Evidence of Pluronic F-68 direct interaction with insect cells: impact on shear protection, recombinant protein, and baculovirus production*

Pluronic F-68 has been widely used to protect animal cells from hydrodynamic stress, but its mechanism of action is still debatable. Published evidence indicates that Pluronic F-68 interacts with cells, yet scarce information exists of its effect on recombinant protein and virus production by insect cells. In this work, the effect of Pluronic F-68 on production of recombinant baculovirus and rotavirus protein VP7 was determined. Evidence of Pluronic F-68 direct interaction with Sf-9 insect cells also was obtained. Maximum recombinant VP7 concentration and yield increased 10x, whereas virus production decreased by 20x, in spinner flask cultures with 0.05% (w/v) Pluronic F-68 compared to controls lacking the additive. No differences were observed in media rheology, nor kinetics of growth and infection (as inferred from cell size) between both cultures. Hence, Pluronic F-68 influenced cell physiology independently of its shear protective effect. Cells subjected to a laminar shear rate of 3000 s(-1) for 15 min, without gas/liquid interfaces, were protected by Pluronic F-68 even after its removal from culture medium. Furthermore, the protective action was immediate in vortexed cells. The results shown here indicate that Pluronic F-68 physically interacts with cells in a direct, strong, and stable mode, not only protecting them from hydrodynamic damage, but also modifying their capacity for recombinant protein and virus production.     

Magnetic field influence on electrical properties of human blood measured by impedance spectroscopy

The impedance spectroscopy technique (IST) was used for studying the effect of a 0.5 T magnetic field on the electrical properties of whole human blood. A Solartron SI 1260 spectrometer was used to measure the impedance spectra of magnetic field exposed blood samples compared to non-exposed samples. An equivalent electrical circuit model, consisting in a resistance Rs in series with a parallel circuit formed by a constant phase element (CPE) and another resistance Rp, is proposed to fit the data in both cases. The experiment used 3 ml human blood samples from 160 healthy donors. A Wilcoxon matched pairs statistical test was applied to the data. The data analysis seems to show a statistically significant increase of the values of resistance Rp (Z = 5.06, P < 0.001) and capacitance CT (Z = 3.32, P < 0.001) of the blood exposed to magnetic field, by approximately 10.4% and 1.9%, respectively.     

Plasmid Transfer Detection in Soil using the Inducible lPR System Fused to Eukaryotic Luciferase Genes

We report a model system for plasmid transfer analysis using the regulated lambda phage right promoter, λPr, fused to luc and lucOR as repoter genes. We have demonstrated that the systems cI857-λPr::luc and cI857-λPr::lucOR are temperature-inducible in Escherichia coli but not in other Gram-negative bacteria analyzed, enabling detection of luminescence when plasmids were mobilized from E. coli to those Gram-negative backgrounds. Using light for the detection, we have observed plasmid transfer from E. coli harboring RK2 and R388 derived plasmids to Pseudomonas putida KT2440 (co-introduced with donors) and to indigenous microorganisms, in vitro and in nonsterile soil microcosms. The importance of nutrients for an efficient plasmid transfer in nonsterile soil microcosms has been confirmed. When plasmid transfer experiments were carried out into nonsterile soil microcosms, significant populations of indigenous transconjugants arose. This system provides efficient marker genes and avoids the use of antibiotics for the selection of transconjugants.     

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.     

Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells

Chinese hamster ovary (CHO) cells are characterized by a low glucose catabolic efficiency, resulting in undesirable lactate production. Here, it is hypothesized that such low efficiency is determined by the transport of pyruvate into the mitochondria. The mitochondrial pyruvate carrier (MPC), responsible for introducing pyruvate into the mitochondria, is formed by two subunits, MPC1 and MPC2. Stable CHO cell lines, overexpressing the genes of both subunits, were constructed to facilitate the entry of pyruvate into the mitochondria and its incorporation into oxidative pathways. Significant overexpression of both genes, compared to the basal level of the control cells, was verified, and subcellular localization of both subunits in the mitochondria was confirmed. Kinetic evaluation of the best MPC overexpressing CHO cells showed a reduction of up to 50% in the overall yield of lactate production with respect to the control. An increase in specific growth rate and maximum viable cell concentration, as well as an increase of up to 40% on the maximum concentration of two recombinant model proteins transiently expressed (alkaline phosphatase or a monoclonal antibody), was also observed. Hybrid cybernetic modeling, that considered 89 reactions, 25 extracellular metabolites, and a network of 62 intracellular metabolites, explained that the best MPC overexpression case resulted in an increased metabolic flux across the mitochondrial membrane, activated a more balanced growth, and reduced the Warburg effect without compromising glucose consumption rate and maximum cell concentration. Overall, this study showed that transport of pyruvate into the mitochondria limits the efficiency of glucose oxidation, which can be overcome by a cell engineering approach.     

Comparative phylogeography of two African carnivorans presumably introduced into Europe: disentangling natural versus human‐mediated dispersal across the Strait of Gibraltar

Aim Natural processes of colonization and human‐mediated introductions have shaped current patterns of biodiversity in the Mediterranean Basin. We use a comparative phylogeographic approach to investigate the genetic structure of Herpestes ichneumon and Genetta genetta (Carnivora) across the Strait of Gibraltar, and test for their supposedly contemporaneous introduction into Iberia. Location Mediterranean Basin and Africa. Methods We sequenced two mitochondrial fragments (cytochrome b and control region) of 91 (H. ichneumon) and 185 (G. genetta) individuals, including the sole archaeological record of G. genetta in Iberia, dating from the Muslim occupation. We used phylogenetic and tokogenetic methods, summary statistics, neutrality tests, geographic–genetic pairwise comparisons and coalescent estimates to explore the history of the two species in the Mediterranean Basin. Results In North Africa, an autochthonous (Clade I) and a western African mtDNA clade, coalescing in the Middle to Late Pleistocene, co‐occurred in both species. Only Clade I was present in Europe. In H. ichneumon, the European pool showed deep coalescence (median = 335 kyr) and high genetic differentiation and diversity compared with its North African counterpart, suggesting long‐term stability of female effective population size. In sharp contrast, G. genetta in Europe exhibited lower genetic diversity, weak differentiation with North Africa and recent demographic expansion; however, Andalusia and Catalonia (Spain) showed distinctly higher genetic diversity, and the archaeological specimen had the predominant European haplotype. Main conclusions The co‐occurrence of autochthonous and sub‐Saharan lineages in North Africa (1) supports a new, emerging biogeographic scenario in North Africa, and (2) suggests a connection through the Sahara, possibly from the Middle Pleistocene onwards. Our results refute the idea that H. ichneumon was introduced into Europe contemporaneously with G. genetta. Instead, they support a scenario of sweepstake dispersal during Late Pleistocene sea‐level fluctuations, followed by long‐term in situ evolution throughout the last glaciation cycles. Genetta genetta appears to have undergone a recent spread from at least two independent introduction ‘hotspots’ in Catalonia and Andalusia, possibly following antique trade routes and/or Muslim invasions. Despite their contrasting histories, the European gene pools of both species represent unusual cases leading to the preservation of autochthonous, North African lineages.     

The Association between Mycobacterium Tuberculosis Genotype and Drug Resistance in Peru

BackgroundThe comparison of Mycobacterium tuberculosis bacterial genotypes with phenotypic, demographic, geospatial and clinical data improves our understanding of how strain lineage influences the development of drug-resistance and the spread of tuberculosis.MethodsTo investigate the association of Mycobacterium tuberculosis bacterial genotype with drug-resistance. Drug susceptibility testing together with genotyping using both 15-loci MIRU-typing and spoligotyping, was performed on 2,139 culture positive isolates, each from a different patient in Lima, Peru. Demographic, geospatial and socio-economic data were collected using questionnaires, global positioning equipment and the latest national census.ResultsThe Latin American Mediterranean (LAM) clade (OR 2.4, p<0.001) was significantly associated with drug-resistance and alone accounted for more than half of all drug resistance in the region. Previously treated patients, prisoners and genetically clustered cases were also significantly associated with drug-resistance (OR''s 2.5, 2.4 and 1.8, p<0.001, p<0.05, p<0.001 respectively).ConclusionsTuberculosis disease caused by the LAM clade was more likely to be drug resistant independent of important clinical, genetic and socio-economic confounding factors. Explanations for this include; the preferential co-evolution of LAM strains in a Latin American population, a LAM strain bacterial genetic background that favors drug-resistance or the "founder effect" from pre-existing LAM strains disproportionately exposed to drugs.