Photoaddition of Ru(tap)₂(bpy)²⁺ to DNA: A New Mode of Covalent Attachment of Metal Complexes to Duplex DNA

Near-UV or visible light irradiation of Ru(tap)2(bpy)2+ (1) (tap = 1,4,5,8-tetraazaphenanthrene; bpy = 2,2‘-bipyridyl) in the presence of duplex DNA induces the formation of covalent adducts with guanine. The adduct has been isolated from the photomodified DNA as both its nucleotide and nucleobase derivatives by using a combination of enzymatic and acid hydrolytic procedures in conjunction with HPLC. Characterization by electrospray mass spectrometry and NMR spectroscopy shows that two isomeric covalent adducts are formed in which the exocyclic amino group of a guanine nucleobase is linked to the C2 or C7 position of one of the tap ligands. It is proposed that the products are generated from the reduced ruthenium complex and the guanine radical cation resulting from photoinduced electron transfer between 1 and guanine

Design, Synthesis, and Trypanocidal Activity of New Aminoadamantane Derivatives

To develop functionalized adamantanes for treating African trypanosomiasis, we report on the synthesis of new 1-alkyl-2-aminoadamantanes 1a−i, 1-alkyltricyclo[3.3.1.13,7]decan-2-guanylhydrazones 2a−g, and their congeneric thiosemicarbazones 3a,b. The potency of these compounds against Trypanosoma brucei was compared to that of amantadine and rimantadine and found to be substantially higher. The most active analogues, 1c, 1d, 2c, 2g, and 3b, illustrate the synergistic effect of the lipophilic character of the C1 side chain and the C2 functionality on trypanocidal activity

Oligonucleotide Functionalization of Hollow Triangular Gold Silver Alloy Nanoboxes

Triangular AuAg nanoboxes functionalized with short DNA molecules (oligodeoxynucleotides, ODNs) have been prepared and their properties compared to those formed from spherical gold particles. The nanoboxes, produced from triangular silver nanoplates (TSNPs) by reduction of AuCl₄^– with ascorbic acid, have been further characterized by EDS mapping and HAADF-STEM measurements. Both AuAg nanoboxes and gold spherical nanoparticles were functionalized with thiol-terminated single-stranded ODNs or their complementary ODN sequences. When the complementary AuAg nanobox–ODN conjugates were combined they were observed to form networks, with the localized surface plasmon resonance (LSPR) band undergoing a red shift and a significant dampening. This assembly process was reversible upon heating and the systems showed sharp melting transitions, which could be monitored at wavelengths throughout the visible and near-IR. Finally, assemblies of the triangular nanoboxes and spherical nanoparticles have been generated

Spontaneous Debundling of Single-Walled Carbon Nanotubes in DNA-Based Dispersions

Natural salmon testes DNA has been used to disperse single-walled carbon nanotubes (SWNTs) in water. It has been found that the primary factor controlling the nanotube bundle size distribution in the dispersion is the nanotube concentration. As measured by AFM, the mean bundle diameter tends to decrease with decreasing concentration. The number fraction of individual nanotubes increases with decreasing concentration. At low nanotube concentrations, number fractions of up to 83% individual SWNTs, equating to a mass fraction of 6.2%, have been obtained. Both the absolute number density and mass per volume of individual nanotubes initially increased with decreasing concentration, displaying a peak at ∼0.027 mg/mL. This concentration thus yields the largest quantities of individually dispersed SWNTs. The AFM data for populations of individual nanotubes was confirmed by infrared photoluminescence spectroscopy. The photoluminescence intensity increased with decreasing concentration, indicating extensive debundling. The concentration dependence of the luminescence intensity matched well to the AFM data on the number density of individual nanotubes. More importantly, it was found that, once initially dispersed, spontaneous debundling occurs upon dilution without the need for sonication. This implies that DNA−SWNT hybrids exist in water as a solution rather than a dispersion. The effects of dilution have been compared to the results obtained by ultracentrifuging the samples, showing dilution methods to be a viable and cost-effective alternative to ultracentrifugation. It was found that even after 4 h of ultracentrifugation at 122 000g, bundles with diameters of up to 4 nm remained in solution. The bundle diameter distribution after ultracentrifugation was very similar to the equilibrium distribution for the appropriate concentration after dilution, showing ultracentrifugation to be equivalent to dilution

The gastrointestinal tract is the major site of <i>T</i>. <i>cruzi</i> persistence in mice infected with either wild type or null mutant parasites.

<p><i>Ex vivo</i> imaging of organs from two representative mice of each group (groups as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003707#pntd.0003707.g004" target="_blank">Fig 4</a>), one sacrificed at day 56 (<b>A</b>) and one at day 63 (<b>B</b>). Each of the images is on the same luminescence scale (right) for radiance where purple indicates low signal intensity and red indicates a high signal. The maximum (5.5x10⁵) and minimum (4.9 x10³) signals are indicated at the top and bottom of the scale bar respectively. The heart is indicated in each image by a white arrow. Foci of infection are visible in the stomach and colon in all cases. Occasional foci were detected in the heart, gut mesenteries and lungs. The schematic at the bottom of the TcΔAPx1 column indicates the layout of organs in each dish, abbreviations: Gut Mes: Gut mesenteries, SKM: skeletal muscle (right hind leg), vis fat: visceral fat, OES: oesophagus, STM: stomach.</p

Novel Lipophilic Acetohydroxamic Acid Derivatives Based on Conformationally Constrained Spiro Carbocyclic 2,6-Diketopiperazine Scaffolds with Potent Trypanocidal Activity

We describe novel acetohydroxamic acid derivatives with potent activity against cultured bloodstream-form <i>Trypanosoma brucei</i> and selectivity indices of >1000. These analogues were derived from conformationally constrained, lipophilic, spiro carbocyclic 2,6-diketopiperazine (2,6-DKP) scaffolds by attaching acetohydroxamic acid moieties to the imidic nitrogen. Optimal activity was achieved by placing benzyl groups adjacent to the basic nitrogen of the 2,6-DKP core. <i>S</i>-Enantiomer <b>7d</b> was the most active derivative against <i>T. brucei</i> (IC₅₀ = 6.8 nM) and <i>T. cruzi</i> (IC₅₀ = 0.21 μM)

Etching-Resistant Silver Nanoprisms by Epitaxial Deposition of a Protecting Layer of Gold at the Edges

The protection of silver nanoprisms against etching by the epitaxial deposition of a thin layer of gold in solution has been investigated. It has been found that at low Au/Ag ratios (∼0.08 to 0.17) a thin layer of gold is deposited on the edges of the nanoprisms as expected, but without the structural damage typically associated with galvanic replacement. Furthermore, this layer of gold provides robust protection against etching of the nanoprisms by chloride and is strong evidence that etching by chloride is face-selective and does not take place at the flat {111} faces of the nanoprisms. Additionally, the deposition of a protecting layer of gold results in only a small red shift in the position of the main plasmon resonance. We have investigated the sensitivity of the localized surface plasmon resonance (LSPR) to changes in the bulk refractive index of the solution and find that the gold-protected silver nanoprisms are promising candidates for the development of new refractive index-based biosensors

Generation of <i>TcAPx</i> null mutants.

<p><b>A</b> Map of the <i>T</i>. <i>cruzi</i> Sylvio X10/6 <i>TcAPx</i> locus (1) indicating the derivation of constructs used for targeted integration. The location of the flanking <i>TcCLPTM1</i> (Cleft-lip and palate transmembrane 1–like protein) and <i>TcG6PDH</i> (glucose-6-phosphate dehydrogenase) genes are indicated and the hatched box represents a degenerate VIPER/SIRE element. The ‘first round’ gene disruption construct is shown in (2), with the ‘second round’ gene deletion vector represented by (3). Restriction sites shown are C: Cla I and S: Sma I. <b>B</b> Strategy used to generate TcAPx null mutants. Briefly, the first allele was disrupted by insertional integration of the <i>PAC</i> gene into the ORF (1). An episomal copy of TcAPx was introduced into the <i>TcAPX</i>^+/- heterozygote line (2). The second endogenous allele was then deleted by homologous recombination using the flanking DNA external to the ORF to insert the <i>BLA</i> gene (3). The parasites were then removed from G418 selective pressure and passaged for up to 125 generations (4). Clones were then isolated and characterised (5). <b>C</b> The pTEX-APx episome is unstable in both wild type and <i>TcAPx</i> null backgrounds. The autoradiographs show Southern blots containing genomic DNA from wild type and null mutant cells isolated before (lanes 0) and after removal of G418 from the growth medium. Generations without G418 selection are indicated above the blot. The blot was probed with the <i>Neo</i>^R ORF. <b>D</b> Western blot showing expression level of TcAPx in parasite populations 125 generations after removal of G418 selection. WT: wild type Sylvio X10/6 lysate; SK: lysate from cells with a single copy of <i>TcAPX</i> disrupted; TcΔAPx: lysate from cells with both genes ablated. The panel below shows the blot probed with anti-TbBiP (a kind gift from Jay Bangs, University of Wisconsin-Madison) to control for loading. <b>E</b> Southern blot of genomic DNA digested with Cla I and Sma I, showing that <i>TcAPx</i> is absent from cloned null mutant cells. The left hand panel was probed with the <i>TcAPx</i> ORF and shows the endogenous gene (lane WT, 2.8 kb Cla I-Sma I fragment) and the gene disrupted by <i>PAC</i> insertion (lanes TcΔAPx 1 and 2, 3.1 kb Cla I fragment). <b>F</b> Western blot indicating that the null mutant clones do not express TcAPx. The wild type population show a single band of ~30kDa (lane WT: wild type) which is absent from the null mutant clones (lanes TcΔAPx 1 and 2). Equivalent loading is indicated by the Coomassie stained gel below.</p

<i>In vitro</i> sensitivity of null mutants to oxidative stress and benznidazole.

<p>Epimastigotes seeded at 5 x 10⁵ ml^-1 were exposed to various concentrations of <b>A</b> hydrogen peroxide and <b>B</b> benznidazole. The number of viable cells after 9 days was measured using resazurin fluorescence. TcΔAPx1comp refers to TcΔAPx1 cells retransformed with pTEX-APx to complement the null phenotype. Data were analysed by sigmoidal curve fitting using GraphPad Prism. The table below shows the EC₅₀ values (μM) for each compound against the various cell lines +/- standard deviation. Significance of differences in the EC₅₀ for H₂O₂ was measured using the F-test. (ND—not done).</p

Phenotypic assessment of null mutants <i>in vitro</i>.

<p><b>A</b> Growth rate of <i>T</i>. <i>cruzi</i> epimastigotes for wild type and null mutant (TcΔAPx1 and 2) clones. Triplicate cultures were followed for 10 days. There was no significant difference in growth rate. <b>B</b> Parasites lacking TcAPx can differentiate to amastigotes (AM) and trypomastigotes (TR). Examples shown are Giemsa stained wild type and null mutant (TcΔAPx2) cells. <b>C</b><i>In vitro</i> infectivity for L6 rat myoblast cells. Metacyclic trypomastigotes were used to infect L6 cells at a ratio of 5 trypanosomes per cell and left for 48 hours (Methods). Cells were Giemsa stained and the number of infected cells counted. Infections were carried out with seven replicates per parasite line. TcΔAPx1comp refers to TcΔAPx1 cells retransformed with pTEX-APx to complement the null phenotype. Data presented as mean <u>+</u> SD. Significance of difference between each pair was assessed by Student’s t-test, (**) corresponds to <i>P</i> = 0.007, (***) <i>P</i> = 0.0006. <i>P</i> values for wild type:TcΔAPx1 indicated by short horizontal line, wild type:TcΔAPx2 indicated by long horizontal line. The difference between the wild type and complemented lines was not significant. <b>D</b><i>In vitro</i> infectivity for Vero epithelial cells. Metacyclic trypomastigotes were used to infect Vero cells at a ratio of 5 trypanosomes per cell and left for 48 hours (Methods). Cells were Giemsa stained and the number of infected cells counted. Infections were carried out with seven replicates per parasite line. Data presented as mean <u>+</u> SD. Significance of difference was assessed by Student’s t-test, (***) corresponds to <i>P</i> = 0.0006. <i>P</i> values for wild type:TcΔAPx1 indicated by short horizontal line, wild type:TcΔAPx2 indicated by long horizontal line.</p