Fluxome study of Pseudomonas fluorescens reveals major reorganisation of carbon flux through centralmetabolic pathways in response to inactivation of the anti-sigma factor MucA.

BackgroundThe bacterium Pseudomonas fluorescens switches to an alginate-producing phenotype when the pleiotropic anti-sigma factor MucA is inactivated. The inactivation is accompanied by an increased biomass yield on carbon sources when grown under nitrogen-limited chemostat conditions. A previous metabolome study showed significant changes in the intracellular metabolite concentrations, especially of the nucleotides, in mucA deletion mutants compared to the wild-type. In this study, the P. fluorescens SBW25 wild-type and an alginate non-producing mucA- ΔalgC double-knockout mutant are investigated through model-based 13C-metabolic flux analysis (13C-MFA) to explore the physiological consequences of MucA inactivation at the metabolic flux level. Intracellular metabolite extracts from three carbon labelling experiments using fructose as the sole carbon source are analysed for 13C-label incorporation in primary metabolites by gas and liquid chromatography tandem mass spectrometry.ResultsFrom mass isotopomer distribution datasets, absolute intracellular metabolic reaction rates for the wild type and the mutant are determined, revealing extensive reorganisation of carbon flux through central metabolic pathways in response to MucA inactivation. The carbon flux through the Entner-Doudoroff pathway was reduced in the mucA- ΔalgC mutant, while flux through the pentose phosphate pathway was increased. Our findings also indicated flexibility of the anaplerotic reactions through down-regulation of the pyruvate shunt in the mucA- ΔalgC mutant and up-regulation of the glyoxylate shunt.ConclusionsAbsolute metabolic fluxes and metabolite levels give detailed, integrated insight into the physiology of this industrially, medically and agriculturally important bacterial species and suggest that the most efficient way of using a mucA- mutant as a cell factory for alginate production would be to use non-growing conditions and nitrogen deprivation

Investigating alginate production and carbon utilization in Pseudomonas fluorescens SBW25 using mass spectrometry-based metabolic profiling

Metabolic profiling of Pseudomonas fluorescens SBW25 and various mutants derived thereof was performed to explore how the bacterium adapt to changes in carbon source and upon induction of alginate synthesis. The experiments were performed at steady-state conditions in nitrogen-limited chemostats using either fructose or glycerol as carbon source. Carbon source consumption was up-regulated in the alginate producing mutant with inactivated anti-sigma factor MucA. The mucA- mutants (also non-alginate producing mucA- control strains) had a higher dry weight yield on carbon source implying a change in carbon and energy metabolism due to the inactivation of the anti-sigma factor MucA. Both LC–MS/MS and GC–MS methods were used for quantitative metabolic profiling, and major reorganization of primary metabolite pools in both an alginate producing and a carbon source dependent manner was observed. Generally, larger changes were observed among the phosphorylated glycolytic metabolites, the pentose phosphate pathway metabolites and the nucleotide pool than among amino acids and citric acid cycle compounds. The most significant observation at the metabolite level was the significantly reduced energy charge of the mucA- mutants (both alginate producing and non-producing control strains) compared to the wild type strain. This reduction was caused more by a strong increase in the AMP pool than changes in the ATP and ADP pools. The alginate-producing mucA- mutant had a slightly increased GTP pool, while the GDP and GMP pools were strongly increased compared to non-producing mucA- strains and to the wild type. Thus, whilst changes in the adenosine phosphate nucleotide pool are attributed to the mucA inactivation, adjustments in the guanosine phosphate nucleotide pool are consequences of the GTP-dependent alginate production induced by the mucA inactivation. This metabolic profiling study provides new insight into carbon and energy metabolism of the alginate producer P. fluorescens

Proliferation.

<p>Ki67-staining revealed a significantly higher number of proliferating cells in the control group compared to the HBOT group in both models (MDA-MB-231, * p = 0.0002. BT-474, * p = 0.0001). Scale bar 500 μm.</p

Oxygen-dependent regulation of tumor growth and metastasis in human breast cancer xenografts

<div><p>Background</p><p>Tumor hypoxia is relevant for tumor growth, metabolism, resistance to chemotherapy and metastasis. We have previously shown that hyperoxia, using hyperbaric oxygen treatment (HBOT), attenuates tumor growth and shifts the phenotype from mesenchymal to epithelial (MET) in the DMBA-induced mammary tumor model. This study describes the effect of HBOT on tumor growth, angiogenesis, chemotherapy efficacy and metastasis in a triple negative MDA-MB-231 breast cancer model, and evaluates tumor growth using a triple positive BT-474 breast cancer model.</p><p>Materials and methods</p><p>5 x 10⁵ cancer cells were injected s.c. in the groin area of NOD/SCID female mice. The BT-474 group was supplied with Progesterone and Estradiol pellets 2-days prior to tumor cell injection. Mice were divided into controls (1 bar, pO₂ = 0.2 bar) or HBOT (2.5 bar, pO₂ = 2.5 bar, 90 min, every third day until termination of the experiments). Treatment effects were determined by assessment of tumor growth, proliferation (Ki67-staining), angiogenesis (CD31-staining), metastasis (immunostaining), EMT markers (western blot), stromal components collagen type I, Itgb1 and FSP1 (immunostaining) and chemotherapeutic efficacy (5FU).</p><p>Results</p><p>HBOT significantly suppressed tumor growth in both the triple positive and negative tumors, and both MDA-MB-231 and BT-474 showed a decrease in proliferation after HBOT. No differences were found in angiogenesis or 5FU efficacy between HBOT and controls. Nevertheless, HBOT significantly reduced both numbers and total area of the metastastatic lesions, as well as reduced expression of N-cadherin, Axl and collagen type I measured in the MDA-MB-231 model. No change in stromal Itgb1 and FSP1 was found in either tumor model.</p><p>Conclusion</p><p>Despite the fact that behavior and prognosis of the triple positive and negative subtypes of cancer are different, the HBOT had a similar suppressive effect on tumor growth, indicating that they share a common oxygen dependent anti-tumor mechanism. Furthermore, HBOT significantly reduced the number and area of metastatic lesions in the triple negative model as well as a significant reduction in the EMT markers N-cadherin, Axl and density of collagen type I.</p></div

The Effect of Prolonged Use of a Wearable Soft-Robotic Glove Post Stroke -: A Proof-of-Principle

Many stroke survivors encounter difficulties in the performance of activities of daily life due to limitations in functional use of the hand. Robotic technology has the potential to compensate for this loss by providing the support that is required to perform activities of daily living, especially when these devices are wearable comfortably for many hours at home. As a first step towards the implementation of assistive technology in the homes of stroke survivors, usability along with the potential effect of prolonged use of a wearable soft-robotic glove during activities of daily life on functional task performance was assessed in this study. Therefore, five chronic stroke survivors were asked to use a wearable soft-robotic glove for four weeks at home during preferred activities of daily life. Before and after the home use of the glove, functional task performance was assessed in a lab environment. After the use of the glove, system usability was assessed. The prolonged use of the glove resulted in an improved supported and unsupported functional performance during tasks related to activities of daily life, as measured with the Jebsen-Taylor Hand Function Test. Promising system usability results were found indicating a good probability for acceptance of the glove. The results from this study indicate the potential of the current glove to be used as assistive tool, which even showed a therapeutic effect. Yet, the glove should be tested in a larger sample for better interpretation and confirmation of these promising results

Tumor growth.

<p>The effect of hyperbaric oxygen treatment on the growth of triple negative MDA-MB-231 (p<0.001) (A) and triple positive BT-474 (p<0.001) (B) tumors. Cells were injected subcutaneously in the mammary fat pad. Mice were treated with 2.5 bar pure oxygen, 90 min each time, every third day, during a period of 24 days (measurements started day 7 post injection) (closed circles) and compared to controls (open circles). Mean ± SEM. n = 7 in each group.</p

5-Fluorouracil.

<p>The effect of hyperbaric oxygen treatment (HBOT) (n = 10), the chemotherapeutic drug 5FU (1.5 mg/kg in 0.1 ml) (n = 10) or 5FU in combination with HBOT (n = 10) on growth of MDA-MB-231 human mammary tumors over a period of 17 days. *p<0.05.</p

Collagen type I.

<p>The total amount of collagen type I quantified as percent of total area in both control (n = 5 tumors) and HBOT (n = 5 tumors) primary tumors (A) is shown. The total amount of ITGB1 as percent of total area in both control (n = 5 tumors) and HBOT (n = 5 tumors) primary tumors (B). A representative immunofluorescence (IF) staining picture from a control (C) and HBOT (D) tumor is shown. Green represents stroma cells (stained with antibody to mouse integrin-β1) red represents collagen type I secreted by the stromal cells (stained with antibody to mouse collagen type I), and blue shows the nuclei stained with DAPI. Scale bar represents 100 μm. Representative IF pictures are also shown to illustrate the compostion/proportion of the tumor cells (red, stained with cytokeratin 7 antibody) and the stromal cells (green, stained with integrin β1 antibody) from the control (E) and and HBOT (F) tumors, respectively. Scale bar represents 100 μm.</p

Blood vessels.

<p>Blood vessels.</p