Identifying and characterising regulatory metabolites with generalised supply-demand analysis

We present the framework of generalised supply-demand analysis (SDA) of a kinetic model of a cellular system, which can be applied to networks of arbitrary complexity. By fixing the concentrations of each of the variable species in turn and varying them in a parameter scan, rate characteristics of supply-demand are constructed around each of these species. By inspecting the shapes of the rate characteristic patterns and comparing the flux-response coefficients of the supply and demand blocks with the elasticities of the enzymes that interact directly with the fixed metabolite, regulatory metabolites in the system can be identified and characterised. The analysis provides information on whether and where the system is functionally differentiated and which of its species are homeostatically buffered. The novelty in our proposed method lies in the fact that all metabolites are considered for SDA (hence the term “generalised”), which removes investigator bias. It supplies an entry point for the further analysis and detailed characterisation of large models of cellular systems, in which the choice of metabolite around which to perform a SDA is not always obvious.     

ECA: control in ecosystems

Although metabolic control analysis (MCA) cannot be applied directly to microbial ecological systems because of mass conservation and stoichiometric constraints, we demonstrate here that Hierarchical Control Analysis (HCA) can be applied to such systems. We illustrate the approach for a particular ecosystem example of the biological synthesis of acetic acid from glucose, and uncover some surprising aspects to the control of this miniature ecosystem.     

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory,GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.     

Pharmacological Inhibition of Glycogen Synthase Kinase 3 Regulates T Cell Development In Vitro

The development of functional T cells requires receptor-mediated transition through multiple checkpoints in the thymus. Double negative 3 (DN3) thymocytes are selected for the presence of a rearranged TCR beta chain in a process termed β-selection which requires signalling via the pre-TCR, Notch1 and CXCL12. Signal integration by these receptors converges on core pathways including the Phosphatidylinositol–3-kinase (PI3K) pathway. Glycogen Synthase Kinase 3 (GSK3) is generally thought to be negatively regulated by the PI3K pathway but its role in β-selection has not been characterised. Here we show that developmental progression of DN3 thymocytes is promoted following inhibition of GSK3 by the synthetic compound CHIR99021. CHIR99021 allows differentiation in the absence of pre-TCR-, Notch1- or CXCL12-mediated signalling. It antagonizes IL-7-mediated inhibition of DP thymocyte differentiation and increases IL-7-promoted cell recovery. These data indicate a potentially important role for inactivation of GSK3 during β-selection. They might help to establish an in vitro stromal cell-free culture system of thymocyte development and offer a new platform for screening regulators of proliferation, differentiation and apoptosis.     

Down-regulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in sugarcane enhances sucrose accumulation in immature internodes

Pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity was successfully down-regulated in sugarcane using constitutively expressed antisense and untranslatable forms of the sugarcane PFP-β gene. In young internodal tissue activity was reduced by up to 70% while no residual activity could be detected in mature tissues. The transgenic plants showed no visible phenotype or significant differences in growth and development under greenhouse and field conditions. Sucrose concentrations were significantly increased in the immature internodes of the transgenic plants but not in the mature internodes. This contributed to an increase in the purity of the immature tissues, resembling an early ripening phenotype. Both the immature and mature internodes of the transgenic plants had significantly higher fibre contents. These findings suggest that PFP influences the ability of young, biosynthetically active sugarcane culm tissue to accumulate sucrose but that the equilibrium of the glycolytic intermediates, including the stored sucrose, is restored when ATP-dependent phosphofructokinase and the residual PFP activity is sufficient to sustain the required glycolytic flux as the tissue matures. Moreover, it suggests a role for PFP in glycolytic carbon flow, which could be rate limiting under conditions of high metabolic activity.     

Mechanisms of protein-ligand association and its modulation by protein mutations

Protein-ligand interactions are essential for nearly all biological processes, and yet the biophysical mechanism that enables potential binding partners to associate before specific binding occurs remains poorly understood. Fundamental questions include which factors influence the formation of protein-ligand encounter complexes, and whether designated association pathways exist. To address these questions, we developed a computational approach to systematically analyze the complete ensemble of association pathways. Here, we use this approach to study the binding of a phosphate ion to the Escherichia coli phosphate-binding protein. Various mutants of the protein are considered, and their effects on binding free-energy profiles, association rates, and association pathway distributions are quantified. The results reveal the existence of two anion attractors, i.e., regions that initially attract negatively charged particles and allow them to be efficiently screened for phosphate, which is subsequently specifically bound. Point mutations that affect the charge on these attractors modulate their attraction strength and speed up association to a factor of 10 of the diffusion limit, and thus change the association pathways of the phosphate ligand. It is demonstrated that a phosphate that prebinds to such an attractor neutralizes its attraction effect to the environment, making the simultaneous association of a second phosphate ion unlikely. This study suggests ways in which structural properties can be used to tune molecular association kinetics so as to optimize the efficiency of binding, and highlights the importance of kinetic properties.     

The regulatory design of an allosteric feedback loop: the effect of saturation by pathway substrate

Aspects of metabolic regulation can be fruitfully studied with a combination of generic modelling, control analysis and graphical analysis using rate characteristics. This paper analyses a prototypical supply-demand system consisting of a biosynthetic subsystem subject to allosteric inhibition by its product and a demand process that consumes this product. The effect of changes in affinity of the committing supply enzyme for the pathway substrate on the regulatory properties of the supply subsystem is compared for the Monod-Wyman-Changeux and the reversible Hill allosteric enzyme models. We found that the Hill model has a distinct advantage in that the steady-state concentration at which it maintains the product is set by the half-saturating product concentration and is independent of changes in the degree of saturation for substrate. In contrast, with the Monod-Wyman-Changeux model this set point varies with affinity for substrate. Explicitly incorporating reversibility in all rate equations made it possible to distinguish between kinetic and thermodynamic aspects of regulation. Combining the supply and demand rate characteristics allows us to explore both the control distribution at steady state and the regulatory performance of the system over a wide range of demand activities.     

Early and Middle Holocene Hunter-Gatherer Occupations in Western Amazonia: The Hidden Shell Middens

We report on previously unknown early archaeological sites in the Bolivian lowlands, demonstrating for the first time early and middle Holocene human presence in western Amazonia. Multidisciplinary research in forest islands situated in seasonally-inundated savannahs has revealed stratified shell middens produced by human foragers as early as 10,000 years ago, making them the oldest archaeological sites in the region. The absence of stone resources and partial burial by recent alluvial sediments has meant that these kinds of deposits have, until now, remained unidentified. We conducted core sampling, archaeological excavations and an interdisciplinary study of the stratigraphy and recovered materials from three shell midden mounds. Based on multiple lines of evidence, including radiocarbon dating, sedimentary proxies (elements, steroids and black carbon), micromorphology and faunal analysis, we demonstrate the anthropogenic origin and antiquity of these sites. In a tropical and geomorphologically active landscape often considered challenging both for early human occupation and for the preservation of hunter-gatherer sites, the newly discovered shell middens provide evidence for early to middle Holocene occupation and illustrate the potential for identifying and interpreting early open-air archaeological sites in western Amazonia. The existence of early hunter-gatherer sites in the Bolivian lowlands sheds new light on the region’s past and offers a new context within which the late Holocene “Earthmovers” of the Llanos de Moxos could have emerged.     

Biochemical characterization of an ulvan lyase from the marine flavobacterium Formosa agariphila KMM 3901T

Applied Microbiology and Biotechnology - Carbohydrates are the product of carbon dioxide fixation by algae in the ocean. Their polysaccharides are depolymerized by marine bacteria, with a vast...