High yield production of four-carbon dicarboxylic acids by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Several metabolic engineered Escherichia coli strains were constructed and evaluated for four-carbon dicarboxylic acid production. Fumarase A, fumarase B and fumarase C single, double and triple mutants were constructed in a ldhA adhE mutant background overexpressing the pyruvate carboxylase from Lactococcus lactis. All the mutants produced succinate as the main four-carbon (C4) dicarboxylic acid product when glucose was used as carbon source with the exception of the fumAC and the triple fumB fumAC deletion strains, where malate was the main C4-product with a yield of 0.61–0.67 mol (mole glucose)^?1. Additionally, a mdh mutant strain and a previously engineered high-succinate-producing strain (SBS550MG-Cms pHL413-Km) were investigated for aerobic malate production from succinate. These strains produced 40.38 mM (5.41 g/L) and 50.34 mM (6.75 g/L) malate with a molar yield of 0.53 and 0.55 mol (mole succinate)^?1, respectively. Finally, by exploiting the high-succinate production capability, the strain SBS550MG-Cms243 pHL413-Km showed significant malate production in a two-stage process from glucose. This strain produced 133 mM (17.83 g/L) malate in 47 h, with a high yield of 1.3 mol (mole glucose)^?1 and productivity of 0.38 g L^?1 h^?1.     

Network structure and biodiversity loss in food webs: robustness increases with connectance

Food-web structure mediates dramatic effects of biodiversity loss including secondary and `cascading' extinctions. We studied these effects by simulating primary species loss in 16 food webs from terrestrial and aquatic ecosystems and measuring robustness in terms of the secondary extinctions that followed. As observed in other networks, food webs are more robust to random removal of species than to selective removal of species with the most trophic links to other species. More surprisingly, robustness increases with food-web connectance but appears independent of species richness and omnivory. In particular, food webs experience `rivet-like' thresholds past which they display extreme sensitivity to removal of highly connected species. Higher connectance delays the onset of this threshold. Removing species with few trophic connections generally has little effect though there are several striking exceptions. These findings emphasize how the number of species removed affects ecosystems differently depending on the trophic functions of species removed.     

Myocardium proteome remodelling after nutritional deprivation of methyl donors

Methyl donor (MD: folate, vitamin B12 and choline) deficiency causes hyperhomocysteinemia, a risk factor for cardiovascular diseases. However, the mechanisms of the association between MD deficiency, hyperhomocysteinemia, and cardiomyopathy remain unclear. Therefore, we performed a proteomic analysis of myocardium of pups from rat dams fed a MD-depleted diet to understand the impact of MD deficiency on heart at the protein level. Two-dimension gel electrophoresis and mass spectrometry-based analyses allowed us to identify 39 proteins with significantly altered abundance in MD-deficient myocardium. Ingenuity Pathway Analysis showed that 87% of them fitted to a single protein network associated with developmental disorder, cellular compromise and lipid metabolism. Concurrently increased protein carbonylation, the major oxidative post-translational protein modification, could contribute to the decreased abundance of many myocardial proteins after MD deficiency. To decipher the effect of MD deficiency on the abundance of specific proteins identified in vivo, we developed an in vitro model using the cardiomyoblast cell line H9c2. After a 4-day exposure to a MD-deprived (vs. complete) medium, cells were deficient of folate and vitamin B12, and released abnormal amounts of homocysteine. Western blot analyses of pup myocardium and H9c2 cells yielded similar findings for several proteins. Of specific interest is the result showing increased and decreased abundances of prohibitin and α-crystallin B, respectively, which underlines mitochondrial injury and endoplasmic reticulum stress within MD deficiency. The in vitro findings validate the MD-deficient H9c2 cells as a relevant model for studying mechanisms of the early metabolic changes occurring in cardiac cells after MD deprivation.     

Early Interactions of Rhizobium leguminosarum bv. phaseoli and Bean Roots: Specificity in the Process of Adsorption and Its Requirement of Ca(sup2+) and Mg(sup2+) Ions

Roots of Phaseolus vulgaris L. were incubated with dilute suspensions (1 x 10(sup3) to 3 x 10(sup3) bacteria ml(sup-1)) of an antibiotic-resistant indicator strain of Rhizobium leguminosarum bv. phaseoli in mineral medium and washed four times by a standardized procedure prior to quantitation of adsorption (G. Caetano-Anolles and G. Favelukes, Appl. Environ. Microbiol. 52:371-376, 1986). The population of rhizobia remaining adsorbed on roots after washing was homogeneous, as indicated by the first-order course of its desorption by hydrodynamic shear. Rhizobia were maximally active for adsorption in the early stationary phase of growth. The process leading to adsorption was rapid, without an initial lag, and slowed down after 1 h. Adsorption of the indicator strain at 10(sup3) bacteria ml(sup-1) was inhibited to different extents in the presence of 10(sup3) to 10(sup8) antibiotic-sensitive competitor rhizobia ml(sup-1). After a steep rise above 10(sup4) bacteria ml(sup-1), inhibition by heterologous competitors in the concentration range of 10(sup5) to 10(sup7) bacteria ml(sup-1) was markedly less than by homologous strains, while at 10(sup8) bacteria ml(sup-1) it approached the high level of inhibition by the latter. At 10(sup7) bacteria ml(sup-1), all of the heterologous strains tested were consistently less inhibitory than homologous competitors (P < 0.001). These differences in competitive behavior indicate that in the process of adsorption of R. leguminosarum bv. phaseoli to its host bean roots, different modes of adsorption occur and that some of these modes are specific for the microsymbiont (as previously reported for the alfalfa system [G. Caetano-Anolles and G. Favelukes, Appl. Environ. Microbiol. 52:377-381, 1986]). Moreover, whereas the nonspecific process occurred either in the absence or in the presence of Ca(sup2+) and Mg(sup2+) ions, expression of specificity was totally dependent on the presence of those cations. R. leguminosarum bv. phaseoli bacteria adsorbed in the presence of Ca(sup2+) and Mg(sup2+) were more resistant to desorption by shear forces than were rhizobia adsorbed in their absence. These results indicate that (i) symbiotic specificity in the P. vulgaris-R. leguminosarum bv. phaseoli system is expressed already during the early process of rhizobial adsorption to roots, (ii) Ca(sup2+) and Mg(sup2+) ions are required by R. leguminosarum bv. phaseoli for that specificity, and (iii) those cations cause tighter binding of rhizobia to roots.     

A simple method to determine evaporation and compensate for liquid losses in small-scale cell culture systems

Objectives

Establish a method to indirectly measure evaporation in microwell-based cell culture systems and show that the proposed method allows compensating for liquid losses in fed-batch processes.

Results

A correlation between evaporation and the concentration of Na⁺ was found (R²?=?0.95) when using the 24-well-based miniature bioreactor system (micro-Matrix) for a batch culture with GS-CHO. Based on these results, a method was developed to counteract evaporation with periodic water additions based on measurements of the Na⁺ concentration. Implementation of this method resulted in a reduction of the relative liquid loss after 15 days of a fed-batch cultivation from 36.7?±?6.7% without volume corrections to 6.9?±?6.5% with volume corrections.

Conclusion

A procedure was established to indirectly measure evaporation through a correlation with the level of Na⁺ ions in solution and deriving a simple formula to account for liquid losses.

     

A Review of the International Atomic Energy Agency (IAEA) International Standards for Tissue Banks

The IAEA International Standards for Tissue Banks published in 2003 were based on the Standards then currently in use in the USA and the European Union, among others, and reflect the best practices associated with the operation of a tissue bank. They cover legal, ethical and regulatory controls as well as requirements and procedures from donor selection and tissue retrieval to processing and distribution of finished tissue for clinical use. The application of these standards allows tissue banks to operate with the current good tissue practice, thereby providing grafts of high quality that satisfy the national and international demand for safe and biologically useful grafts. The objective of this article is to review the IAEA Standards and recommend new topics that could improve the current version.     

Usefulness of autocidal gravid ovitraps for the surveillance and control of Aedes (Stegomyia) aegypti (Diptera: Culicidae) in eastern Colombia

In the past decade, new strategies have been developed to control the Aedes aegypti (Diptera: Culicidae) mosquito vector, as well as a broad range of arboviral agents. Vector control surveillance programmes in Puerto Rico and Australia have implemented the Centers for Disease Control and Prevention autocidal gravid ovitrap (AGO), which has had an impact on vector density and, consequently, the epidemiology of arboviral infections. Colombia intends to establish the AGO as a new tool for the surveillance and control of the A. aegypti vector. AGOs were evaluated in a hyperendemic area for dengue virus during an 8-week period in Villavicencio city, eastern Colombia. The results indicated that the AGOs detect a high density of A. aegypti, with positive results for these traps of over 80% and an average catch of six individuals per trap per week. Acceptance of AGOs in the community exceeded 95%, and adherence was around 89%. This study's results demonstrate, for the first time in Colombia, that traps are a useful tool for the surveillance of A. aegypti. Future studies must consider the implementation of AGOs in the region.     

Specific regions within the embryonic midbrain and cerebellum require different levels of FGF signaling during development

Prospective midbrain and cerebellum formation are coordinated by FGF ligands produced by the isthmic organizer. Previous studies have suggested that midbrain and cerebellum development require different levels of FGF signaling. However, little is known about the extent to which specific regions within these two parts of the brain differ in their requirement for FGF signaling during embryogenesis. Here, we have explored the effects of inhibiting FGF signaling within the embryonic mouse midbrain (mesencephalon) and cerebellum (rhombomere 1) by misexpressing sprouty2 (Spry2) from an early stage. We show that such Spry2 misexpression moderately reduces FGF signaling, and that this reduction causes cell death in the anterior mesencephalon, the region furthest from the source of FGF ligands. Interestingly, the remaining mesencephalon cells develop into anterior midbrain, indicating that a low level of FGF signaling is sufficient to promote only anterior midbrain development. Spry2 misexpression also affects development of the vermis, the part of the cerebellum that spans the midline. We found that, whereas misexpression of Spry2 alone caused loss of the anterior vermis, reducing FGF signaling further, by decreasing Fgf8 gene dose, resulted in loss of the entire vermis. Our data suggest that cell death is not responsible for vermis loss, but rather that it fails to develop because reducing FGF signaling perturbs the balance between vermis and roof plate development in rhombomere 1. We suggest a molecular explanation for this phenomenon by providing evidence that FGF signaling functions to inhibit the BMP signaling that promotes roof plate development.     

Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability

Isocitrate dehydrogenase (IDH) has been studied extensively due to its central role in the Krebs cycle, catalyzing the oxidative NAD(P)(+)-dependent decarboxylation of isocitrate to alpha-ketoglutarate and CO(2). Here, we present the first crystal structure of IDH from a psychrophilic bacterium, Desulfotalea psychrophila (DpIDH). The structural information is combined with a detailed biochemical characterization and a comparative study with IDHs from the mesophilic bacterium Desulfitobacterium hafniense (DhIDH), porcine (PcIDH), human cytosolic (HcIDH) and the hyperthermophilic Thermotoga maritima (TmIDH). DpIDH was found to have a higher melting temperature (T(m)=66.9 degrees C) than its mesophilic homologues and a suboptimal catalytic efficiency at low temperatures. The thermodynamic activation parameters indicated a disordered active site, as seen also for the drastic increase in K(m) for isocitrate at elevated temperatures. A methionine cluster situated at the dimeric interface between the two active sites and a cluster of destabilizing charged amino acids in a region close to the active site might explain the poor isocitrate affinity. On the other hand, DpIDH was optimized for interacting with NADP(+) and the crystal structure revealed unique interactions with the cofactor. The highly acidic surface, destabilizing charged residues, fewer ion pairs and reduced size of ionic networks in DpIDH suggest a flexible global structure. However, strategic placement of ionic interactions stabilizing the N and C termini, and additional ionic interactions in the clasp domain as well as two enlarged aromatic clusters might counteract the destabilizing interactions and promote the increased thermal stability. The structure analysis of DpIDH illustrates how psychrophilic enzymes can adjust their flexibility in dynamic regions during their catalytic cycle without compromising the global stability of the protein.