Structural and Mechanical Properties of “Peelable” Organoaqueous Dispersions with Partially Hydrolyzed Poly(vinyl acetate)-Borate Networks: Applications to Cleaning Painted Surfaces

The preparation and structural characterization of a family of viscoelastic dispersions of borate cross-linked, 80% hydrolyzed poly(vinyl acetate) (80PVAc) in aqueous–organic liquids are presented. Correlations between mechanical properties (from rheological measurements) and the degree and nature of cross-linking (from ¹¹B NMR spectroscopy) are reported, and the results are used to assess their potential as low-impact cleaning agents for the surfaces of paintings. Because the dispersions can be prepared at room temperature by simple procedures from readily available materials and can contain up to 50% (w/w) of an organic liquid, they offer important advantages over previously described cleaning agents that are based on fully hydrolyzed PVAc (i.e., poly(vinyl alcohol). The mechanical properties of the various aqueous–organic dispersions, as determined quantitatively by rheological investigations and qualitatively by their ease of removal from a solid surface (i.e., the so-called “peel-off” ability) have been tuned systematically by varying the amount of organic liquid, its structure, and the concentrations of borax and 80PVAc. The ¹¹B NMR studies demonstrate that the concentration of borate ions actively participating in cross-linking increases significantly with the amount of organic liquid in the mixture. The degree of cross-linking remains constant when the 80PVAc and borax concentrations are varied, as long as their ratios are kept constant. Some of the 80PVAc–borax dispersions have been tested successfully as cleaning agents on the surface of a 16th–17th century oil-on-wood painting by Lodovico Cardi, “Il Cigoli”, that was covered by a brown patina and on the surface of a Renaissance wall painting by Vecchietta in Santa Maria della Scala, Siena, Italy, that had a degraded polyacrylate coating from a previous conservation treatment

SiRNA-Induced Mutation in HIV-1 Polypurine Tract Region and Its Influence on Viral Fitness

<div><p>Converting single-stranded viral RNA into double stranded DNA for integration is an essential step in HIV-1 replication. Initial polymerization of minus-strand DNA is primed from a host derived tRNA, whereas subsequent plus-strand synthesis requires viral primers derived from the 3′ and central polypurine tracts (3′ and cPPTs). The 5′ and 3′ termini of these conserved RNA sequence elements are precisely cleaved by RT-associated RNase H to generate specific primers that are used to initiate plus-strand DNA synthesis. In this study, siRNA wad used to produce a replicative HIV-1 variant contained G(-1)A and T(-16)A substitutions within/adjacent to the 3′PPT sequence. Introducing either or both mutations into the 3′PPT region or only the G(-1)A substitution in the cPPT region of NL4-3 produced infectious virus with decreased fitness relative to the wild-type virus. In contrast, introducing the T(-16)A or both mutations into the cPPT rendered the virus(es) incapable of replication, most likely due to the F185L integrase mutation produced by this nucleotide substitution. Finally, the effects of G(-1)A and T(-16)A mutations on cleavage of the 3′PPT were examined using an in vitro RNase H cleavage assay. Substrate containing both mutations was mis-cleaved to a greater extent than either wild-type substrate or substrate containing the T(-16)A mutation alone, which is consistent with the observed effects of the equivalent nucleotide substitutions on the replication fitness of NL4-3 virus. In conclusion, siRNA targeting of the HIV-1 3′PPT region can substantially suppress virus replication, and this selective pressure can be used to generate infectious virus containing mutations within or near the HIV-1 PPT. Moreover, in-depth analysis of the resistance mutations demonstrates that although virus containing a G(-1)A mutation within the 3′PPT is capable of replication, this nucleotide substitution shifts the 3′-terminal cleavage site in the 3′PPT by one nucleotide (nt) and significantly reduces viral fitness.</p></div

Sensitivity of 3′PPT and cPPT mutant viruses to siRNA-PPT1 and siRNA-PPTmu.

<p>(A) PPT and flanking sequences of infectious 3′PPT and cPPT mutants together with siRNA-PPTmu. (C)-(F) Sensitivities of infectious PPT mutants to siRNA-PPT1 and siRNA-PPTmu: (C) 3′PPT_G(-1)A, (D) 3′PPT_T(-16)A (E) 3′PPT_{G(-1)A/T(-16)A} and (F) cPPT _G(-1)A. (B) Sensitivity of wild-type NL4-3 to siRNA-PPT1 and siRNAmu. Data was based on three independent experiments.</p

The influence of PPT-region mutations on viral fitness.

<p>(A) Example of heteroduplex tracking assay (HTA) gel used to measure the viral fitness of PPT Δnef mutants. Migration of heteroduplexes produced in single or dual/competitive infections are shown on the left and right sides of the panel, respectively. (B) Relative fitness values of PPT and Δnef mutants compared with NL4-3.</p

Generation of replication-competent PPT mutants through siRNA treatment.

<p>(A) Schematic illustrating the process of generating PPT mutants through siRNA treatment; (B) Mutated PPT in the emergent v120-A strain together with the wild-type 3′PPT and siRNA-PPT1 sequences; (C) Inhibitory efficiency of siRNA-PPT1 on wild-type and mutated v120-A.</p

In vitro PPT RNA/DNA hybrids cleavage assay.

<p>(A) PPT RNA-DNA hybrid substrates (wild type, G(-1)A, T(-16)A, and G(-1)A/T(-16)A double mutants) were subjected to RNase H-mediated cleavage by HIV-1 RT in the absence of nucleotides. Reactions were terminated at 0, 1, 3, 10, or 30 min; (B) Quantitative analysis of PPT RNA-DNA hybrid cleavage.</p

Design of siRNA-ppts and measuring their inhibition of HIV-1 replication.

<p>(A) Three siRNAs were designed to target the HIV-1 3′PPT region: siRNA-PPT1, siRNA-PPT2 and siRNA-PPT3 contain sequence matching nt9069-9089, nt9071-9091, and nt9076-9096 of HXB2, respectively. The sense, passenger strands of the respective siRNA duplexes are indicated and aligned with the corresponding 3’PPT and flanking sequences of NL4-3. Importantly, the targeted sequences within NL4-3, v120 and v126 virus strains are identical. (B) The efficiency of siRNA-PPT inhibition of HIV replication was monitored by RT activity assay.</p

Replication of 3′PPT and cPPT mutant viruses.

<p>(A) Introduction of single and double mutations into the 3′PPT or cPPT region in an NL4-3 backbone through yeast based/HIV-1 cloning technology [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122953#pone.0122953.ref033" target="_blank">33</a>]; (B) Replication of various PPT mutants, as well as Δnef mutant (with premature stop codon in nef but outside of PPT region) in U87.CD4.CXCR4 cells. The input viruses were normalized according to RT activity.</p