Tatiana Zaikova

Single-Molecule Atomic Force Microscopy Force Spectroscopy Study of Aβ-40 Interactions

Misfolding and aggregation of amyloid β-40 (Aβ-40) peptide play key roles in the development of Alzheimers disease (AD). However, very little is known about the molecular mechanisms underlying these molecular processes. We developed a novel experimental approach that can directly probe aggregation-prone states of proteins and their interactions. In this approach, the proteins are anchored to the surface of the atomic force microscopy substrate (mica) and the probe, and the interaction between anchored molecules is measured in the approach-retraction cycles. We used dynamic force spectroscopy (DFS) to measure the stability of transiently formed dimers. One of the major findings from DFS analysis of α-synuclein (α-Syn) is that dimeric complexes formed by misfolded α-Syn protein are very stable and dissociate over a range of seconds. This differs markedly from the dynamics of monomers, which occurs on a microsecond to nanosecond time scale. Here we applied the same approach to quantitatively characterize interactions of Aβ-40 peptides over a broad range of pH values. These studies showed that misfolded dimers are characterized by lifetimes in the range of seconds. This value depends on pH and varies between 2.7 s for pH 2.7 and 0.1 s for pH 7, indicating that the aggregation properties of Aβ-40 are modulated by the environmental conditions. The analysis of the contour lengths revealed the existence of various pathways for dimer dissociation, suggesting that dimers with different conformations are formed. These structural variations result in different aggregation pathways, leading to different types of oligomers and higher-order aggregates, including fibrils.

Gold Nanoparticles Disrupt Zebrafish Eye Development and Pigmentation

Systematic toxicological study is still required to fully understand the hazard potentials of gold nanoparticles (AuNPs). Because their biomedical applications are rapidly evolving, we investigated developmental toxicity of AuNPs in an in vivo embryonic zebrafish model at exposure concentration ranges from 0.08 to 50mg/l. Exposure of zebrafish embryos to 1.3 nm AuNPs functionalized with a cationic ligand, N,N,N-trimethylammoniumethanethiol (TMAT-AuNPs), resulted in smaller malpigmented eyes. We determined that TMAT-AuNPs caused a significant increase of cell death in the eye, which was correlated with an increase in gene expression of p53 and bax. Expression patterns of key transcription factors regulating eye development (pax6a, pax6b, otx2, and rx1) and pigmentation (sox10) were both repressed in a concentration-dependent manner in embryos exposed to TMAT-AuNPs. Reduced spatial localization of pax6a, rx1, sox10, and mitfa was observed in embryos by whole-mount in situ hybridization. The swimming behavior of embryos exposed to sublethal concentrations of TMAT-AuNPs showed hypoactivity, and embryos exhibited axonal growth inhibition. Overall, these results demonstrated that TMAT-AuNPs disrupt the progression of eye development and pigmentation that continues to behavioral and neuronal damage in the developing zebrafish.

Structurally Similar Triphenylphosphine-Stabilized Undecagolds, Au11(PPh3)7Cl3 and [Au11(PPh3)8Cl2]Cl, Exhibit Distinct Ligand Exchange Pathways with Glutathione

Ligand exchange is frequently used to introduce new functional groups on the surface of inorganic nanoparticles or clusters while preserving the core size. For one of the smallest clusters, triphenylphosphine (TPP)-stabilized undecagold, there are conflicting reports in the literature regarding whether core size is retained or significant growth occurs during exchange with thiol ligands. During an investigation of these differences in reactivity, two distinct forms of undecagold were isolated. The X-ray structures of the two forms, Au11(PPh3)7Cl3 and [Au11(PPh3)8Cl2]Cl, differ only in the number of TPP ligands bound to the core. Syntheses were developed to produce each of the two forms, and their spectroscopic features correlated with the structures. Ligand exchange on [Au11(PPh3)8Cl2]Cl yields only small clusters, whereas exchange on Au11(PPh3)7Cl3 (or mixtures of the two forms) yields the larger Au25 cluster. The distinctive features in the optical spectra of the two forms made it possible to evaluate which of the cluster forms were used in the previously published papers and clarify the origin of the differences in reactivity that had been reported. The results confirm that reactions of clusters and nanoparticles may be influenced by small variations in the arrangement of ligands and suggest that the role of the ligand shell in stabilizing intermediates during ligand exchange may be essential to preventing particle growth or coalescence.

Media ionic strength impacts embryonic responses to engineered nanoparticle exposure

Abstract Embryonic zebrafish were used to assess the impact of solution ion concentrations on agglomeration and resulting in vivo biological responses of gold nanoparticles (AuNPs). The minimum ion concentration necessary to support embryonic development was determined. Surprisingly, zebrafish exhibit no adverse outcomes when raised in nearly ion-free media. During a rapid throughput screening of AuNPs, 1.2-nm 3-mercaptopropionic acid-functionalized AuNPs (1.2-nm 3-MPA-AuNPs) rapidly agglomerate in exposure solutions. When embryos were exposed to 1.2-nm 3-MPA-AuNPs dispersed in low ionic media, both morbidity and mortality were induced, but when suspended in high ionic media, there was little to no biological response. We demonstrated that the media ionic strength greatly affects agglomeration rates and biological responses. Most importantly, the insensitivity of the zebrafish embryo to external ions indicates that it is possible, and necessary, to adjust the exposure media conditions to optimize NP dispersion prior to assessment.

Surface functionalities of gold nanoparticles impact embryonic gene expression responses

Abstract Incorporation of gold nanoparticles (AuNPs) into consumer products is increasing; however, there is a gap in available toxicological data to determine the safety of AuNPs. In this study, we utilised the embryonic zebrafish to investigate how surface functionalisation and charge influence molecular responses. Precisely engineered AuNPs with 1.5 nm cores were synthesised and functionalized with three ligands: 2-mercaptoethanesulfonic acid (MES), N,N,N-trimethylammoniumethanethiol (TMAT), or 2-(2-(2-mercaptoethoxy)ethoxy)ethanol. Developmental assessments revealed differential biological responses when embryos were exposed to the functionalised AuNPs at the same concentration. Using inductively coupled plasma–mass spectrometry, AuNP uptake was confirmed in exposed embryos. Following exposure to MES- and TMAT-AuNPs from 6 to 24 or 6 to 48 h post fertilisation, pathways involved in inflammation and immune response were perturbed. Additionally, transport mechanisms were misregulated after exposure to TMAT and MES-AuNPs, demonstrating that surface functionalisation influences many molecular pathways.

Synthesis of Ligand-Stabilized Metal Oxide Nanocrystals and Epitaxial Core/Shell Nanocrystals via a Lower-Temperature Esterification Process

The properties of metal oxide nanocrystals can be tuned by incorporating mixtures of matrix metal elements, adding metal ion dopants, or constructing core/shell structures. However, high-temperature conditions required to synthesize these nanocrystals make it difficult to achieve the desired compositions, doping levels, and structural control. We present a lower temperature synthesis of ligand-stabilized metal oxide nanocrystals that produces crystalline, monodisperse nanocrystals at temperatures well below the thermal decomposition point of the precursors. Slow injection (0.2 mL/min) of an oleic acid solution of the metal oleate complex into an oleyl alcohol solvent at 230 °C results in a rapid esterification reaction and the production of metal oxide nanocrystals. The approach produces high yields of crystalline, monodisperse metal oxide nanoparticles containing manganese, iron, cobalt, zinc, and indium within 20 min. Synthesis of tin-doped indium oxide (ITO) can be accomplished with good control of the tin doping levels. Finally, the method makes it possible to perform epitaxial growth of shells onto nanocrystal cores to produce core/shell nanocrystals.

Potential Environmental Impacts and Antimicrobial Efficacy of Silver- and Nanosilver-Containing Textiles

For textiles containing nanosilver, we assessed benefit (antimicrobial efficacy) in parallel with potential to release nanosilver (impact) during multiple life cycle stages. The silver loading and method of silver attachment to the textile highly influenced the silver release during washing. Multiple sequential simulated household washing experiments for fabric swatches in deionized water with or without detergent showed a range of silver release. The toxicity of washing experiment supernatants to zebrafish (Danio rerio) embryos was negligible, with the exception of the very highest Ag releases (∼1 mg/L Ag). In fact, toxicity tests indicated that residual detergent exhibited greater adverse response than the released silver. Although washing the fabrics did release silver, it did not affect their antimicrobial efficacy, as demonstrated by >99.9% inhibition of E. coli growth on the textiles, even for textiles that retained as little as 2 μg/g Ag after washing. This suggests that very little nanosilver is required to control bacterial growth in textiles. Visible light irradiation of the fabrics reduced the extent of Ag release for textiles during subsequent washings. End-of-life experiments using simulated landfill conditions showed that silver remaining on the textile is likely to continue leaching from textiles after disposal in a landfill.

Allosteric Interactions within Subsites of a Monomeric Enzyme: Kinetics of Fluorogenic Substrates of PI-Specific Phospholipase C

Two novel water-soluble fluorescein myo-inositol phosphate (FLIP) substrates, butyl-FLIP and methyl-FLIP, were used to examine the kinetics and subsite interactions of Bacillus cereus phosphatidylinositol-specific phospholipase C. Butyl-FLIP exhibited sigmoidal kinetics when initial rates are plotted versus substrate concentration. The data fit a Hill coefficient of 1.2-1.5, suggesting an allosteric interaction between two sites. Two substrate molecules bind to this enzyme, one at the active site and one at a subsite, causing an increase in activity. The kinetic behavior is mathematically similar to that of well-known cooperative multimeric enzymes even though this phosphatidylinositol-specific phospholipase C is a small, monomeric enzyme. The less hydrophobic substrate, methyl-FLIP, binds only to the active site and not the activator site, and thus exhibits standard hyperbolic kinetics. An analytical expression is presented that accounts for the kinetics of both substrates in the absence and presence of a nonsubstrate short-chain phospholipid, dihexanoylphosphatidylcholine. The fluorogenic substrates detect activation at much lower concentrations of dihexanoylphosphatidylcholine than previously reported.

Synthesis of a new fluorogenic substrate for the continuous assay of mammalian phosphoinositide-specific phospholipase C

The synthesis of a fluorogenic substrate for mammalian phosphoinositide-specific phospholipase C is described. The substrate, based on the widely used fluorescein molecule, is a water-soluble substrate analog of phosphatidylinositol-4-phosphate. The fluorogenic substrate 2 is shown to be a sensitive substrate for human PI-PLC-delta1 in a continuous assay.

Environmental impacts of reusable nanoscale silver-coated hospital gowns compared to single-use, disposable gowns

Nanoscale silver has been incorporated into a variety of products where its antimicrobial properties enhance their functionality. One particular application is hospital linens, potential vectors of disease transmission. There is an on-going debate as to whether it is more beneficial to use disposable versus reusable hospital gowns in efforts to prevent nosocomial infections. This work models the life cycle impacts of nanoscale silver (nAg)-enabled, reusable hospital gowns from a life cycle assessment perspective and then compares the midpoint environmental impact data to the use of disposable hospital gowns. A key finding of this work is the environmental parity (when the environmental impact of nAg and disposable gowns are equal) of a nAg-enabled gown is 12 wearings. These results suggest that nAg textiles may be key in reducing the environmental impact of hospitals, while still preventing infection.