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Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing betathalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent proteinbeta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule. Enhanced recombination, with frequencies up to 0.4%, was observed with use of the lysomotropic agent chloroquine. Finally, we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells.beta-thalassemia ͉ gene correction ͉ triplex-forming oligonucleotides ͉ gene targeting M utations in the beta-globin gene that affect any stage in beta-globin biogenesis can cause beta-thalassemia. Identified mutations include single base pair changes that lead to frameshift mutations or changes in canonical sequences that affect mRNA stability and processing (1). As monogenic disorders, betathalassemia and sickle cell anemia have attracted substantial efforts directed at gene therapy by gene replacement, and there has been ongoing progress in this regard. In one approach specific to the thalassemias in which the genetic defect affects mRNA splicing, antisense oligonucleotides have been used to manipulate the splice site choice in beta-globin premRNA to prevent aberrant splicing. Restoration of proper beta-globin splicing has been demonstrated in human erythroid cells derived from beta-thalassemic patients, and in transgenic mouse models containing splicing mutations in the beta-globin gene (2, 3).In this study, we use an antigene approach to correct a thalassemia-causing splice-site mutation at the level of chromosomal DNA in cultured cells, generating heritable, site-specific modification of the beta-globin gene. We have used peptide nucleic acids (PNAs), a class of triplex-forming molecules shown to be effective at provoking recombination and repair at chromosomal sites near PNA binding sites (4). PNAs contain standard nucleobases linked to a peptide-like backbone, and their advantages include resistance to nucl...

Early diagnosis of esophageal perforations following LACA may allow temporary esophageal stenting with successful esophageal healing. Prompt chest CT scans with oral and i.v. contrast should be considered in any patient with sub-sternal chest pain or dysphagia following LACA.

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Zebra dolomites, characterized by a repetition of dark grey (a) and light (b) coloured dolomite sheets building up abbabba‐sequences, occur in Dinantian strata from deep boreholes (> 2000 m) south of the Brabant‐Wales Massif in Belgium. These zebra dolomite sequences are several tens of metres thick. The dark grey dolomite sheets (a) consist of non‐planar crystals, 80–150 μm in diameter. These crystals display a mottled red–orange luminescence and are interpreted to be replacive in origin. The white dolomite sheets (b) consist of coarse crystalline nonplanar b1 dolomite, which evolves outwards into transparent saddle shaped b2 dolomite. The b1 dolomites possess a mottled red–orange luminescence similar to the a dolomites, while the saddle shaped b2 rims display red to dark brown luminescent‐zones. The b1 dolomites are possibly partly replacive and partly cavity filling. Their b2 rims display criteria typical for a cement origin. Locally, cavities exist between two succeeding white dolomite sheets. These cavities make up ≈5% of the zebra rocks and are locally filled by saddle shaped ankerite and/or xenomorphic ferroan calcite. Geochemical and fluid inclusion data (Th ≈ 120 °C) indicate a burial diagenetic origin for these zebra dolomites. The a and b1 dolomites are characterized by similar geochemical compositions and fluid inclusion data pointing toward a related origin. To explain the development of the zebra textures, suprahydrostatic pressures in conjunction with late Variscan tectonic compression are invoked. A model involving dolomitizing fluids expelled during the Variscan orogeny is proposed. An overpressured system is also invoked to explain the important porosity development, the creation of centimetre‐scale subvertical displacements of the zebra pattern and the microfractures affecting the b1–b2 dolomite crystals.

Phospbolipid vesicles (liposomes) as membrane models have been used to study the penetration properties of peptide nucleic acid (PNA), a new DNA analog in which the nucleobases are attached to a pseudo-peptide backbone. The liposomes were characterised by carboxyfluorescein efflux, light-scattering and cryogenic transmission electron microscopy. The liposome structure was found not to be affected by the incorporation of PNA or an oligonucleotide. Two 10-mer fluorescein-labelled PNAs were found to have low efflux rates (half-times of 5.5 and 11 days), comparable to a 10-met oligonucleotide (half-time of 7 days). We conclude that passive diffusion of unmodified PNA is not an effective way of transport into biological cells.

The in situ hybridization kinetics of label-free DNA on mixed monolayers of peptide nucleic acid (PNA) and 6-mercapto-1-hexanol (MCH) on Au electrodes was investigated by electrochemical impedance spectroscopy (EIS) and used to discriminate the fully complementary DNA from the single-base mismatched hybrids.