wayne State). The result identified the calcium signaling pathway as the most significant overrepresented pathway, followed by focal adhesion. In the calcium signaling pathway, 56 genes were found to be significantly differentially expressed out of 180 genes listed in the KEGG calcium signaling pathway. Nineteen of these genes were consistently identified by both statistical tests, 11 of which were unique to the test, and 26 were unique to the ANOVA test, using the cutoffs noted above. ^ In conclusion, this finding suggested that hypercholesterolemia drives the disease progression by altering the expression of calcium channels and regulators which subsequently results in cell differentiation, growth, adhesion, cytoskeletal change and death. Clinically, this pathway may serve as an important target for future therapeutic intervention, and thus the calcium signaling pathway may serve as an important target for future diagnostic and therapeutic intervention. ^

An analysis of the revenue policy -making process of the Texas Legislature

The purpose of this dissertation was to develop a conceptual framework which can be used to account for policy decisions made by the House Ways and Means Committee (HW&MC) of the Texas House of Representatives. This analysis will examine the actions of the committee over a ten-year period with the goal of explaining and predicting the success of failure of certain efforts to raise revenue.^ The basis framework for modelling the revenue decision-making process includes three major components--the decision alternatives, the external factors and two competing contingency theories. The decision alternatives encompass the particular options available to increase tax revenue. The options were classified as non-innovative or innovative. The non-innovative options included the sales, franchise, property and severance taxes. The innovative options were principally the personal and corporate income taxes.^ The external factors included political and economic constraints that affected the actions of the HW&MC. Several key political constraints on committee decision-making were addressed--including public attitudes, interest groups, political party strength and tradition and precedents. The economic constraints that affected revenue decisions included court mandates, federal mandates and the fiscal condition of the nation and the state.^ The third component of the revenue decision-making framework included two alternative contingency theories. The first alternative theory postulated that the committee structure, including the individual member roles and the overall committee style, resulted in distinctive revenue decisions. This theory will be favored if evidence points to the committee acting autonomously with less concern for the policies of the Speaker of the House. The Speaker assignment theory, postulated that the assignment of committee members shaped or changed the course of committee decision-making. This theory will be favored if there was evidence that the committee was strictly a vehicle for the Speaker to institute his preferred tax policies.^ The ultimate goal of this analysis is to develop an explanation for legislative decision-making about tax policy. This explanation will be based on the linkages across various tax options, political and economic constraints, member roles and committee style and the patterns of committee assignment. ^

The experience of flow in adolescence : Its relationship with personality traits and age

The aim of this study is to assess the experience of flow and its relationship with the personality traits and the age of the adolescents. For this purpose, 224 participants of both sexes were selected, aged 12-20 years, who were examined with various tools: Flow State in adolescents (Leibovich de Figueroa; Schmidt, 2013). This is a self-report technique of 28 items that assesses the Flow State, covering all the aspects theoretically listed as components in the optimal experience of enjoyment. And a self-report Being a teenager nowadays, which evaluates 33 pairs of opposite personality characteristics that represent the personality domains of the NEO-PI-R (Costa; McCrae, 1992. Costa; McCrae, 2005, Leibovich; Schmidt, 2005). Among the found results, it was observed that in the adolescents with high scores on the scale of Flow State, the main personality trait was extroversion. Also, the influence of age on optimal flow experience appears in the chosen activities

The experience of flow in adolescence : Its relationship with personality traits and age

The aim of this study is to assess the experience of flow and its relationship with the personality traits and the age of the adolescents. For this purpose, 224 participants of both sexes were selected, aged 12-20 years, who were examined with various tools: Flow State in adolescents (Leibovich de Figueroa; Schmidt, 2013). This is a self-report technique of 28 items that assesses the Flow State, covering all the aspects theoretically listed as components in the optimal experience of enjoyment. And a self-report Being a teenager nowadays, which evaluates 33 pairs of opposite personality characteristics that represent the personality domains of the NEO-PI-R (Costa; McCrae, 1992. Costa; McCrae, 2005, Leibovich; Schmidt, 2005). Among the found results, it was observed that in the adolescents with high scores on the scale of Flow State, the main personality trait was extroversion. Also, the influence of age on optimal flow experience appears in the chosen activities

The experience of flow in adolescence : Its relationship with personality traits and age

The aim of this study is to assess the experience of flow and its relationship with the personality traits and the age of the adolescents. For this purpose, 224 participants of both sexes were selected, aged 12-20 years, who were examined with various tools: Flow State in adolescents (Leibovich de Figueroa; Schmidt, 2013). This is a self-report technique of 28 items that assesses the Flow State, covering all the aspects theoretically listed as components in the optimal experience of enjoyment. And a self-report Being a teenager nowadays, which evaluates 33 pairs of opposite personality characteristics that represent the personality domains of the NEO-PI-R (Costa; McCrae, 1992. Costa; McCrae, 2005, Leibovich; Schmidt, 2005). Among the found results, it was observed that in the adolescents with high scores on the scale of Flow State, the main personality trait was extroversion. Also, the influence of age on optimal flow experience appears in the chosen activities

(Table 2) d13C from the Lusklint section

The carbon isotope composition (d13C) of bulk organic matter and two palynomorph groups (scolecodonts and chitinozoans) from the Llandovery-Wenlock strata of Gotland (E Sweden) are compared to gain knowledge about carbon cycling in the Silurian (sub)tropical shelf environment. The d13C values of the palynomorphs are mostly lower than the d13C values of the bulk organic matter, and the d13C values of the benthic scolecodonts are lower than those of the planktonic chitinozoans. While the difference between bulk and palynomorph d13C may be in part a function of trophic state, the lower values of the scolecodonts relative to those of chitinozoans, which are assumed to live in the well-mixed water column, might imply an infaunal mode of life for the polychaetes that carried the scolecodonts. Lower d13C for the scolecodonts in the middle of the section may represent variations in primary marine productivity (supported by acritarch abundance data), oxidation of organic matter in the bottom waters, or genera effects. In general, however, trends between the three data sets are parallel, indicating similarities in the low frequency, environmentally forced controls. The d13C data show a decreasing trend from the base of the section, up to a horizon well below the base of the Upper Visby Formation. At this level, and therefore probably several 10 kyr before the d13C increase in the carbonates, the d13C organic values increase by ~1 per mil. This perhaps is an expression of a changed composition of the bulk organic matter associated with the extinction events prior to the Llandovery-Wenlock boundary.

Ground maritime structure interaction. Recent failures in inner gravity constructions on the Spanish Coastline

A maritime construction is usually a slender line in the ocean.It is usual to see just its narrow surface strip and not analyse the large amount of submerged material the latter is supporting.Without doubt,it is the ground to which a notable load is transmitted in an environment subjected to periodic,alternating stresses,dynamic forces which the sea's media constitute. Both an outer and inner maritime construction works in a complex fashion.A granular solid(breakwater)breathes with the incident wave flow,dissipating part of the wave energy between its gaps.The backflow tries to extract the different items from the solid block,setting a balance between effective and neutral tensions that follow Terzaghui's principle. On some occasions,fluidification of the armour layer has caused the breakwater to collapse(Sines,Portugal,February 1978).On others,siphoning or liquefaction of sand supporting monoliths(vertical breakwaters)lead them to destruction or collapse(New Barcelona Harbour Mouth,Spain,November 2001). This is why the ground-force-structure interaction is a complicated analysis with joint design tools still in an incipient state. The purpose of this article is to describe two singular failures in inner maritime constructions in Spain deriving from ground problems(Malaga,July 2004and Barcelona,January 2007).They occurred recently and the causes are the subject of reflection and analysis.