Development and Application of Pseudoreceptor Modeling

Quantitative Structure-Activity Relationship (QSAR) methods are a commonly used tool in the drug discovery process. These methods attempt to form a statistical model that relates descriptor properties of a ligand to the activity of that ligand compound towards a specific desired physiological response. QSAR methods are known as a ligand-based method, as they specifically use information from ligands and not protein structural data. However, a derivation of QSAR methods are pseudoreceptor methods. Pseudoreceptor methods go beyond standard QSAR by building a model representation of the protein pocket. However, the ability of pseudoreceptors to accurately replicate natural protein surfaces has not been studied. The goal of this thesis work is to investigate the necessary descriptors to map a protein binding pocket and a method to accurately recreate the 3-D spatial structure of the binding pocket. In addition, additional applications of existing pseudoreceptor methods are explored

Secondary Electron Yield Measurements of Carbon Nanotube Forests: Dependence on Morphology and Substrate

Total, secondary, and backscatter electron yield data were taken with beam energies between 15 eV and 30 keV, in conjunction with energy emission data, to determine the extent of suppression of yield caused by carbon nanotube (CNT) forest coatings on substrates. CNT forests can potentially lower substrate yield due to both its inherently low-yield, low-atomic number (Z) carbon composition, and its bundled, high-aspect ratio structure. Rough surfaces, and in particular, surfaces with deep high-aspect-ratio voids, can suppress yields, as the electrons emitted from lower lying surfaces are recaptured by surface protrusions rather than escaping the near-surface region. Yields of multilayered materials can be modeled essentially serially as a combination of the constituents. However, it is shown that suppression of yields due to CNT forest morphology is more significant than simple predicted contributions of homogeneous layered components. This effect is found to be most pronounced at low energies, where the incident electrons interact preferentially with the CNTs. CNT forests between 20 and 50 μm tall were grown on a thick silicon substrate capped with a 3-nm diffusion barrier of evaporated aluminum using a wet injection chemical vapor deposition (CVD) method. Yields of an annealed substrate and constituent bulk materials were also investigated. At incident electron energies above ~1200 eV, the substrate secondary yield dominated those of the CNT forests, as incident electrons penetrated through the low-density, low-Z CNT forests, and backscattered from the higher-Z substrate. At lower energies \u3c1200 \u3eeV, the CNT forests substantially reduced the overall yields of the substrate, and for \u3c500 eV CNT forest yields were \u3c1, well below the already low yields of bulk graphite. This suppressed yield at low energies is attributed to the porosity and preferred vertical alignment of the CNT forest. The yield’s dependence on the height and density of the CNT forest is also discussed

Influence of Vibrationally-Induced Structural Changes on Carbon Nanotube Forests Suppression of Electron Yield

Carbon nanotube (CNT) forest coatings have been found to lower electron yield from material surfaces. The suppressed yields have been attributed to both the lower inherent yields of low-atomic number carbon and the enhanced electron recapture resulting from the morphology of the carbon layer. To explore the relative contributions of these two causes of yield suppression, tests have been made on CNT forest-coated conducting substrate samples subjected to vibrationally-induced changes of the coating structure. The extent of vibrationally-induced structural changes—due, for example, to shear-force conditions during space-vehicle transit—are of interest, as CNT have been a frequent topic of scientific curiosity and space applications due to their high tensile strength, high aspect ratio geometry, and unique electromagnetic characteristics. Their use has also been beneficial for sensor equipment, both terrestrial and space-faring, due to their extremely low photon and electron reflectivity

Suppresion of Electron Yield With Carbon Nanotube Forests: A Case Study

Electron emission of carbon nanotube (CNT) forests grown on silicon substrates was measured to investigate possible electron yield suppression due to the composition and morphology of CNT forests. CNT forests are vertically-oriented tubular formations of graphitic carbon grown on a substrate; these have been widely investigated for their extreme properties in optical, electrical, and mechanical aspects of physics and material sciences. CNT coatings are good candidates for yield reduction, in analogy with the near-ideal blackbody optical properties of CNT forests. Carbon with its low atomic number has an inherent low yield due to its low density of bulk electrons. Furthermore, the large aspect ratio of this vertically-aligned CNT allows for easy penetration of the high energy incident electrons, but enhanced recapture of lower-energy secondary electrons due to their wider angular distribution of emission. Total (TEY), secondary (SEY) and backscattered (BSEY) yield curves using 15 eV to 30 keV electron beams, along with energy emission spectra, were acquired for three CNT forest samples to determine the extent of yield suppression of the substrate due to the CNT forests [Wood, 2018]

MSL1 is a mechanosensitive ion channel that dissipates mitochondrial membrane potential and maintains redox homeostasis in mitochondria during abiotic stress

Mitochondria must maintain tight control over the electrochemical gradient across their inner membrane to allow ATP synthesis while maintaining a redox-balanced electron transport chain and avoiding excessive reactive oxygen species production. However, there is a scarcity of knowledge about the ion transporters in the inner mitochondrial membrane that contribute to control of membrane potential. We show that loss of MSL1, a member of a family of mechanosensitive ion channels related to the bacterial channel MscS, leads to increased membrane potential of Arabidopsis mitochondria under specific bioenergetic states. We demonstrate that MSL1 localises to the inner mitochondrial membrane. When expressed in Escherichia coli, MSL1 forms a stretch-activated ion channel with a slight preference for anions and provides protection against hypo-osmotic shock. In contrast, loss of MSL1 in Arabidopsis did not prevent swelling of isolated mitochondria in hypo-osmotic conditions. Instead, our data suggest that ion transport by MSL1 leads to dissipation of mitochondrial membrane potential when it becomes too high. The importance of MSL1 function was demonstrated by the observation of a higher oxidation state of the mitochondrial glutathione pool in msl1-1 mutants under moderate heat- and heavy-metal-stress. Furthermore, we show that MSL1 function is not directly implicated in mitochondrial membrane potential pulsing, but is complementary and appears to be important under similar conditions

Modulation of topoisomerase IIα expression and chemosensitivity through targeted inhibition of NF-Y:DNA binding by a diamino p-anisyl-benzimidazole (Hx) polyamide

BACKGROUND: Sequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα (topo IIα) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxI*P 2 and HxIP* 3) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole. METHODS: DNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs. Topo IIα mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18h polyamide treatment of confluent A549 cells. γH2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer Glo®. RESULTS: Introduction of the N1-alkyl amino group reduced selectivity for the target sequence 5'-TACGAT-3' on the topo IIα promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet HxI*P 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIα. In contrast, inhibition of NF-Y binding by HxIP* 3 stimulated dose-dependent (0.1-2μM) re-induction of topo IIα and potentiated cytotoxicity of topo II poisons by enhancing DNA damage. CONCLUSIONS: Polyamide functionalisation at the N1-position offers a design strategy to improve drug-like properties. Dicationic HxIP* 3 increased topo IIα expression and chemosensitivity to topo II-targeting agents. GENERAL SIGNIFICANCE: Pharmacological modulation of topo IIα expression has the potential to enhance cellular sensitivity to clinically-used anticancer therapeutics. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani

Design of small molecule-responsive microRNAs based on structural requirements for Drosha processing

MicroRNAs (miRNAs) are prevalent regulatory RNAs that mediate gene silencing and play key roles in diverse cellular processes. While synthetic RNA-based regulatory systems that integrate regulatory and sensing functions have been demonstrated, the lack of detail on miRNA structure–function relationships has limited the development of integrated control systems based on miRNA silencing. Using an elucidated relationship between Drosha processing and the single-stranded nature of the miRNA basal segments, we developed a strategy for designing ligand-responsive miRNAs. We demonstrate that ligand binding to an aptamer integrated into the miRNA basal segments inhibits Drosha processing, resulting in titratable control over gene silencing. The generality of this control strategy was shown for three aptamer–small molecule ligand pairs. The platform can be extended to the design of synthetic miRNAs clusters, cis-acting miRNAs and self-targeting miRNAs that act both in cis and trans, enabling fine-tuning of the regulatory strength and dynamics. The ability of our ligand-responsive miRNA platform to respond to user-defined inputs, undergo regulatory performance tuning and display scalable combinatorial control schemes will help advance applications in biological research and applied medicine

Intracellular bacillary burden reflects a burst size for Mycobacterium tuberculosis in vivo

We previously reported that Mycobacterium tuberculosis triggers macrophage necrosis in vitro at a threshold intracellular load of ~25 bacilli. This suggests a model for tuberculosis where bacilli invading lung macrophages at low multiplicity of infection proliferate to burst size and spread to naïve phagocytes for repeated cycles of replication and cytolysis. The current study evaluated that model in vivo, an environment significantly more complex than in vitro culture. In the lungs of mice infected with M. tuberculosis by aerosol we observed three distinct mononuclear leukocyte populations (CD11b(-) CD11c(+/hi), CD11b(+/lo) CD11c(lo/-), CD11b(+/hi) CD11c(+/hi)) and neutrophils hosting bacilli. Four weeks after aerosol challenge, CD11b(+/hi) CD11c(+/hi) mononuclear cells and neutrophils were the predominant hosts for M. tuberculosis while CD11b(+/lo) CD11c(lo/-) cells assumed that role by ten weeks. Alveolar macrophages (CD11b(-) CD11c(+/hi)) were a minority infected cell type at both time points. The burst size model predicts that individual lung phagocytes would harbor a range of bacillary loads with most containing few bacilli, a smaller proportion containing many bacilli, and few or none exceeding a burst size load. Bacterial load per cell was enumerated in lung monocytic cells and neutrophils at time points after aerosol challenge of wild type and interferon-γ null mice. The resulting data fulfilled those predictions, suggesting a median in vivo burst size in the range of 20 to 40 bacilli for monocytic cells. Most heavily burdened monocytic cells were nonviable, with morphological features similar to those observed after high multiplicity challenge in vitro: nuclear condensation without fragmentation and disintegration of cell membranes without apoptotic vesicle formation. Neutrophils had a narrow range and lower peak bacillary burden than monocytic cells and some exhibited cell death with release of extracellular neutrophil traps. Our studies suggest that burst size cytolysis is a major cause of infection-induced mononuclear cell death in tuberculosis

Bispectrum speckle interferometry observations and radiative transfer modelling of the red supergiant NML Cyg: Multiple dust-shell structures evidencing previous superwind phases

(abridged) NML Cyg is a highly evolved OH/IR supergiant and supposed to be among the most luminous supergiants in the galaxy. We present the first diffraction limited 2.13micron observations of NML Cyg with 73mas resolution. The speckle interferograms were obtained with the SAO 6m telescope, image reconstruction is based on the bispectrum speckle interferometry method. Radiative transfer calculations have been carried out to model the spectral energy distribution, our 2.13micron visibility function, and mid-infrared visibility functions. The observed dust shell properties do not appear to be in accordance with single-shell models but seem to require multiple components. Considering previous periods of enhanced mass-loss, various density enhancements in the dust shell were taken into account. An extensive grid of models was calculated for different locations and strenghts of such superwind regions in the dust shell. To match the observations from the optical to the sub-mm domain requires at least two superwind regions embedded in the shell. The best model includes a dust shell with a temperature of 1000K at its inner radius of 6.2Rstar, a close embedded superwind shell extending from 15.5Rstar to 21.7Rstar with amplitude 10 (factor of density enhancement), and a far-out density enhancement at 186Rstar with amplitude 5. The angular diameter of the inner dust-shell rim amounts to 105mas. Within the various parts of the dust shell, 1/r^2 density distributions could be maintained differing only in their amplitude A. The present-day mass-loss rate was determined to be 1.2 10^-4 Msol/yr. The inner embedded superwind shell corresponds to a phase of enhanced mass-loss which began ~59yr ago and lasted for ~18yr, and the outer superwind region to a high mass-loss period which terminated 529yr ago.Comment: 12 pages including 13 PostScript figures, also available from http://www.mpifr-bonn.mpg.de/div/ir-interferometry/publications.html; accepted for publication in Astronomy & Astrophysic

A robust system for RNA interference in the chicken using a modified microRNA operon

AbstractRNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo