CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression

Sjuksköterskeyrket innebär ofta långvarig stress och bristfällig arbetsmiljö. Personalbrist, hög arbetsbelastning och bristande inflytande på arbetsplatsen är orsaker till att sjuksköterskor väljer att sluta inom yrket. För lite forskning gällande den upplevda stressen på arbetsplatsen inom sjuksköterskeyrket råder. Därför finns anledning till ökad kunskap kring ämnet. Syftet med studien är att undersöka i hur hög utsträckning sjuksköterskor upplever stress i sin arbetsmiljö, och studien är baserad på 1044 yrkesverksamma sjuksköterskor över hela landet som är medlemmar i Facebookgruppen ”Sjuksköterskan”. Dessa personer har fått svara på enkäten Work Stress Questionnaire med 21 frågor. Resultatet från denna studie visar att arbetsbelastningen ökat och personalen har inte möjlighet att påverka beslut som tas på arbetsplatsen. Konflikter är också förekommande där chefen, i de flesta fall, inte gör något för att lösa dessa konflikter. Sjuksköterskorna sätter höga krav på sig själva och är mycket engagerade i arbetet, men har ofta svårt att sätta gränser. Resultatet visar också att en hög andel har svårt att hinna med familj, vänner och fritidsintressen. Det finns tidigare studier med samma mätinstrument som visar att arbetsbelastningen och ansvarstagandet har ökat under de senaste åren. Eventuellt kan detta i slutändan innebära en risk för ökad utbrändhet och fler sjukskrivningar

CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression

Huntington's disease (HD) is a fatal, neurodegenerative disorder in which patients suffer from mobility, psychological and cognitive impairments. Existing therapeutics are only symptomatic and do not significantly alter the disease progression or increase life expectancy. HD is caused by expansion of the CAG trinucleotide repeat region in exon 1 of the Huntingtin gene (HTT), leading to the formation of mutant HTT transcripts (muHTT). The toxic gain-of-function of muHTT protein is a major cause of the disease. In addition, it has been suggested that the muHTT transcript contributes to the toxicity. Thus, reduction of both muHTT mRNA and protein levels would ideally be the most useful therapeutic option. We herein present a novel strategy for HD treatment using oligonucleotides (ONs) directly targeting the HTT trinucleotide repeat DNA. A partial, but significant and potentially long-term, HTT knock-down of both mRNA and protein was successfully achieved. Diminished phosphorylation of HTT gene-associated RNA-polymerase II is demonstrated, suggestive of reduced transcription downstream the ON-targeted repeat. Different backbone chemistries were found to have a strong impact on the ON efficiency. We also successfully use different delivery vehicles as well as naked uptake of the ONs, demonstrating versatility and possibly providing insights for in vivo applications.</p

Genome-Wide Studies of Histone Demethylation Catalysed by the Fission Yeast Homologues of Mammalian LSD1

In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO) domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex. However, we find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) and both up- and down-regulates expression of different groups of genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1+ gene. Using hyper-geometric probability comparisons we uncover genetic links between lysine-specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. The data presented here demonstrate that in fission yeast the SWIRM/PAO domain proteins Swm1 and Swm2 are associated in complexes that can remove methyl groups from lysine 9 methylated histone H3. In vitro, we show that bacterially expressed Swm1 also possesses lysine 9 demethylase activity. In vivo, loss of Swm1 increases the global levels of both H3K9me2 and H3K4me2. A significant accumulation of H3K4me2 is observed at genes that are up-regulated in a swm1 deletion strain. In addition, H3K9me2 accumulates at some genes known to be direct Swm1/2 targets that are down-regulated in the swm1¿ strain. The in vivo data indicate that Swm1 acts in concert with the HDAC Clr6 and the chromatin remodeller Hrp1 to repress gene expression. In addition, our in vitro analyses suggest that the H3K9 demethylase activity requires an unidentified post-translational modification to allow it to act. Thus, our results highlight complex interactions between histone demethylase, deacetylase and chromatin remodelling activities in the regulation of gene expression

Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA

The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 5′-monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably well-conserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1Δ cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity

Factor structure and construct validity of the Adult Social Care Outcomes Toolkit for Carers (ASCOT-Carer)

Background: The ASCOT-Carer is a self-report instrument designed to measure social care-related quality of life (SCRQoL). This article presents the psychometric testing and validation of the ASCOT-Carer four response-level interview (INT4) in a sample of unpaid carers of adults who receive publicly-funded social care services in England. Methods: Unpaid carers were identified through a survey of users of publicly-funded social care services in England. 387 carers completed a face-to-face or telephone interview. Data on variables hypothesised to be related to SCRQoL (for example, characteristics of the carer, cared-for person and care situation) and measures of carer experience, strain, health-related quality of life and overall QoL were collected. Relationships between these variables and overall SCRQoL score were evaluated through correlation, ANOVA and regression analysis to test the construct validity of the scale. Internal reliability was assessed using Cronbach’s alpha and feasibility by the number of missing responses. Results: The construct validity was supported by statistically significant relationships between SCRQoL and scores on instruments of related constructs, as well as with characteristics of the carer and care recipient in univariate and multivariate analyses. A Cronbach’s alpha of 0.87 (7 items) indicates that the internal reliability of the instrument is satisfactory and a low number of missing responses (<1%) indicates a high level of acceptance. Conclusions: The results provide evidence to support the construct validity, factor structure, internal reliability and feasibility of the ASCOT-Carer INT4 as an instrument for measuring social care-related quality of life of unpaid carers who care for adults with a variety of long-term conditions, disability or problems related to old age

A two-step mechanism for epigenetic specification of centromere identity and function

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.National Institutes of Health grant: (GM 074150); Ludwig Institute for Cancer Research; European Molecular Biology Organization (EMBO) long-term fellowship

H3K9me-Independent Gene Silencing in Fission Yeast Heterochromatin by Clr5 and Histone Deacetylases

Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me) and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well-characterized heterochromatic region, is hardly affected in cells lacking the H3K9 methyltransferase Clr4. We document the existence of a pathway parallel to H3K9me ensuring gene repression in the absence of Clr4 and identify a silencing factor central to this pathway, Clr5. We find that Clr5 controls gene expression at multiple chromosomal locations in addition to affecting the mating-type region. The histone deacetylase Clr6 acts in the same pathway as Clr5, at least for its effects in the mating-type region, and on a subset of other targets, notably a region recently found to be prone to neo-centromere formation. The genomic targets of Clr5 also include Ste11, a master regulator of sexual differentiation. Hence Clr5, like the multi-functional Atf1 transcription factor which also modulates chromatin structure in the mating-type region, controls sexual differentiation and genome integrity at several levels. Globally, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci

A DNA Polymerase α Accessory Protein, Mcl1, Is Required for Propagation of Centromere Structures in Fission Yeast

Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-ACnp1 kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase α (Polα) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-ACnp1 in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7+, which encodes a catalytic subunit of Polα. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Polα. These results suggest that Mcl1 and Polα are required for propagation of centromere chromatin structures during DNA replication

Absence of autoantibodies connected to autoimmune polyendocrine syndrome type I and II and Addison's disease in girls and women with Turner syndrome

<p>Abstract</p> <p>Background</p> <p>A disturbance in the immune system has been described in Turner syndrome (45,X), with an association to low levels of IgG and IgM and decreased levels of T- and B-lymphocytes. Also different autoimmune diseases have been connected to Turner syndrome (45,X), thyroiditis being the most common. Other autoimmune diseases seen are inflammatory bowel disease, insulin dependent diabetes mellitus, Addison's disease, rheumatoid arthritis, myasthenia gravis, vitiligo, alopecia, pernicious anaemia and hypoparathyroidism, but the association to Turner syndrome is not definite.</p> <p>Besides the typical features of Turner syndrome (short stature, failure to enter puberty spontaneously and infertility due to ovarian insufficiency) ear problems are common. Otitis media and a progressive sensorineural hearing disorder are commonly seen. In the normal population there are known inner ear disorders related to autoimmune diseases. The aim of this study was to investigate patients with Turner syndrome regarding autoantibodies connected to the autoimmune disorders; autoimmune polyendocrine syndrome type I and II and Addison's disease, to screen for overlapping profile of autoantibodies.</p> <p>Blood samples from 110 Turner patients (7–65 years) were investigated using <it>in vitro </it>transcription, translation and immunoprecipitation techniques regarding autoantibodies connected to autoimmune polyendocrine syndrome type I and II and Addison's disease (21-hydroxylase, 17α-hydroxylase, side-chain cleavage enzyme, aromatic L-amino acid decarboxylase, tyrosine hydroxylase and tryptophan hydroxylase).</p> <p>Results</p> <p>The autoantibodies investigated were not overrepresented among the Turner patients.</p> <p>Conclusion</p> <p>The autoimmune disorders associated with Turner syndrome do not seem to be of the same origin as Addison's disease, the type I or II autoimmune polyendocrine syndrome.</p