Equine cytochrome P450 2B6--genomic identification, expression and functional characterization with ketamine

Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V(max) for S-/and R-norketamine formation was 0.49 and 0.45nmol/h/mg cellular protein and K(m) was 3.41 and 2.66μM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC(50) of 5.63 and 6.26μM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in ketamine and norketamine metabolism, thus confirming results from inhibition studies with horse liver microsomes. Clopidogrel seems to be a feasible inhibitor for equine CYP2B6. The specificity still needs to be established with other single equine CYPs. Heterologous expression of single equine CYP enzymes opens new possibilities to substantially improve the understanding of drug metabolism and drug interactions in horses.

An examination of the metabolic processes underpinning critical swimming in Atlantic cod (Gadus morhua L.) using in vivo 31P-NMR spectroscopy.

Traditionally, critical swimming speed has been defined as the speed when a fish can no longer propel itself forward, and is exhausted. To gain a better understanding of the metabolic processes at work during a U(crit) swim test, and that lead to fatigue, we developed a method using in vivo (31)P-NMR spectroscopy in combination with a Brett-type swim tunnel. Our data showed that a metabolic transition point is reached when the fish change from using steady state aerobic metabolism to non-steady state anaerobic metabolism, as indicated by a significant increase in inorganic phosphate levels from 0.3+/-0.3 to 9.5+/-3.4 mol g(-1), and a drop in intracellular pH from 7.48+/-0.03 to 6.81+/-0.05 in muscle. This coincides with the point when the fish change gait from subcarangiform swimming to kick-and-glide bursts. As the number of kicks increased, so too did the Pi concentration, and the pH(i) dropped. Both changes were maximal at U(crit). A significant drop in Gibbs free energy change of ATP hydrolysis from -55.6+/-1.4 to -49.8+/-0.7 kJ mol(-1) is argued to have been involved in fatigue. This confirms earlier findings that the traditional definition of U(crit), unlike other critical points that are typically marked by a transition from aerobic to anaerobic metabolism, is the point of complete exhaustion of both aerobic and anaerobic resources.

Chemical analysis and Microbial community composition of surface water samples from the Southern Lagoon of Venice

The Lagoon of Venice is a large water basin that exchanges water with the Northern Adriatic Sea through three large inlets. We examined two adjacent sites within the Southern Basin and at the Chioggia inlet in autumn 2007 and summer 2008. A pilot study in June 2007 on a surface water sample from Chioggia with a rather high salinity of 36.9 PSU had revealed a conspicuous bloom of CF319a-positive cells likely affiliated with the Cytophaga /Flavobacteria cluster of Bacteroidetes. These flavobacterial abundances were one to two orders of magnitude higher than in other marine surface waters. DAPI-stained cells were identified as bacteria with the general bacterial probe mixture EUB338 I-III. CARD-FISH counts with group-specific probes confirmed the dominance of Bacteroidetes (CF319a), Alphaproteobacteria (ALF968), and Gammaproteobacteria (GAM42a). CARD-FISH showed thatBetaproteobacteria and Planctomycetes were minor components of the bacterioplankton in the Lagoon of Venice.

NOR5/OM60 clade of the Yangtze River estuary

The phylogeny, abundance, and biogeography of the NOR5/OM60 clade was investigated. This clade includes "Congregibacter litoralis" strain KT71, the first cultured representative of marine aerobic anoxygenic phototrophic Gammaproteobacteria. Most of the NOR5/OM60 sequences were retrieved from marine coastal settings, whereas there were fewer from open-ocean surface waters, deep-sea sediment, freshwater, saline lakes and soil. The abundance of members of the NOR5/OM60 clade in various marine sites was determined by fluorescence in situ hybridization using a newly designed and optimized probe set. Relative abundances in coastal marine waters off the Yangtze estuary were up to 3% of the total 4',6-diamidino-2-phenylindole (DAPI) counts. A small cruise was undertaken from 2006-09-06 to 2006-09-08 in the Yangtze River estuary. Samples were taken from surface water, and immediately fixed with 1% paraformaldehyde (PFA) for 1 h, filtered onto polycarbonate filters (Millipore, 47 mm in diameter, 0.2 µm pore size) and stored frozen at -20 °C.

Mineralogy of cold seep sediment inhabited by vesicomyid clams in the Japan Deep Sea Trench

Sediment sampling was performed at the center of the clam colony. Immediately after sample recovery onboard, the sediment core was sub-sampled and preserved for later analyses. Pyrite and carbonate content of the sediment was measured by X-ray refraction analysis as previously described (Ertefai et al., 2010).

Investigación sobre fermentaciones metánicas de desechos estacionales

En el campo agrícola se producen una serie de desechos orgánicos, que por un lado representan serios problemas de contaminación ambiental y por otro el desperdicio de valores energéticos importantes. Es decir una acción contraria a la sostenibilidad que debe buscarse en este siglo XXI. Entre estos productos agrícolas pueden citarse la pulpa de café, residuos herbáceos, bagazo de caña y la fracción insoluble de estiércol de ganado porcino conocida como cerdaza. Un problema añadido para dar solución adecuada es la disponibilidad de estos solo en cortas épocas del año. Todo lo anterior ha sido el origen de la presente investigación, para dar solución adecuada tanto en el aprovechamiento de biogás como en la reducción de la contaminación. La investigación descrita en este documento contempla el desarrollo de los siguientes aspectos: 1) Caracterización y problemática de cada uno de los productos señalados, 2) la solución al problema mediante el proceso de digestión anaerobia con fases separadas con el aprovechamiento del biogás generados y 3) recomendaciones para el arranque del proceso de digestión anaerobia y su mantenimiento en una alternancia de los productos citados. En la primera etapa de la fase experimental se estimó el rendimiento específico de metano para los diferentes sustratos, utilizando reactores batch configurados en una y dos fases concluyendo que la digestión anaerobia en dos fases presenta diferentes ventajas sobre la digestión monoetapa. En general se obtuvo un mayor rendimiento en la producción de metano, una reducción en los tiempos de retención, mayor eficiencia en la eliminación de los sólidos volátiles agregados, y una mayor estabilidad en el proceso reflejado en el mantenimiento de valores de pH en los rangos de operación recomendados. Seguidamente, al comparar dos procesos para la puesta en marcha de digestores metanogénicos operados en forma continua, se concluye que las variables determinantes en la estabilidad del sistema son la alcalinidad total presente en el digestor, el establecimiento de la población de microorganismos y la carga orgánica aplicada. Las dos primeras están determinadas por la calidad y proporción del inóculo suministrado al inicio del proceso. La alternación de sustratos suministrados al sistema de digestión en dos fases, permitió determinar el impacto sobre el desempeño del mismo, registrando una reducción en la producción de biogás, la riqueza de metano y la eficiencia de eliminación de sólidos volátiles durante los primeros días de operación luego del cambio de sustrato. Este periodo corresponde al proceso de aclimatación de los microorganismos el cual requirió de 20 días para asimilar los componentes del nuevo sustrato. Finalmente, entre los sustratos analizados, la menor carga orgánica de operación para mantener la operación del sistema en continuo corresponde a la pulpa de café con 0.1 kg SV/m3. La composición de este sustrato favorece la rápida acumulación de acidez volátil en el sistema, proporcionando una tendencia a la acidificación. Sin embargo, al controlar las cargas orgánicas volumétricas, el sistema permaneció operando sin necesidad de adición de alcalinizantes. La aplicación de los resultados de la presente investigación a la problemática de residuos de café es alentadora, comprobando que el sistema puede ser operado en continuo alternando residuos boreales y pulpa de café, ambos sustratos disponibles en las plantas de procesamiento de la cereza de café. ABSTRACT In the agricultural field there are series of organic wastes, which in one hand are the source of serious problems of environmental pollution and in the other, they represent a residue that could be used as a feedstock with significant energy values. These actions are contrary to efforts towards sustainability, which should be a priority in this century. Among agricultural residues with significant abundance, the coffee pulp, herbaceous waste, sugarcane bagasse and the insoluble fraction of pig manure can be mentioned. An added problem to the development of appropriate treatment systems, which provides a solution to the disposal of such wastes, is the limited availability of these feedstocks only in short seasons. These arguments have been the source of our research, in order to provide properly measures to biogas usage and pollution reduction. The research presented in this document includes the approaches to the following aspects. 1) Characterization and problems regarding the selected feedstocks 2) the solution to the problem by anaerobic digestion process with separate phases and 3) recommendations for starting the process of anaerobic digestion and its maintenance with alternation of the products listed For the first stage of the experimental phase, the specific methane yield of the selected feedstocks was estimated using batch reactors configured in one and two phases. It was concluded that two-phase anaerobic digestion offered distinct advantages over the single-stage digestion. In general a higher methane production yields, lower retention times, higher efficiency in volatile solids removal, and increased stability among the process were obtained. When comparing two processes for starting up methanogenic digesters, it is concluded that the variables that determine the stability of the system are the total alkalinity in the digester, the establishment of the population of microorganisms and the organic load. The first variables are influenced by the proportion and quality of the inoculum supplied at the beginning of the process. The alternation of substrates gave as a result a negative impact on system performance, recording a reduction on biogas production, the methane concentration and the efficiency of volatile solids removal. The situation was observed during the first days of operation after the change of feeding. This period corresponds to the process of acclimatization of the microorganisms which required 20 days to assimilate new substrate components. Finally, among substrates studied, the lowest organic load applied to maintain a continuous operation of the system, corresponds to the coffee pulp with 0.1 kg VS / m3. The composition of this substrate promotes a rapid accumulation of volatile acidity within the system, providing a tendency to acidification. However, by controlling organic loads, the operating system remained stable without addition of alkalizing components. The application of the results of this research to the problem of coffee waste is promising, proving that an anaerobic system can be operated continuously by alternating boreal waste and coffee pulp, both substrates available in coffee processing plants.

Delivery of antisense oligodeoxyribonucleotides against the human epidermal growth factor receptor into cultured KB cells with liposomes conjugated to folate via polyethylene glycol.

Antisense oligodeoxyribonucleotides targeted to the epidermal growth factor (EGF) receptor were encapsulated into liposomes linked to folate via a polyethylene glycol spacer (folate-PEG-liposomes) and efficiently delivered into cultured KB cells via folate receptor-mediated endocytosis. The oligonucleotides were a phosphodiester 15-mer antisense to the EGF receptor (EGFR) gene stop codon (AEGFR2), the same sequence with three phosphorothioate linkages at each terminus (AEGFR2S), a randomized 15-mer control of similar base composition to AEGFR2 (RC15), a 14-mer control derived from a symmetrized Escherichia coli lac operator (LACM), and the 5'-fluorescein-labeled homologs of several of the above. Cellular uptake of AEGFR2 encapsulated in folate-PEG-liposomes was nine times higher than AEGFR2 encapsulated in nontargeted liposomes and 16 times higher than unencapsulated AEGFR2. Treatment of KB cells with AEGFR2 in folate-PEG-liposomes resulted in growth inhibition and significant morphological changes. Curiously, AEGFR2 and AEGFR2S encapsulated in folate-PEG-liposomes exhibited virtually identical growth inhibitory effects, reducing KB cell proliferation by > 90% 48 hr after the cells were treated for 4 hr with 3 microM oligonucleotide. Free AEGFR2 caused almost no growth inhibition, whereas free AEGFR2S was only one-fifth as potent as the folate-PEG-liposome-encapsulated oligonucleotide. Growth inhibition of the oligonucleotide-treated cells was probably due to reduced EGFR expression because indirect immunofluorescence staining of the cells with a monoclonal antibody against the EGFR showed an almost quantitative reduction of the EGFR in cells treated with folate-PEG-liposome-entrapped AEGFR2. These results suggest that antisense oligonucleotide encapsulation in folate-PEG-liposomes promise efficient and tumor-specific delivery and that phosphorothioate oligonucleotides appear to offer no major advantage over native phosphodiester DNA when delivered by this route.

Depth profiles of dissolved sulfide and sulfate in the pore water of hydrocarbon seep sediments offshore Pakistan

The interaction between fluid seepage, bottom water redox, and chemosynthetic communities was studied at cold seeps across one of the world's largest oxygen minimum zones (OMZ) located at the Makran convergent continental margin. Push cores were obtained from seeps within and below the core-OMZ with a remotely operated vehicle. Extracted sediment pore water was analyzed for sulfide and sulfate concentrations. Depending on oxygen availability in the bottom water, seeps were either colonized by microbial mats or by mats and macrofauna. The latter, including ampharetid polychaetes and vesicomyid clams, occurred in distinct benthic habitats, which were arranged in a concentric fashion around gas orifices. At most sites colonized by microbial mats, hydrogen sulfide was exported into the bottom water. Where macrofauna was widely abundant, hydrogen sulfide was retained within the sediment. Numerical modeling of pore water profiles was performed in order to assess rates of fluid advection and bioirrigation. While the magnitude of upward fluid flow decreased from 11 cm yr**-1 to <1 cm yr**-1 and the sulfate/methane transition (SMT) deepened with increasing distance from the central gas orifice, the fluxes of sulfate into the SMT did not significantly differ (6.6-9.3 mol m**-2 yr**-1). Depth-integrated rates of bioirrigation increased from 120 cm yr**-1 in the central habitat, characterized by microbial mats and sparse macrofauna, to 297 cm yr**-1 in the habitat of large and few small vesicomyid clams. These results reveal that chemosynthetic macrofauna inhabiting the outer seep habitats below the core-OMZ efficiently bioirrigate and thus transport sulfate down into the upper 10 to 15 cm of the sediment. In this way the animals deal with the lower upward flux of methane in outer habitats by stimulating rates of anaerobic oxidation of methane (AOM) with sulfate high enough to provide hydrogen sulfide for chemosynthesis. Through bioirrigation, macrofauna engineer their geochemical environment and fuel upward sulfide flux via AOM. Furthermore, due to the introduction of oxygenated bottom water into the sediment via bioirrigation, the depth of the sulfide sink gradually deepens towards outer habitats. We therefore suggest that - in addition to the oxygen levels in the water column, which determine whether macrofaunal communities can develop or not - it is the depth of the SMT and thus of sulfide production that determines which chemosynthetic communities are able to exploit the sulfide at depth. We hypothesize that large vesicomyid clams, by efficiently expanding the sulfate zone down into the sediment, could cut off smaller or less mobile organisms, as e.g. small clams and sulfur bacteria, from the sulfide source.

Identification, detection, and spatial resolution of Clostridium populations responsible for cellulose degradation in a methanogenic landfill leachate bioreactor

An anaerobic landfill leachate bioreactor was operated with crystalline cellulose and sterile landfill leacbate until a steady state was reached. Cellulose hydrolysis, acidogenesis, and methanogenesis were measured. Microorganisms attached to the cellulose surfaces were hypothesized to be the cellulose hydrolyzers. 16S rRNA gene clone libraries were prepared from this attached fraction and also from the mixed fraction (biomass associated with cellulose particles and in the planktonic phase). Both clone libraries were dominated by Firmicutes phylum sequences (100% of the attached library and 90% of the mixed library), and the majority fell into one of five lineages of the clostridia. Clone group 1 (most closely related to Clostridium stercorarium), clone group 2 (most closely related to Clostridium thermocellum), and clone group 5 (most closely related to Bacteroides cellulosolvens) comprised sequences in Clostridium group III. Clone group 3 sequences were in Clostridium group XIVa (most closely related to Clostridium sp. strain XB90). Clone group 4 sequences were affiliated with a deeply branching clostridial lineage peripherally associated with Clostridium group VI. This monophyletic group comprises a new Clostridium cluster, designated cluster VIa. Specific fluorescence in situ hybridization (FISH) probes for the five groups were designed and synthesized, and it was demonstrated in FISH experiments that bacteria targeted by the probes for clone groups 1, 2, 4, and 5 were very abundant on the surfaces of the cellulose particles and likely the key cellulolytic microorganisms in the landfill bioreactor. The FISH probe for clone group 3 targeted cells in the planktonic phase, and these organisms were hypothesized to be glucose fermenters.

Pore water and solid-phase measurements on sediment cores from the Black Sea

The surface sediments in the Black Sea are underlain by extensive deposits of iron (Fe) oxide-rich lake sediments that were deposited prior to the inflow of marine Mediterranean Sea waters ca. 9000 years ago. The subsequent downward diffusion of marine sulfate into the methane-bearing lake sediments has led to a multitude of diagenetic reactions in the sulfate-methane transition zone (SMTZ), including anaerobic oxidation of methane (AOM) with sulfate. While the sedimentary cycles of sulfur (S), methane and Fe in the SMTZ have been extensively studied, relatively little is known about the diagenetic alterations of the sediment record occurring below the SMTZ. Here we combine detailed geochemical analyses of the sediment and pore water with multicomponent diagenetic modeling to study the diagenetic alterations below the SMTZ at two sites in the western Black Sea. We focus on the dynamics of Fe, S and phosphorus (P) and demonstrate that diagenesis has strongly overprinted the sedimentary burial records of these elements. Our results show that sulfate-mediated AOM substantially enhances the downward diffusive flux of sulfide into the deep limnic deposits. During this downward sulfidization, Fe oxides, Fe carbonates and Fe phosphates (e.g. vivianite) are converted to sulfide phases, leading to an enrichment in solid phase S and the release of phosphate to the pore water. Below the sulfidization front, high concentrations of dissolved ferrous Fe (Fe2+) lead to sequestration of downward diffusing phosphate as authigenic vivianite, resulting in a transient accumulation of total P directly below the sulfidization front. Our model results further demonstrate that downward migrating sulfide becomes partly re-oxidized to sulfate due to reactions with oxidized Fe minerals, fueling a cryptic S cycle and thus stimulating slow rates of sulfate-driven AOM (~ 1-100 pmol/cm**3/d) in the sulfate-depleted limnic deposits. However, this process is unlikely to explain the observed release of dissolved Fe2+ below the SMTZ. Instead, we suggest that besides organoclastic Fe oxide reduction, AOM coupled to the reduction of Fe oxides may also provide a possible mechanism for the high concentrations of Fe2+ in the pore water at depth. Our results reveal that methane plays a key role in the diagenetic alterations of Fe, S and P records in Black Sea sediments. The downward sulfidization into the limnic deposits is enhanced through sulfate-driven AOM with sulfate and AOM with Fe oxides may provide a deep source of dissolved Fe2+ that drives the sequestration of P in vivianite below the sulfidization front.

(Table 2) Carbonate chemistry and isotopes from northern Cascadia margin samples

Most authigenic carbonates previously recovered from the Cascadia slope have 87Sr/86Sr signatures that reflect shallow precipitation in equilibrium with coeval seawater. There is also evidence for carbonate formation supported by fluids that have been modified by reactions with the incoming Juan de Fuca plate (87Sr/86Sr = 0.7071; Teichert et al., 2005, doi:10.1016/j.epsl.2005.08.002) or with terrigenous turbidites (87Sr/86Sr = 0.70975 to 0.71279; Sample et al., 1993, doi:10.1130/0091-7613(1993)021<0507:CCICFF>2.3.CO;2). We report on the strontium isotopic composition of carbonates and fluids from IODP Site U1329 and nearby Barkley Canyon (offshore Vancouver Island), which have strontium isotope ratios as low as 0.70539. Whereas the strontium and oxygen isotopic compositions of carbonates from paleoseeps in the uplifted Coast Range forearc indicate formation in ambient bottom seawater, several samples from the Pysht/Sooke Fm. show a 87Sr-depleted signal (87Sr/86Sr = 0.70494 and 0.70511) similar to that of the anomalous Site U1329 and Barkley Canyon carbonates. Our data, when analyzed in the context of published elemental and isotopic composition of these carbonates (Joseph et al., 2012, doi:10.1016/j.palaeo.2013.01.012 ), point to two formation mechanisms: 1) shallow precipitation driven by the anaerobic oxidation of methane (AOM) with d13C values as low as -50 per mil and contemporaneous 87Sr/86Sr seawater ratios, and 2) carbonate precipitation driven by fluids that have circulated through the oceanic crust, which are depleted in 87Sr. Carbonates formed from the second mechanism precipitate both at depth and at sites of deep-sourced fluid seepage on the seafloor. The 87Sr-depleted carbonates and pore fluids found at Barkley Canyon represent migration of a deep, exotic fluid similar to that found in high permeability conglomerate layers at 188 mbsf of Site U1329, and which may have fed paleoseeps in the Pysht/Sooke Fm. These exotic fluids likely reflect interaction with the 52-57 Ma igneous Crescent Terrane, which supplies fluids with high calcium, manganese and strontium enriched in the non-radiogenic nucleide. Tectonic compression and dehydration reactions then force these fluids updip, where they pick up the thermogenic hydrocarbons and 13C-enriched dissolved inorganic carbon that are manifested in fluids and carbonates sampled at Barkley Canyon and at Site U1329. The Crescent Terrane may have sourced cold seeps in this margin since at least the late Oligocene.