Proliferating bloodstream-form Trypanosoma brucei use a negligible part of consumed glucose for anabolic processes

Our quantitative knowledge of carbon fluxes in the long slender bloodstream form (BSF) Trypanosoma brucei is mainly based on non-proliferating parasites, isolated from laboratory animals and kept in buffers. In this paper we present a carbon balance for exponentially growing bloodstream form trypanosomes. The cells grew with a doubling time of 5.3h, contained 46 μ mol of carbon (10(8) cells)(-1) and had a glucose consumption flux of 160 nmol min(-1) (10(8) cells)(-1). The molar ratio of pyruvate excreted versus glucose consumed was 2.1. Furthermore, analysis of the (13)C label distribution in pyruvate in (13)C-glucose incubations of exponentially growing trypanosomes showed that glucose was the sole substrate for pyruvate production. We conclude that the glucose metabolised in glycolysis was hardly, if at all, used for biosynthetic processes. Carbon flux through glycolysis in exponentially growing trypanosomes was 10 times higher than the incorporation of carbon into biomass. This biosynthetic carbon is derived from other precursors present in the nutrient rich growth medium. Furthermore, we found that the glycolytic flux was unaltered when the culture went into stationary phase, suggesting that most of the ATP produced in glycolysis is used for processes other than growth.     

In Silico Prediction of Horizontal Gene Transfer Events in Lactobacillus bulgaricus and Streptococcus thermophilus Reveals Protocooperation in Yogurt Manufacturing

Lactobacillus bulgaricus and Streptococcus thermophilus, used in yogurt starter cultures, are well known for their stability and protocooperation during their coexistence in milk. In this study, we show that a close interaction between the two species also takes place at the genetic level. We performed an in silico analysis, combining gene composition and gene transfer mechanism-associated features, and predicted horizontally transferred genes in both L. bulgaricus and S. thermophilus. Putative horizontal gene transfer (HGT) events that have occurred between the two bacterial species include the transfer of exopolysaccharide (EPS) biosynthesis genes, transferred from S. thermophilus to L. bulgaricus, and the gene cluster cbs-cblB(cglB)-cysE for the metabolism of sulfur-containing amino acids, transferred from L. bulgaricus or Lactobacillus helveticus to S. thermophilus. The HGT event for the cbs-cblB(cglB)-cysE gene cluster was analyzed in detail, with respect to both evolutionary and functional aspects. It can be concluded that during the coexistence of both yogurt starter species in a milk environment, agonistic coevolution at the genetic level has probably been involved in the optimization of their combined growth and interactions.Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus) and Streptococcus thermophilus have been used in starter cultures for yogurt manufacturing for thousands of years. Both species are known to stably coexist in a milk environment and interact beneficially. This so-called protocooperation, previously defined as biochemical mutualism, involves the exchange of metabolites and/or stimulatory factors (38). Examples of biochemical protocooperation between L. bulgaricus and S. thermophilus include the action of cell wall-bound proteases, produced by L. bulgaricus strains, and formate, required for growth of L. bulgaricus and supplied by S. thermophilus (6, 7). An overview of the interactions between the two yogurt bacteria, including the exchange of CO₂, pyruvate, folate, etc., can be found in a recently published review by Sieuwerts et al. (43). Putative genetic mechanisms underlying protocooperation, however, so far have not been studied in detail.The genomes of two L. bulgaricus strains and three S. thermophilus strains, all used in yogurt manufacturing, have been fully sequenced (3, 32, 33, 34, 39, 44, 46). The available genomic information could provide new insights into the genetic aspects of protocooperation between L. bulgaricus and S. thermophilus through the identification of putative horizontal gene transfer (HGT) events at the genome scale. HGT can be defined as the exchange of genetic material between phylogenetically unrelated organisms (23). It is considered to be a major factor in the process of environmental adaptation, for both individual species and entire microbial populations. Especially HGT events between two species existing in the same niche can reflect their interrelated activities and dependencies (13, 17). Nicolas et al. (36) predicted HGT events between Lactobacillus acidophilus and Lactobacillus johnsonii by analyzing 401 phylogenetic trees, also including the genes of L. bulgaricus. Several HGT events have been predicted in the S. thermophilus strains CNRZ1066 and LMG 18311 (3, 10, 18) as well as in L. bulgaricus ATCC 11842 (46). Moreover, a core genome of S. thermophilus and possibly acquired genes were identified by a comparative genome hybridization study of 47 strains (40).In this study, we describe an in-depth bioinformatics analysis in which we combined gene composition (GC content and dinucleotide composition) and gene transfer mechanism-associated features. Thus, we predicted horizontally transferred genes and gene clusters in the five sequenced L. bulgaricus and S. thermophilus genomes, with a focus on niche-specific genes and genes required for bacterial growth. Identification of HGT events led to a list of putative transferred genes, some of which could be important for bacterial protocooperation and the adaptation to their environment. The evolution and function of the transferred gene cluster cbs-cblB(cglB)-cysE (originally called cysM2-metB2-cysE2 in S. thermophilus), involved in the metabolism of sulfur-containing amino acids, were analyzed in detail. On the basis of our analysis, it can be concluded that both species probably agonistically coevolved at the genetic level to optimize their combined growth in a milk environment and that protocooperation thus includes both biochemical and genetic aspects.     

Environmental impact of thin-film GaInP/GaAs and multicrystalline silicon solar modules produced with solar electricity

Background, aim, and scope  The environmental burden of photovoltaic (PV) solar modules is currently largely determined by the cumulative input of fossil energy used for module production. However, with an increased focus on limiting the emission of CO₂ coming from fossil fuels, it is expected that renewable resources, including photovoltaics, may well become more important in producing electricity. A comparison of the environmental impacts of PV modules in case their life cycle is based on the use of PV electricity in contrast to conventional electricity can elucidate potential environmental drawbacks in an early stage of development of a solar-based economy. The goal of this paper is to show for ten impact categories the environmental consequences of replacing fossil electricity with solar electricity into the life cycle of two types of PV modules. Materials and methods  Using life cycle assessment (LCA), we evaluated the environmental impacts of two types of PV modules: a thin-film GaInP/GaAs tandem module and a multicrystalline silicon (multi-Si) module. For each of the modules, the total amount of fossil electricity required in the life cycle of the module was substituted with electricity that is generated by a corresponding PV module. The environmental impacts of the modules on the midpoint level were compared with those of the same modules in case their life cycle is based on the use of conventional electricity. The environmental impacts were assessed for Western European circumstances with an annual solar irradiation of 1000 kWh/m². For the GaInP/GaAs module, the environmental impacts of individual production steps were also analysed. Results  Environmental burdens decreased when PV electricity was applied in the life cycle of the two PV modules. The impact score reductions of the GaInP/GaAs module were up to a factor of 4.9 (global warming). The impact score reductions found for the multi-Si module were up to a factor of 2.5 (abiotic depletion and global warming). Reductions of the toxicity scores of both module types were smaller or negligible. This is caused by a decreased use of fossil fuels, on the one hand, and an increased consumption of materials for the production of the additional solar modules used for generating the required PV electricity on the other. Overall, the impact scores of the GaInP/GaAs module were reduced more than the corresponding scores of the multi-Si module. The contribution analysis of the GaInP/GaAs module production steps indicated that for global warming, the cell growth process is dominant for supply with conventional electricity, while for the solar scenario, the frame becomes dominant. Regarding freshwater aquatic ecotoxicity scores associated with the life cycle of the GaInP/GaAs module, the cell growth process is dominant for supply with conventional electricity, while the reactor system for the cell growth with the associated gas scrubbing system is dominant for the solar scenario. Discussion  There are uncertainties regarding the calculated environmental impact scores. This paper describes uncertainties associated with the used economic allocation method, and uncertainties because of missing life cycle inventory data. For the GaInP/GaAs module, it was found that the global warming impact scores range from −66% to +41%, and the freshwater aquatic ecotoxicity scores (for an infinite time horizon) range from −40% to +300% compared to the default estimates. For both impact categories, the choices associated with the allocation of gallium, with the electricity mix, with the conversion efficiency of the commercially produced GaInP/GaAs cells, and with the yield of the cell growth process are most influential. For freshwater aquatic ecotoxicity, the uncertainty concerning the lifetime of the reactor system for the GaInP/GaAs cell growth process and the gas scrubbing system is particularly relevant. Conclusions  Use of PV electricity instead of fossil electricity significantly reduces the environmental burdens of the GaInP/GaAs and the multi-Si module. The reductions of the toxicity scores, however, are smaller or negligible. Toxicity impacts of the GaInP/GaAs cells can be reduced by improvement of the yield of the cell growth process, a reduced energy demand in the cell growth process, reduction of the amount of stainless steel in the cell growth reactor system and the gas scrubbing system, and a longer lifetime of these systems. Recommendations and perspectives  Because the greenhouse gas emissions associated with the production of fossil-fuel-based electricity have an important share in global warming on a world-wide scale, switching to a more extensive use of solar power is helpful to comply with the present international legislation on the area of global warming reduction. As reductions in toxicity impact scores are smaller or negligible when fossil electricity is replaced by PV electricity, it is desirable to give specific attention to the processes which dominantly contribute to these impact categories. Furthermore, in this study, a shift in ranking of several environmental impacts of the modules has been found when PV electricity is used instead of fossil electricity. The results of a comparative LCA can thus be dependent of the electricity mix used in the life cycles of the assessed products. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Plant expression of chicken secretory antibodies derived from combinatorial libraries

Delivery of secretory IgA antibodies (sIgA) to mucosal surfaces is a promising strategy to passively prevent infectious diseases. Plants have been proposed as biofactories for such complex immunoglobulin molecules. Recently, the molecular characterization of all four monomers of chicken sIgA (IgA immunoglobulin heavy and light chains, J-chain and secretory component) has been completed, allowing recombinant, up scaled production of chicken sIgA and extension of passive immune strategies to poultry. To test the suitability of the plant cell factory for bulk production of chicken sIgA, we studied the expression of chicken IgA, dIgA and sIgA in planta. To that end, new cassettes were designed that allowed the grafting of immunoglobulin variable regions derived from combinatorial libraries into full-size chicken IgA frames ready for plant expression. Using this system, 10 individual phage display clones, which had previously been selected against Eimeria acervulina antigens, were transferred "from phage to plant". Plant-made chicken antibodies showed strong differences in expression levels, which seemed governed mainly by the stability of their respective light chains. Finally, with the co-expression of chicken IgA heavy and light chains, J-chain and secretory component in N. benthamiana leaves we showed that plant cells are suitable biofactories for the production of assembled chicken sIgA complexes.     

Direct interaction with Rab11a targets the epithelial Ca2+ channels TRPV5 and TRPV6 to the plasma membrane

TRPV5 and TRPV6 are the most Ca2+-selective members of the transient receptor potential (TRP) family of cation channels and play a pivotal role in the maintenance of Ca2+ balance in the body. However, little is known about the mechanisms controlling the plasma membrane abundance of these channels to regulate epithelial Ca2+ transport. In this study, we demonstrated the direct and specific interaction of GDP-bound Rab11a with TRPV5 and TRPV6. Rab11a colocalized with TRPV5 and TRPV6 in vesicular structures underlying the apical plasma membrane of Ca2+-transporting epithelial cells. This GTPase recognized a conserved stretch in the carboxyl terminus of TRPV5 that is essential for channel trafficking. Furthermore, coexpression of GDP-locked Rab11a with TRPV5 or TRPV6 resulted in significantly decreased Ca2+ uptake, caused by diminished channel cell surface expression. Together, our data demonstrated the important role of Rab11a in the trafficking of TRPV5 and TRPV6. Rab11a exerts this function in a novel fashion, since it operates via direct cargo interaction while in the GDP-bound configuration.     

In vitro degradation behavior of microspheres based on cross-linked dextran

The aim of this study was to investigate the in vitro degradation of hydroxyl ethyl methacrylated dextran (dex-HEMA) microspheres. Dextran microspheres were incubated in phosphate buffer pH 7.4 at 37 degrees C, and the dry mass, mechanical strength, and chemical composition of the microspheres were monitored in time. The amount and nature of the formed degradation products were established for microspheres with different cross-link densities by FT-IR (Fourier transformed infrared spectroscopy), NMR, mass spectrometry, SEC analysis, and XPS (X-ray photoelectron microscopy). The dex-HEMA microspheres DS 12 (degree of HEMA substitution; the number of HEMA groups per 100 glucose units) incubated at pH 7.4 and 37 degrees C showed a continuous mass loss, leaving after 6 months a residue of about 10% (w/w) of water-insoluble products. NMR, mass spectrometry, and SEC showed that the water-soluble degradation products consisted of dextran, low molecular weight pHEMA (M(n) approximately 15 kg/mol), and small amounts of unreacted HEMA and HEMA-DMAP (intermediate reaction product of the Baylis-Hillman reaction of HEMA with DMAP (4-dimethyl aminopyridine)). Microscopy revealed that the water-insoluble residue consisted of particles with shape and size similar to that of nondegraded microspheres. However, these particles had lost their mechanical strength as evidenced from micromanipulation experiments. FT-IR and XPS (X-ray photoelectron microscopy) revealed that these particles consisted of pHEMA, of which a small fraction was soluble in methanol (M(n) ranging between 27 and 82 kg/mol). The insoluble material likely consisted of lightly cross-linked pHEMA. In conclusion, in vitro degradation of dex-HEMA microspheres results in the formation of water-soluble degradation products (mainly dextran), leaving a small water-insoluble residue mainly consisting of pHEMA.     

Microorganisms and nematodes increase levels of secondary metabolites in roots and root exudates of Plantago lanceolata

Plant secondary metabolites play an important role in constitutive and inducible direct defense of plants against their natural enemies. While induction of defense by aboveground pathogens and herbivores is well-studied, induction by belowground organisms is less explored. Here, we examine whether soil microorganisms and nematodes can induce changes in levels of the secondary metabolites aucubin and catalpol (iridoid glycosides, IG) in roots and root exudates of two full-sib families of Plantago lanceolata originating from lines selected for low and high constitutive levels of IG in leaves. Addition of soil microorganisms enhanced the shoot and root biomass, and the concentration of aucubin in roots of both Plantago lines without affecting IG levels in the rhizosphere. By contrast, nematode addition tended to reduce the root biomass and enhanced the stalk biomass, and increased the levels of aucubin and catalpol in root exudates of both Plantago lines, without affecting root IG concentrations. The Plantago lines did not differ in constitutive levels of aucubin and total IG in roots, while the concentration of catalpol was slightly higher in roots of plants originally selected for low constitutive levels of IG in leaves. Root exudates of “high IG line” plants contained significantly higher levels of aucubin, which might be explained by their higher root biomass. We conclude that soil microorganisms can induce an increase of aucubin concentrations in the roots, whereas nematodes (probably plant feeders) lead to an enhancement of aucubin and catalpol levels in root exudates of P. lanceolata. A potential involvement of secondary metabolites in belowground interactions between plants and soil organisms is discussed.     

Epithelial p38α Controls Immune Cell Recruitment in the Colonic Mucosa

Intestinal epithelial cells (IECs) compose the first barrier against microorganisms in the gastrointestinal tract. Although the NF-κB pathway in IECs was recently shown to be essential for epithelial integrity and intestinal immune homeostasis, the roles of other inflammatory signaling pathways in immune responses in IECs are still largely unknown. Here we show that p38α in IECs is critical for chemokine expression, subsequent immune cell recruitment into the intestinal mucosa, and clearance of the infected pathogen. Mice with p38α deletion in IECs suffer from a sustained bacterial burden after inoculation with Citrobacter rodentium. These animals are normal in epithelial integrity and immune cell function, but fail to recruit CD4⁺ T cells into colonic mucosal lesions. The expression of chemokines in IECs is impaired, which appears to be responsible for the impaired T cell recruitment. Thus, p38α in IECs contributes to the host immune responses against enteric bacteria by the recruitment of immune cells.