Role of gluconic acid production in the regulation of biocontrol traits of Pseudomonas fluorescens CHA0.

The rhizobacterium Pseudomonas fluorescens CHA0 promotes the growth of various crop plants and protects them against root diseases caused by pathogenic fungi. The main mechanism of disease suppression by this strain is the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). Direct plant growth promotion can be achieved through solubilization of inorganic phosphates by the production of organic acids, mainly gluconic acid, which is one of the principal acids produced by Pseudomonas spp. The aim of this study was to elucidate the role of gluconic acid production in CHA0. Therefore, mutants were created with deletions in the genes encoding glucose dehydrogenase (gcd) and gluconate dehydrogenase (gad), required for the conversion of glucose to gluconic acid and gluconic acid to 2-ketogluconate, respectively. These enzymes should be of predominant importance for rhizosphere-colonizing biocontrol bacteria, as major carbon sources provided by plant root exudates are made up of glucose. Our results show that the ability of strain CHA0 to acidify its environment and to solubilize mineral phosphate is strongly dependent on its ability to produce gluconic acid. Moreover, we provide evidence that the formation of gluconic acid by CHA0 completely inhibits the production of PLT and partially inhibits that of DAPG. In the Deltagcd mutant, which does not produce gluconic acid, the enhanced production of antifungal compounds was associated with improved biocontrol activity against take-all disease of wheat, caused by Gaeumannomyces graminis var. tritici. This study provides new evidence for a close association of gluconic acid metabolism with antifungal compound production and biocontrol activity in P. fluorescens CHA0.

Microbiological and biochemical properties of an agricultural Mexican soil amended with sewage sludge

The application of sewage sludge is a concern because it may affect the quality of organic matter and microbiological and biochemical soil properties. The effects of surface application of sewage sludge to an agricultural soil (at 18 and 36 t ha-1 dry basis) were assessed in one maize (Zea mays L.) growing season. The study evaluated microbial biomass, basal respiration and selected enzymatic activities (catalase, urease, acid and alkaline phosphatase, and β-glucosidase) 230 days after sewage sludge application and infrared spectroscopy was used to assess the quality of dissolved organic matter and humic acids. Sewage sludge applications increased the band intensity assigned to polysaccharides, carboxylic acids, amides and lignin groups in the soil. The organic matter from the sewage sludge had a significant influence on the soil microbial biomass; nevertheless, at the end of the experiment the equilibrium of the soil microbial biomass (defined as microbial metabolic quotient, qCO2) was recovered. Soil urease, acid and alkaline phosphatase activity were strongly influenced by sewage sludge applications.

Extractants to assess zinc phytoavailability in mineral fertilizer and industrial by-products

Efficient analytical methods for the quantification of plant-available Zn contained in mineral fertilizers and industrial by-products are fundamental for the control and marketing of these inputs. In this sense, there are some doubts on the part of the scientific community as well as of the fertilizer production sector, whether the extractor requested by the government (Normative Instruction No. 28, called 2nd extractor), which is citric acid 2 % (2 % CA) (Brasil, 2007b), is effective in predicting the plant availability of Zn via mineral fertilizers and about the agronomic significance of the required minimal solubility of 60 % compared to the total content (HCl) (Brasil, 2007a). The purpose of this study was to evaluate the alternative extractors DTPA, EDTA, neutral ammonium citrate (NAC), buffer solution pH 6.0, 10 % HCl, 10 % sulfuric acid, 1 % acetic acid, water, and hot water to quantify the contents of Zn available for maize and compare them with indices of agronomic efficiency of fertilizers and industrial by-products when applied to dystrophic Clayey Red Latosol and Dystrophic Alic Red Yellow Latosol with medium texture. The rate of Zn applied to the soil was 5 mg kg-1, using the sources zinc sulfate, commercial granular zinc, ash and galvanic sludge, ash and two brass slags. Most Zn was extracted from the sources by DTPA, 10 % HCl, NAC, 1% acetic acid, and 10 % sulfuric acid. Recovery by the extractors 2 % CA, EDTA, water, and hot water was low. The agronomic efficiency index was found to be high when using galvanic sludge (238 %) and commercial granular zinc (142 %) and lower with brass slag I and II (67 and 27 %, respectively). The sources galvanizing ash and brass ash showed solubility lower than 60 % in 2 % CA, despite agronomic efficiency indices of 78 and 125 %, respectively. The low agronomic efficiency index of industrial by-products such as brass slag I and galvanizing ash can be compensated by higher doses, provided there is no restriction, as well as for all other sources, in terms of contaminant levels of arsenic, cadmium, chromium, lead, and mercury as required by law (Normative Instruction No 27/2006). The implementation of 2nd extractor 2 % CA and the requirement of minimum solubility for industrial by-products could restrict the use of alternative sources as potential Zn sources for plants.

The role of fungi in the precipitation of calcite : relationships between fungal filaments, nanofibres, and needle fibre calcite

RésuméLes champignons sont impliqués dans les cycles biogéochimiques de différentes manières. En particulier, ils sont reconnus en tant qu'acteurs clés dans la dégradation de la matière organique, comme fournisseurs d'éléments nutritifs via l'altération des minéraux mais aussi comme grands producteurs d'acide oxalique et de complexes oxalo-métalliques. Toutefois, peu de choses sont connues quant à leur contribution à la genèse d'autres types de minéraux, tel que le carbonate de calcium (CaCO3). Le CaCO3 est un minéral ubiquiste dans de nombreux écosystèmes et il joue un rôle essentiel dans les cycles biogéochimiques du carbone (C) et du calcium (Ca). Le CaCO3 peut être d'origine physico-chimique ou biogénique et de nombreux organismes sont connus pour contrôler ou induire sa biominéralisation. Les champignons ont souvent été soupçonnés d'être impliqué dans ce processus, cependant il existe très peu d'informations pour étayer cette hypothèse.Cette thèse a eu pour but l'étude de cet aspect négligé de l'impact des champignons dans les cycles biogéochimiques, par l'exploration de leur implication potentielle dans la formation d'un type particulier de CaCO3 secondaires observés dans les sols et dans les grottes des environnements calcaires. Dans les grottes, ces dépôts sont appelés moonmilk, alors que dans les sols on les appelle calcite en aiguilles. Cependant ces deux descriptions correspondent en fait au même assemblage microscopique de deux habitus particulier de la calcite: la calcite en aiguilles (au sens strict du terme cette fois-ci) et les nanofibres. Ces deux éléments sont des habitus aciculaires de la calcite, mais présentent des dimensions différentes. Leur origine, physico-chimique ou biologique, est l'objet de débats intenses depuis plusieurs années déjà.L'observation d'échantillons environnementaux avec des techniques de microscopie (microscopie électronique et micromorphologie), ainsi que de la microanalyse EDX, ont démontré plusieurs relations intéressantes entre la calcite en aiguilles, les nanofibres et des éléments organiques. Premièrement, il est montré que les nanofibres peuvent être organiques ou minérales. Deuxièmement, la calcite en aiguilles et les nanofibres présentent de fortes analogies avec des structures hyphales, ce qui permet de confirmer l'hypothèse de leur origine fongique. En outre, des expériences en laboratoire ont confirmé l'origine fongique des nanofibres, par des digestions enzymatiques d'hyphes fongiques. En effet, des structures à base de nanofibres, similaires à celles observées dans des échantillons naturels, ont pu être produites par cette approche. Finalement, des enrichissements en calcium ont été mesurés dans les parois des hyphes et dans des inclusions intrahyphales provenant d'échantillons naturels de rhizomorphes. Ces résultats suggèrent une implication de la séquestration de calcium dans la formation de la calcite en aiguilles et/ou des nanofibres.Plusieurs aspects restent à élucider, en particulier la compréhension des processus physiologiques impliqués dans la nucléation de calcite dans les hyphes fongiques. Cependant, les résultats obtenus dans cette thèse ont permis de confirmer l'implication des champignons dans la formation de la calcite en aiguilles et des nanofibres. Ces découvertes sont d'une grande importance dans les cycles biogéochimiques puisqu'ils apportent de nouveaux éléments dans le cycle couplé C-Ca. Classiquement, les champignons sont considérés comme étant impliqués principalement dans la minéralisation de la matière organique et dans l'altération minérale. Cette étude démontre que les champignons doivent aussi être pris en compte en tant qu'agents majeurs de la genèse de minéraux, en particulier de CaCO3. Ceci représente une toute nouvelle perspective en géomycologie quant à la participation des champignons au cycle biologique du C. En effet, la présence de ces précipitations de CaCO3 secondaires représente un court-circuit dans le cycle biologique du C puisque du C inorganique du sol se retrouve piégé dans de la calcite plutôt que d'être retourné dans l'atmosphère.AbstractFungi are known to be involved in biogeochemical cycles in numerous ways. In particular, they are recognized as key players in organic matter recycling, as nutrient suppliers via mineral weathering, as well as large producers of oxalic acid and metal-oxalate. However, little is known about their contribution to the genesis of other types of minerals such as calcium carbonate (CaCO3). Yet, CaC03 are ubiquitous minerals in many ecosystems and play an essential role in the biogeochemical cycles of both carbon (C) and calcium (Ca). CaC03 may be physicochemical or biogenic in origin and numerous organisms have been recognized to control or induce calcite biomineralization. While fungi have often been suspected to be involved in this process, only scarce information support this hypothesis.This Ph.D. thesis aims at investigating this disregarded aspect of fungal impact on biogeochemical cycles by exploring their possible implication in the formation of a particular type of secondary CaC03 deposit ubiquitously observed in soils and caves from calcareous environments. In caves, these deposits are known as moonmilk, whereas in soils, they are known as Needle Fibre Calcite (NFC - sensu lato). However, they both correspond to the same microscopic assemblage of two distinct and unusual habits of calcite: NFC {sensu stricto) and nanofibres. Both features are acicular habits of calcite displaying different dimensions. Whether these habits are physicochemical or biogenic in origin has been under discussion for a long time.Observations of natural samples using microscopic techniques (electron microscopy and micromorphology) and EDX microanalyses have demonstrated several interesting relationships between NFC, nanofibres, and organic features. First, it has shown that nanofibres can be either organic or minera! in nature. Second, both nanofibres and NFC display strong structural analogies with fungal hyphal features, supporting their fungal origin. Furthermore, laboratory experiments have confirmed the fungal origin of nanofibres through an enzymatic digestion of fungal hyphae. Indeed, structures made of nanofibres with similar features as those observed in natural samples have been produced. Finally, calcium enrichments have been measured in both cell walls and intrahyphal inclusions of hyphae from rhizomorphs sampled in the natural environment. These results point out an involvement of calcium sequestration in nanofibres and/or NFC genesis.Several aspects need further investigation, in particular the understanding of the physiological processes involved in hyphal calcite nucleation. However, the results obtained during this study have allowed the confirmation of the implication of fungi in the formation of both NFC and nanofibres. These findings are of great importance regarding global biogeochemical cycles as they bring new insights into the coupled C and Ca cycles. Conventionally, fungi are considered to be involved in organic matter mineralization and mineral weathering. In this study, we demonstrate that they must also be considered as major agents in mineral genesis, in particular CaC03. This is a completely new perspective in geomycology regarding the role of fungi in the short-term (or biological) C cycle. Indeed, the presence of these secondary CaC03 precipitations represents a bypass in the short- term carbon cycle, as soil inorganic C is not readily returned to the atmosphere.

Hygroscopicity and ammonia volatilization losses from nitrogen sources in coated urea

Hygroscopic fertilizers tend to absorb moisture from the air and may have undesirable characteristics such as moistness, clumping and lower fluidity, hampering the application. The increasing use of urea is due to its numerous advantages, although this nitrogen (N) source is highly susceptible to volatilization losses, particularly when applied to the soil surface of management systems with conservation of crop residues. The volatilization losses can be minimized by slow or controlled-release fertilizers, with controlled water solubility of the urea-coating materials; and by stabilized fertilizers, which prolong the period during which N remains in the amide or ammonia forms by urease inhibitors. This study evaluated the hygroscopicity of and ammonia volatilization from urea coated with boric acid and copper sulfate or with sulfur. The hygroscopicity of the sources was evaluated over time after exposure to five levels of relative humidity (RH) and volatilization evaluated after application to the soil surface covered with sugarcane trash. Ammonium nitrate has a low potential for volatilization losses, but is highly hygroscopic. Although coating with boric acid and copper sulfate or elemental sulfur reduced the critical humidity level of urea, the delay in the volatilization process is a potential positive factor.

Cover plants and mineral nitrogen: effects on organic matter fractions in an oxisol under no-tillage in the cerrado

Cover plants are essential for the sustainability of no-tillage systems in tropical regions. However, information on the effects of these plants and N fertilization on soil organic matter fractions is still scarce. This study evaluated the effect of cover crops with different chemical composition and of N topdressing on the labile and humified organic matter fractions of an Oxisol of the Cerrado (savanna-like vegetation). The study in a randomized complete block design was arranged in split-plots with three replications. Four cover species were tested in the plots and the presence or absence of N topdressing in the subplot. The following cover species were planted in succession to corn for eight years: Urochloa ruziziensis; Canavalia brasiliensis M. ex Benth; Cajanus cajan (L.) Millsp; and Sorghum bicolor (L.) Moench. In general, the cultivation of U. ruziziensis increased soil C levels, particularly of C in the humic acid and particulate organic C fractions, which are quality indicators of soil organic matter. The C in humic substances and mineral organic C accounted for the highest proportions of total organic C, demonstrating the strong interaction between organic matter, Fe and Al oxides and kaolinite, which are predominant in these weathered soils of the Cerrado.

CHROMIUM IN SOIL ORGANIC MATTER AND COWPEA AFTER FOUR CONSECUTIVE ANNUAL APPLICATIONS OF COMPOSTED TANNERY SLUDGE

Tannery sludge contains high concentrations of inorganic elements, such as chromium (Cr), which may lead to environmental pollution and affect human health The behavior of Cr in organic matter fractions and in the growth of cowpea (Vigna unguiculata L.) was studied in a sandy soil after four consecutive annual applications of composted tannery sludge (CTS). Over a four-year period, CTS was applied on permanent plots (2 × 5 m) and incorporated in the soil (0-20 cm) at the rates of 0, 2.5, 5.0, 10.0, and 20.0 Mg ha-1 (dry weight basis). These treatments were replicated four times in a randomized block design. In the fourth year, cowpea was planted and grown for 50 days, at which time we analyzed the Cr concentrations in the soil, in the fulvic acid, humic acid, and humin fractions, and in the leaves, pods, and grains of cowpea. Composted tannery sludge led to an increase in Cr concentration in the soil. Among the humic substances, the highest Cr concentration was found in humin. The application rates of CTS significantly increased Cr concentration in leaves and grains.

pH Sensitive surfactants from lysine: assessment of their cytotoxicity and environmental behavior

The toxicity and environmental behavior of new pH-sensitive surfactants from lysine are presented. Three different chemical structures are studied: surfactants with one amino acid and one alkyl chain, surfactants with two amino acids on the polar head and one alkyl chain, and gemini surfactants. The pH sensitivity of these compounds can be tuned by modifying their chemical structures. Cytotoxicity has been evaluated using erythrocytes and fibroblast cells. The toxic effects against these cells depend on the hydrophobicity of the molecules as well as their cationic charge density. The effect of hydrophobicity and cationic charge density on toxicity is different for each type of cells. For erythrocytes, the toxicity increases as hydrophobicity and charge density increases. Nevertheless, for fibroblasts cationic charge density affects cytotoxicity in the opposite way: the higher charge density, the lower the toxicity. The effect of the pH on hemolysis has been evaluated in detail. The aquatic toxicity was established using Daphnia magna. All surfactants yielded EC50 values considerably higher than that reported for cationic surfactants based on quaternary ammonium groups. Finally, their biodegradability was evaluated using the CO2 headspace test (ISO 14593). These lysine derivatives showed high levels of biodegradation under aerobic conditions and can be classified as"readily biodegradable compounds".

Simultaneous determination of Pu and Am radioisotopes in environmental samples: a tool for determining origin and release date

A radiochemical procedure was developed for the sequential determination of Pu and Am radioisotopes in environmental samples. The radioisotope activities were then used to assess the origin and release date of the environmental plutonium. The radioanalytical procedure is based on the separation of Pu and Am on selective extraction chromatographic resins (Eichrom TEVA and DGA). Alpha sources were prepared by electrodeposition on stainless steel discs, and the alpha emitting radionuclides (238Pu, 239,240Pu and 241Am) were measured by alpha spectrometry. For the determination of the beta emitting 241Pu, the Pu alpha source was leached in hot concentrated nitric acid and the Pu fraction further purified by extraction chromatography on a small column of TEVA resin (100 μg of resin in a pipette tip). 241Pu is then measured by ultra low level liquid scintillation counting. Due to the lack of reference material for 241Pu, the proposed radiochemical method was nevertheless validated using four IAEA reference sediments with information values of 241Pu. The proposed method was then used to determine the 238Pu, 239,240Pu, 241Pu and 241Am activity concentrations in alpine soils of France and Switzerland. The soil is the primary receptor of the atmospheric radioactive fallout and, because of the strong binding interaction with soils particles, the isotopes are little fractionated. Therefore, the activity ratios 241Pu/239+240Pu and 238Pu/239,240Pu in soil samples were used to determine the origin (source) and date of the Pu contamination in the investigated alpine sites. The 241Pu/239,240Pu and 238Pu/239,240Pu activity ratios confirmed that the main origin of Pu in the alpine soils was the global fallout from the nuclear bomb tests (NBT) in the fifties and sixties. Furthermore, the 241Pu/241Am activity ratios were used to determine the age of the Pu contamination, which is also an important data for distinguishing the Pu sources. The estimation of the date of the contamination, by the 241Pu/241Am age-dating method, further confirmed the NBT as the Pu source. However, the 241Pu/241Am dating method was limited to samples where Pu-Am fractionation was insignificant. If any, the contribution of the Chernobyl accident in the studied sites is negligible.

Detection of live and antibiotic-killed bacteria by quantitative real-time PCR of specific fragments of rRNA.

Assessing bacterial viability by molecular markers might help accelerate the measurement of antibiotic-induced killing. This study investigated whether rRNA could be suitable for this purpose. Cultures of penicillin-susceptible and penicillin-tolerant (Tol1 mutant) Streptococcus gordonii were exposed to mechanistically different penicillin and levofloxacin. Bacterial survival was assessed by viable counts and compared to quantitative real-time PCR amplification of either the 16S rRNA genes or the 16S rRNA, following reverse transcription. Penicillin-susceptible S. gordonii lost > or =4 log(10) CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Tol1 mutant lost < or =1 log(10) CFU/ml. Amplification of a 427-bp fragment of 16S rRNA genes yielded amplicons that increased proportionally to viable counts during bacterial growth but did not decrease during drug-induced killing. In contrast, the same 427-bp fragment amplified from 16S rRNA paralleled both bacterial growth and drug-induced killing. It also differentiated between penicillin-induced killing of the parent and the Tol1 mutant (> or =4 log(10) CFU/ml and < or =1 log(10) CFU/ml, respectively) and detected killing by mechanistically unrelated levofloxacin. Since large fragments of polynucleotides might be degraded faster than smaller fragments, the experiments were repeated by amplifying a 119-bp region internal to the original 427-bp fragment. The amount of 119-bp amplicons increased proportionally to viability during growth but remained stable during drug treatment. Thus, 16S rRNA was a marker of antibiotic-induced killing, but the size of the amplified fragment was critical for differentiation between live and dead bacteria.

Induction of the acyl-coenzyme A synthetase gene by fibrates and fatty acids is mediated by a peroxisome proliferator response element in the C promoter.

The long-chain acyl-coenzyme A synthetase (ACS) gene gives rise to three transcripts containing different first exons preceded by specific regulatory regions A, B, and C. Exon-specific oligonucleotide hybridization indicated that only A-ACS mRNA is expressed in rat liver. Fibrate administration induced liver C-ACS strongly and A-ACS mRNA to a lesser extent. B-ACS mRNA remained undetectable. In primary rat hepatocytes and Fa-32 hepatoma cells C-ACS mRNA increased after treatment with fenofibric acid, alpha-bromopalmitate, tetradecylthioacetic acid, or alpha-linolenic acid. Nuclear run-on experiments indicated that fenofibric acid and alpha-bromopalmitate act at the transcriptional level. Transient transfections showed a 3.4-, 2.3-, and 2.2-fold induction of C-ACS promoter activity after fenofibric acid, alpha-bromopalmitate, and tetradecylthioacetic acid, respectively. Unilateral deletion and site-directed mutagenesis identified a peroxisome proliferator activator receptor (PPAR)-responsive element (PPRE) mediating the responsiveness to fibrates and fatty acids. This ACS PPRE contains three imperfect half sites spaced by 1 and 3 oligonucleotides and binds PPAR.retinoid X receptor heterodimers in gel retardation assays. In conclusion, the regulation of C-ACS mRNA expression by fibrates and fatty acids is mediated by PPAR.retinoid X receptor heterodimers interacting through a PPRE in the C-ACS promoters. PPAR therefore occupies a key position in the transcriptional control of a pivotal enzyme controlling the channeling of fatty acids into various metabolic pathways.

Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers.

The nuclear hormone receptors called PPARs (peroxisome proliferator-activated receptors alpha, beta, and gamma) regulate the peroxisomal beta-oxidation of fatty acids by induction of the acyl-CoA oxidase gene that encodes the rate-limiting enzyme of the pathway. Gel retardation and cotransfection assays revealed that PPAR alpha heterodimerizes with retinoid X receptor beta (RXR beta; RXR is the receptor for 9-cis-retinoic acid) and that the two receptors cooperate for the activation of the acyl-CoA oxidase gene promoter. The strongest stimulation of this promoter was obtained when both receptors were exposed simultaneously to their cognate activators. Furthermore, we show that natural fatty acids, and especially polyunsaturated fatty acids, activate PPARs as potently as does the hypolipidemic drug Wy 14,643, the most effective activator known so far. Moreover, we discovered that the synthetic arachidonic acid analogue 5,8,11,14-eicosatetraynoic acid is 100 times more effective than Wy 14,643 in the activation of PPAR alpha. In conclusion, our data demonstrate a convergence of the PPAR and RXR signaling pathways in the regulation of the peroxisomal beta-oxidation of fatty acids by fatty acids and retinoids.

Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Background Plant hormones play a pivotal role in several physiological processes during a plant's life cycle, from germination to senescence, and the determination of endogenous concentrations of hormones is essential to elucidate the role of a particular hormone in any physiological process. Availability of a sensitive and rapid method to quantify multiple classes of hormones simultaneously will greatly facilitate the investigation of signaling networks in controlling specific developmental pathways and physiological responses. Due to the presence of hormones at very low concentrations in plant tissues (10-9 M to 10-6 M) and their different chemistries, the development of a high-throughput and comprehensive method for the determination of hormones is challenging. Results The present work reports a rapid, specific and sensitive method using ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UPLC/ESI-MS/MS) to analyze quantitatively the major hormones found in plant tissues within six minutes, including auxins, cytokinins, gibberellins, abscisic acid, 1-amino-cyclopropane-1-carboxyic acid (the ethylene precursor), jasmonic acid and salicylic acid. Sample preparation, extraction procedures and UPLC-MS/MS conditions were optimized for the determination of all plant hormones and are summarized in a schematic extraction diagram for the analysis of small amounts of plant material without time-consuming additional steps such as purification, sample drying or re-suspension. Conclusions This new method is applicable to the analysis of dynamic changes in endogenous concentrations of hormones to study plant developmental processes or plant responses to biotic and abiotic stresses in complex tissues. An example is shown in which a hormone profiling is obtained from leaves of plants exposed to salt stress in the aromatic plant, Rosmarinus officinalis.

Production of Acetic Acid by Fermentation with Propionibacteria, HR-321, 1993

This project was undertaken jointly with a project supported by the Iowa Corn Promotion Board. Together the projects aimed at producing the organic acids, propionic acid and acetic acid, by fermentation. The impacts were to provide agriculturally-based alternatives to production of these acids, currently produced mainly as petrochemicals. The potentially high-demand use for acetic acid is as the "acetate" in Calcium Magnesium Acetate (CMA), the non-corrosive road deicer. Fermentation was, however, far from being an economically acceptable alternative. Gains were made in this work toward making this a feasible route. These advances included (1) development of a variant strain of propionibacteria capable of producing higher concentrations of acids; (2) comparison of conditions for several ways of cultivating free cells and establishment of the relative benefits of each; (3) achievement of the highest productivity in fermentations using immobilized cells; (4) identification of corn steep liquor as a lower cost substrate for the fermentation; (5) application of a membrane extraction system for acid recovery and reduction of product inhibition; and (6) initial use of more detailed economic analysis of process alternatives to guide in the identification of where the greatest payback potential is for future research. At this point, the fermentation route to these acids using the propionibacteria is technically feasible, but economically unfeasible. Future work with integration of the above process improvements can be expected to lead to further gains in economics. However, such work can not be expected to make CMA a less expensive deicer than common road salt.

A LC-tandem MS assay for the simultaneous measurement of new antiretroviral agents: Raltegravir, maraviroc, darunavir, and etravirine.

Raltegravir (RAL), maraviroc (MVC), darunavir (DRV), and etravirine (ETV) are new antiretroviral agents with significant potential for drug interactions. This work describes a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of plasma drug levels. Single-step extraction of RAL, MVC, DRV, ETV and RTV from plasma (100 microl) is performed by protein precipitation using 600 microl of acetonitrile, after the addition of 100 microl darunavir-d(9) (DRV-d(9)) at 1000 ng/ml in MeOH/H(2)O 50/50 as internal standard (I.S.). The mixture is vortexed, sonicated for 10 min, vortex-mixed again and centrifuged. An aliquot of supernatant (150 microl) is diluted 1:1 with a mixture of 20 mM ammonium acetate/MeOH 40/60 and 10 microl is injected onto a 2.1 x 50 mm Waters Atlantis-dC18 3 microm analytical column. Chromatographic separations are performed using a gradient program with 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile with 0.1% formic acid. Analytes quantification is performed by electrospray ionisation-triple quadrupole mass spectrometry using the selected reaction monitoring detection in the positive mode. The method has been validated over the clinically relevant concentrations ranging from 12.5 to 5000 ng/ml, 2.5 to 1000 ng/ml, 25 to 10,000 ng/ml, 10 to 4000 ng/ml, and 5 to 2000 ng/ml for RAL, MRV, DRV, ETV and RTV, respectively. The extraction recovery for all antiretroviral drugs is always above 91%. The method is precise, with mean inter-day CV% within 5.1-9.8%, and accurate (range of inter-day deviation from nominal values -3.3 to +5.1%). In addition our method enables the simultaneous assessment of raltegravir-glucuronide. This is the first analytical method allowing the simultaneous assay of antiretroviral agents targeted to four different steps of HIV replication. The proposed method is suitable for the Therapeutic Drug Monitoring Service of these new regimen combinations administered as salvage therapy to patients having experienced treatment failure, and for whom exposure, tolerance and adherence assessments are critical.