Systems Biology Approaches to Probe Gene Regulation in Bacteria

Mechanisms that allow pathogens to colonize the host are not the product of isolated genes, but instead emerge from the concerted operation of regulatory networks. Therefore, identifying components and the systemic behavior of networks is necessary to a better understanding of gene regulation and pathogenesis. To this end, I have developed systems biology approaches to study transcriptional and post-transcriptional gene regulation in bacteria, with an emphasis in the human pathogen Mycobacterium tuberculosis (Mtb). First, I developed a network response method to identify parts of the Mtb global transcriptional regulatory network utilized by the pathogen to counteract phagosomal stresses and survive within resting macrophages. As a result, the method unveiled transcriptional regulators and associated regulons utilized by Mtb to establish a successful infection of macrophages throughout the first 14 days of infection. Additionally, this network-based analysis identified the production of Fe-S proteins coupled to lipid metabolism through the alkane hydroxylase complex as a possible strategy employed by Mtb to survive in the host. Second, I developed a network inference method to infer the small non-coding RNA (sRNA) regulatory network in Mtb. The method identifies sRNA-mRNA interactions by integrating a priori knowledge of possible binding sites with structure-driven identification of binding sites. The reconstructed network was useful to predict functional roles for the multitude of sRNAs recently discovered in the pathogen, being that several sRNAs were postulated to be involved in virulence-related processes. Finally, I applied a combined experimental and computational approach to study post-transcriptional repression mediated by small non-coding RNAs in bacteria. Specifically, a probabilistic ranking methodology termed rank-conciliation was developed to infer sRNA-mRNA interactions based on multiple types of data. The method was shown to improve target prediction in Escherichia coli, and therefore is useful to prioritize candidate targets for experimental validation.

(Table 1) Lipid and organochlorine content in blood of common eiders (Somateria mollissima) in Svalbard

Female common eiders (Somateria mollissima) starve during the nesting stage and may lose 30-45% of their initial body mass, mostly through lipid mobilization. In this study, the effects of fasting on the blood concentrations of three lipid-soluble organochlorines (OCs: polychlorinated biphenyl [PCB]-153; 1-dichloro-2,2-bis (p-chlorophenyl) ethylene [p,p'-DDE]; and hexachlorobenzene [HCB]) were examined in eiders breeding in the high Arctic. Blood samples were taken from females (n = 47) at day 5 and day 20 of the incubation period. The mean wet weight concentrations of PCB-153 and p,p'-DDE increased strongly between day 5 and day 20 (3.6 and 8.2-fold, respectively), while HCB increased less (1.7-fold). There was a strong negative association between daily increase in PCB-153 and clutch size, and a weaker relationship for p,p'-DDE, suggesting that maternal transfer to the eggs is a significant pathway of elimination of OCs in eiders. Moreover, poor body condition (body mass controlled for body size) late in the incubation period was associated with strong daily increase of both p,p'-DDE and PCB-153, which may suggest that the release of these compounds increases when lipid reserves become depleted. For HCB, the increase was mainly associated with increase in blood lipid concentrations, and weakly to the amount of burned lipids. The causes for the differences between the compounds are, however, poorly understood. Although the absolute levels of OCs in eiders were relatively low, their rapid build-up during incubation is worrying as it coincides with poor body condition and weakened immune systems.

Simulation of the deformation of polycrystalline nanostructured Ti by computational homogenization

Computational homogenization by means of the finite element analysis of a representative volume element of the microstructure is used to simulate the deformation of nanostructured Ti. The behavior of each grain is taken into account using a single crystal elasto-viscoplastic model which includes the microscopic mechanisms of plastic deformation by slip along basal, prismatic and pyramidal systems. Two different representations of the polycrystal were used. Each grain was modeled with one cubic finite element in the first one while many cubic elements were used to represent each grain in the second one, leading to a model which includes the effect of grain shape and size in a limited number of grains due to the computational cost. Both representations were used to simulate the tensile deformation of nanostructured Ti processed by ECAP-C as well as the drawing process of nanostructured Ti billets. It was found that the first representation based in one finite element per grain led to a stiffer response in tension and was not able to predict the texture evolution during drawing because the strain gradient within each grain could not be captured. On the contrary, the second representation of the polycrystal microstructure with many finite elements per grain was able to predict accurately the deformation of nanostructured Ti.

The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems.

The experiments reported here were designed to test the hypothesis that the two-electron quinone reductase DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] functions to maintain membrane-bound coenzyme Q (CoQ) in its reduced antioxidant state, thereby providing protection from free radical damage. DT-diaphorase was isolated and purified from rat liver cytosol, and its ability to reduce several CoQ homologs incorporated into large unilamellar vesicles was demonstrated. Addition of NADH and DT-diaphorase to either large unilamellar or multilamellar vesicles containing homologs of CoQ, including CoQ9 and CoQ10, resulted in the essentially complete reduction of the CoQ. The ability of DT-diaphorase to maintain the reduced state of CoQ and protect membrane components from free radical damage as lipid peroxidation was tested by incorporating either reduced CoQ9 or CoQ10 and the lipophylic azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile) into multilamellar vesicles in the presence of NADH and DT-diaphorase. The presence of DT-diaphorase prevented the oxidation of reduced CoQ and inhibited lipid peroxidation. The interaction between DT-diaphorase and CoQ was also demonstrated in an isolated rat liver hepatocyte system. Incubation with adriamycin resulted in mitochondrial membrane damage as measured by membrane potential and the release of hydrogen peroxide. Incorporation of CoQ10 provided protection from adriamycin-induced mitochondrial membrane damage. The incorporation of dicoumarol, a potent inhibitor of DT-diaphorase, interfered with the protection provided by CoQ. The results of these experiments provide support for the hypothesis that DT-diaphorase functions as an antioxidant in both artificial membrane and natural membrane systems by acting as a two-electron CoQ reductase that forms and maintains the antioxidant form of CoQ. The suggestion is offered that DT-diaphorase was selected during evolution to perform this role and that its conversion of xenobiotics and other synthetic molecules is secondary and coincidental.

Molecular organization of histidine-tagged biomolecules at self-assembled lipid interfaces using a novel class of chelator lipids.

In molecular biology, the expression of fusion proteins is a very useful and well-established technique for the identification and one-step purification of gene products. Even a short fused sequence of five or six histidines enables proteins to bind to an immobilized metal ion chelate complex. By synthesis of a class of chelator lipids, we have transferred this approach to the concept of self-assembly. The specific interaction and lateral organization of a fluorescent fusion molecule containing a C-terminal oligohistidine sequence was studied by film balance techniques in combination with epifluorescence microscopy. Due to the phase behavior of the various lipid mixtures used, the chelator lipids can be laterally structured, generating two-dimensional arrays of histidine-tagged biomolecules. Because of the large variety of fusion proteins already available, this concept represents a powerful technique for orientation and organization of proteins at lipid interfaces with applications in biosensing, biofunctionalization of nanostructured interfaces, two-dimensional crystallization, and studies of lipid-anchored proteins.

Incorporation of ovalbumin into ISCOMs and related colloidal particles prepared by the lipid film hydration method

The ann of this study was to investigate the incorporation of a model antigen, fluorescently labelled ovalbumin (FITC-OVA), into various colloidal particles including immune stimulating complexes (ISCOMs), liposomes, ring and worm-like micelles, lamellae and lipidic/layered structures that are formed from various combinations of the triterpene saponin Quil A, cholesterol and phosphatidylethanolamine (PE) following hydration of PE/cholesterol lipid films with aqueous Solutions of Quil A. Colloidal dispersions of these three components were also prepared by the dialysis method for comparison. FITC-OVA was conjugated with palmitic acid (P) and PE to produce P-FITC-OVA and PE-FITC-OVA, respectively. Both P-FITC-OVA and PE-FITC-OVA could be incorporated in all colloidal structures whereas FITC-OVA was incorporated only into liposomes. The incorporation of PE-FITC-OVA into all colloidal structures was significantly higher than P-FITC-OVA (P < 0.05). The degree of incorporation of protein was in the order: ring and worm-like micelles < liposomes and lipidic/layered structures < ISCOMs and lamellae. The incorporation of protein into the various particles prepared by the lipid film hydration method was similar to those for colloidal particles prepared by the dialysis method (provided both methods lead to the formation of the same colloidal structures). In the case of different colloidal structures arising due to the preparation method, differences in encapsulation efficiency were found (P < 0.05) for formulations with the same polar lipid composition. This study demonstrates that the various colloidal particles formed as a result of hydrating PE/cholesterol lipid films with different amounts of Quil A are capable of incorporating antigen, provided it is amphipathic. Some of these colloidal particles may be used as effective vaccine delivery systems. (C) 2004 Elsevier B.V. All rights reserved.

Pseudo-ternary phase diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by the lipid-film hydration method

Pseudo-ternary phase diagrams of the polar lipids Quil A, cholesterol (Chol) and phosphatidylcholine (PC) in aqueous mixtures prepared by the lipid film hydration method (where dried lipid film of phospholipids and cholesterol are hydrated by an aqueous solution of Quil A) were investigated in terms of the types of particulate structures formed therein. Negative staining transmission electron microscopy and polarized light microscopy were used to characterize the colloidal and coarse dispersed particles present in the systems. Pseudo-ternary phase diagrams were established for lipid mixtures hydrated in water and in Tris buffer (pH 7.4). The effect of equilibration time was also studied with respect to systems hydrated in water where the samples were stored for 2 months at 4degreesC. Depending on the mass ratio of Quil A, Chol and PC in the systems, various colloidal particles including ISCOM matrices, liposomes, ring-like micelles and worm-like micelles were observed. Other colloidal particles were also observed as minor structures in the presence of these predominant colloids including helices, layered structures and lamellae (hexagonal pattern of ring-like micelles). In terms of the conditions which appeared to promote the formation of ISCOM matrices, the area of the phase diagrams associated with systems containing these structures increased in the order: hydrated in water/short equilibration period < hydrated in buffer/short equilibration period < hydrated in water/prolonged equilibration period. ISCOM matrices appeared to form over time from samples, which initially contained a high concentration of ring-like micelles suggesting that these colloidal structures may be precursors to ISCOM matrix formation. Helices were also frequently found in samples containing ISCOM matrices as a minor colloidal structure. Equilibration time and presence of buffer salts also promoted the formation of liposomes in systems not containing Quil A. These parameters however, did not appear to significantly affect the occurrence and predominance of other structures present in the pseudo-binary systems containing Quil A. Pseudo-ternary phase diagrams of PC, Chol and Quil A are important to identify combinations which will produce different colloidal structures, particularly ISCOM matrices, by the method of lipid film hydration. Colloidal structures comprising these three components are readily prepared by hydration of dried lipid films and may have application in vaccine delivery where the functionality of ISCOMs has clearly been demonstrated. (C) 2003 Elsevier B.V. All rights reserved.

A comparison of pseudo-ternary diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by lipid-film hydration and dialysis

Pseudo-ternary diagrams for Quil A, phospholipid (phosphatidylcholine (PC) or phosphatidylethanolamine (PE)) and cholesterol were established in order to identify combinations that result in the formation of immune-stimulating complex (ISCOM) matrices and other colloidal structures produced by these three components in aqueous systems following lipid-film hydration or dialysis (methods that can be used to produce ISCOMs). In addition, the effect of equilibration time (1 month at 4degreesC) on the structures formed by the various combinations of the three components was investigated. Depending on the ratio of Quil A, cholesterol and phospholipid, different colloidal particles, including ISCOM matrices, liposomes and ring-like micelles, were found irrespective of the preparation method used. In contrast, worm-like micelles were only observed in systems prepared by lipid-film hydration. For samples prepared by dialysis, ISCOM matrices were predominantly found near the Quil A apex of the pseudo-ternary diagram (> 50% Quil A). On the other hand, for samples prepared by lipid-film hydration, ISCOM matrices were predominantly found near the phospholipid apex of the pseudo-ternary diagram (> 50% phospholipid). The regions in the pseudo-ternary diagrams in which ISCOM matrices were observed increased following an extended equilibration time, particularly for samples prepared by lipid-film hydration. Differences were also observed between pseudoternary diagrams prepared using either PE or PC as phospholipids.

The cyclic antimicrobial peptide RTD-1 induces stabilized lipid-peptide domains more efficiently than its open-chain analogue

The effects of a mammalian cyclic antimicrobial peptide, rhesus theta defensin 1 (RTD-1) and its open chain analogue (oRTD-1), on the phase behaviour and structure of model membrane systems (dipalmitoyl phosphatidylcholine, DPPC and dipalmitoyl phosphatidylglycerol, DPPG) were studied. The increased selectivity of RTD-1 for anionic DPPG over zwitterionic DPPC was shown by differential scanning calorimetry. RTD-1, at a molar peptide-lipid ratio of 1:100, induced considerable changes in the phase behaviour of DPPG, but not of DPPC. The main transition temperature, T-m, Was unchanged, but additional phase transitions appeared above T-m. oRTD-1 induced similar effects. However, the effects were not observable below a peptide:lipid molar ratio of 1:50, which correlates with the weaker biological activity of oRTD-1. Small-and wide-angle X-ray scattering revealed for DPPG the appearance of additional structural features induced by RTP-1 above T-m, which were interpreted as correlated lamellar structures, with increased order of the fatty acyl side chains of the lipid. It is proposed that after initial electrostatic interaction of the cationic rim of the peptide with the anionic DPPG headgroups, leading to stabilized lipid-peptide clusters, the hydrophobic face of the peptide assists in its interaction with the fatty acyl side chains eventually leading to membrane disruption. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Effects of pravastatin on coronary events in 2073 patients with low levels of both low-density lipoprotein cholesterol and high-density lipoprotein cholesterol: results from the LIPID study

Aims Fibrates or nicotinic acid are usually recommended for secondary prevention of coronary heart disease in patients with low plasma levels of both low-density tipoprotein cholesterol (LDL-C) less than or equal to140 mg/dL (less than or equal to3.6 mmol/L) and high-density lipoprotein cholesterol (HDL-C) less than or equal to40 mg/dL (less than or equal to1.03 mmol/L). The LIPID trial, a randomised, placebo-controlled trial in 9014 patients at 87 centres in Australia and New Zealand, provided an opportunity to investigate the effects of an HMG-CoA reductase inhibitor in patients with tow LDL-C and tow HDL-C. Methods and results Participants in this post hoc substudy were 2073 patients aged 31-75 years with baseline LDL-C less than or equal to140 mg/dL (less than or equal to3.6 mmoL/L), HDL-C less than or equal to40 mg/dL (less than or equal to1.03 mmol/L), and triglyceride less than or equal to300 mg/dL (less than or equal to3.4 mmol/L). The relative risk reduction with pravastatin treatment was 27% for major coronary events (95% Cl 8-42%), 27% for coronary heart disease mortality (95% CI 0-47%), 21% for all-cause mortality (95% Cl 0-38%), and 51% for stroke (95% CI 24-69%). The number needed to treat to prevent a major coronary event over 6 years was 22. Conclusions Treatment with pravastatin in patients with both low LDL-C and low HDL-C significantly reduced major coronary events, stroke, and all-cause mortality. The level of HDL-C is crucial to the risk of recurrent CHD events and, consequently, the benefit of lowering LDL-C. (C) 2004 Published by Elsevier Ltd on behalf of The European Society of Cardiology.

Encapsulation of lipopeptides within liposomes: Effect of number of lipid chains, chain length and method of liposome preparation

The purpose of this study was to systematically investigate the effect of lipid chain length and number of lipid chains present on lipopeptides on their ability to be incorporated within liposomes. The peptide KAVYNFATM was synthesized and conjugated to lipoamino acids having acyl chain lengths of C-8, C-12 and C-16. The C-12 construct was also prepared in the monomeric, dimeric and trimeric form. Liposomes were prepared by two techniques: hydration of dried lipid films (Bangham method) and hydration of freeze-dried monophase systems. Encapsulation of lipopeptide within liposomes prepared by hydration of dried lipid films was incomplete in all cases ranging from an entrapment efficiency of 70% for monomeric lipoamino acids at a 5% (w/w) loading to less than 20% for di- and trimeric forms at loadings of 20% (w/w). The incomplete entrapment of lipopeptides within liposomes appeared to be a result of the different solubilities of the lipopeptide and the phospholipids in the solvent used for the preparation of the lipid film. In contrast, encapsulation of lipopeptide within liposomes prepared by hydration of freeze-dried monophase systems was high, even up to a loading of 20% (w/w) and was much less affected by the acyl chain length and number than when liposomes were prepared by hydration of dried lipid films. Freeze drying of monophase systems is better at maintaining a molecular dispersion of the lipopeptide within the solid phospholipid matrix compared to preparation of lipid film by evaporation, particularly if the solubility of the lipopeptide in solvents is markedly different from that of the polar lipids used for liposome preparation. Consequently, upon hydration, the lipopeptide is more efficiently intercalated within the phospholipid bilayers. (C) 2005 Elsevier B.V. All rights reserved.

Quil A-lipid powder formulations releasing ISCOMs and related colloidal stuctures upon hydration

The aim of the present study was to prepare solid Quil A-cholesterol-phospholid formulations (as powder mixtures or compressed to pellets) by physical mixing or by freeze-drying of aqueous dispersions of these components in ratios that allow spontaneous formation of ISCOMs and other colloidal stuctures upon hydration. The effect of addition of excess cholesterol to the lipid mixtures on the release of a model antigen (PE-FITC-OVA) from the pellets was also investigated. Physical properties were evaluated by X-ray powder diffractometry (XPRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and polarized light microscopy (PLM). Characterization of aqueous colloidal dispersions was performed by negative staining transmission electron microscopy (TEM). Physically mixed powders (with or without PE-FITC-OVA) and pellets prepared from the same powders did not spontaneously form ISCOM matrices and related colloidal structures such as worm-like micelles, ring-like micelles, lipidic/layered structures and lamellae (hexagonal array of ring-like micelles) upon hydration as expected from the pseudo-temary diagram for aqueous mixtures of Quil A, cholesterol and phospholipid. In contrast, spontaneous formation of the expected colloids was demonstrated for the freeze-dried lipid mixtures. Pellets prepared by compression of freeze-dried powders released PE-FITC-OVA slower than those prepared from physically mixed powders. TEM investigations revealed that the antigen was released in the form of colloidal particles (ISCOMs) from pellets prepared by compression of freeze-dried powders. The addition of excess cholesterol slowed down the release of antigen. The findings obtained in this study are important for the formulation of solid Quil A-containing lipid articles as controlled particulate adjuvant containing antigen delivery systems. (c) 2004 Elsevier B.V. All rights reserved.

Studies on the membrane interactions of the cyclotides kalata B1 and kalata B6 on model membrane systems by surface plasmon resonance

In this study we have demonstrated the interactions of kalata B1 and its naturally occurring analogue kalata B6 with five model lipid membranes and have analyzed the binding kinetics using surface plasmon resonance. Two kalata peptides showed a higher affinity for the phosphatidylethanolamine-containing membranes, indicating that the peptides would bind selectively to bacterial membranes. Also we have optimized the procedure for the immobilization of five liposome mixtures and have shown that the procedure provides reproducible levels of immobilized liposomes and could be used to screen the selective binding of putative antimicrobial peptides to model mammalian or microbial phospholipid membranes. (C) 2004 Elsevier Inc. All rights reserved.