Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Transmission electron microscopy has provided most of what is known about the ultrastructural organization of tissues, cells, and organelles. Due to tremendous advances in crystallography and magnetic resonance imaging, almost any protein can now be modeled at atomic resolution. To fully understand the workings of biological "nanomachines" it is necessary to obtain images of intact macromolecular assemblies in situ. Although the resolution power of electron microscopes is on the atomic scale, in biological samples artifacts introduced by aldehyde fixation, dehydration and staining, but also section thickness reduces it to some nanometers. Cryofixation by high pressure freezing circumvents many of the artifacts since it allows vitrifying biological samples of about 200 mum in thickness and immobilizes complex macromolecular assemblies in their native state in situ. To exploit the perfect structural preservation of frozen hydrated sections, sophisticated instruments are needed, e.g., high voltage electron microscopes equipped with precise goniometers that work at low temperature and digital cameras of high sensitivity and pixel number. With them, it is possible to generate high resolution tomograms, i.e., 3D views of subcellular structures. This review describes theory and applications of the high pressure cryofixation methodology and compares its results with those of conventional procedures. Moreover, recent findings will be discussed showing that molecular models of proteins can be fitted into depicted organellar ultrastructure of images of frozen hydrated sections. High pressure freezing of tissue is the base which may lead to precise models of macromolecular assemblies in situ, and thus to a better understanding of the function of complex cellular structures.

Dual-energy X-ray absorptiometry for measuring total bone mineral content in the rat: study of accuracy and precision

Sequential studies of osteopenic bone disease in small animals require the availability of non-invasive, accurate and precise methods to assess bone mineral content (BMC) and bone mineral density (BMD). Dual-energy X-ray absorptiometry (DXA), which is currently used in humans for this purpose, can also be applied to small animals by means of adapted software. Precision and accuracy of DXA was evaluated in 10 rats weighing 50-265 g. The rats were anesthetized with a mixture of ketamine-xylazine administrated intraperitoneally. Each rat was scanned six times consecutively in the antero-posterior incidence after repositioning using the rat whole-body software for determination of whole-body BMC and BMD (Hologic QDR 1000, software version 5.52). Scan duration was 10-20 min depending on rat size. After the last measurement, rats were sacrificed and soft tissues were removed by dermestid beetles. Skeletons were then scanned in vitro (ultra high resolution software, version 4.47). Bones were subsequently ashed and dissolved in hydrochloric acid and total body calcium directly assayed by atomic absorption spectrophotometry (TBCa[chem]). Total body calcium was also calculated from the DXA whole-body in vivo measurement (TBCa[DXA]) and from the ultra high resolution measurement (TBCa[UH]) under the assumption that calcium accounts for 40.5% of the BMC expressed as hydroxyapatite. Precision error for whole-body BMC and BMD (mean +/- S.D.) was 1.3% and 1.5%, respectively. Simple regression analysis between TBCa[DXA] or TBCa[UH] and TBCa[chem] revealed tight correlations (n = 0.991 and 0.996, respectively), with slopes and intercepts which were significantly different from 1 and 0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrochemical current-sensing atomic force microscopy in conductive solutions

Insulated atomic force microscopy probes carrying gold conductive tips were fabricated and employed as bifunctional force and current sensors in electrolyte solutions under electrochemical potential control. The application of the probes for current-sensing imaging, force and current–distance spectroscopy as well as scanning electrochemical microscopy experiments was demonstrated.

Atomic Quantum Simulation of U(N) and SU(N) Abelian Lattice Gauge Theories

Using ultracold alkaline-earth atoms in optical lattices, we construct a quantum simulator for U(N) and SU(N) lattice gauge theories with fermionic matter based on quantum link models. These systems share qualitative features with QCD, including chiral symmetry breaking and restoration at nonzero temperature or baryon density. Unlike classical simulations, a quantum simulator does not suffer from sign problems and can address the corresponding chiral dynamics in real time.

End-tidal CO2 pressure: an important parameter for a correct interpretation of changes in cerebral hemodynamics and oxygenation measured with functional near infrared spectrophotometry (fNIRS)

The aim of the present study was to investigate the effects of different speech tasks (recitation of prose (PR), alliteration (AR) and hexameter (HR) verses) and a control task (mental arithmetic (MA) with voicing of the result) on endtidal CO2 (ET-CO2), cerebral hemodynamics; i.e. total hemoglobin (tHb) and tissue oxygen saturation (StO2). tHb and StO2 were measured with a frequency domain near infrared spectrophotometer (ISS Inc., USA) and ET-CO2 with a gas analyzer (Nellcor N1000). Measurements were performed in 24 adult volunteers (11 female, 13 male; age range 22 to 64 years) during task performance in a randomized order on 4 different days to avoid potential carry over effects. Statistical analysis was applied to test differences between baseline, 2 recitation and 5 recovery periods. The two brain hemispheres and 4 tasks were tested separately. Data analysis revealed that during the recitation tasks (PR, AR and HR) StO2 decreased statistically significant (p < 0.05) during PR and AR in the right prefrontal cortex (PFC) and during AR and HR in the left PFC. tHb showed a significant decrease during HR in the right PFC and during PR, AR and HR in the left PFC. During the MA task, StO2 increased significantly. A significant decrease in ET-CO2 was found during all 4 tasks with the smallest decrease during the MA task. In conclusion, we hypothesize that the observed changes in tHb and StO2 are mainly caused by an altered breathing during the tasks that led a lowering of the CO2 content in the blood provoked a cerebral CO2 reaction, i.e. a vasoconstriction of blood vessels due to decreased CO2 pressure and thereby decrease in cerebral blood volume. Therefore, breathing changes should be monitored during brain studies involving speech when using functional near infrared spectroscopy (fNIRS) to ensure a correct interpretation of changes in hemodynamics and oxygenation.

A quantitative analysis of microcirculation in sore-prone pressure areas on conventional and pressure relief hospital mattresses using laser Doppler flowmetry and tissue spectrophotometry

BACKGROUND Pressure ulcers are associated with severe impairment for the patients and high economic load. With this study we wanted to gain more insight to the skin perfusion dynamics due to external loading. Furthermore, we evaluated the effect of different types of pressure relief mattresses. METHODS A total of 25 healthy volunteers were enrolled in the study. Perfusion dynamics of the sacral and the heel area were assessed using the O2C-device, which combines a laser light, to determine blood flow, and white light to determine the relative amount of hemoglobin. Three mattresses were evaluated compared to a hard surface: a standard hospital foam mattress bed, a visco-elastic foam mattress, and an air-fluidized bed. RESULTS In the heel area, only the air-fluidized bed was able to maintain the blood circulation (mean blood flow of 13.6 ± 6 versus 3.9 ± 3 AU and mean relative amount of hemoglobin of 44.0 ± 14 versus 32.7 ± 12 AU.) In the sacral area, all used mattresses revealed an improvement of blood circulation compared to the hard surface. CONCLUSION The results of this study form a more precise pattern of perfusion changes due to external loading on various pressure relief mattresses. This knowledge may reduce the incidence of pressure ulcers and may be an influencing factor in pressure relief mattress selection.

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.