In situ continuous monitoring of dissolved gases (N2, O2, CO2, H2) prior to H2 injection in an aquifer (Catenoy, France) by on-site Raman and infrared spectroscopies: instrumental assessment and geochemical baseline establishment.

The establishment of a baseline of gases from an aquifer appears to be an essential prerequisite for monitoring and securing underground storage operations such as the storage of carbon dioxide (carbon capture and storage: CCS), methane or hydrogen. This study describes an innovative metrological technique dedicated to the in situ and continuous quantification of dissolved gases (CO2, O2, N2, CH4 and H2) in a shallow aquifer, on the site of Catenoy (Paris Basin) with a water table at a depth of 13 m. Monitoring was carried out from May 7, 2019 to November 19, 2019, before the simulation of H2 injection. Gases as vapors were collected from the aquifer through a nine-meter long, half-permeable polymer membrane positioned below a packer in a 25-meter deep well. Collected gases were analyzed simultaneously at the surface by fiber Raman (CO2, O2, N2, CH4 and H2) and infrared sensors (CO2). Gas concentrations were determined from Raman and infrared data, and then converted into dissolved concentrations using Henry's law. The dissolved gas concentrations were about constant over the 6 months period with average values of 31-40 mg L-1 (CO2), 8 mg L-1 (O2), 17 mg L-1 (N2), and 0 mg L-1 (H2, CH4) indicating a very low variability in the aquifer. This is believed to allow for rapid detection of any possible abnormal concentration variation, in particular linked to an accidental arrival of gases such as hydrogen. Such an online gas measurement system can be deployed as is on any site type of underground storage without any need for adaptation.

Folic acid supplementation during the preconception period: A systematic review and meta-analysis.

Guidelines recommend that women take folic acid supplements in the preconception period to prevent neural tube defects (NTDs) in their offspring. Estimates of adherence to this recommendation across different countries worldwide have not been synthesized. Medline, CINAHL, and EMBASE were systematically searched to identify studies reporting the prevalence of preconception folic acid supplementation. Pooled prevalence estimates for each country (where data were available) were calculated; and differences based on demographic, methodological, and study quality characteristics were examined. Of 3372 titles and abstracts screened, 722 full-texts were reviewed and 105 articles that reported 106 estimates of preconception folic acid supplementation in 34 countries were included. Pooled prevalence estimates were 32-51% in North America, 9-78% in Europe, 21-46% in Asia, 4-34% in the Middle East, 32-39% in Australia/New Zealand, and 0% in Africa. No South American studies were identified. Higher supplementation prevalence was observed in studies that had more highly educated samples, were conducted in fertility clinics, and assessed folic acid use via self-report. Of note, only 32% and 28% of studies reported timing of folic acid use and adherence to folic acid, respectively. Preconception folic acid supplementation is highly variable worldwide and many women may not achieve sufficient folate levels to prevent NTDs. To better understand non-adherence, recommendations for future research include: more explicit reporting of methodology, more detailed assessment of folic acid use, assessment of variables potentially relevant to folic acid use, and surveillance of folic acid use in a greater diversity of countries, especially in the developing world.

Fuel poverty as a major determinant of perceived health: the case of France.

OBJECTIVES: The number of households in fuel poverty is growing. Individuals increasingly struggle to heat their homes, and therefore, a growing number of individuals are exposed to low temperatures, which can affect their health. This study sought to determine the link between a subjective measure of fuel poverty (self-reported feeling cold) and self-reported health. The impact of other particular individual and environmental features on self-reported health were also analysed. STUDY DESIGN: Econometric analysis. METHODS: The study method uses self-reported perception of thermal discomfort (self-reported feeling cold) as a proxy for fuel poverty. The French database of the Healthcare and Insurance survey carried by the Institute for Research and Information on Health Economics (IRDES) was used to estimate a dichotomous probit model. RESULTS: The estimation allows us to infer a negative impact of fuel poverty on self-reported health. Thus, a person in fuel poverty is 2.36 percentage points more likely to report poor or fair health status than a person who is not in fuel poverty. CONCLUSION: It may be appropriate to reduce the impacts of fuel poverty to provide support for the most vulnerable categories of individuals with respect to the health impacts of fuel poverty and cold homes, e.g., chronic patients who experience difficulty heating their homes.

Biostimulation and bioaugmentation for on-site treatment of weathered diesel fuel in Arctic soil.

Bioremediation of weathered diesel fuel in Arctic soil at low temperature was studied both on-site in small-scale biopiles and in laboratory microcosms. The field study site was on Ellesmere Island (82 degrees 30'N, 62 degrees 20'W). Biostimulation was by fertilization with phosphorous and nitrogen. Bioaugmentation was with an enrichment culture originating from the field site. In biopiles, total petroleum hydrocarbons (TPH) were reduced from 2.9 to 0.5 mg/g of dry soil over a period of 65 days. In microcosms at 7 degrees C, TPH were reduced from 2.4 to 0.5 mg/g of dry soil over a period of 90 days. Inoculation had no effect on hydrocarbon removal in biopiles or in microcosms. Maximum TPH removal rates in the biopiles were approximately 90 micro g of TPH g(-1) of soil day(-1), occurring during the first 14 days when ambient temperature ranged from 0 to 10 degrees C. The fate of three phylotypes present in the inoculum was monitored using most-probable-number PCR, targeting 16S rRNA genes. Populations of all three phylotypes increased more than 100-fold during incubation of both uninoculated and inoculated biopiles. The inoculum increased the initial populations of the phylotypes but did not significantly affect their final populations. Thus, biostimulation on site enriched populations that were also selected in laboratory enrichment cultures.

Computer-assisted re-design of spectrin SH3 residue clusters.

We have developed a protein design computer program, called Perla, which performs searches in sequence space to uncover optimal amino acid sequences for desired protein three-dimensional structures. Optimal sequences are localised at the minima of a sequence-structure energy landscape defined using a complex scoring function (an all-atom molecular mechanics force field plus statistical terms including entropy and solvation) measured with respect to a reference state simulating a denatured protein. Sequence choices eventually optimise side chain packing, secondary structure propensities, and hydrogen bonding and electrostatics interactions. Perla was used to re-design clusters of residues of the SH3 domain of alpha-spectrin. Several mutant proteins were produced and characterised. Some of our designed proteins have significantly higher stabilities (stability enhancements about 0.25, 0.70 and 1.0 kcal mol(-1)) than the wild-type protein. These successful protein re-designs, and similar examples found in the literature, establish the quality of the structure-based computational approach to protein design.

Computer-aided design of beta-sheet peptides.

The design of beta-sheet proteins is still a challenge in the field of de novo protein design. Here, we have tested the validity of automatic design methods to create and/or improve beta-sheet peptides and proteins. We chose Betanova, a three-stranded beta-sheet peptide, as target system, and, as an automatic design tool, a protein design algorithm called PERLA (protein engineering rotamer library algorithm). PERLA was used to define both stabilising and destabilising single- and multiple-residue mutations of Betanova. Conformational analysis by NMR spectroscopy and far-UV circular dichroism (CD) allowed us to evaluate population differences among the set of designed peptides. Some of the new mutants are approximately 1 kcal/mol more stable than the wild-type peptide. Comparison of the scale of predicted and observed stabilities demonstrates that they are in good agreement for most peptides studied. Our results show that automatic design algorithms can be successfully applied to the design of beta-sheet peptides.

Multifunctionalized alpha,beta-cyclopentenones from C-2 and C-4-ulopyranosyl compounds: a stereospecific rearrangement initiated by base.

Base treatment of O-benzyl protected C-2- or C-4-ulopyranosyl compounds (4 alpha, 4 beta, and 11) by either 10% Et(3)N or 1% K(2)CO(3) in MeOH initiated a beta elimination to afford alpha,beta-unsaturated C-ulopyranosyl compounds (5 alpha, 5 beta, and 12), which further rearranged in a stereocontrolled manner to multifuctionalized alpha,beta-cyclopentenones (6 and 14) in 70-80% yield. Both C-alpha- and C-beta-2-ulosides (5 alpha and 5 beta) produced the same cyclopentenone 6, indicating that a 1,2-enolate is formed prior to the cleavage of the C-5--O bond. Because 6 is racemic, it was probably formed by the intramolecular cycloaldolization of two equally populated enantiomeric intermediates. When treated with 90% Et(3)N in MeOH, 5 alpha yielded almost exclusively 15 (isomer of 6), which was formed by a migration of the double bond in 5 alpha during the previously described rearrangement. Thus either 6 or 15 was the major product, depending on the base used.

Computational estimation of specific side chain interaction energies in alpha helices.

We have used a structure energy-based computer program developed for protein design, Perla, to provide theoretical estimates of all specific side chain-side chain interaction energies occurring in alpha helices. The computed side chain-side chain interaction energies were used as substitutes for the corresponding values used by the helix/coil transition algorithm, AGADIR. Predictions of peptide helical contents were nearly as successful as those obtained with the originally calibrated set of parameters; a correlation to experimentally observed alpha-helical populations of 0.91 proved that our theoretical estimates are reasonably correct for amino acid pairs that are frequent in our database of peptides. Furthermore, we have determined experimentally the previously uncharacterized interaction energies for Lys-Ile, Thr-Ile, and Phe-Ile amino acid pairs at i,i + 4 positions. The experimental values compare favorably with the computed theoretical estimates. Importantly, the computed values for Thr-Ile and Phe-Ile interactions are better than the energies based on chemical similarity, whereas for Lys-Ile they are similar. Thus, computational techniques can be used to provide precise energies for amino acid pairwise interactions, a fact that supports the development of structure energy-based computational tools for structure predictions and sequence design.

DNA-based and culture-based characterization of a hydrocarbon-degrading consortium enriched from Arctic soil.

A hydrocarbon-degrading consortium was enriched from fuel-contaminated soil from the northeastern tip of Ellesmere Island (82 degrees 30'N, 62 degrees 19'W). The enrichment culture was grown on Jet A-1 fuel at 7 degrees C. Bacterial 16S RNA gene (rDNA) fragments were amplified by polymerase chain reaction (PCR) from members of the above consortium and cloned into a plasmid vector. Partial sequences (approximately 500 bp) were determined for 29 randomly selected rDNA clones. The majority of sequences were most similar to the corresponding rDNA sequences of Rhodococcus erythropolis (15 sequences), Sphingomonas spp. (six sequences), and Pseudomonas synxantha (four sequences). Amplified ribosomal DNA restriction analysis confirmed that a larger set of 50 clones had frequencies of the three phylotypes similar to those above. Phylotype-specific PCR assays were developed and validated for the above three phylotypes. The consortium was plated and grown on Jet A-1 fuel vapors, and randomly selected isolated colonies were screened with the above PCR assays. Of 17 colonies, six matched the Rhodococcus phylotype, and three matched the Pseudomonas phylotype. A representative strain of each phylotype was physiologically characterized. Both isolates grew on alkanes at low temperature and had general characteristics consistent with their respective phylotypes. During growth of the consortium, the three phylotype populations were monitored by a most probable number PCR assay. All three phylotypes were detected, but their relative abundance was not consistent with that of the phylotypes in the clone library. The relative abundance of all three phylotypes changed substantially during long-term incubation of the consortium. The DNA-based approach used identified phylotypes consistently present in the consortium, but it failed to predict the relative abundance of their populations.

Towards understanding a molecular switch mechanism: thermodynamic and crystallographic studies of the signal transduction protein CheY.

The signal transduction protein CheY displays an alpha/beta-parallel polypeptide folding, including a highly unstable helix alpha4 and a strongly charged active site. Helix alpha4 has been shown to adopt various positions and conformations in different crystal structures, suggesting that it is a mobile segment. Furthermore, the instability of this helix is believed to have functional significance because it is involved in protein-protein contacts with the transmitter protein kinase CheA, the target protein FliM and the phosphatase CheZ. The active site of CheY comprises a cluster of three aspartic acid residues and a lysine residue, all of which participate in the binding of the Mg(2+) needed for the protein activation. Two steps were followed to study the activation mechanism of CheY upon phosphorylation: first, we independently substituted the three aspartic acid residues in the active site with alanine; second, several mutations were designed in helix alpha 4, both to increase its level of stability and to improve its packing against the protein core. The structural and thermodynamic analysis of these mutant proteins provides further evidence of the connection between the active-site area and helix alpha 4, and helps to understand how small movements at the active site are transmitted and amplified to the protein surface.

MEDLINEplus: building and maintaining the National Library of Medicine's consumer health Web service.

MEDLINEplus is a Web-based consumer health information resource, made available by the National Library of Medicine (NLM). MEDLINEplus has been designed to provide consumers with a well-organized, selective Web site facilitating access to reliable full-text health information. In addition to full-text resources, MEDLINEplus directs consumers to dictionaries, organizations, directories, libraries, and clearinghouses for answers to health questions. For each health topic, MEDLINEplus includes a preformulated MEDLINE search created by librarians. The site has been designed to match consumer language to medical terminology. NLM has used advances in database and Web technologies to build and maintain MEDLINEplus, allowing health sciences librarians to contribute remotely to the resource. This article describes the development and implementation of MEDLINEplus, its supporting technology, and plans for future development.

The design of linear peptides that fold as monomeric beta-sheet structures.

Current knowledge about the determinants of beta-sheet formation has been notably improved by the structural and kinetic analysis of model peptides, by mutagenesis experiments in proteins and by the statistical analysis of the protein structure database (Protein Data Bank; PDB). In the past year, several peptides comprising natural and non-natural amino acids have been designed to fold as monomeric three-stranded beta-sheets. In all these cases, the design strategy has involved both the statistical analysis of the protein structure database and empirical information obtained in model beta-hairpin systems and in proteins. Only in one case was rotamer analysis performed to check for the compatibility of the sidechain packing. It is foreseeable that, in future designs, algorithms exploring the sequence and conformational space will be employed. For the design of small proteins (less than 30 amino acids), questions remain about the demonstration of two-state behavior, the formation of a well-defined network of mainchain hydrogen bonds and the quantification of the structured populations.

Clopidogrel and drug metabolism: absence of effect on hepatic enzymes in healthy volunteers.

The influence of clopidogrel 75 mg, given once daily for 10 days on hepatic P-450 mixed function oxidases, was examined by assessing its effect on the disposition of antipyrine, on urinary 6-betahydroxycortisol (6beta-OHC) and on the plasma activity of gamma-glutamyl transpeptidase. This double-blind, randomized, placebo-controlled study was conducted in two parallel groups of 10 healthy young volunteers. Subjects were required to fast for 12 hours before and for 4 hours after dosing. Antipyrine 10 mg/kg was administered in the morning, two days before treatment (day -2) and 24 hours after the last dose of clopidogrel or placebo. Plasma levels of antipyrine, and urinary excretion of antipyrine, 3-hydroxymethyl-antipyrine and nor-antipyrine were measured over 36 hours post-drug for pharmacokinetic determinations. Bleeding time and platelet aggregation induced by 5 microM of ADP were measured before treatment (baseline) and at regular intervals after dosing during treatment. Clopidogrel treatment had a marked effect on platelet aggregation and bleeding time. No significant change in the disposition of antipyrine was observed after the ingestion of clopidogrel over 10 days: mean AUC ratio (+/-SEM) for plasma antipyrine was 1.021+/-0.023 for the clopidogrel group versus 1.001+/-0.019 for the placebo group; mean day 10/day -2 t 1/2 ratios were 1.019+/-0.018 and 1.027+/-0.023, respectively. Urinary excretions of antipyrine and metabolites were unchanged by clopidogrel compared to placebo. The changes in plasma cortisol concentrations, 6beta-OHC excretion and serum gamma-glutamyl transpeptidase activities observed at the end of treatment were fully comparable between the two treatment groups. Thus, the different tests showed no evidence of hepatic enzyme induction by clopidogrel in a pharmacologically effective dose regimen.

Clopidogrel antiplatelet activity is independent of age and presence of atherosclerosis.

The pharmacodynamic and pharmacokinetic effects of clopidogrel 75 mg taken once daily in the morning before breakfast for 10 days were compared among three groups: 12 healthy young subjects, 10 healthy elderly subjects (>65 years) and 10 otherwise healthy elderly subjects with atherosclerosis, manifested by intermittent claudication. Platelet aggregation induced by 5 microM of ADP was measured in plasma samples taken at screening, 2 hours after dosing on day 1 to day 9; 2, 5, and 24 hours after dosing on day 10; and on alternate days until day 24. The inhibition of platelet aggregation was expressed as the percent reduction in maximum platelet aggregation with respect to baseline. The bleeding time was measured at screening, 5 hours after dosing on day 10, and on day 18. Plasma concentrations of SR26334, the main circulating metabolite of clopidogrel, were determined before dosing on day 1 to day 10 and at regular intervals over 72 hours after dosing on day 10. Inhibition of platelet aggregation appeared 2 hours after the first dose, became significant after the second dose, and progressed to a steady-state value of 55 to 57% by day 7 in the three groups, with no statistically significant difference between groups. A moderate, statistically significant prolongation of bleeding time of similar extent (prolongation factor of 1.5 to 1.6) was found on day 10 in the three groups. The pharmacodynamic parameters generally returned to baseline within 8 days after treatment. Based on AUC(0-24th) values, drug exposures were very similar for the two groups of elderly subjects but approximately twice that for the young group. The pharmacodynamic effects of clopidogrel were comparable in all three groups.

[Comparative study of the ideas about causes, disease mechanisms and therapies of the plague on the basis of the plague treatises of the Medical Faculty of Paris (1348-1349), of Joannes de Vesalia (after 1454) and of Thomas Montanus (1669)]. / Vergelijkende studie van de opvattingen omtrent oorzaken, ziektemechanismen en therapieën van pest op basis van de pesttraktaten van de medische faculteit te Parijs (1348-1349), van Joannes de Vesalia (na 1454), en van Thomas Montanus (1669).

The comparison between the consilium of the Faculty of Paris (14th century), and the treatises by Joannes de Vesalia (15th century) and by Thomas Montanus (17th century) shows that the concepts with regard to the causes and the mechanisms of and the proposed preventive and curative measures against the plague did in essence not change for over three centuries. In fact this is not very surprising, since the development of modern physiology and physiopathology only started gradually in the second half of the 19th century. Furthermore, the plague bacillus has only been identified in 1894. However, the preventive and curative measures against plague that are proposed in the three treatises, do not result from an uncontrolled imagination. Most of these measures have a rational basis and are the result of--although erroneous--concepts with regard to the causes and mechanisms of the disease.

Elucidating the folding problem of alpha-helices: local motifs, long-range electrostatics, ionic-strength dependence and prediction of NMR parameters.

The information about the conformational behavior of monomeric helical peptides in solution, as well as the alpha-helix stability in proteins, has been previously utilized to derive a database with the energy contributions for various interactions taking place in an alpha-helix: intrinsic helical propensities, side-chain-side-chain interactions, main-chain-main-chain hydrogen bonds, and capping effects. This database was implemented in an algorithm based on the helix/coil transition theory (AGADIR). Here, we have modified this algorithm to include previously described local motifs: hydrophobic staple, Schellman motif and Pro-capping motif, new variants of these, and newly described side-chain-side-chain interactions. Based on recent experimental data we have introduced a position dependence of the helical propensities for some of the 20 amino acid residues. A new electrostatic model that takes into consideration all electrostatic interactions up to 12 residues in distance in the helix and random-coil conformations, as well as the effect of ionic strength, has been implemented. We have synthesized and analyzed several peptides, and used data from peptides already analysed by other groups, to test the validity of our electrostatic model. The modified algorithm predicts, with an overall standard deviation value of 6.6 (maximum helix is 100%), the helical, content of 778 peptides of which 223 correspond to wild-type and modified protein fragments. To improve the prediction potential of the algorithm and to have a direct comparison with nuclear magnetic resonance data, the algorithm now predicts the conformational shift of the CalphaH protons, 13Calpha and 3JalphaN values. We have found that for those peptides correctly predicted from the point of view of circular dichroism, the prediction of the NMR parameters is very good.

Reading protein sequences backwards.

BACKGROUND: Reading a protein sequence backwards provides a new polypeptide that does not align with its parent sequence. The foldability of this new sequence is questionable. On one hand, structure prediction at low resolution using lattice simulations for such a protein provided a model close to the native parent fold or to a topological mirror image of it. On the other hand, there is no experimental evidence yet to tell whether such a retro protein folds (and to which structure) or not. RESULTS: In this work, we have analysed the possibility of a retro protein folding in two different ways. First, we modelled the retro sequence of the alpha-spectrin SH3 domain through distance geometry and molecular dynamics. This contradicted the plausibility of a mirror image of the native domain, whereas basic considerations opposed the likelihood of the native fold. Second, we obtained experimental evidence that the retro sequences of the SH3 domain, as well as the B domain of Staphylococcal protein A and the B1 domain of Streptococcal protein G, are unfolded proteins, even though some propensities for the formation of secondary structures might remain. CONCLUSIONS: Retro proteins are no more similar to their parent sequences than any random sequence despite their common hydrophobic/hydrophilic pattern, global amino acid composition and possible tertiary contacts. Although simple folding models contribute to our global understanding of protein folding, they cannot yet be used to predict the structure of new proteins.

Pre-exercise branched-chain amino acid administration increases endurance performance in rats.

This study investigated the effects of pre-exercise branched-chain amino acid (BCAA) administration on blood ammonia levels and on time to exhaustion during treadmill exercise in rats. Adult female Wistar rats were trained on a motor driven treadmill. After a 24-h fast, rats were injected intraperitoneally (i.p.) with 1 mL of placebo or BCAA (30 mg), 5 min before performing 30 min of submaximal exercise (N = 18) or running to exhaustion (N = 12). In both cases, rats were sacrificed immediately following exercise, and blood was collected for the measurement of glucose, nonesterified fatty acid (NEFA), lactic acid, BCAA, ammonia, and free-tryptophan (free-TRP) levels. Control values were obtained from sedentary rats that were subjected to identical treatments and procedures (N = 30). Plasma BCAA levels increased threefold within 5 min after BCAA administration. Mean run time to exhaustion was significantly longer (P < 0.01) after BCAA administration (99 +/- 9 min) compared with placebo (76 +/- 4 min). During exercise, blood ammonia levels were significantly higher (P < 0.01) in the BCAA treated compared with those in the placebo treated rats both in the 30-min exercise bout (113 +/- 25 mumol.L-1 (BCAA) vs 89 +/- 16 mumol.L-1) and following exercise to exhaustion (186 +/- 44 mumol.L-1 (BCAA) vs 123 +/- 19 mumol.L-1). These data demonstrate that BCAA administration in rats results in enhanced endurance performance and an increase in blood ammonia during exercise.

Amide hydrogen exchange and internal dynamics in the chemotactic protein CheY from Escherichia coli.

The backbone internal dynamics of the wild-type 129 amino acid alpha/beta parallel protein CheY and its double mutant F14N/P110G are analysed here by the hydrogen-exchange method. The F14N mutation is known to stabilise the protein and to accelerate refolding while P110G is destabilising and accelerates unfolding. We first assigned and characterised the double mutant by nuclear magnetic resonance (NMR), to try and discover any possible conformational change induced by the two mutations. The main difference between the two proteins is a favourable N-capping interaction of the newly introduced Asn14 side-chain at the beginning of the first alpha-helix (alpha-helix A). Second, we have measured the exchange rates in the wild-type and mutant CheY. In the first case the observed protection factors are slightly dispersed around an average value. According to their distribution in the structure, protein stability is highest on one face of the central beta-sheet, in the surroundings of the main hydrophobic core formed by side-chains of residues in beta-strands I, II and III and helices A and E. The mutations in the double mutant protein affect two distinct subdomains differently (from beta-strand I to III and from alpha-helix C to the end). In the second subdomain the number of protected protons is reduced with respect to those in the wild-type. This differential behaviour can be explained by a selective decrease in stability of the second folding subdomain produced by the P110G mutation and the opposite effect in the first subdomain, produced by the F14N mutation. alpha-Helix A, which is involved together with beta-strands I and III in the folding nucleus of CheY, shows the largest protection factors in both proteins.