The effect of pH on the glucose metabolism of non-growing cells of L. lactis MG1363 was studied by in vivo NMR in the range 4.8 to 6.5. Immediate pH effects on glucose transporters and/or enzyme ...activities were distinguished from transcriptional/translational effects by using cells grown at the optimal pH of 6.5 or pre-adjusted to low pH by growth at 5.1. In cells grown at pH 5.1, glucose metabolism proceeds at a rate 35% higher than in non-adjusted cells at the same pH. Besides the upregulation of stress-related genes (such as dnaK and groEL), cells adjusted to low pH overexpressed H(+)-ATPase subunits as well as glycolytic genes. At sub-optimal pHs, the total intracellular pool of lactic acid reached approximately 500 mM in cells grown at optimal pH and about 700 mM in cells grown at pH 5.1. These high levels, together with good pH homeostasis (internal pH always above 6), imply intracellular accumulation of the ionized form of lactic acid (lactate anion), and the concomitant export of the equivalent protons. The average number, n, of protons exported with each lactate anion was determined directly from the kinetics of accumulation of intra- and extracellular lactic acid as monitored online by (13)C-NMR. In cells non-adjusted to low pH, n varies between 2 and 1 during glucose consumption, suggesting an inhibitory effect of intracellular lactate on proton export. We confirmed that extracellular lactate did not affect the lactate: proton stoichiometry. In adjusted cells, n was lower and varied less, indicating a different mix of lactic acid exporters less affected by the high level of intracellular lactate. A qualitative model for pH effects and acid stress adaptation is proposed on the basis of these results.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Wild-type Corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO2 on the production of organic ...acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO2 caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense of l-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO2 was estimated by using (13)C-labeled glucose and (13)C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (~5%) regardless of the presence or absence of CO2. Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured by in vivo NMR, and the stoichiometry (H(+):organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate with C. glutamicum without genetic manipulation.
A major factor contributing to the etiology of depression is a neurochemical imbalance of the dopaminergic and serotonergic systems, which is caused by persistently high levels of circulating stress ...hormones. Here, a computational model is proposed to investigate the interplay between dopaminergic and serotonergic-kynurenine metabolism under cortisolemia and its consequences for the onset of depression. The model was formulated as a set of nonlinear ordinary differential equations represented with power-law functions. Parameter values were obtained from experimental data reported in the literature, biological databases, and other general information, and subsequently fine-tuned through optimization. Model simulations predict that changes in the kynurenine pathway, caused by elevated levels of cortisol, can increase the risk of neurotoxicity and lead to increased levels of 3,4-dihydroxyphenylaceltahyde (DOPAL) and 5-hydroxyindoleacetaldehyde (5-HIAL). These aldehydes contribute to alpha-synuclein aggregation and may cause mitochondrial fragmentation. Further model analysis demonstrated that the inhibition of both serotonin transport and kynurenine-3-monooxygenase decreased the levels of DOPAL and 5-HIAL and the neurotoxic risk often associated with depression. The mathematical model was also able to predict a novel role of the dopamine and serotonin metabolites DOPAL and 5-HIAL in the ethiology of depression, which is facilitated through increased cortisol levels. Finally, the model analysis suggests treatment with a combination of inhibitors of serotonin transport and kynurenine-3-monooxygenase as a potentially effective pharmacological strategy to revert the slow-down in monoamine neurotransmission that is often triggered by inflammation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ethanolic fermentation of lignocellulose raw materials requires industrial xylose-fermenting strains capable of complete and efficient
d-xylose consumption. A central question in xylose fermentation ...by
Saccharomyces cerevisiae engineered for xylose fermentation is to improve the xylose uptake. In the current study, the glucose/xylose facilitator Gxf1 from
Candida intermedia, was expressed in three different xylose-fermenting
S. cerevisiae strains of industrial origin. The
in vivo effect on aerobic xylose growth and the initial xylose uptake rate were assessed. The expression of Gxf1 resulted in enhanced aerobic xylose growth only for the TMB3400 based strain. It displayed more than a 2-fold higher affinity for
d-xylose than the parental strain and approximately 2-fold higher initial specific growth rate at 4
g/L
d-xylose. Enhanced xylose consumption was furthermore observed when the GXF1-strain was assessed in simultaneous saccharification and co-fermentation (SSCF) of pretreated wheat straw. However, the ethanol yield remained unchanged due to increased by-product formation. Metabolic flux analysis suggested that the expression of the Gxf1 transporter had shifted the control of xylose catabolism from transport to the NAD
+ dependent oxidation of xylitol to xylulose.
According to previous reports, Lactococcus lactis imports glucose via two distinct phosphoenolpyruvate:phosphotransferase systems (mannose-PTS and cellobiose-PTS) and one or more unknown non-PTS ...permease(s). GlcU was identified as the sole non-PTS permease involved in the transport of glucose. Additionally, the biochemical properties of PTSMan, PTSCel and GlcU were characterized in double knockout mutants with glucose uptake restricted to a single system. Transport susceptibility to protonophores indicated that glucose uptake via GlcU is proton-motive force dependent. Competition assays revealed a high specificity of GlcU for glucose. Furthermore, the permease has low affinity for glucose and displays strong preference for the β-anomer as shown by the profiles of consumption of the two glucose anomers studied by ¹³C-NMR. Similar kinetic properties were found for PTSCel, while PTSMan is a high-affinity system recognizing equally well the two anomeric forms of glucose. Transcripts of the genes encoding the three transporters are present simultaneously in the parent strain NZ9000 as shown by reverse transcription-PCR. Investigation of the distribution of GlcU homologues among bacteria showed that these proteins are restricted to the low-GC Gram-positive Firmicutes. This work completes the identification of the glucose transport systems in L. lactis MG1363.
Transcript data obtained by RNA-Seq were used to identify differentially expressed alternatively spliced genes in ribeye muscle tissue between Nelore cattle that differed in their ribeye area (REA) ...or intramuscular fat content (IF). A total of 166 alternatively spliced transcripts from 125 genes were significantly differentially expressed in ribeye muscle between the highest and lowest REA groups (p ≤ 0.05). For animals selected on their IF content, 269 alternatively spliced transcripts from 219 genes were differentially expressed in ribeye muscle between the highest and lowest IF animals. Cassette exons and alternative 3' splice sites were the most frequently found alternatively spliced transcripts for REA and IF content. For both traits, some differentially expressed alternatively spliced transcripts belonged to myosin and myotilin gene families. The hub transcripts were identified for REA (LRRFIP1, RCAN1 and RHOBTB1) and IF (TRIP12, HSPE1 and MAP2K6) have an important role to play in muscle cell degradation, development and motility. In general, transcripts were found for both traits with biological process GO terms that were involved in pathways related to protein ubiquitination, muscle differentiation, lipids and hormonal systems. Our results reinforce the biological importance of these known processes but also reveal new insights into the complexity of the whole cell muscle mRNA of Nelore cattle.
Comparing metabolic profiles under different biological perturbations has become a powerful approach to investigating the functioning of cells. The profiles can be taken as single snapshots of a ...system, but more information is gained if they are measured longitudinally over time. The results are short time series consisting of relatively sparse data that cannot be analyzed effectively with standard time series techniques, such as autocorrelation and frequency domain methods. In this work, we study longitudinal time series profiles of glucose consumption in the yeast Saccharomyces cerevisiae under different temperatures and preconditioning regimens, which we obtained with methods of in vivo nuclear magnetic resonance (NMR) spectroscopy. For the statistical analysis we first fit several nonlinear mixed effect regression models to the longitudinal profiles and then used an ANOVA likelihood ratio method in order to test for significant differences between the profiles.
The proposed methods are capable of distinguishing metabolic time trends resulting from different treatments and associate significance levels to these differences. Among several nonlinear mixed-effects regression models tested, a three-parameter logistic function represents the data with highest accuracy. ANOVA and likelihood ratio tests suggest that there are significant differences between the glucose consumption rate profiles for cells that had been--or had not been--preconditioned by heat during growth. Furthermore, pair-wise t-tests reveal significant differences in the longitudinal profiles for glucose consumption rates between optimal conditions and heat stress, optimal and recovery conditions, and heat stress and recovery conditions (p-values <0.0001).
We have developed a nonlinear mixed effects model that is appropriate for the analysis of sparse metabolic and physiological time profiles. The model permits sound statistical inference procedures, based on ANOVA likelihood ratio tests, for testing the significance of differences between short time course data under different biological perturbations.
Plasmodium knowlesi, a model malaria parasite, is responsible for a significant portion of zoonotic malaria cases in Southeast Asia and must be controlled to avoid disease severity and fatalities. ...However, little is known about the host-parasite interactions and molecular mechanisms in play during the course of P. knowlesi malaria infections, which also may be relevant across Plasmodium species. Here we contrast P. knowlesi sporozoite-initiated infections in Macaca mulatta and Macaca fascicularis using whole blood RNA-sequencing and transcriptomic analysis. These macaque hosts are evolutionarily close, yet malaria-naïve M. mulatta will succumb to blood-stage infection without treatment, whereas malaria-naïve M. fascicularis controls parasitemia without treatment. This comparative analysis reveals transcriptomic differences as early as the liver phase of infection, in the form of signaling pathways that are activated in M. fascicularis, but not M. mulatta. Additionally, while most immune responses are initially similar during the acute stage of the blood infection, significant differences arise subsequently. The observed differences point to prolonged inflammation and anti-inflammatory effects of IL10 in M. mulatta, while M. fascicularis undergoes a transcriptional makeover towards cell proliferation, consistent with its recovery. Together, these findings suggest that timely detection of P. knowlesi in M. fascicularis, coupled with control of inflammation while initiating the replenishment of key cell populations, helps contain the infection. Overall, this study points to specific genes and pathways that could be investigated as a basis for new drug targets that support recovery from acute malaria.
Plasmodium vivax can cause severe malaria. The total parasite biomass during infections is correlated with the severity of disease but not necessarily quantified accurately by microscopy. This ...finding has raised the question whether there could be sub-populations of parasites that are not observed in peripheral blood smears but continue to contribute to the increase in parasite numbers that drive pathogenesis. Non-human primate infection models utilizing the closely related simian malaria parasite Plasmodium cynomolgi hold the potential for quantifying the magnitude of possibly unobserved infected red blood cell (iRBC) populations and determining how the presence of this hidden reservoir correlates with disease severity.
Time series data tracking the longitudinal development of parasitaemia in five Macaca mulatta infected with P. cynomolgi were used to design a computational model quantifying iRBCs that circulate in the blood versus those that are not detectable and are termed here as 'concealed'. This terminology is proposed to distinguish such observations from the deep vascular and widespread 'sequestration' of Plasmodium falciparum iRBCs, which is governed by distinctly different molecular mechanisms.
The computational model presented here clearly demonstrates that the observed growth data of iRBC populations are not consistent with the known biology and blood-stage cycle of P. cynomolgi. However, the discrepancies can be resolved when a sub-population of concealed iRBCs is taken into account. The model suggests that the early growth of a hidden parasite sub-population has the potential to drive disease. As an alternative, the data could be explained by the sequential release of merozoites from the liver over a number of days, but this scenario seems less likely.
Concealment of a non-circulating iRBC sub-population during P. cynomolgi infection of M. mulatta is an important aspect of this successful host-pathogen relationship. The data also support the likelihood that a sub-population of iRBCs of P. vivax has a comparable means to become withdrawn from the peripheral circulation. This inference has implications for understanding vivax biology and pathogenesis and stresses the importance of considering a concealed parasite reservoir with regard to vivax epidemiology and the quantification and treatment of P. vivax infections.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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