Evolutionary conservation of residues in vertebrate DNA polymerase N conferring low fidelity and bypass activity.

POLN is a nuclear A-family DNA polymerase encoded in vertebrate genomes. POLN has unusual fidelity and DNA lesion bypass properties, including strong strand displacement activity, low fidelity favoring incorporation of T for template G and accurate translesion synthesis past a 5S-thymine glycol (5S-Tg). We searched for conserved features of the polymerase domain that distinguish it from prokaryotic pol I-type DNA polymerases. A Lys residue (679 in human POLN) of particular interest was identified in the conserved 'O-helix' of motif 4 in the fingers sub-domain. The corresponding residue is one of the most important for controlling fidelity of prokaryotic pol I and is a nonpolar Ala or Thr in those enzymes. Kinetic measurements show that K679A or K679T POLN mutant DNA polymerases have full activity on nondamaged templates, but poorly incorporate T opposite template G and do not bypass 5S-Tg efficiently. We also found that a conserved Tyr residue in the same motif not only affects sensitivity to dideoxynucleotides, but also greatly influences enzyme activity, fidelity and bypass. Protein sequence alignment reveals that POLN has three specific insertions in the DNA polymerase domain. The results demonstrate that residues have been strictly retained during evolution that confer unique bypass and fidelity properties on POLN.

Treatment evolution in high-risk congenital diaphragmatic hernia: ten years' experience with diaphragmatic agenesis.

OBJECTIVE: The objective of this study was to evaluate the impact of newer therapies on the highest risk patients with congenital diaphragmatic hernia (CDH), those with agenesis of the diaphragm. SUMMARY BACKGROUND DATA: CDH remains a significant cause of neonatal mortality. Many novel therapeutic interventions have been used in these infants. Those children with large defects or agenesis of the diaphragm have the highest mortality and morbidity. METHODS: Twenty centers from 5 countries collected data prospectively on all liveborn infants with CDH over a 10-year period. The treatment and outcomes in these patients were examined. Patients were followed until death or hospital discharge. RESULTS: A total of 1,569 patients with CDH were seen between January 1995 and December 2004 in 20 centers. A total of 218 patients (14%) had diaphragmatic agenesis and underwent repair. The overall survival for all patients was 68%, while survival was 54% in patients with agenesis. When patients with diaphragmatic agenesis from the first 2 years were compared with similar patients from the last 2 years, there was significantly less use of ECMO (75% vs. 52%) and an increased use of inhaled nitric oxide (iNO) (30% vs. 80%). There was a trend toward improved survival in patients with agenesis from 47% in the first 2 years to 59% in the last 2 years. The survivors with diaphragmatic agenesis had prolonged hospital stays compared with patients without agenesis (median, 68 vs. 30 days). For the last 2 years of the study, 36% of the patients with agenesis were discharged on tube feedings and 22% on oxygen therapy. CONCLUSIONS: There has been a change in the management of infants with CDH with less frequent use of ECMO and a greater use of iNO in high-risk patients with a potential improvement in survival. However, the mortality, hospital length of stay, and morbidity in agenesis patients remain significant.

Impact of Acetazolamide and CPAP on Cortical Activity in Obstructive Sleep Apnea Patients

STUDY OBJECTIVES 1) To investigate the impact of acetazolamide, a drug commonly prescribed for altitude sickness, on cortical oscillations in patients with obstructive sleep apnea syndrome (OSAS). 2) To examine alterations in the sleep EEG after short-term discontinuation of continuous positive airway pressure (CPAP) therapy. DESIGN Data from two double-blind, placebo-controlled randomized cross-over design studies were analyzed. SETTING Polysomnographic recordings in sleep laboratory at 490 m and at moderate altitudes in the Swiss Alps: 1630 or 1860 m and 2590 m. PATIENTS Study 1: 39 OSAS patients. Study 2: 41 OSAS patients. INTERVENTIONS Study 1: OSAS patients withdrawn from treatment with CPAP. Study 2: OSAS patients treated with autoCPAP. Treatment with acetazolamide (500-750 mg) or placebo at moderate altitudes. MEASUREMENTS AND RESULTS An evening dose of 500 mg acetazolamide reduced slow-wave activity (SWA; approximately 10%) and increased spindle activity (approximately 10%) during non-REM sleep. In addition, alpha activity during wake after lights out was increased. An evening dose of 250 mg did not affect these cortical oscillations. Discontinuation of CPAP therapy revealed a reduction in SWA (5-10%) and increase in beta activity (approximately 25%). CONCLUSIONS The higher evening dose of 500 mg acetazolamide showed the "spectral fingerprint" of Benzodiazepines, while 250 mg acetazolamide had no impact on cortical oscillations. However, both doses had beneficial effects on oxygen saturation and sleep quality.

A Wavelet Analysis of Pliopleistocene Climate Indicators: A New View of Periodicity Evolution

Wavelet analysis offers an alternative to Fourier based time-series analysis, and is particularly useful when the amplitudes and periods of dominant cycles are time dependent. We analyse climatic records derived from oxygen isotopic ratios of marine sediment cores with modified Morlet wavelets. We use a normalization of the Morlet wavelets which allows direct correspondence with Fourier analysis. This provides a direct view of the oscillations at various frequencies, and illustrates the nature of the time-dependence of the dominant cycles.

A 13,600-year diatom oxygen isotope record from the South Carpathians (Romania): Reflection of winter conditions and possible links with North Atlantic circulation changes

This study provides a continuous lateglacial and Holocene record of diatom silica oxygen isotope changes (delta O-18(DIAT)) in a subalpine lake sediment sequence obtained from the Retezat Mts (Taul dintre Brazi, 1740 m a.s.l.). This through-flow, shallow, high-altitude lake with a surface area of only 0.4 ha has short water residence time and is predominantly fed by snowmelt and rainwater. Its delta O-18(DIAT) record principally reflects the oxygen isotope composition of the winter and spring precipitation, as diatom blooms occur mainly in the spring and early summer. Hence, changes in delta O-18(DIAT) are interpreted as seasonal scale changes: in the amount of winter precipitation. Low oxygen isotope values (27-28.5 parts per thousand) occurred during the lateglacial until 12,300 cal BP, followed by a sharp increase thereafter. In the Holocene delta O-18(DIAT) values ranged from 29 to 31 parts per thousand until 3200 cal BP, followed by generally lower values during the late Holocene (27-30 parts per thousand). Short-term decreases in the isotopic values were found between 10,140-9570, 9000-8500, 7800-7300, 6300-5800, 5500-5000 and at 8015, 4400, 4000 cal BP. After 3200 cal BP a decreasing trend was visible with the lowest values between 3100-2500 and after 2100 cal BP The general trend in the record suggests that contribution of winter precipitation was generally lower between 11,680 and 3200 cal BP, followed by increased contribution during the last millennia. The late Holocene decrease in delta O-18(DIAT) shows good agreement with the speleothem delta O-18, lake level and testate amoebae records from the Carpathian Mountains that also display gradual delta O-18 decrease and lake level/mire water table level rise after 3200 cal BR Strong positive correlation with North Atlantic circulation and solar activity proxies, such as the Austrian and Hungarian speleothem records, furthermore suggested that short-term increases in the isotopic ratios in the early and mid Holocene are likely connectable to high solar activity phases and high frequency of positive North Atlantic Oscillation indexes that may have resulted in decreased winter precipitation in this region.

Oxygen consumption while standing iwth and unstable shoe design

This study explored the effects of unstable shoe design on oxygen consumption. Methods. Oxygen consumption (VO2) and heart rate (HR) were measured in 16 individuals while barefoot, wearing unstable shoes (Masai Barefoot Technology) and wearing conventional sport shoes while standing and walking on a treadmill and for 5 individuals while walking around a 400 m track. Results. When wearing the MBT shoes, a significant (p < 0.01) increase of 9.3 ± 5.2% in VO2 was measured while standing quietly for 6 min. No differences in VO2 and HR were observed between the MBT shoes or weight-adjusted conventional shoes (to match the weight of the MBT shoes) while walking on a treadmill. However, significant increases (p < 0.01) in VO2 (4.4 ± 8.2%) and HR (3.6 ± 7.3%) were observed for the MBT shoes compared with being barefoot. No significant differences in VO2 and HR were recorded while walking around a 400 m track either with MBT shoes, weight-adjusted conventional shoes or barefoot. Nonetheless, a comparison of the MBT shoes with barefoot revealed a tendency for VO2 to be higher when wearing the MBT shoes (7.1 ± 6.5%, p < 0.1) although HR was not significantly affected. Conclusions. The unstable shoe design predominantly effects oxygen consumption while standing, most likely due to increased muscle activity of the lower extremities.

Probing the Electrocatalytic Oxygen Reduction Reaction Reactivity of Immobilized Multicopper Oxidase CueO

The bioelectrocatalytic (oxygen reduction reaction, ORR) properties of the multicopper oxidase CueO immobilized on gold electrodes were investigated. Macroscopic electrochemical techniques were combined with in situ scanning tunneling microscopy (STM) and surface-enhanced Raman spectroscopy at the ensemble and at the single-molecule level. Self-assembled monolayer of mercaptopropionic acid, cysteamine, and p-aminothiophenol were chosen as redox mediators. The highest ORR activity was observed for the protein attached to amino-terminated adlayers. In situ STM experiments revealed that the presence of oxygen causes distinct structure and electronic changes in the metallic centers of the enzyme, which determine the rate of intramolecular electron transfer and, consequently, affect the rate of electron tunneling through the protein. Complementary Raman spectroscopy experiments provided access for monitoring structural changes in the redox state of the type 1 copper center of the immobilized enzyme during the CueO-catalyzed oxygen reduction cycle. These results unequivocally demonstrate the existence of a direct electronic communication between the electrode substrate and the type 1 copper center.

Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

Experimental dissolution of molybdenum-sulphides at low oxygen concentrations: A first-order approximation of late Archean atmospheric conditions

The abundance of atmospheric oxygen and its evolution through Earth's history is a highly debated topic. The earliest change of the Mo concentration and isotope composition of marine sediments are interpreted to be linked to the onset of the accumulation of free O2 in Earth's atmosphere. The O2 concentration needed to dissolve significant amounts of Mo in water is not yet quantified, however. We present laboratory experiments on pulverized and surface-cleaned molybdenite (MoS2) and a hydrothermal breccia enriched in Mo-bearing sulphides using a glove box setup. Duration of an experiment was 14 days, and first signs of oxidation and subsequent dissolution of Mo compounds start to occur above an atmospheric oxygen concentration of 72 ± 20 ppmv (i.e., 2.6 to 4.6 × 10−4 present atmospheric level (PAL)). This experimentally determined value coincides with published model calculations supporting atmospheric O2 concentrations between 1 × 10−5 to 3 × 10−4 PAL prior to the Great Oxidation Event and sets an upper limit to the molecular oxygen needed to trigger Mo accumulation and Mo isotope variations recorded in sediments. In combination with the published Mo isotope composition of the rock record, this result implies an atmospheric oxygen concentration prior to 2.76 Ga of below 72 ± 20 ppmv.

Genome profiling of sterol synthesis shows convergent evolution in parasites and guides chemotherapeutic attack

Sterols are an essential class of lipids in eukaryotes, where they serve as structural components of membranes and play important roles as signaling molecules. Sterols are also of high pharmacological significance: cholesterol-lowering drugs are blockbusters in human health, and inhibitors of ergosterol biosynthesis are widely used as antifungals. Inhibitors of ergosterol synthesis are also being developed for Chagas's disease, caused by Trypanosoma cruzi. Here we develop an in silico pipeline to globally evaluate sterol metabolism and perform comparative genomics. We generate a library of hidden Markov model-based profiles for 42 sterol biosynthetic enzymes, which allows expressing the genomic makeup of a given species as a numerical vector. Hierarchical clustering of these vectors functionally groups eukaryote proteomes and reveals convergent evolution, in particular metabolic reduction in obligate endoparasites. We experimentally explore sterol metabolism by testing a set of sterol biosynthesis inhibitors against trypanosomatids, Plasmodium falciparum, Giardia, and mammalian cells, and by quantifying the expression levels of sterol biosynthetic genes during the different life stages of T. cruzi and Trypanosoma brucei. The phenotypic data correlate with genomic makeup for simvastatin, which showed activity against trypanosomatids. Other findings, such as the activity of terbinafine against Giardia, are not in agreement with the genotypic profile.

Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered hydrolysis

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. (1) In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered hydrolysis

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. (1) In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

Sublimation of water ice mixed with silicates and tholins: Evolution of surface texture and reflectance spectra, with implications for comets

The surfaces of many objects in the Solar System comprise substantial quantities of water ice sometimes mixed with minerals and/or organic molecules. The sublimation of the ice changes the structural and optical properties of these objects. We present laboratory data on the evolution of the structure and the visible and near-infrared spectral reflectance of icy surface analogues of cometary ices, made of water ice, complex organic matter (tholins) and silicates, as they undergo sublimation under low temperature (<-70°C) and pressure (10-⁵mbar) conditions inside the SCITEAS simulation chamber. As the water ice sublimated, we observed in situ the formation of a porous sublimation lag deposit, or sublimation mantle, at the top of the ice. This mantle is a network of filaments made of the non-volatile particles. Organics or phyllosilicates grains, able to interact via stronger inter-particulate forces than olivine grains, can form a foam-like structure having internal cohesiveness, holding olivine grains together. As this mantle builds-up, the band depths of the sub-surface water ice are attenuated until complete extinction under only few millimeters of mantle. Optically thick sublimation mantles are mainly featureless in the near infrared. The absorption bands of the minerals present in the mantle are weak, or even totally absent if minerals are mixed with organics which largely dominate the VIS–NIR reflectance spectrum. During sublimation, ejections of large fragments of mantle, triggered by the gas flow, expose ice particles to the surface. The contrast of brightness between mantled and ice-exposed areas depends on the wavelength range and the dust/ice ratio considered. We describe how the chemical nature of the non-volatiles, the size of their particles, the way they are mixed with the ice and the dust/ice mass ratio influence the texture, activity and spectro-photometric properties of the sublimation mantles. These data provide useful references for interpreting remote-sensing observations of comets and also icy satellites or trans-neptunian objects.

Dating prograde fluid pulses during subduction by in situ U–Pb and oxygen isotope analysis

Keywords High-pressure fluids · Whiteschists · U–Pb dating · Oxygen isotopes · Ion microprobe · Metasomatism Introduction The subduction of crustal material to mantle depths and its chemical modification during burial and exhumation contribute to element recycling in the mantle and the formation of new crust through arc magmatism. Crustal rocks that Abstract The Dora-Maira whiteschists derive from metasomatically altered granites that experienced ultrahighpressure metamorphism at ~750 °C and 40 kbar during the Alpine orogeny. In order to investigate the P–T–time– fluid evolution of the whiteschists, we obtained U–Pb ages from zircon and monazite and combined those with trace element composition and oxygen isotopes of the accessory minerals and coexisting garnet. Zircon cores are the only remnants of the granitic protolith and still preserve a Permian age, magmatic trace element compositions and δ18O of ~10 ‰. Thermodynamic modelling of Si-rich and Si-poor whiteschist compositions shows that there are two main fluid pulses during prograde subduction between 20 and 40 kbar. In Si-poor samples, the breakdown of chlorite to garnet + fluid occurs at ~22 kbar. A first zircon rim directly overgrowing the cores has inclusions of prograde phlogopite and HREE-enriched patterns indicating zircon growth at the onset of garnet formation. A second main fluid pulse is documented close to peak metamorphic conditions in both Si-rich and Si-poor whiteschist when talc + kyanite react to garnet + coesite + fluid. A second metamorphic overgrowth on zircon with HREE depletion was observed in the Si-poor whiteschists, whereas a single metamorphic overgrowth capturing phengite and talc inclusions was observed in the Si-rich whiteschists. Garnet rims, zircon rims and monazite are in chemical and isotopic equilibrium for oxygen, demonstrating that they all formed at peak metamorphism at 35 Ma as constrained by the age of monazite (34.7 ± 0.4 Ma) and zircon rims (35.1 ± 0.8 Ma). The prograde zircon rim in Si-poor whiteschists has an age that is within error indistinguishable from the age of peak metamorphic conditions, consistent with a minimum rate of subduction of 2 cm/year for the Dora-Maira unit. Oxygen isotope values for zircon rims, monazite and garnet are equal within error at 6.4 ± 0.4 ‰, which is in line with closed-system equilibrium fractionation during prograde to peak temperatures. The resulting equilibrium Δ18Ozircon-monazite at 700 ± 20 °C is 0.1 ± 0.7 ‰. The in situ oxygen isotope data argue against an externally derived input of fluids into the whiteschists. Instead, fluidassisted zircon and monazite recrystallisation can be linked to internal dehydration reactions during prograde subduction. We propose that the major metasomatic event affecting the granite protolith was related to hydrothermal seafloor alteration post-dating Jurassic rifting, well before the onset of Alpine subduction.

Constraints on the thermal evolution of the Adriatic margin during Jurassic continental break-up: U–Pb dating of rutile from the Ivrea–Verbano Zone, Italy

The Ivrea–Verbano Zone (IVZ), northern Italy, exposes an attenuated section through the Permian lower crust that records high-temperature metamorphism under lower crustal conditions and a protracted history of extension and exhumation associated partly with the Jurassic opening of the Alpine Tethys ocean. This study presents SHRIMP U–Pb geochronology of rutile from seven granulite facies metapelites from the base of the IVZ, collected from locations spanning ~35 km along the strike of Paleozoic fabrics. Rutile crystallised during Permian high-temperature metamorphism and anatexis, yet all samples give Jurassic rutile U–Pb ages that record cooling through 650–550 °C. Rutile age distributions are dominated by a peak at ~160 Ma, with a subordinate peak at ~175 Ma. Both ~160 and ~175 Ma age populations show excellent agreement between samples, indicating that the two distinctive cooling stages they record were synchronous on a regional scale. The ~175 Ma population is interpreted to record cooling in the footwall of rift-related faults and shear zones, for which widespread activity in the Lower Jurassic has been documented along the western margin of the Adriatic plate. The ~160 Ma age population postdates the activity of all known rift-related structures within the Adriatic margin, but coincides with extensive gabbroic magmatism and exhumation of sub-continental mantle to the floor of the Alpine Tethys, west of the Ivrea Zone. We propose that this ~160 Ma early post-rift age population records regional cooling following episodic heating of the distal Adriatic margin, likely related to extreme lithospheric thinning and associated advection of the asthenosphere to shallow levels. The partial preservation of the ~175 Ma age cluster suggests that the post-rift (~160 Ma) heating pulse was of short duration. The regional consistency of the data presented here, which is in contrast to many other thermochronometers in the IVZ, demonstrates the value of the rutile U–Pb technique for probing the thermal evolution of high-grade metamorphic terrains. In the IVZ, a significant decoupling between Zr-in-rutile temperatures and U–Pb ages of rutile is observed, with the two systems recording events ~120 Ma apart.