Short-term metabolome dynamics and carbon, electron, and ATP balances in chemostat-grown Saccharomyces cerevisiae CEN.PK 113-7D following a glucose pulse

The in vivo kinetics in Saccharomyces cerevisiae CEN.PK 113-7D was evaluated during a 300-second transient period after applying a glucose pulse to an aerobic, carbon-limited chemostat culture. We quantified the responses of extracellular metabolites, intracellular intermediates in primary metabolism, intracellular free amino acids, and in vivo rates of O(2) uptake and CO(2) evolution. With these measurements, dynamic carbon, electron, and ATP balances were set up to identify major carbon, electron, and energy sinks during the postpulse period. There were three distinct metabolic phases during this time. In phase I (0 to 50 seconds after the pulse), the carbon/electron balances closed up to 85%. The accumulation of glycolytic and storage compounds accounted for 60% of the consumed glucose, caused an energy depletion, and may have led to a temporary decrease in the anabolic flux. In phase II (50 to 150 seconds), the fermentative metabolism gradually became the most important carbon/electron sink. In phase III (150 to 300 seconds), 29% of the carbon uptake was not identified in the measurements, and the ATP balance had a large surplus. These results indicate an increase in the anabolic flux, which is consistent with macroscopic balances of extracellular fluxes and the observed increase in CO(2) evolution associated with nonfermentative metabolism. The identified metabolic processes involving major carbon, electron, and energy sinks must be taken into account in in vivo kinetic models based on short-term dynamic metabolome responses.     

13C-labeled gluconate tracing as a direct and accurate method for determining the pentose phosphate pathway split ratio in Penicillium chrysogenum

In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-13C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a 13C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of 13C-labeled primary metabolites are reported for P. chrysogenum and used for a 13C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h(-1) yielded comparable values for the gluconate tracer method and the 13C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the 13C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the 13C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.     

Topology characterization by MALDI-ToF-MS of enzymatically synthesized poly(lactide-co-glycolide)

Lipase catalyzed copolymerization of the monomers lactide and glycolide by Pseudomonas cepacia employing a molar ratio of 80L/20G has been studied. The copolymers were characterized by MALDI-ToF-MS, DSC, SEC and NMR. MALDI-ToF-MS has successfully been used not only to determine end groups and chemical composition but even the microstructure of the copolymers. We demonstrated that for this lipase catalyzed copolymerization, the main product of the reaction at 100 degrees C was linear homopolymer of lactide while at 130 degrees C the main product was cyclic random copolymer.     

An appraisal of simple tree-mounted shelters for non-lethal monitoring of weta (Orthoptera: Anostostomatidae and Rhaphidophoridae) in New Zealand nature reserves

When monitoring rare insect species, or when surveying faunas within nature reserves, it is desirable not to use indiscriminate lethal sampling techniques. In this investigation we assessed the usefulness of simple tree-mounted wooden shelters to monitor endemic weta (Orthoptera) in nature reserves in Canterbury, New Zealand. Fifty shelters were placed out at six sites and examined at three-monthly intervals for a year. A wide variety of invertebrates were found utilizing the shelters, with Arachnida, Blattodea and Collembola being the most common occupants. After three months over 80% of the shelters exhibited signs of use by invertebrates, increasing to 96% after 12 months. Only seven tree weta (Anostostomatidae) and one (dead) ground weta (Hemiandrus sp.) were observed in the shelters over the full 12 month period. There were 52 observations of cave weta (Rhaphidophoridae) in the shelters, 36 of which occurred at one site, Orton Bradley Park. Occupation of the shelters by cave weta was not affected by soil conditions, light intensity or aspect of the shelter. However, cave weta exhibited a preference for shelters less than 50 cm above the ground and for shelters attached to kanuka and vines. Although weta were found in only a small proportion (9%) of the shelters, this method proved useful in confirming the presence of weta without risk of harming vulnerable populations. These shelters are inexpensive and easy to manufacture and have potential for long-term non-lethal monitoring of weta and as a collection/carriage device for live specimens used in conservation translocations.     

MAIT Cells Detect and Efficiently Lyse Bacterially-Infected Epithelial Cells

Mucosal associated invariant T cells (MAIT) are innate T lymphocytes that detect a large variety of bacteria and yeasts. This recognition depends on the detection of microbial compounds presented by the evolutionarily conserved major-histocompatibility-complex (MHC) class I molecule, MR1. Here we show that MAIT cells display cytotoxic activity towards MR1 overexpressing non-hematopoietic cells cocultured with bacteria. The NK receptor, CD161, highly expressed by MAIT cells, modulated the cytokine but not the cytotoxic response triggered by bacteria infected cells. MAIT cells are also activated by and kill epithelial cells expressing endogenous levels of MRI after infection with the invasive bacteria Shigella flexneri. In contrast, MAIT cells were not activated by epithelial cells infected by Salmonella enterica Typhimurium. Finally, MAIT cells are activated in human volunteers receiving an attenuated strain of Shigella dysenteriae-1 tested as a potential vaccine. Thus, in humans, MAIT cells are the most abundant T cell subset able to detect and kill bacteria infected cells.     

Structural and magnetic phase transitions in simple oxides using hybrid functionals

We present the structural as well as elastic properties of the alkaline earth oxides and FeO, calculated using hybrid exchange functionals within DFT. We show that by empirically fitting the amount of Fock-exchange in the hybrid functionals, we can accurately reproduce the pressure-induced phase transitions for MgO, CaO, SrO and BaO. For FeO the hybrid functionals predict an insulator?metal transition at ca. 150?GPa, associated with an i-B8?B8 structural phase transition. The structural phase transition is accompanied by a spin transition from a high- to low-spin electron configuration on the Fe²⁺ ions. Hence, FeO undergoes a magnetic phase transition from an anti-ferromagnetic to non-magnetic structure. We also find that as the ionicity of the polymorphs increases a higher fraction of Fock-exchange is required to reproduce the structural volumes reported from experiments.     

Predicting demographically sustainable rates of adaptation: can great tit breeding time keep pace with climate change?

Populations need to adapt to sustained climate change, which requires micro-evolutionary change in the long term. A key question is how the rate of this micro-evolutionary change compares with the rate of environmental change, given that theoretically there is a ‘critical rate of environmental change’ beyond which increased maladaptation leads to population extinction. Here, we parametrize two closely related models to predict this critical rate using data from a long-term study of great tits (Parus major). We used stochastic dynamic programming to predict changes in optimal breeding time under three different climate scenarios. Using these results we parametrized two theoretical models to predict critical rates. Results from both models agreed qualitatively in that even ‘mild’ rates of climate change would be close to these critical rates with respect to great tit breeding time, while for scenarios close to the upper limit of IPCC climate projections the calculated critical rates would be clearly exceeded with possible consequences for population persistence. We therefore tentatively conclude that micro-evolution, together with plasticity, would rescue only the population from mild rates of climate change, although the models make many simplifying assumptions that remain to be tested.     

Analysis of the genetic variability of PvMSP-3α among Plasmodium vivax in Brazilian field isolates

Reliable molecular markers are essential for a better understanding of the molecular epidemiology of Plasmodium vivax, which is a neglected human malaria parasite. The aim of this study was to analyze the genetic diversity of P. vivax isolates from the Brazilian Amazon using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the highly polymorphic merozoite surface protein-3alpha (PvMSP-3α) gene. To accomplish this, 60 isolates of P. vivax from different endemic areas in the Brazilian Amazon were collected. The PvMSP-3α gene was amplified by nested-PCR. Three major types of the PvMSP-3α locus were detected at different frequencies: type A (68%), B (15%) and C (17%). A single sample showed two PCR fragments, which corresponded to infection with types A and C. PCR-RFLP analysis using the HhaI restriction enzyme for 52 isolates clearly identified 11 haplotypes, eight of which were from type A, two from type B and only one from type C. Seven other isolates did not show a clear pattern using PCR-RFLP. This result might be due to multiple clone infections. This study showed a high diversity of the PvMSP-3α gene among P. vivax isolates from the Brazilian Amazon, but also indicated that the detection performance of PCR-RFLP of the PvMSP-3α gene may not be sufficient to detect multiple clone infections.     

Angiopoietin-2 and Angiopoietin-2/Angiopoietin-1 Ratio as Indicators of Potential Severity of Plasmodium vivax Malaria in Patients with Thrombocytopenia

Introduction

Angiogenic factors such as angiopoietin 1 (Ang-1) and angiopoietin 2 (Ang-2) are biomarkers produced during activation and dysfunction of the vascular endothelium in several infectious diseases. The aim of this study was to determine the serum levels of Ang-1 and Ang-2 and to establish their relationship with the main indicators of worst-case prognosis in patients with P. vivax malaria.

Methods

This is a retrospective case-control study nested within a cohort of symptomatic malaria patients. A potentially severe case was defined as a patient that presented at least one of the main indicators of the worst-case prognosis for falciparum malaria, as established by the World Health Organization. Ang-2 and Ang-1 and the Ang-2/Ang-1 ratio were used to analyze the role of angiopoietins as biomarkers in signaling potentially severe vivax malaria. ROC curves were generated to identify a cut-off point discriminating between the angiopoietin concentrations that were most strongly associated with potential infection severity.

Results

The serum levels of Ang-2 and the Ang-2/Ang-1 ratio were higher in the case group. In contrast, the serum levels of Ang-1 were lower in the cases than in the control patients. The blood count for platelets showed a positive correlation with Ang-1 and a negative correlation with Ang-2 and with the Ang-2/Ang-1 ratio. The area under the ROC curve (AUC) for serum angiopoietins, as an indicator of worst-case prognosis in a potentially severe P. vivax malarial infection, was larger in the subgroup of patients with platelet counts <75,000/µL.

Conclusion

This study showed that patients with predictors of worst-case prognoses for P. vivax malaria have lower Ang-1 and higher Ang-2 serum levels (and higher values for the Ang-2/Ang-1 ratio) than controls. Elevated serum levels of Ang-2 and high values for the Ang-2/Ang-1 ratio may potentially be used as predictors of worst-case prognoses for P. vivax malaria, especially in patients with thrombocytopenia.