Targeted correction of a thalassemia-associated β-globin mutation induced by pseudo-complementary peptide nucleic acids

β-Thalassemia is a genetic disorder caused by mutations in the β-globin gene. Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. However, the use of these molecules for gene targeting requires homopurine tracts to facilitate triple helix formation. Alternatively, to achieve binding to mixed-sequence target sites for the induced gene correction, we have used pseudo-complementary PNAs (pcPNAs). Due to steric hindrance, pcPNAs are unable to form pcPNA–pcPNA duplexes but can bind to complementary DNA sequences via double duplex-invasion complexes. We demonstrate here that pcPNAs, when co-transfected with donor DNA fragments, can promote single base pair modification at the start of the second intron of the beta-globin gene. This was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta globin fusion gene. We also demonstrate that pcPNAs are effective in stimulating recombination in human fibroblast cells in a manner dependent on the nucleotide excision repair factor, XPA. These results suggest that pcPNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells without purine sequence restriction

Long homopurine•homopyrimidine sequences are characteristic of genes expressed in brain and the pseudoautosomal region

Homo(purine•pyrimidine) sequences (R•Y tracts) with mirror repeat symmetries form stable triplexes that block replication and transcription and promote genetic rearrangements. A systematic search was conducted to map the location of the longest R•Y tracts in the human genome in order to assess their potential function(s). The 814 R•Y tracts with ≥250 uninterrupted base pairs were preferentially clustered in the pseudoautosomal region of the sex chromosomes and located in the introns of 228 annotated genes whose protein products were associated with functions at the cell membrane. These genes were highly expressed in the brain and particularly in genes associated with susceptibility to mental disorders, such as schizophrenia. The set of 1957 genes harboring the 2886 R•Y tracts with ≥100 uninterrupted base pairs was additionally enriched in proteins associated with phosphorylation, signal transduction, development and morphogenesis. Comparisons of the ≥250 bp R•Y tracts in the mouse and chimpanzee genomes indicated that these sequences have mutated faster than the surrounding regions and are longer in humans than in chimpanzees. These results support a role for long R•Y tracts in promoting recombination and genome diversity during evolution through destabilization of chromosomal DNA, thereby inducing repair and mutation

Relationship between Anaemia, Haemolysis, Inflammation and Haem Oxygenase-1 at Admission with Sepsis: a pilot study

Upregulation of haem oxygenase-1 (HO-1), due to haemolysis and/or inflammation, can lead to impaired immune function. Anaemia is common among sepsis patients, but the consequences of sepsis-associated anaemia are poorly understood. Here, our objective was to determine the prevalence and extent of anaemia, haemolysis, inflammation, and HO-1 induction after early hospital admission. We hypothesised that inflammation- or infection-induced haemolysis contributes to sepsis-associated anaemia and that this will lead to expression of HO-1. In this study, plasma obtained from seventy adult patients within 12 hours of admission to intensive care due to sepsis were analysed for anaemia, haemolysis and inflammatory markers by ELISA and microbead array. The majority (82.6%) of patients were anaemic with evidence of haemolysis (raised haem, haptoglobin, haemopexin, and HO-1 concentrations). Interestingly, concentrations of both haemoglobin and IL-10 were moderately positively correlated with HO-1 concentration (Hb: r = 0.32, p = 0.007; IL-10 r = 0.39, p = 0.0008) whereas HO-1 concentration was weakly negatively correlated with haemopexin (r = -0.23, p = 0.055). Anaemia, while common, was not associated with HO-1 concentration. After adjusting for confounding, HO-1 induction appears to be associated primarily with IL-10 concentration rather than haemolysis. Disease severity at diagnosis was correlated with early plasma IL-10 (r = 0.35, p = 0.003) and HO-1 (r = 0.24, p = 0.048) concentrations. Notably, admission levels of haem, HO-1, and IL-10 were indicators of survival

Association of intensive care unit delirium with sleep disturbance and functional disability after critical illness: an observational cohort study

Abstract Background In medical intensive care unit (MICU) patients, the predictors of post-discharge sleep disturbance and functional disability are poorly understood. ICU delirium is a risk factor with a plausible link to sleep disturbance and disability. This study evaluated the prevalence of self-reported post-ICU sleep disturbance and increased functional disability, and their association with MICU delirium and other ICU factors. Methods This was an observational cohort study of MICU patients enrolled in a biorepository and assessed upon MICU admission by demographics, comorbidities, and baseline characteristics. Delirium was assessed daily using the Confusion Assessment Method for the ICU. Telephone follow-up interview instruments occurred after hospital discharge and included the Pittsburgh Sleep Quality Index (PSQI), and basic and instrumental activities of daily living (BADLs, IADLs) for disability. We define sleep disturbance as a PSQI score > 5 and increased disability as an increase in composite BADL/IADL score at follow-up relative to baseline. Multivariable regression modeled the associations of delirium and other MICU factors on follow-up PSQI scores and change in disability scores. Results PSQI and BADL/IADL instruments were completed by 112 and 122 participants, respectively, at mean 147 days after hospital discharge. Of those surveyed, 63% had sleep disturbance by PSQI criteria, and 37% had increased disability by BADL/IADL scores compared to their pre-MICU baseline. Total days of MICU delirium (p = 0.013), younger age (p = 0.013), and preexisting depression (p = 0.025) were significantly associated with higher PSQI scores at follow-up. Lower baseline disability (p < 0.001), older age (p = 0.048), and less time to follow-up (p = 0.024) were significantly associated with worsening post-ICU disability, while the occurrence of MICU delirium showed a trend toward association (p = 0.077). Conclusions After adjusting for important covariates, total days of MICU delirium were significantly associated with increased post-discharge sleep disturbance. Delirium incidence showed a trend toward association with increased functional disability in the year following discharge

Targeted correction of a thalassemia-associated

b-globin mutation induced by pseudocomplementary peptide nucleic acid