Drag coefficients with applications to satellite orbits

In the last twenty or so years the results of theory and experiment have produced much information on the characteristics of gas-surface interactions relevant to a satellite in hyperthermal free-molecular flow. This thesis contains reviews of the rarefied gas dynamics applicable to satellites and has attempted to compare existing models of gas-surface interaction with contemporary knowledge of such systems. It is shown that a more natural approach would be to characterise the gas-surface interaction using the normal and tangential momentum accommodation coefficients, igma' and igma respectively, specifically in the form igma = constant , igma' = igma'0 -igma'1sec i where i is the angle subtended between the incident flow and the surface normal and igma,igma'0 and igma'1 are constants. Adopting these relationships, the effects of atmospheric lift on inclination, i, and atmospheric drag on the semi-major axis, a, and eccentricity, e, have been investigated. Applications to ANS-1 (1974-70A) show that the observed perturbation in i can be ascribed primarily to non-zero igma'1 whilst perturbations in a and e produce constraint equations between the three parameters. The numerical results seem to imply that a good theoretical orbit is achieved despite a much lower drag coefficient than anticipated by earlier theories.

Molecular and cellular effects of ultraviolet light-induced genotoxicity

Exposure to the solar ultraviolet spectrum that penetrates the Earth's stratosphere (UVA and UVB) causes cellular DNA damage within skin cells. This damage is elicited directly through absorption of energy (UVB), and indirectly through intermediates such as sensitizer radicals and reactive oxygen species (UVA). DNA damage is detected as strand breaks or as base lesions, the most common lesions being 8-hydroxydeoxyguanosine (8OHdG) from UVA exposure and cyclobutane pyrimidine dimers from UVB exposure. The presence of these products in the genome may cause misreading and misreplication. Cells are protected by free radical scavengers that remove potentially mutagenic radical intermediates. In addition, the glutathione-S-transferase family can catalyze the removal of epoxides and peroxides. An extensive repair capacity exists for removing (1) strand breaks, (2) small base modifications (8OHdG), and (3) bulky lesions (cyclobutane pyrimidine dimers). UV also stimulates the cell to produce early response genes that activate a cascade of signaling molecules (e.g., protein kinases) and protective enzymes (e.g., haem oxygenase). The cell cycle is restricted via p53-dependent and -independent pathways to facilitate repair processes prior to replication and division. Failure to rescue the cell from replication block will ultimately lead to cell death, and apoptosis may be induced. The implications for UV-induced genotoxicity in disease are considered.

Structure, dynamics, and energetics of siRNA-cationic vector complexation:a molecular dynamics study

The design and synthesis of safe and efficient nonviral vectors for gene delivery has attracted significant attention in recent years. Previous experiments have revealed that the charge density of a polycation (the carrier) plays a crucial role in complexation and the release of the gene from the complex in the cytosol. In this work, we adopt an atomistic molecular dynamics simulation approach to study the complexation of short strand duplex RNA with six cationic carrier systems of varying charge and surface topology. The simulations reveal detailed molecular-level pictures of the structures and dynamics of the RNA-polycation complexes. Estimates for the binding free energy indicate that electrostatic contributions are dominant followed by van der Waals interactions. The binding free energy between the 8(+)polymers and the RNA is found to be larger than that of the 4(+)polymers, in general agreement with previously published data. Because reliable binding free energies provide an effective index of the ability of the polycationic carrier to bind the nucleic acid and also carry implications for the process of gene release within the cytosol, these novel simulations have the potential to provide us with a much better understanding of key mechanistic aspects of gene-polycation complexation and thereby advance the rational design of nonviral gene delivery systems.

Structure and dynamics of multiple cationic vectors-siRNA complexation by all-atomic molecular dynamics simulations

Understanding the molecular mechanism of gene condensation is a key component to rationalizing gene delivery phenomena, including functional properties such as the stability of the gene-vector complex and the intracellular release of the gene. In this work, we adopt an atomistic molecular dynamics simulation approach to study the complexation of short strand duplex RNA with four cationic carrier systems of varying charge and surface topology at different charge ratios. At lower charge ratios, polymers bind quite effectively to siRNA, while at high charge ratios, the complexes are saturated and there are free polymers that are unable to associate with RNA. We also observed reduced fluctuations in RNA structures when complexed with multiple polymers in solution as compared to both free siRNA in water and the single polymer complexes. These novel simulations provide a much better understanding of key mechanistic aspects of gene-polycation complexation and thereby advance progress toward rational design of nonviral gene delivery systems.

The effect of pH on PAMAM dendrimer-siRNA complexation:endosomal considerations as determined by molecular dynamics simulation

Intracellular degradation of genes, most notably within the endo-lysosomal compartment is considered a significant barrier to (non-viral) gene delivery in vivo. Previous reports based on in vitro studies claim that carriers possessing a mixture of primary, secondary and tertiary amines are able to buffer the acidic environment within the endosome, allowing for timely release of their contents, leading to higher transfection rates. In this report, we adopt an atomistic molecular dynamics (MD) simulation approach, comparing the complexation of 21-bp siRNA with low-generation polyamidoamine (PAMAM) dendrimers (G0 and G1) at both neutral and acidic pHs, the latter of which mimics the degradative environment within maturing 'late-endosomes'. Our simulations reveal that the time taken for the dendrimer-gene complex (dendriplex) to reach equilibrium is appreciably longer at low pH and this is accompanied by more compact packaging of the dendriplex, as compared to simulations performed at neutral pH. We also note larger absolute values of calculated binding free energies of the dendriplex at low pH, indicating a higher dendrimer-nucleic acid affinity in comparison with neutral pH. These novel simulations provide a more detailed understanding of low molecular-weight polymer-siRNA behavior, mimicking the endosomal environment and provide input of direct relevance to the "proton sponge theory", thereby advancing the rational design of non-viral gene delivery systems.

Molecular phase space transport in water:non-stationary random walk model

Molecular transport in phase space is crucial for chemical reactions because it defines how pre-reactive molecular configurations are found during the time evolution of the system. Using Molecular Dynamics (MD) simulated atomistic trajectories we test the assumption of the normal diffusion in the phase space for bulk water at ambient conditions by checking the equivalence of the transport to the random walk model. Contrary to common expectations we have found that some statistical features of the transport in the phase space differ from those of the normal diffusion models. This implies a non-random character of the path search process by the reacting complexes in water solutions. Our further numerical experiments show that a significant long period of non-stationarity in the transition probabilities of the segments of molecular trajectories can account for the observed non-uniform filling of the phase space. Surprisingly, the characteristic periods in the model non-stationarity constitute hundreds of nanoseconds, that is much longer time scales compared to typical lifetime of known liquid water molecular structures (several picoseconds).

Molecular regulation of iron uptake in pseudomonas aeruginosa

The microbial demand for iron is often met by the elaboration of siderophores into the surrounding medium and expression of cognate outer membrane receptors for the ferric siderophore complexes. Conditions of iron limitation, such as those encountered in vivo, cause Pseudomonas aeruginosa to express two high-affinity iron-uptake systems based on pyoverdin and pyochelin. These systems will operate both in the organism's natural habitat, soil and water, where the solubility of iron at neutral pH is extremely low, and in the human host where the availability of free iron is too low to sustain bacterial growth due to the iron-binding glycoproteins transferrin and lactoferrin. Cross-feeding and radiolabelled iron uptake experiments demonstrated that pyoverdin biosynthesis and uptake were highly heterogeneous amongst P.aeruginosa strains, that growth either in the presence of pyoverdin or pyochelin resulted in induction of specific IROMPs, and that induction of iron uptake is siderophore-specific. The P.aeruginosa Tn5 mutant PH1 is deficient in ferripyoverdin uptake and resistant to pyocin Sa, suggesting that the site of interaction of pyocin Sa is a ferripyoverdin receptor. Additional Tn5 mutants appeared to exploit different strategies to achieve pyocin Sa-resistance, involving modifications in expression of pyoverdin-mediated iron uptake, indicating that complex regulatory systems exist to enable these organisms to compete effectively for iron. Modulation of expression of IROMPs prompted a study of the mechanism of uptake of a semi-synthetic C(7) α-formamido substituted cephalosporin BRL 41897A. Sensitivity to this agent correlated with expression of the 75 kDa ferri-pyochelin receptor and demonstrated the potential of high-affinity iron uptake systems for targeting of novel antibiotics. Studies with ferri-pyoverdin uptake-deficient mutant PH1 indicated that expression of outer membrane protein G (OprG), which is usually expressed under iron-rich conditions and repressed under iron-deficient conditions, was perturbed. Attempts were made to clone the oprG gene using a degenerate probe based on the N-terminal amino acid sequence. A strongly hybridising HindIll restriction fragment was cloned and sequenced, but failed to reveal an open reading frame correspondmg to OprG. However, there appears to be good evidence that a part of the gene codmg for the hydrophilic membrane-associated ATP-binding component of a hitherto uncharacterised periplasmic- binding-protein-dependent transport system has been isolated. The full organisation and sequence of the operon, and substrate for this putative transport system, are yet: to be elucidated,

Toward the atomistic simulation of T cell epitopes automated construction of MHC:peptide structures for free energy calculations

Epitopes mediated by T cells lie at the heart of the adaptive immune response and form the essential nucleus of anti-tumour peptide or epitope-based vaccines. Antigenic T cell epitopes are mediated by major histocompatibility complex (MHC) molecules, which present them to T cell receptors. Calculating the affinity between a given MHC molecule and an antigenic peptide using experimental approaches is both difficult and time consuming, thus various computational methods have been developed for this purpose. A server has been developed to allow a structural approach to the problem by generating specific MHC:peptide complex structures and providing configuration files to run molecular modelling simulations upon them. A system has been produced which allows the automated construction of MHC:peptide structure files and the corresponding configuration files required to execute a molecular dynamics simulation using NAMD. The system has been made available through a web-based front end and stand-alone scripts. Previous attempts at structural prediction of MHC:peptide affinity have been limited due to the paucity of structures and the computational expense in running large scale molecular dynamics simulations. The MHCsim server (http://igrid-ext.cryst.bbk.ac.uk/MHCsim) allows the user to rapidly generate any desired MHC:peptide complex and will facilitate molecular modelling simulation of MHC complexes on an unprecedented scale.

Molecular dynamics simulations:bring biomolecular structures alive on a computer

The molecular dynamics (MD) simulations play a very important role in science today. They have been used successfully in binding free-energy calculations and rational design of drugs and vaccines. MD simulations can help visualize and understand structures and dynamics at an atomistic level when combined with molecular graphics programs. The molecular and atomistic properties can be displayed on a computer in a time-dependent way, which opens a road toward a better understanding of the relationship of structure, dynamics, and function. In this chapter, the basics of MD are explained, together with a step-by-step description of setup and running an MD simulation.

RT-PCR for the pseudogene-free amplification of the glyceraldehyde-3-phosphate dehydrogenase gene (gapd)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme which catalyses the conversion of glyceraldehyde-3-phosphate to 1,3 diphosphoglycerate. It is considered to be constitutively expressed in all cells, and as such the gene for GAPDH (gapd) is commonly used as a benchmark reference in expression studies. However, previous investigations have demonstrated that gapd may show altered gene expression in a number of disease states and under certain experimental conditions, suggesting that results of experiments using gapd as a control should be interpreted with caution. Furthermore, consideration must be given to the potential co-amplification of pseudogenes of gapd during RT-PCR. Here, we describe a method to avoid the amplification of contaminating pseudogenes through the design of primers that bind only to genuine gapd mRNA transcript. © 2003 Elsevier Ltd. All rights reserved.

Detergent-free membrane protein purification

Membrane proteins are localised within a lipid bilayer; in order to purify them for functional and structural studies the first step must involve solubilising or extracting the protein from these lipids. To date this has been achieved using detergents which disrupt the bilayer and bind to the protein in the transmembrane region. However finding conditions for optimal extraction, without destabilising protein structure is time consuming and expensive. Here we present a recently-developed method using a styrene maleic acid (SMA) co-polymer instead of detergents. The SMA co-polymer extracts membrane proteins in a small disc of lipid bilayer which can be used for affinity chromatography purification, thus enabling the purification of membrane proteins while maintaining their native lipid bilayer environment.

Exploratory catalyst screening studies on the base free conversion of glycerol to lactic acid and glyceric acid in water using bimetallic Au-Pt nanoparticles on acidic zeolites

The base free oxidation of glycerol with molecular oxygen in water using bimetallic Au-Pt catalysts on three different acidic zeolite supports (H-mordenite, H-β and H-USY) was explored in a batch setup. At temperatures between 140 and 180 °C, lactic acid formation was significant and highest selectivity (60 % lactic acid at 80 % glycerol conversion) was obtained using Au-Pt/USY-600 (180 °C). A selectivity switch to glyceric acid (GLYA) was observed when the reactions were performed at 100 °C. Highest conversion and selectivity towards GLYA were obtained with Au-Pt/H-β as the catalyst (68 % selectivity at 68 % conversion).