Hypoxia-induced transcriptional stress is mediated by ROS-induced R-loops.

Hypoxia is a common feature of solid tumors and is associated with poor patient prognosis, therapy resistance and metastasis. Radiobiological hypoxia (<0.1% O2) is one of the few physiologically relevant stresses that activates both the replication stress/DNA damage response and the unfolded protein response. Recently, we found that hypoxia also leads to the robust accumulation of R-loops, which led us to question here both the mechanism and consequence of hypoxia-induced R-loops. Interestingly, we found that the mechanism of R-loop accumulation in hypoxia is dependent on non-DNA damaging levels of reactive oxygen species. We show that hypoxia-induced R-loops play a critical role in the transcriptional stress response, evidenced by the repression of ribosomal RNA synthesis and the translocation of nucleolin from the nucleolus into the nucleoplasm. Upon depletion of R-loops, we observed a rescue of both rRNA transcription and nucleolin translocation in hypoxia. Mechanistically, R-loops accumulate on the rDNA in hypoxia and promote the deposition of heterochromatic H3K9me2 which leads to the inhibition of Pol I-mediated transcription of rRNA. These data highlight a novel mechanistic insight into the hypoxia-induced transcriptional stress response through the ROS-R-loop-H3K9me2 axis. Overall, this study highlights the contribution of transcriptional stress to hypoxia-mediated tumorigenesis.

Induction of the alternative lengthening of telomeres pathway by trapping of proteins on DNA.

Telomere maintenance is a hallmark of malignant cells and allows cancers to divide indefinitely. In some cancers, this is achieved through the alternative lengthening of telomeres (ALT) pathway. Whilst loss of ATRX is a near universal feature of ALT-cancers, it is insufficient in isolation. As such, other cellular events must be necessary - but the exact nature of the secondary events has remained elusive. Here, we report that trapping of proteins (such as TOP1, TOP2A and PARP1) on DNA leads to ALT induction in cells lacking ATRX. We demonstrate that protein-trapping chemotherapeutic agents, such as etoposide, camptothecin and talazoparib, induce ALT markers specifically in ATRX-null cells. Further, we show that treatment with G4-stabilising drugs cause an increase in trapped TOP2A levels which leads to ALT induction in ATRX-null cells. This process is MUS81-endonuclease and break-induced replication dependent, suggesting that protein trapping leads to replication fork stalling, with these forks being aberrantly processed in the absence of ATRX. Finally, we show ALT-positive cells harbour a higher load of genome-wide trapped proteins, such as TOP1, and knockdown of TOP1 reduced ALT activity. Taken together, these findings suggest that protein trapping is a fundamental driving force behind ALT-biology in ATRX-deficient malignancies.

A key feature of all cancer cells is their ability to divide indefinitely, and this is dependent on circumvention of telomere shortening through induction of a telomere maintenance mechanism, such as the telomerase-independent, Alternative Lengthening of Telomeres (ALT) pathway. The ALT pathway is characterised by loss of the ATRX chromatin remodeler. The current study provides evidence that, in the absence of ATRX, increased trapping of proteins on DNA leads to replication fork stalling and collapse. At telomeres, this leads to ALT pathway activity. These results help to better understand ALT tumours and might, eventually, be instrumental in developing new therapeutic strategies.

Assessing the co-variability of DNA methylation across peripheral cells and tissues: Implications for the interpretation of findings in epigenetic epidemiology.

Most epigenome-wide association studies (EWAS) quantify DNA methylation (DNAm) in peripheral tissues such as whole blood to identify positions in the genome where variation is statistically associated with a trait or exposure. As whole blood comprises a mix of cell types, it is unclear whether trait-associated DNAm variation is specific to an individual cellular population. We collected three peripheral tissues (whole blood, buccal epithelial and nasal epithelial cells) from thirty individuals. Whole blood samples were subsequently processed using fluorescence-activated cell sorting (FACS) to purify five constituent cell-types (monocytes, granulocytes, CD4+ T cells, CD8+ T cells, and B cells). DNAm was profiled in all eight sample-types from each individual using the Illumina EPIC array. We identified significant differences in both the level and variability of DNAm between different sample types, and DNAm data-derived estimates of age and smoking were found to differ dramatically across sample types from the same individual. We found that for the majority of loci variation in DNAm in individual blood cell types was only weakly predictive of variance in DNAm measured in whole blood, although the proportion of variance explained was greater than that explained by either buccal or nasal epithelial samples. Covariation across sample types was much higher for DNAm sites influenced by genetic factors. Overall, we observe that DNAm variation in whole blood is additively influenced by a combination of the major blood cell types. For a subset of sites, however, variable DNAm detected in whole blood can be attributed to variation in a single blood cell type providing potential mechanistic insight about EWAS findings. Our results suggest that associations between whole blood DNAm and traits or exposures reflect differences in multiple cell types and our data will facilitate the interpretation of findings in epigenetic epidemiology.

Two cases of SMA syndrome after neurosurgical injury to the frontal aslant tract.

Supplementary motor area (SMA) syndrome is characterised by transient disturbance in volitional movement and speech production which classically occurs after injury to the medial premotor area. We present two cases of SMA syndrome following isolated surgical injury to the frontal aslant tract (FAT) with the SMA intact. The first case occurred after resection of a left frontal operculum tumour. The second case occurred after a transcortical approach to a ventricular neurocytoma. The clinical picture and fMRI activation patterns during recovery were typical for SMA syndrome and support the theory that the FAT is a critical bundle in the SMA complex function.

Circulating bile acids as a link between the gut microbiota and cardiovascular health: impact of prebiotics, probiotics and polyphenol-rich foods.

Beneficial effects of probiotic, prebiotic and polyphenol-rich interventions on fasting lipid profiles have been reported, with changes in the gut microbiota composition believed to play an important role in lipid regulation. Primary bile acids, which are involved in the digestion of fats and cholesterol metabolism, can be converted by the gut microbiota to secondary bile acids, some species of which are less well reabsorbed and consequently may be excreted in the stool. This can lead to increased hepatic bile acid neo-synthesis, resulting in a net loss of circulating low-density lipoprotein. Bile acids may therefore provide a link between the gut microbiota and cardiovascular health. This narrative review presents an overview of bile acid metabolism and the role of probiotics, prebiotics and polyphenol-rich foods in modulating circulating cardiovascular disease (CVD) risk markers and bile acids. Although findings from human studies are inconsistent, there is growing evidence for associations between these dietary components and improved lipid CVD risk markers, attributed to modulation of the gut microbiota and bile acid metabolism. These include increased bile acid neo-synthesis, due to bile sequestering action, bile salt metabolising activity and effects of short-chain fatty acids generated through bacterial fermentation of fibres. Animal studies have demonstrated effects on the FXR/FGF-15 axis and hepatic genes involved in bile acid synthesis (CYP7A1) and cholesterol synthesis (SREBP and HMGR). Further human studies are needed to determine the relationship between diet and bile acid metabolism and whether circulating bile acids can be utilised as a potential CVD risk biomarker.

Risk and Lag-time for Development of Lacrimal Gland Carcinoma Expleomorphic Adenoma: Observations and Conjectural Study.

AIMS: To estimate the proportion of de novo lacrimal gland pleomorphic adenomas (PAs) and carcinomas expleomorphic adenomas (CEPAs), together with age at presentation and first symptom. Conjectural models of tumor growth are considered. METHODS: Retrospective review of patients with orbital lobe PA or CEPA. The presenting age was examined for conformation to a Gaussian distribution and the cumulative distribution function derived for both tumor types. The risk of CEPA with age was estimated by logistic regression. RESULTS: About one-sixth (27/172; 16%) of these primary orbital lobe tumors were CEPAs, with 145 PAs (76/145 male; 52%) and 27 CEPAs (12/27 male; 44%). The mean presenting age for PAs was 48.3 years (median 47.7; range 11-84 years) and 57.7 years for CEPAs (median 61.2, range 27-91 years) ( p = 0.0062), and the standard deviations for each group are almost identical (16.3 for PAs, 15.9 for CEPAs; p = 0.92). Five (3.4%) PAs and 1 (3.7%) CEPA were asymptomatic: otherwise, the median symptom duration was 24 months for both PAs and CEPAs ( U test: p = 0.65). The odds of CEPA rises significantly with age, increasing 1.04-fold annually ( p = 0.0079). CONCLUSION: The almost identical measures of dispersion for the presenting ages of PA and CEPA suggests that, once malignant transformation occurs, there might be a relatively constant period before it is evident. CEPAs present about a decade after PAs, this unexpectedly later presentation for the malignancy possibly being explained by a gradual replacement of the PA by the newly arising carcinoma within the preceding benign tumor.

Newly graduated nurses' experiences of the intervention graduate guidance nurses: A qualitative interview study.

AIMS: This study is describing newly graduated nurses' experiences of the intervention graduate guidance nurses. BACKGROUND: Newly graduated nurses need support to become established in the profession. The intervention was initiated to empower and support in the professional role. METHODS: A qualitative case study was conducted with semi-structured interviews, using a thematic content analysis. RESULTS: One overarching theme 'Organizational prerequisites', consisting of three themes, occurred: 'Activator' involved that the graduate guidance nurse was the activator creating a clear structure, and the wards became more attractive workplaces. 'Supportive nursing' meant that the graduate guidance nurse constituted an important support function which ensured patient safety. 'Professional development' created the opportunity for professional growth. CONCLUSION: Newly graduated nurses' experiences show that the creation of an organisational structure enabled the graduate guidance nurses to be an important support and contributed to professional development. IMPLICATIONS FOR NURSING MANAGEMENT: In health care organisations, strategic decisions, management support and clear goals are important to create the organisational conditions to improve safer care. Support from experienced nurses is a large enabler in supporting newly graduated nurses developing in their profession. The results of the current study can be transferred to other similar health care organisations and can be supporting managers who plan to initiate support to newly graduated nurses.

Recapitulation of erythropoiesis in congenital dyserythropoietic anaemia type I (CDA-I) identifies defects in differentiation and nucleolar abnormalities.

The investigation of inherited disorders of erythropoiesis has elucidated many of the principles underlying the production of normal red blood cells and how this is perturbed in human disease. Congenital Dyserythropoietic Anaemia type 1 (CDA-I) is a rare form of anaemia caused by mutations in two genes of unknown function: CDAN1 and CDIN1 (previously called C15orf41), whilst in some cases, the underlying genetic abnormality is completely unknown. Consequently, the pathways affected in CDA-I remain to be discovered. To enable detailed analysis of this rare disorder we have validated a culture system which recapitulates all of the cardinal haematological features of CDA-I, including the formation of the pathognomonic 'spongy' heterochromatin seen by electron microscopy. Using a variety of cell and molecular biological approaches we discovered that erythroid cells in this condition show a delay during terminal erythroid differentiation, associated with increased proliferation and widespread changes in chromatin accessibility. We also show that the proteins encoded by CDAN1 and CDIN1 are enriched in nucleoli which are structurally and functionally abnormal in CDA-I. Together these findings provide important pointers to the pathways affected in CDA-I which for the first time can now be pursued in the tractable culture system utilised here.

Satisfaction and experiences of patients taking fingolimod and involved in a pharmacy-based patient support program in Switzerland - a qualitative study.

BACKGROUND: Fingolimod is an oral multiple sclerosis drug that is considered a specialty drug due to its high cost and safety issues. The Fingolimod Patient Support Program (F-PSP) is a specialty pharmacy service developed to ensure the responsible use of fingolimod by promoting patient safety and medication adherence. This study aims to explore the satisfaction, experiences and perceptions regarding the F-PSP among patients currently involved in this program or recently withdrawn. METHODS: A qualitative study was conducted via individual, face-to-face semistructured interviews with patients involved in the F-PSP. The interviews were audio-recorded, transcribed verbatim, coded and analyzed via thematic content analysis. RESULTS: The main themes identified from the interviews (n = 17) were overall perception of the F-PSP, perception of the pharmacist-led consultations, perception of the tools (electronic monitor and drug intake graph), reasons to participate or potentially withdraw, and suggestions for improvements. Participants perceived the F-PSP as a reassuring support that complemented their medical care, providing a more human, personalized and person-centered approach than usual pharmacy care. Pharmacist-led consultations were valued for the medication-related and holistic support they provided. The importance of the pharmacist's attitude was emphasized. The electronic monitor was valued for promoting daily medication adherence and allowing the involvement of relatives, which reassured participants and their relatives. The participants appreciated the drug intake graph because it provided an objective overview of medication adherence, thereby reassuring, rewarding, and motivating them. The main reason to join the program was to be supported, especially with respect to medication adherence. CONCLUSIONS: Participants were satisfied with the F-PSP, each for different reasons. Their feedback enabled the identification of measures for the optimization of the F-PSP and should facilitate its dissemination and transfer to other drugs/diseases/populations. Essential elements of generic pharmacist-led patient support programs considered valuable from the patients' perspective were identified.

Trajectoires de personnes souffrant de lombalgie chronique : ruptures et reconstructions après un programme de rééducation.

Purpose of research: The objective of this article is to investigate, from the perspective of patients, the disruptions of the biographical trajectories induced by chronic low back pain and the impact of a multidisciplinary rehabilitation program on their reconstruction. METHODS: Based on an interdisciplinary qualitative research, we investigated the experience of 20 participants with chronic low back pain following a three-week rehabilitation program at the hospital. Semi-directive interviews were conducted before and after inclusion in the program. RESULTS: Although affecting each person in a singular way, chronic low back pain induces biographical linearity disruptions related to the apparition of pain, and the disruption of daily and professional activities. For the majority of participants, the rehabilitation program provided a repairative space to restore continuity between past, present and future life. Whether or not there is a significant improvement in pain, most participants report benefits that give them the feeling of getting back to normality. Nevertheless, they identify those more for the domestic, family, and social spheres than at the professional level, effects remaining moderate to mitigated in that area. CONCLUSIONS: The rehabilitation program influences the dynamics of biographical trajectories and promotes a return to what is perceived as normality. By providing individuals with theoretical and practical tools and increasing their functional capacities, it promotes autonomous pain and problem management. Immediate effects are seen as restorative for domestic, family and social activities, but remain limited on the professional level.

[Deprescribing in nursing homes: comparative views of residents, their relatives, and healthcare professionals]. / Déprescrire en EMS: regards croisés entre les résidents, leurs proches et les professionnels de la santé.

Deprescribing, in order to reduce both polypharmacy and the use of potentially inappropriate medications, remains a challenge, especially in nursing homes. Healthcare professionals perceive residents of these homes as wary of change and reluctant to take part in such endeavours. The results of two studies, one qualitative and the other quantitative, show that, on the contrary, nursing home residents and their relatives would be ready to consider a treatment reduction, provided that time is invested to explain the expected benefits of such changes.

Déprescrire pour réduire l'usage de médicaments inappropriés et, plus généralement, diminuer la polymédication reste un défi, en particulier chez les résidents d'établissements médico-sociaux. Les professionnels de la santé actifs en institution perçoivent cette population comme réticente au changement et peu encline à s'engager dans une telle démarche. Pourtant, les résultats de trois études, les deux premières qualitatives, la dernière quantitative, indiquent que ces résidents, ainsi que leurs proches, seraient prêts à tester une réduction de leur traitement, pour autant que l'on prenne le temps de discuter avec eux des bénéfices potentiels.

Detection of mutations in SF3B1, EIF1AX and GNAQ in primary orbital melanoma by candidate gene analysis.

BACKGROUND: Ocular melanoma is a rare but often deadly malignancy that arises in the uvea (commonest primary site), conjunctiva or the orbit. Primary orbital melanoma (POM) is exceedingly rare, with approximately 60 cases reported to date. Despite recent advances in our understanding of the genetics of primary uveal and conjunctival melanomas, this information is lacking for POM. METHODS: DNA was extracted from 12 POM tissues, with matched germline DNA (where available). MLPA was conducted to detect chromosomal alterations and Sanger sequencing used to identify point mutations in candidate melanoma driver genes (BRAF, NRAS, KRAS, GNA11, GNAQ), and other genes implicated in melanoma prognosis (EIF1AX, SF3B1). Immunohistochemistry was performed to analyse BAP1 nuclear expression. RESULTS: MLPA detected copy number alterations in chromosomes 1p, 3, 6 and 8. Sequencing of melanoma driver genes revealed GNAQ (p.Q209L) mutations in two samples; although it is possible that these samples represent extraocular spread of an occult uveal melanoma. A recurrent mutation in SF3B1 (p.R625H) was observed in indolent, but not aggressive, tumours; a mutation in EIF1AX (p.N4S) was detected in one patient with non-aggressive disease. CONCLUSIONS: EIF1AX and SF3B1 mutations appear have a role in determining the clinical course of POM and detection of these changes could have clinical significance. Further in depth analysis of this rare group using differing 'omic technologies will provide novel insights into tumour pathogenesis.

Intervention thresholds and cesarean section rates: A time-trends analysis.

INTRODUCTION: To improve understanding of rising cesarean section (CS) rates in the UK, this study assessed the relation between clinician thresholds for performing CS for delayed labor progress or suspected fetal distress and corresponding CS rates in Aberdeen, UK. MATERIAL AND METHODS: Time-trends analysis of term births from 1988 to 2012 in a population of nulliparous women (N = 53 745) in Aberdeen, UK, using Chi-square test for trend, and binary logistic regression. Data were obtained from the Aberdeen Maternity and Neonatal Databank. RESULTS: Unplanned CS rates per quintile increased from 11.0% (1391/12 686) to 21.1% (2383/11 273) between 1988 and 2012, while planned CS rates increased from 2.7% (338/12 686) to 5.2% (591/11 273). The median duration of labor before CS for delayed progress per quintile decreased from 17.2 (IQR 12.5-22.3) to 13.1 hours (9.6-16.9) before first stage CS and from 17.1 (12.6-22.3) to 15.3 (11.5-19.1) hours before second stage CS (P < .001). The proportion of CS for suspected fetal distress performed with evidence of fetal acidosis declined from 23.4% (98/418) to 17.4% (106/608) per quintile (P < .01). Neonatal unit admission (adjusted OR 1.99, 95% CI 1.85-2.14) was more likely following unplanned CS than vaginal births. Birth trauma was less likely following both unplanned (adjusted OR 0.48, 95% CI 0.39-0.60) and planned (adjusted OR 0.33, 95% CI 0.18-0.63) CS. CONCLUSION: Increased CS rates can be partly attributed to lowered clinical thresholds for intrapartum CS. Higher CS rates are associated with less birth trauma for the offspring.

Hb Penang [ß78(EF2)Leu→Pro, HBB: c.236T>C]: a Novel ß-Globin Variant.

We report a novel hemoglobin (Hb) variant with a ß chain amino acid substitution at codon 78 (CTG>CCG) (HBB: c.236T>C), detected through prenatal screening via capillary electrophoresis (CE) in an otherwise healthy and asymptomatic 38-year-old female of Southeast Asian ancestry. The variant, named Hb Penang after the proband's Malaysian city of origin, underwent further characterization through high performance liquid chromatography (HPLC), reversed phase HPLC, Sanger sequencing, isopropanol stability testing and isoelectric focusing (IEF).

[Chronic low back pain: from the uncertain medical diagnosis to the profane etiologies]. / Lombalgie chronique : du diagnostic médical incertain aux étiologies profanes.

This qualitative research article is based on interviews with 20 participants to a low back pain rehabilitation program in a Swiss hospital. It shows that, in the absence of the obvious cause that can explain pain, patients construct their own interpretations and explanations in order to give meaning to their experience. Their explanatory models mainly include the lifestyle and the physical aspects related to the body function, what leaves little room for the psychosocial component. Their interpretation is consequently discordant with the current medical approach, which considers that chronic low back pain results from bio-psycho-social factors. This discrepancy implies negotiation between patients and professionals about the objectives to achieve in order to treat pain.

Cet article, issu d'une recherche qualitative menée dans un hôpital suisse, est basé sur des entretiens avec 20 participants à un programme de rééducation de la lombalgie. Il montre qu'en l'absence de causes objectivables permettant d'expliquer la douleur, les patients élaborent leurs propres interprétations et explications pour donner sens à leur vécu. Leurs modèles explicatifs incluent principalement le mode de vie et les aspects physiques liés au fonctionnement du corps et laissent peu de place à la composante psychologique. Leur interprétation est, par conséquent, en décalage avec la vision médicale actuelle qui considère que les douleurs dans la lombalgie chronique sont d'origine bio-psychosociale. Ce décalage implique une concertation entre patients et professionnels sur les objectifs à poursuivre pour traiter la douleur.

[Oncology trajectories of elderly patients in a palliative situation : « The long and winding road ¼]. / Trajectoires oncologiques de personnes âgées en situation palliative: « The long and winding road ¼.

This paper focuses on trajectories of elderly patients with metastatic cancer who experience several lines of systemic palliative cancer treatments. Based on photographs representing paths, the representations between patients and professional caregivers vary. Where the latter see wearisome treatments and spaces of negotiation, the patients wish to be seen as fighters, a figure that ought to be adopted to face cancer and its treatments, day after day, to meet medical and social expectations.

Cet article s'intéresse aux trajectoires des personnes âgées atteintes de cancer métastatique qui font l'expérience de plusieurs lignes de traitements oncologiques systémiques palliatifs. Sur la base d'un choix de photos évoquant des parcours, les représentations entre les patients et les soignants diffèrent. Là où ces derniers voient la pénibilité des traitements et des espaces de négociation, les patients veulent être appréhendés comme des battants, figure qu'il convient d'adopter pour faire face au cancer et aux traitements, jour après jour, et correspondre aux attentes médicales et sociales.

CNOT3 is a modifier of PRPF31 mutations in retinitis pigmentosa with incomplete penetrance.

Heterozygous mutations in the PRPF31 gene cause autosomal dominant retinitis pigmentosa (adRP), a hereditary disorder leading to progressive blindness. In some cases, such mutations display incomplete penetrance, implying that certain carriers develop retinal degeneration while others have no symptoms at all. Asymptomatic carriers are protected from the disease by a higher than average expression of the PRPF31 allele that is not mutated, mainly through the action of an unknown modifier gene mapping to chromosome 19q13.4. We investigated a large family with adRP segregating an 11-bp deletion in PRPF31. The analysis of cell lines derived from asymptomatic and affected individuals revealed that the expression of only one gene among a number of candidates within the 19q13.4 interval significantly correlated with that of PRPF31, both at the mRNA and protein levels, and according to an inverse relationship. This gene was CNOT3, encoding a subunit of the Ccr4-not transcription complex. In cultured cells, siRNA-mediated silencing of CNOT3 provoked an increase in PRPF31 expression, confirming a repressive nature of CNOT3 on PRPF31. Furthermore, chromatin immunoprecipitation revealed that CNOT3 directly binds to a specific PRPF31 promoter sequence, while next-generation sequencing of the CNOT3 genomic region indicated that its variable expression is associated with a common intronic SNP. In conclusion, we identify CNOT3 as the main modifier gene determining penetrance of PRPF31 mutations, via a mechanism of transcriptional repression. In asymptomatic carriers CNOT3 is expressed at low levels, allowing higher amounts of wild-type PRPF31 transcripts to be produced and preventing manifestation of retinal degeneration.