Sulfide oxidation at halo-alkaline conditions in a fed-batch bioreactor

A biotechnological process is described to remove hydrogen sulfide (H(2)S) from high-pressure natural gas and sour gases produced in the petrochemical industry. The process operates at halo-alkaline conditions and combines an aerobic sulfide-oxidizing reactor with an anaerobic sulfate (SO(4) (2-)) and thiosulfate (S(2)O(3) (2-)) reducing reactor. The feasibility of biological H(2)S oxidation at pH around 10 and total sodium concentration of 2 mol L(-1) was studied in gas-lift bioreactors, using halo-alkaliphilic sulfur-oxidizing bacteria (HA-SOB). Reactor operation at different oxygen to sulfide (O(2):H(2)S) supply ratios resulted in a stable low redox potential that was directly related with the polysulfide (S(x) (2-)) and total sulfide concentration in the bioreactor. Selectivity for SO(4) (2-) formation decreased with increasing S(x) (2-) and total sulfide concentrations. At total sulfide concentrations above 0.25 mmol L(-1), selectivity for SO(4) (2-) formation approached zero and the end products of H(2)S oxidation were elemental sulfur (S(0)) and S(2)O(3) (2-). Maximum selectivity for S(0) formation (83.3+/-0.7%) during stable reactor operation was obtained at a molar O(2):H(2)S supply ratio of 0.65. Under these conditions, intermediary S(x) (2-) plays a major role in the process. Instead of dissolved sulfide (HS(-)), S(x) (2-) seemed to be the most important electron donor for HA-SOB under S(0) producing conditions. In addition, abiotic oxidation of S(x) (2-) was the main cause of undesirable formation of S(2)O(3) (2-). The observed biomass growth yield under SO(4) (2-) producing conditions was 0.86 g N mol(-1) H(2)S. When selectivity for SO(4) (2-) formation was below 5%, almost no biomass growth was observed.     

Reversal of coenzyme specificity of 2,3‐butanediol dehydrogenase from Saccharomyces cerevisae and in vivo functional analysis

Saccharomyces cerevisiae NAD(H)‐dependent 2,3‐butanediol dehydrogenase (Bdh1), a medium chain dehydrogenase/reductase is the main enzyme catalyzing the reduction of acetoin to 2,3‐butanediol. In this work we focused on altering the coenzyme specificity of Bdh1 from NAD(H) to NADP(H). Based on homology studies and the crystal structure of the NADP(H)‐dependent yeast alcohol dehydrogenase Adh6, three adjacent residues (Glu²²¹, Ile²²², and Ala²²³) were predicted to be involved in the coenzyme specificity of Bdh1 and were altered by site‐directed mutagenesis. Coenzyme reversal of Bdh1 was obtained with double Glu221Ser/Ile222Arg and triple Glu221Ser/Ile222Arg/Ala223Ser mutants. The performance of the triple mutant for NADPH was close to that of native Bdh1 for NADH. The three engineered mutants were able to restore the growth of a phosphoglucose isomerase deficient strain (pgi), which cannot grow on glucose unless an alternative NADPH oxidizing system is provided, thus demonstrating their in vivo functionality. These mutants are interesting tools to reduce the excess of acetoin produced by engineered brewing or wine yeasts overproducing glycerol. In addition, they represent promising tools for the manipulation of the NADP(H) metabolism and for the development of a powerful catalyst in biotransformations requiring NADPH regeneration. Biotechnol. Bioeng. 2009; 104: 381–389 © 2009 Wiley Periodicals, Inc.     

Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells

BACKGROUND: Human mesenchymal stem cells (hMSCs) are a promising target for ex vivo gene therapy and lentiviruses are excellent gene transfer vehicles in hMSCs since they achieve high transduction rates with long-term gene expression. Nevertheless, senescence of hMSCs may limit therapeutic applications due to time-consuming cell selection and viral titration. Here, we describe a fast and reliable method to determine functional lentiviral titer by quantitative polymerase chain reaction (qPCR) after highly efficient ex vivo gene transfer in hMSCs. METHODS: Lentivirus production was tested with different types of packaging systems. Using p24 ELISA remaining viral particles were detected in the cell culture supernatant. The lentiviral gene transfer efficiency was quantified by FACS analysis. Lentiviral titers were determined by qPCR of expressed transgenes. RESULTS: Third-generation self-inactivating vectors showed highly efficient gene transfer in hMSCs. No viral antigen was detected in the cell culture supernatant after four media changes, suggesting the absence of infectious particles after 4 days. We observed a linear correlation between virus dilution and level of transgene expression by qPCR analysis, therefore allowing viral titering by quantification of transgene expression. Finally, we demonstrated that transduced hMSCs retained their stem cell character by differentiation towards adipogenic, osteogenic and chondrogenic lineages. CONCLUSIONS: Quantification of transgene copy numbers by qPCR is a fast and reliable method to determine functional lentiviral titer after ex vivo gene transfer in hMSCs.     

Enzyme‐immunoassay for the measurement of luteinizing hormone in the serum of African elephants (Loxodonta africana)

Circulating patterns of progesterone and luteinizing hormone (LH) in the elephant have been well characterized, and routine monitoring of these hormones is now viewed as a valuable tool for making informed decisions about the reproductive management of elephants in captivity. Currently, LH monitoring in elephants is done with radio‐immunoassays (RIAs); unfortunately, the use of radioactive materials in RIAs limits their application to institutions with laboratory facilities equipped for the storage and disposal of radioactive waste. Enzyme‐immunoassays (EIAs) offer an inexpensive and more zoo‐friendly alternative to RIA. This work reports on an EIA capable of quantifying circulating LH in African elephants. The EIA employs a biotin label and microtiter plates coated with goat anti‐mouse gamma globulin. LH surges in African elephants (n=3) increased fivefold over baseline concentrations (1.00±0.1 ng/ml vs. 0.2±0.1 ng/ml) and occurred 19.3±0.2 days apart. Ovulatory LH surges were associated with an increase in serum progestogens from 4.8±0.4 ng/ml to 11.7±0.4 ng/ml. The ability to quantify reproductive hormones in elephants via EIA is an important step in the process of making endocrine monitoring more accessible to zoos housing these species. Zoo Biol 21:403–408, 2002. © 2002 Wiley‐Liss, Inc.     

Proteomic analysis of the wing imaginal discs of Drosophila melanogaster

We have combined high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry with the aim of identifying proteins represented in the 2-D gel database of the wing imaginal discs of Drosophila melanogaster. First, we obtained a high-resolution 2-D gel pattern of [35S]methionine + [35S]cysteine-labeled polypeptides of Schneider cells, a permanent cell line of Drosophila embryonic origin, and compared it with the standard pattern of polypeptides of the wing imaginal disc. These studies reveal qualitative and quantitative differences between the two samples, but have more than 600 polypeptides in common. Second, we carried out preparative 2-D polyacrylamide gel electrophoresis using Schneider cells mixed with radioactively labeled wing imaginal discs in order to isolate some of the shared polypeptides and characterize them by matrix-assisted laser desorption/ionization-time of flight MALDI-TOF analysis. Using this strategy we identified 100 shared proteins represented in the database, and in each case confirmed their identity by MALDI-TOF/TOF analysis.     

Association mapping of cold‐induced sweetening in potato using historical phenotypic data

In this study, historical phenotypic data from a potato breeding programme were used with an association mapping approach to identify alleles of candidate genes associated with cold‐induced sweetening of potato. Molecular marker analysis was used to determine allelic variation of candidate genes potentially involved in cold‐induced sweetening. Variations in the UDP‐glucose pyrophosphorylase (UGPase, EC 2.7.7.9) and apoplastic invertase genes (EC 3.2.1.26) were significantly associated with cold‐induced sweetening, and a possible interaction of apoplastic invertase and apoplastic invertase inhibitor was identified. This demonstrates that breeding programme phenotypic data collected over multiple years and environments can be used successfully with pedigree information for association mapping. It also confirms that the UGPase and apoplastic invertase markers are transferable across breeding programmes with distinct germplasm.     

A review of the occurrence of inter‐colony segregation of seabird foraging areas and the implications for marine environmental impact assessment

Understanding the determinants of species’ distributions is a fundamental aim in ecology and a prerequisite for conservation but is particularly challenging in the marine environment. Advances in bio‐logging technology have resulted in a rapid increase in studies of seabird movement and distribution in recent years. Multi‐colony studies examining the effects of intra‐ and inter‐colony competition on distribution have found that several species exhibit inter‐colony segregation of foraging areas, rather than overlapping distributions. These findings are timely given the increasing rate of human exploitation of marine resources and the need to make robust assessments of likely impacts of proposed marine developments on biodiversity. Here we review the occurrence of foraging area segregation reported by published tracking studies in relation to the density‐dependent hinterland (DDH) model, which predicts that segregation occurs in response to inter‐colony competition, itself a function of colony size, distance from the colony and prey distribution. We found that inter‐colony foraging area segregation occurred in 79% of 39 studies. The frequency of occurrence was similar across the four seabird orders for which data were available, and included species with both smaller (10–100 km) and larger (100–1000 km) foraging ranges. Many predictions of the DDH model were confirmed, with examples of segregation in response to high levels of inter‐colony competition related to colony size and proximity, and enclosed landform restricting the extent of available habitat. Moreover, as predicted by the DDH model, inter‐colony overlap tended to occur where birds aggregated in highly productive areas, often remote from all colonies. The apparent prevalence of inter‐colony foraging segregation has important implications for assessment of impacts of marine development on protected seabird colonies. If a development area is accessible from multiple colonies, it may impact those colonies much more asymmetrically than previously supposed. Current impact assessment approaches that do not consider spatial inter‐colony segregation will therefore be subject to error. We recommend the collection of tracking data from multiple colonies and modelling of inter‐colony interactions to predict colony‐specific distributions.