Prediction of protein-ligand binding affinity using neural networks

Master'sMASTER OF SCIENC

Analysis of the role of outer surface protein C (OspC) in Borrelia burgdorferi pathogenesis

Lyme disease is an emerging infection that is caused by the Borrelia burgdorferi sensu lato complex. These bacteria exist in nature through an enzootic life cycle involving Ixodes ticks and various reservoir hosts. One way that this bacterium adapts to the different hosts in the enzootic cycle is through the expression of outer surface protein C (OspC). OspC is a surface exposed lipoprotein encoded on circular plasmid 26 that forms homodimers on the bacterial surface and has distinct conserved and variable portions of sequence. When ospC is deleted, the spirochetes are unable to cause mammalian infection although the mechanism of this is unknown. Additionally, OspC is thought to be involved in reservoir host specificity/association and in tissue dissemination. In order to better understand the functional domains of OspC, the different conserved and variable portions of this protein were investigated. Three conserved portions of OspC were investigated: (1) the conserved cysteine residue at position 130 (C130), (2) the last ten C-terminal amino acids (C10), and (3) ligand binding domain 1 (LBD1). The C130 residue was mutated and this substitution disrupted OspC oligomerization in vitro and in vivo. A B. burgdorferi strain lacking the C10 retained full infectivity and plasminogen binding. The mutation of a single residue within LBD1 rendered B. burgdorferi noninfectious, indicating the importance of this domain in infection establishment. The variable portion of OspC was investigated by: (1) altering the surface charge of ligand binding domain 2 (LBD2), (2) inserting different OspC types into a constant genetic background, and (3) creating OspC hybrids. Alteration of the surface charge of LBD2 by site directed mutagenesis resulted in a lack of persistence in mice. By inserting an OspC type known to be noninfectious in mice into an infectious strain, infectivity was abolished. Strains expressing OspC hybrids indicated that multiple domains of OspC are involved in species specificity. Together these analyses demonstrated that OspC is as important protein that plays multiple roles in pathogenesis. The work presented here helps to increase the understanding of this crucial protein and the strains described can be used to decipher the full function of OspC

Gene Regulation by the transcription factor ZEB1 in Glioblastoma Multiforme

Glioblastoma Multiforme (GBM) is the most prevalent type of glioma, bearing the highest incidence rate of brain and Central Nervous System (CNS) malignant tumors and the lowest survival rate. GBMs distinguish themselves from lower grade glial tumors by the presence of certain hallmark histological features such as the presence of central necrosis in the tumor mass, marginal proliferation of endothelial cells and the presence of palisading cells around the area of necrosis. Several hallmark features contribute to the poor responsiveness that GBM tumors have to treatment including their high inter- and intratumoral heterogeneity at a phenotypic, cellular, genetic and epigenetic level. Most importantly, the existence of cancer stem cell (CSC) populations within GBM tumors is crucial for driving invasive tumor growth due to their potential to proliferate in vascular conditions, while becoming highly invasive in hypoxic conditions. Moreover, the ability of GBM CSCs to infiltrate surrounding brain parenchyma means that even the smallest number of such cells left after surgery will cause tumor recurrence.(...

Simulation and Modeling of Nanomaterials

This Special Issue focuses on computational detailed studies (simulation, modeling, and calculations) of the structures, main properties, and peculiarities of the various nanomaterials (nanocrystals, nanoparticles, nanolayers, nanofibers, nanotubes, etc.) based on various elements, including organic and biological components, such as amino acids and peptides. For many practical applications in nanoelectronics., such materials as ferroelectrics and ferromagnetics, having switching parameters (polarization, magnetization), are highly requested, and simulation of dynamics and kinetics of their switching are a very important task. An important task for these studies is computer modeling and computational research of the properties on the various composites of the other nanostructures with polymeric ferroelectrics and with different graphene-like 2-dimensional structures. A wide range of contemporary computational methods and software are used in all these studies

Enhancer viruses and a transgenic platform for combinatorial cell subclass-specific labeling

The rapid pace of cell type identification by new single-cell analysis methods has not been met with efficient experimental access to the newly discovered types. To enable flexible and efficient access to specific neural populations in the mouse cortex, we collected chromatin accessibility data from individual cells and clustered the single-cell data to identify enhancers specific for cell classes and subclasses. When cloned into adeno-associated viruses (AAVs) and delivered to the brain by retro-orbital injections, these enhancers drive transgene expression in specific cell subclasses in the cortex. We characterize several enhancer viruses in detail to show that they result in labeling of different projection neuron subclasses in mouse cortex, and that one of them can be used to label the homologous projection neuron subclass in human cortical slices. To enable the combinatorial labeling of more than one cell type by enhancer viruses, we developed a three-color Cre-, Flp- and Nigri- recombinase dependent reporter mouse line, Ai213. The delivery of three enhancer viruses driving these recombinases via a single retroorbital injection into a single Ai213 transgenic mouse results in labeling of three different neuronal classes/subclasses in the same brain tissue. This approach combines unprecedented flexibility with specificity for investigation of cell types in the mouse brain and beyond

Novel Cell Penetrating Peptides Effect Endosomal Escape and Deliver Protein Cargos into Living Cells

Over the last decade a number of peptides that are rapidly internalized by mammalian cells have been discovered or designed. Cell-penetrating peptides (CPPs) are capable of mediating penetration of the plasma membrane, allowing delivery of macromolecular cargoes to the cell interior. We have developed a novel CPP-adaptor protein technology that allows any user-defined cargo delivery and release into the cytoplasm. Our hypothesis is that a CPP-adaptor with a moiety allowing high-affinity but reversible non-covalent cargo binding would lead to more efficient penetration and release than current CPP delivery strategies. Delivery of proteins to the interiors of cells has many applications. In addition to detecting and mapping the location of the components of living cells with fluorescent tags in real time, the availability of our system will likely enable the manipulation of signaling pathways and gene expression by allowing the introduction of components, e.g. constitutively active kinases, repressors or enhancers. CPP-adaptor, TaT-Calmodulin, and cargo proteins (horse radish peroxidase, myoglobin and beta-galactosidase) were expressed and purified from E. coli BL21 (DE3)pLysS. Optical biosensing experiments demonstrated that affinity and kinetics between the novel CPP and cargo proteins did not significantly differ from wild-type interactions; all had subnanomolar affinities. Cargo proteins were labelled with DyLight 550. CPP-cargo complexes or cargo alone were incubated with subconfluent baby hamster kidney, HEK 293T and HT-3 cells. After washing, cells were imaged by fluorescence confocal microscopy. All users define cargos exhibited penetration and release to the cytoplasm whereas cargo-only controls exhibited no measurable penetration (though some adherence to the outside of the cells was observed). Time courses and dose-dependency studies characterizing penetration and release kinetics will be presented as will initial efforts to deliver cargo that may alter cell-signaling pathways. The results presented herein demonstrate the feasibility of delivering a wide variety of cargo proteins to the intracellular environment; creating an array of potential research, diagnostic and therapeutic applications

The promoter of ZmMRP-1, a maize transfer cell-specific transcriptional activator, is induced at solute exchange surfaces and responds to transport demands

Transfer cells have specializations that facilitate the transport of solutes across plant exchange surfaces. ZmMRP-1 is a maize (Zea mays) endosperm transfer cell-specific transcriptional activator that plays a central role in the regulatory pathways controlling transfer cell differentiation and function. The present work investigates the signals controlling the expression of ZmMRP-1 through the production of transgenic lines of maize, Arabidopsis, tobacco and barley containing ZmMRP-1promoter:GUS reporter constructs. The GUS signal predominantly appeared in regions of active transport between source and sink tissues, including nematode-induced feeding structures and at sites of vascular connection between developing organs and the main plant vasculature. In those cases, promoter induction was associated with the initial developmental stages of transport structures. Significantly, transfer cells also differentiated in these regions suggesting that, independent of species, location or morphological features, transfer cells might differentiate in a similar way under the influence of conserved induction signals. In planta and yeast experiments showed that the promoter activity is modulated by carbohydrates, glucose being the most effective inducer

Structural Investigations of Creatine Kinase

X-ray crystallographic and other structural studies have been carried out on creatine kinase isolated from rabbit skeletal muscle. As a consequence of the exploration of a wide range of crystallisation conditions, a previously unreported crystal form of creatine kinase has been characterised. This crystal form is monoclinic C2 with three subunits in the asymmetric unit and cell dimensions a = 248 A, b = 149 A, c = 52 A, beta = 9

Regulator of Gene Silencing Calmodulins: Components of RNA granules and autophagy during plant stress

The central focus of this work is to understand how rgsCaM homologs regulate mRNP bodies under cellular stress. While rgsCaM is best known as an endogenous suppressor of gene silencing in tobacco, our findings for a homologous protein in Arabidopsis, CML38, suggest that a broader function in the regulation of mRNP bodies may be common to this family of calmodulin-like proteins. CML38 is induced by hypoxia stress, contributes to plant survival under hypoxia, and localizes to stress-induced mRNP bodies called stress granules (SGs), as well as to processing bodies (PBs). Members of this protein family target viral and endogenous proteins for degradation through the autophagy pathway. We propose that rgsCaM and its relatives may localize to stress-induced mRNP bodies and target them for autophagic degradation (granulophagy). In our investigations for rgsCaM, we used hypoxia stress as a means to induce stress granule formation and found that rgsCaM localizes to hypoxia-induced cytosolic granules which are both independent of and bound to SGs and PBs. We further show that rgsCaM colocalizes with the autophagosome cargo-binding protein ATG8e, and interacts with ATG8e in planta by BiFC assay. Mutations disrupting the N-terminus of rgsCaM, or ones affecting the ability of ATG8 to bind cargo adaptors caused a loss in BiFC interaction. This suggests that the N-terminus contains a site for binding to the cargo binding protein ATG8e, and which might mediate the targeting of rgsCaM and bound cargo to autophagosomes as part of the granulophagy process. For future studies, a FRET-based approach for probing rgsCaM interactions in vivo is demonstrated.In a second trust, we report the development of a novel, fluorescence-based, quantitative oxygen biosensor to facilitate the non-invasive assessment of the oxygen status of cells of living cells by fluorescent imaging

Ultrastructural studies on parasitic flagellates

Summary available: p.4