Gene Specificity of Suppression of Transgene-Mediated Insertional Transcriptional Activation by the Chicken HS4 Insulator

Insertional mutagenesis has emerged as a major obstacle for gene therapy based on vectors that integrate randomly in the genome. Reducing the genotoxicity of genomic viral integration can, in first approximation, be equated with reducing the risk of oncogene activation, at least in the case of therapeutic payloads that have no known oncogenic potential, such as the globin genes. An attractive solution to the problem of oncogene activation is the inclusion of insulators/enhancer-blockers in the viral vectors. In this study we have used Recombinase-Mediated Cassette Exchange to characterize the effect of integration of globin therapeutic cassettes in the presence or absence of the chicken HS4 and three other putative insulators inserted near Stil, Tal1 and MAP17, three well-known cellular proto-oncogenes in the SCL/Tal1 locus. We show that insertion of a Locus Control Region-driven globin therapeutic globin transgene had a dramatic activating effect on Tal1 and Map17, the two closest genes, a minor effect on Stil, and no effect on Cyp4x1, a non-expressed gene. Of the four element tested, cHS4 was the only one that was able to suppress this transgene-mediated insertional transcriptional activation. cHS4 had a strong suppressive effect on the activation expression of Map17 but has little or no effect on expression of Tal1. The suppressive activity of cHS4 is therefore promoter specific. Importantly, the observed suppressive effect of cHS4 on Map17 activation did not depend on its intercalation between the LCR and the Map 17 promoter. Rather, presence of one or two copies of cHS4 anywhere within the transgene was sufficient to almost completely block the activation of Map17. Therefore, at this complex locus, suppression of transgene-mediated insertional transcriptional activation by cHS4 could not be adequately explained by models that predict that cHS4 can only suppress expression through an enhancer-blocking activity that requires intercalation between an enhancer and a promoter. This has important implications for our theoretical understanding of the possible effects of the insertion of cHS4 on gene therapy vectors. We also show that cHS4 decreased the level of expression of the globin transgene. Therefore, the benefits of partially preventing insertional gene activation are in part negated by the lower expression level of the transgene. A cost/benefit analysis of the utility of incorporation of insulators in gene therapy vectors will require further studies in which the effects of insulators on both the therapeutic gene and the flanking genes are determined at a large number of integration sites. Identification of insulators with minimal promoter specificity would also be of great value

Flight Lieutenant Peach’s observations on Burning Feet Syndrome in Far Eastern Prisoners of War 1942-45

This historical review analyses ‘Burning Feet Syndr ome’, a condition suffered by Far Eastern Prisoners of War in the Second World War. Case reco rds from RAF doctor Nowell Peach, written at the time, are retrospectively assessed a gainst modern diagnostic criteria to determine if the syndrome can be retrospectively cl assed as neuropathic pain

Stem cell factor and erythropoietin-independent production of cultured reticulocytes

Cultured reticulocytes can supplement transfusion needs and offer promise for drug delivery and immune tolerization. They can be produced from induced pluripotent stem cells (iPSCs), but the 45-day culture time and cytokine costs make large-scale production prohibitive. To overcome these limitations, we have generated IPSCs that express constitutive SCF receptor and jak2 adaptor alleles. We show that iPSC lines carrying these alleles can differentiate into self-renewing erythroblast (SRE) that can proliferate for up to 70 cell-doubling in a cost-effective, chemically-defined, albumin- and cytokine-free medium. These kitjak2 SREs retain the ability to enucleate at a high rate up to senescence. Kitjak2 derived cultured reticulocytes should be safe for transfusion because they can be irradiated to eliminate residual nucleated cells. The kitjak2 cells express blood group 0 and test negative for RhD and other clinically significant RBCs antigens and have sufficient proliferation capacity to meet global RBC needs

Flight Lieutenant Peach's observations on Burning Feet Syndrome in Far Eastern Prisoners of War 1942-45.

Introduction: ‘Burning Feet Syndrome’ affected up to one third of Far Eastern Prisoners of War in World War 2. Recently discovered medical records, produced by RAF Medical Officer Nowell Peach whilst in captivity, are the first to detail neurological examinations of patients with this condition. Methods: The 54 sets of case notes produced at the time were analysed using modern diagnostic criteria to determine if the syndrome can be retrospectively classed as neuropathic pain. Results: With a history of severe malnutrition raising the possibility of a peripheral polyneuropathy, and a neuroanatomically plausible pain distribution, this analysis showed that Burning Feet Syndrome can now be described as a ‘possible’ neuropathic pain syndrome. Conclusion: After 70 years, the data painstakingly gathered under the worst of circumstances have proved to be of interest and value in modern diagnostics of neuropathic pain

Histone H1 Depletion in Mammals Alters Global Chromatin Structure but Causes Specific Changes in Gene Regulation

SummaryLinker histone H1 plays an important role in chromatin folding in vitro. To study the role of H1 in vivo, mouse embryonic stem cells null for three H1 genes were derived and were found to have 50% of the normal level of H1. H1 depletion caused dramatic chromatin structure changes, including decreased global nucleosome spacing, reduced local chromatin compaction, and decreases in certain core histone modifications. Surprisingly, however, microarray analysis revealed that expression of only a small number of genes is affected. Many of the affected genes are imprinted or are on the X chromosome and are therefore normally regulated by DNA methylation. Although global DNA methylation is not changed, methylation of specific CpGs within the regulatory regions of some of the H1 regulated genes is reduced. These results indicate that linker histones can participate in epigenetic regulation of gene expression by contributing to the maintenance or establishment of specific DNA methylation patterns

Chronic pain associated with the Chikungunya Fever: long lasting burden of an acute illness

<p>Abstract</p> <p>Background</p> <p>Chikungunya virus (CHIKV) is responsible for major epidemics worldwide. Autochthonous cases were recently reported in several European countries. Acute infection is thought to be monophasic. However reports on chronic pain related to CHIKV infection have been made. In particular, the fact that many of these patients do not respond well to usual analgesics suggests that the nature of chronic pain may be not only nociceptive but also neuropathic. Neuropathic pain syndromes require specific treatment and the identification of neuropathic characteristics (NC) in a pain syndrome is a major step towards pain control.</p> <p>Methods</p> <p>We carried out a cross-sectional study at the end of the major two-wave outbreak lasting 17 months in Réunion Island. We assessed pain in 106 patients seeking general practitioners with confirmed infection with the CHIK virus, and evaluated its impact on quality of life (QoL).</p> <p>Results</p> <p>The mean intensity of pain on the visual-analogical scale (VAS) was 5.8 ± 2.1, and its mean duration was 89 ± 2 days. Fifty-six patients fulfilled the definition of chronic pain. Pain had NC in 18.9% according to the DN4 questionnaire. Conversely, about two thirds (65%) of patients with NC had chronic pain. The average pain intensity was similar between patients with or without NC (6.0 ± 1.7 vs 6.1 ± 2.0). However, the total score of the Short Form-McGill Pain Questionnaire (SF-MPQ)(15.5 ± 5.2 vs 11.6 ± 5.2; p < 0.01) and both the affective (18.8 ± 6.2 vs 13.4 ± 6.7; p < 0.01) and sensory subscores (34.3 ± 10.7 vs 25.0 ± 9.9; p < 0.01) were significantly higher in patients with NC. The mean pain interference in life activities calculated from the Brief Pain Inventory (BPI) was significantly higher in patients with chronic pain than in patients without it (6.8 ± 1.9 vs 5.9 ± 1.9, p < 0.05). This score was also significantly higher in patients with NC than in those without such a feature (7.2 ± 1.5 vs 6.1 ± 1.9, p < 0.05).</p> <p>Conclusions</p> <p>There exists a specific chronic pain condition associated to CHIKV. Pain with NC seems to be associated with more aggressive clinical picture, more intense impact in QoL and more challenging pharmacological treatment.</p

DNA Methylation Supports Intrinsic Epigenetic Memory in Mammalian Cells

We have investigated the role of DNA methylation in the initiation and maintenance of silenced chromatin in somatic mammalian cells. We found that a mutated transgene, in which all the CpG dinucleotides have been eliminated, underwent transcriptional silencing to the same extent as the unmodified transgene. These observations demonstrate that DNA methylation is not required for silencing. The silenced CpG-free transgene exhibited all the features of heterochromatin, including silencing of transcriptional activity, delayed DNA replication, lack of histone H3 and H4 acetylation, lack of H3-K4 methylation, and enrichment in tri-methyl-H3-K9. In contrast, when we tested for transgene reactivation using a Cre recombinase-mediated inversion assay, we observed a marked difference between a CpG-free and an unmodified transgene: the CpG-free transgene resumed transcription and did not exhibit markers of heterochromatin whereas the unmodified transgene remained silenced. These data indicate that methylation of CpG residues conferred epigenetic memory in this system. These results also suggest that replication delay, lack of histone H3 and H4 acetylation, H3-K4 methylation, and enrichment in tri-methyl-H3-K9 are not sufficient to confer epigenetic memory. We propose that DNA methylation within transgenes serves as an intrinsic epigenetic memory to permanently silence transgenes and prevent their reactivation

CG dinucleotide clustering is a species-specific property of the genome

Cytosines at cytosine-guanine (CG) dinucleotides are the near-exclusive target of DNA methyltransferases in mammalian genomes. Spontaneous deamination of methylcytosine to thymine makes methylated cytosines unusually susceptible to mutation and consequent depletion. The loci where CG dinucleotides remain relatively enriched, presumably due to their unmethylated status during the germ cell cycle, have been referred to as CpG islands. Currently, CpG islands are solely defined by base compositional criteria, allowing annotation of any sequenced genome. Using a novel bioinformatic approach, we show that CG clusters can be identified as an inherent property of genomic sequence without imposing a base compositional a priori assumption. We also show that the CG clusters co-localize in the human genome with hypomethylated loci and annotated transcription start sites to a greater extent than annotations produced by prior CpG island definitions. Moreover, this new approach allows CG clusters to be identified in a species-specific manner, revealing a degree of orthologous conservation that is not revealed by current base compositional approaches. Finally, our approach is able to identify methylating genomes (such as Takifugu rubripes) that lack CG clustering entirely, in which it is inappropriate to annotate CpG islands or CG clusters

Flight Lieutenant Peach’s observations on Burning Feet Syndrome in Far Eastern Prisoners of War 1942–45

Introduction: ‘Burning Feet Syndrome’ affected up to one third of Far Eastern Prisoners of War in World War 2. Recently discovered medical records, produced by RAF Medical Officer Nowell Peach whilst in captivity, are the first to detail neurological examinations of patients with this condition. Methods: The 54 sets of case notes produced at the time were analysed using modern diagnostic criteria to determine if the syndrome can be retrospectively classed as neuropathic pain. Results: With a history of severe malnutrition raising the possibility of a peripheral polyneuropathy, and a neuroanatomically plausible pain distribution, this analysis showed that Burning Feet Syndrome can now be described as a ‘possible’ neuropathic pain syndrome. Conclusion: After 70 years, the data painstakingly gathered under the worst of circumstances have proved to be of interest and value in modern diagnostics of neuropathic pain

Pain thresholds and intensities of CRPS type I and neuropathic pain in respect to sex

Abstract Background and aims Healthy women have generally been found to have increased experimental pain perception and chronic pain has a higher prevalence in female as compared to male patients. However, no study has investigated whether pain intensity and pain perception thresholds are distinct or similar between sexes within various chronic pain entities. We investigated whether average pain intensities and pain thresholds assessed using quantitative sensory testing (QST) differed between women and men suffering from three distinct chronic pain conditions: Complex Regional Pain Syndrome (CRPS type I), peripheral nerve injury (PNI) or polyneuropathy (PNP), as compared to paired healthy volunteers. Methods QST data of 1252 patients (669 female, 583 male) with PNI (n=342), PNP (n=571) or CRPS (n=339), and average pain intensity reports from previously published studies were included. Absolute and z-values (adjusted for age and body region) of cold, heat, pressure (PPT) and pinprick pain thresholds were compared in generalized linear models with aetiology, duration of underlying pain disease and average pain intensity as fixed effects. Results Average pain intensity during the past four weeks did not differ between women and men, in both mean and range. In women absolute pain thresholds for cold, heat and pinprick were lower than in males across all diagnoses (pPeer reviewe