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Iron, supplemented as iron-loaded transferrin (holotransferrin), is an essential nutrient in mammalian cell cultures, particularly for erythroid cultures. The high cost of human transferrin represents a challenge for large scale production of red blood cells (RBCs) and for cell therapies in general. We evaluated the use of deferiprone, a cell membrane-permeable drug for iron chelation therapy, as an iron carrier for erythroid cultures. Iron-loaded deferiprone (Def3·Fe3+, at 52 µmol/L) could eliminate the need for holotransferrin supplementation during in vitro expansion and differentiation of erythroblast cultures to produce large numbers of enucleated RBC. Only the first stage, when hematopoietic stem cells committed to erythroblasts, required holotransferrin supplementation. RBCs cultured in presence of Def3·Fe3+ or holotransferrin (1000 µg/mL) were similar with respect to differentiation kinetics, expression of cell-surface markers CD235a and CD49d, hemoglobin content, and oxygen association/dissociation. Replacement of holotransferrin supplementation by Def3·Fe3+ was also successful in cultures of myeloid cell lines (MOLM13, NB4, EOL1, K562, HL60, ML2). Thus, iron-loaded deferiprone can partially replace holotransferrin as a supplement in chemically defined cell culture medium. This holds promise for a significant decrease in medium cost and improved economic perspectives of the large scale production of red blood cells for transfusion purposes. [ABSTRACT FROM AUTHOR]

Both the identity and the amount of a carbon source present in laboratory or industrial cultivation media have major impacts on the growth and physiology of a microbial species. In the case of the yeast Saccharomyces cerevisiae, sucrose is arguably the most important sugar used in industrial biotechnology, whereas glucose is the most common carbon and energy source used in research, with many well-known and described regulatory effects, e.g. glucose repression. Here we compared the label-free proteomes of exponentially growing S. cerevisiae cells in a defined medium containing either sucrose or glucose as the sole carbon source. For this purpose, bioreactor cultivations were employed, and three different strains were investigated, namely: CEN.PK113-7D (a common laboratory strain), UFMG-CM-Y259 (a wild isolate), and JP1 (an industrial bioethanol strain). These strains present different physiologies during growth on sucrose; some of them reach higher specific growth rates on this carbon source, when compared to growth on glucose, whereas others display the opposite behavior. It was not possible to identify proteins that commonly presented either higher or lower levels during growth on sucrose, when compared to growth on glucose, considering the three strains investigated here, except for one protein, named Mnp1—a mitochondrial ribosomal protein of the large subunit, which had higher levels on sucrose than on glucose, for all three strains. Interestingly, following a Gene Ontology overrepresentation and KEGG pathway enrichment analyses, an inverse pattern of enriched biological functions and pathways was observed for the strains CEN.PK113-7D and UFMG-CM-Y259, which is in line with the fact that whereas the CEN.PK113-7D strain grows faster on glucose than on sucrose, the opposite is observed for the UFMG-CM-Y259 strain. [ABSTRACT FROM AUTHOR]

Social participation is an important factor for students' socio-emotional/academic development. The literature on the topic discusses four domains in this regard: friendships, interactions, social acceptance, and self-perception of social inclusion. It shows that marginalized groups (e.g., students with behavioral problems/learning deficiencies/physical disabilities) are experience difficulties in those domains. Little, however, is known about the effect of a migration background (one of the most commonly marginalized groups) in this context. Therefore, self-ratings and peer ratings of 818 fourth graders (148 with a migration background, defined by their first learned language not being German) were assessed. The results of the ANOVA indicate that students with a migration background show a decreased level of friendships, interactions, and social acceptance. Gender effects on self-perceived social inclusion were also found. This suggests that social participation is a rather complex concept, which is also impaired for marginalized groups due to social factors like a migration background. Highlights: Friendships, interactions and social acceptance are negatively affected by a migration background. Self-perception of social inclusion is not affected by a migration background. Self-perception of social inclusion is negatively affected by Gender (boys). Social participation is impaired for students with a migration background. [ABSTRACT FROM AUTHOR]

α-Ketoglutarate (αKG) is a metabolite of the tricarboxylic acid cycle, important for biomass synthesis and a precursor for biotechnological products like 1,4-butanediol. In the eukaryote Saccharomyces cerevisiae αKG is present in different compartments. Compartmentation and (intra-)cellular transport could interfere with heterologous product pathways, generate futile cycles and reduce product yields. Batch and chemostat cultivations at low pH (≤ 5) showed that αKG can be transported, catabolized and used for biomass synthesis. The uptake mechanism of αKG was further investigated under αKG limited chemostat conditions at different pH (3, 4, 5, and 6). At very low pH (3, 4) there is a fraction of undissociated αKG that could diffuse over the periplasmic membrane. At pH 5 this fraction is very low, and the observed growth and residual concentration requires a permease/facilitated uptake mechanism of the mono-dissociated form of αKG. Consumption of αKG under mixed substrate conditions was only observed for low glucose concentrations in chemostat cultivations, suggesting that the putative αKG transporter is repressed by glucose. Fully 13C-labeled αKG was introduced as a tracer during a glucose/αKG co-feeding chemostat to trace αKG transport and utilization. The measured 13C enrichments suggest the major part of the consumed 13C αKG was used for the synthesis of glutamate, and the remainder was transported into the mitochondria and fully oxidized. There was no enrichment observed in glycolytic intermediates, suggesting that there was no gluconeogenic activity under the co-feeding conditions. 13C based flux analysis suggests that the intracellular transport is bi-directional, i.e. there is a fast exchange between the cytosol and mitochondria. The model further estimates that most intracellular αKG (88%) was present in the cytosol. Using literature reported volume fractions, the mitochondria/cytosol concentration ratio was 1.33. Such ratio will not require energy investment for transport towards the mitochondria (based on thermodynamic driving forces calculated with literature pH values). Growth on αKG as sole carbon source was observed, suggesting that S. cerevisiae is not fully Krebs-negative. Using 13C tracing and modelling the intracellular use of αKG under co-feeding conditions showed a link with biomass synthesis, transport into the mitochondria and catabolism. For the engineering of strains that use cytosolic αKG as precursor, both observed sinks should be minimized to increase the putative yields. [ABSTRACT FROM AUTHOR]

Biotechnological industry strives to develop anaerobic bioprocesses fueled by abundant and cheap carbon sources, like sucrose. However, oxygen-limiting conditions often lead to by-product formation and reduced ATP yields. While by-product formation is typically decreased by gene deletion, the breakdown of oligosaccharides with inorganic phosphate instead of water could increment the ATP yield. To observe the effect of oxygen limitation during sucrose consumption, a non-fermentative Escherichia coli K-12 strain was transformed with genes enabling sucrose assimilation. It was observed that the combined deletion of the genes adhE, adhP, mhpF, ldhA, and pta abolished the anaerobic growth using sucrose. Therefore, the biomass-specific conversion rates were obtained using oxygen-limited continuous cultures. Strains performing the breakdown of the sucrose by hydrolysis (SUC-HYD) or phosphorolysis (SUC-PHOSP) were studied in such conditions. An experimentally validated in silico model, modified to account for plasmid and protein burdens, was employed to calculate carbon and electron consistent conversion rates. In both strains, the biomass yields were lower than expected and, strikingly, SUC-PHOSP showed a yield lower than SUC-HYD. Flux balance analyses indicated a significant increase in the non-growth-associated ATP expenses by comparison with the growth on glucose. The observed fructose-1,6-biphosphatase and phosphoglucomutase activities, as well as the concentrations of glycogen, suggest the operation of ATP futile cycles triggered by a combination of the oxygen limitation and the metabolites released during the sucrose breakdown. [ABSTRACT FROM AUTHOR]

Afforestation of the Sahara has been proposed as a climate engineering method to sequester a substantial amount of carbon dioxide, potentially effective to mitigate climate change. Earlier studies predicted changes in the atmospheric circulation system. These atmospheric feedbacks raise questions about the self-sustainability of such an intervention, but have not been investigated in detail. Here, we investigate changes in precipitation and circulation in response to Saharan large-scale afforestation and irrigation with NCAR’s CESM-WACCM Earth system model. Our model results show a Saharan temperature reduction by 6 K and weak precipitation enhancement by 267 mm/year over the Sahara. Only 26% of the evapotranspirated water re-precipitates over the Saharan Desert, considerably large amounts are advected southward to the Sahel zone and enhance the West African monsoon (WAM). Different processes cause circulation and precipitation changes over North Africa. The increase in atmospheric moisture leads to radiative cooling above the Sahara and increased high-level cloud coverage as well as atmospheric warming above the Sahel zone. Both lead to a circulation anomaly with descending air over the Sahara and ascending air over the Sahel zone. Together with changes in the meridional temperature gradient, this results in a southward shift of the inner-tropical front. The strengthening of the Tropical easterly jet and the northward displacement of the African easterly jet is associated with a northward displacement and strengthening of the WAM precipitation. Our results suggest complex atmospheric circulation feedbacks, which reduce the precipitation potential over an afforested Sahara and enhance WAM precipitation. [ABSTRACT FROM AUTHOR]

Most of the current coupled general circulation models show a strong warm bias in the eastern Tropical Atlantic. In this paper, various sensitivity experiments with the Kiel Climate Model (KCM) are described. A largely reduced warm bias and an improved seasonal cycle in the eastern Tropical Atlantic are simulated in one particular version of KCM. By comparing the stable and well-tested standard version with the sensitivity experiments and the modified version, mechanisms contributing to the reduction of the eastern Atlantic warm bias are identified and compared to what has been proposed in literature. The error in the spring and early summer zonal winds associated with erroneous zonal precipitation seems to be the key mechanism, and large-scale coupled ocean-atmosphere feedbacks play an important role in reducing the warm bias. Improved winds in boreal spring cause the summer cooling in the eastern Tropical Atlantic (ETA) via shoaling of the thermocline and increased upwelling, and hence reduced sea surface temperature (SST). Reduced SSTs in the summer suppress convection and favor the development of low-level cloud cover in the ETA region. Subsurface ocean structure is shown to be improved, and potentially influences the development of the bias. The strong warm bias along the southeastern coastline is related to underestimation of low-level cloud cover and the associated overestimation of surface shortwave radiation in the same region. Therefore, in addition to the primarily wind forced response at the equator both changes in surface shortwave radiation and outgoing longwave radiation contribute significantly to reduction of the warm bias from summer to fall. [ABSTRACT FROM AUTHOR]

Introduction: The switch from quiescence (G0) into G1 and cell cycle progression critically depends on specific nutrients and metabolic capabilities. Conversely, metabolic networks are regulated by enzyme–metabolite interaction and transcriptional regulation that lead to flux modifications to support cell growth. How cells process and integrate environmental information into coordinated responses is challenging to analyse and not yet described quantitatively. Objectives: To quantitatively monitor the central carbon metabolism during G0 exit and the first 2 h after reentering the cell cycle from synchronized Saccharomyces cerevisiae. Methods: Dynamic tailored 13C metabolic flux analysis was used to observe the intracellular metabolite flux changes, and the metabolome and proteome were observed to identify regulatory mechanisms. Results: G0 cells responded immediately to an extracellular increase of glucose. The intracellular metabolic flux changed in time and specific events were observed. High fluxes into trehalose and glycogen synthesis were observed during the G0 exit. Both fluxes then decreased, reaching a minimum at t = 65 min. Here, storage degradation contributed significantly (i.e. 21%) to the glycolytic flux. In contrast to these changes, the glucose uptake rate remained constant after the G0 exit. The flux into the oxidative pentose phosphate pathway was highest (29-fold increase, 36.4% of the glucose uptake) at t = 65 min, while it was very low at other time points. The maximum flux seems to correlate with a late G1 state preparing for the S phase transition. In the G1/S phase (t = 87 min), anaplerotic reactions such as glyoxylate shunt increased. Protein results show that during this transition, proteins belonging to clusters related with ribosome biogenesis and assembly, and initiation transcription factors clusters were continuously synthetised. Conclusion: The intracellular flux distribution changes dynamically and these major rearrangements highlight the coordinate reorganization of metabolic flux to meet requirements for growth during different cell state. [ABSTRACT FROM AUTHOR]