A common transcriptional mechanism involving R-loop and RNA abasic site regulates an enhancer RNA of APOE.

RNA is modified by hundreds of chemical reactions and folds into innumerable shapes. However, the regulatory role of RNA sequence and structure and how dysregulation leads to diseases remain largely unknown. Here, we uncovered a mechanism where RNA abasic sites in R-loops regulate transcription by pausing RNA polymerase II. We found an enhancer RNA, AANCR, that regulates the transcription and expression of apolipoprotein E (APOE). In some human cells such as fibroblasts, AANCR is folded into an R-loop and modified by N-glycosidic cleavage; in this form, AANCR is a partially transcribed nonfunctional enhancer and APOE is not expressed. In contrast, in other cell types including hepatocytes and under stress, AANCR does not form a stable R-loop as its sequence is not modified, so it is transcribed into a full-length enhancer that promotes APOE expression. DNA sequence variants in AANCR are associated significantly with APOE expression and Alzheimer's Disease, thus AANCR is a modifier of Alzheimer's Disease. Besides AANCR, thousands of noncoding RNAs are regulated by abasic sites in R-loops. Together our data reveal the essentiality of the folding and modification of RNA in cellular regulation and demonstrate that dysregulation underlies common complex diseases such as Alzheimer's disease.

RNA abasic sites in yeast and human cells.

RNA abasic sites and the mechanisms involved in their regulation are mostly unknown; in contrast, DNA abasic sites are well-studied. We found surprisingly that, in yeast and human cells, RNA abasic sites are prevalent. When a base is lost from RNA, the remaining ribose is found as a closed-ring or an open-ring sugar with a reactive C1' aldehyde group. Using primary amine-based reagents that react with the aldehyde group, we uncovered evidence for abasic sites in nascent RNA, messenger RNA, and ribosomal RNA from yeast and human cells. Mass spectroscopic analysis confirmed the presence of RNA abasic sites. The RNA abasic sites were found to be coupled to R-loops. We show that human methylpurine DNA glycosylase cleaves N-glycosidic bonds on RNA and that human apurinic/apyrimidinic endonuclease 1 incises RNA abasic sites in RNA-DNA hybrids. Our results reveal that, in yeast and human cells, there are RNA abasic sites, and we identify a glycosylase that generates these sites and an AP endonuclease that processes them.

Provision of acute care pathways for older major trauma patients in the UK.

BACKGROUND: The introduction of specific pathways of care for older trauma patients has been shown to decrease hospital length of stay and the overall rate of complications. The extent and scope of pathways and services for older major trauma patients in the UK is not currently known. OBJECTIVE: The primary objective of this study was to map the current care pathways and provision of services for older people following major trauma in the UK. METHODS: A cross-sectional survey of UK hospitals delivering care to major trauma patients (major trauma centres and trauma units). Data were collected on respondent and site characteristics, and local definitions of older trauma patients. To explore pathways for older people with major trauma, four clinical case examples were devised and respondents asked to complete responses that best illustrated the admission pathway for each. RESULTS: Responses from 56 hospitals were included in the analysis, including from 25 (84%) of all major trauma centres (MTCs) in the UK. The majority of respondents defined 'old' by chronological age, most commonly patients 65 years and over. The specialty team with overall responsibility for the patient in trauma units was most likely to be acute medicine or acute surgery. Patients in MTCs were not always admitted under the care of the major trauma service. Assessment by a geriatrician within 72 hours of admission varied in both major trauma centres and trauma units and was associated with increased age. CONCLUSIONS: This survey highlights variability in the admitting specialty team and subsequent management of older major trauma patients across hospitals in the UK. Variability appears to be related to patient condition as well as provision of local resources. Whilst lack of standardisation may be a result of local service configuration this has the potential to impact negatively on quality of care, multi-disciplinary working, and outcomes.

High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase.

Cellular levels of ribonucleoside triphosphates (rNTPs) are much higher than those of deoxyribonucleoside triphosphates (dNTPs), thereby influencing the frequency of incorporation of ribonucleoside monophosphates (rNMPs) by DNA polymerases (Pol) into DNA. RNase H2-initiated ribonucleotide excision repair (RER) efficiently removes single rNMPs in genomic DNA. However, processing of rNMPs by Topoisomerase 1 (Top1) in absence of RER induces mutations and genome instability. Here, we greatly increased the abundance of genomic rNMPs in Saccharomyces cerevisiae by depleting Rnr1, the major subunit of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides. We found that in strains that are depleted of Rnr1, RER-deficient, and harbor an rNTP-permissive replicative Pol mutant, excessive accumulation of single genomic rNMPs severely compromised growth, but this was reversed in absence of Top1. Thus, under Rnr1 depletion, limited dNTP pools slow DNA synthesis by replicative Pols and provoke the incorporation of high levels of rNMPs in genomic DNA. If a threshold of single genomic rNMPs is exceeded in absence of RER and presence of limited dNTP pools, Top1-mediated genome instability leads to severe growth defects. Finally, we provide evidence showing that accumulation of RNA/DNA hybrids in absence of RNase H1 and RNase H2 leads to cell lethality under Rnr1 depletion.

Aicardi-Goutières syndrome gene Rnaseh2c is a metastasis susceptibility gene in breast cancer.

Breast cancer is the second leading cause of cancer-related deaths in the United States, with the majority of these deaths due to metastatic lesions rather than the primary tumor. Thus, a better understanding of the etiology of metastatic disease is crucial for improving survival. Using a haplotype mapping strategy in mouse and shRNA-mediated gene knockdown, we identified Rnaseh2c, a scaffolding protein of the heterotrimeric RNase H2 endoribonuclease complex, as a novel metastasis susceptibility factor. We found that the role of Rnaseh2c in metastatic disease is independent of RNase H2 enzymatic activity, and immunophenotyping and RNA-sequencing analysis revealed engagement of the T cell-mediated adaptive immune response. Furthermore, the cGAS-Sting pathway was not activated in the metastatic cancer cells used in this study, suggesting that the mechanism of immune response in breast cancer is different from the mechanism proposed for Aicardi-Goutières Syndrome, a rare interferonopathy caused by RNase H2 mutation. These results suggest an important novel, non-enzymatic role for RNASEH2C during breast cancer progression and add Rnaseh2c to a panel of genes we have identified that together could determine patients with high risk for metastasis. These results also highlight a potential new target for combination with immunotherapies and may contribute to a better understanding of the etiology of Aicardi-Goutières Syndrome autoimmunity.

Mapping the Patient and Family Liaison Role in UK Helicopter Emergency Medical Services: A Service Evaluation.

OBJECTIVE: Patient and family liaison practitioners are a relatively recent addition to UK helicopter emergency medical services to support patients with their recovery. A service evaluation was completed that mapped the current provision of patient and family liaison practitioner roles in helicopter emergency medical services in the United Kingdom. METHODS: An online survey was distributed to key stakeholders involved with UK helicopter emergency medical service patient and family liaison practitioner roles. Quantitative survey results were described, and open-ended questions were analyzed using content analysis. RESULTS: Twenty UK helicopter emergency medical services responded to the survey. Nine of these services employ patient and family liaison practitioners with 4 additional helicopter emergency medical services planning to initiate the role. There is variation in the employment models used between the services. The patient and family liaison practitioner role provides important benefits to patients and their families, clinicians, and the helicopter emergency medical service. CONCLUSION: Nine UK helicopter emergency medical services employ patient and family liaison practitioners. This role benefits patients, their families, helicopter emergency medical service clinicians, and helicopter emergency medical service charities. Further research is required to understand how the role works in practice and to understand how to maximize the benefits to stakeholders.

The Balancing Act of Ribonucleotides in DNA.

The abundance of ribonucleotides in DNA remained undetected until recently because they are efficiently removed by the ribonucleotide excision repair (RER) pathway, a process similar to Okazaki fragment (OF) processing after incision by Ribonuclease H2 (RNase H2). All DNA polymerases incorporate ribonucleotides during DNA synthesis. How many, when, and why they are incorporated has been the focus of intense work during recent years by many labs. In this review, we discuss recent advances in ribonucleotide incorporation by eukaryotic DNA polymerases that suggest an evolutionarily conserved role for ribonucleotides in DNA. We also review the data that indicate that removal of ribonucleotides has an important role in maintaining genome stability.

Unlike the Escherichia coli counterpart, archaeal RNase HII cannot process ribose monophosphate abasic sites and oxidized ribonucleotides embedded in DNA.

The presence of ribonucleoside monophosphates (rNMPs) in nuclear DNA decreases genome stability. To ensure survival despite rNMP insertions, cells have evolved a complex network of DNA repair mechanisms, in which the ribonucleotide excision repair pathway, initiated by type 2 RNase H (RNase HII/2), plays a major role. We recently demonstrated that eukaryotic RNase H2 cannot repair damage, that is, ribose monophosphate abasic (both apurinic or apyrimidinic) site (rAP) or oxidized rNMP embedded in DNA. Currently, it remains unclear why RNase H2 is unable to repair these modified nucleic acids having either only a sugar moiety or an oxidized base. Here, we compared the endoribonuclease specificity of the RNase HII enzymes from the archaeon Pyrococcus abyssi and the bacterium Escherichia coli, examining their ability to process damaged rNMPs embedded in DNA in vitro We found that E. coli RNase HII cleaves both rAP and oxidized rNMP sites. In contrast, like the eukaryotic RNase H2, P. abyssi RNase HII did not display any rAP or oxidized rNMP incision activities, even though it recognized them. Notably, the archaeal enzyme was also inactive on a mismatched rNMP, whereas the E. coli enzyme displayed a strong preference for the mispaired rNMP over the paired rNMP in DNA. On the basis of our biochemical findings and also structural modeling analyses of RNase HII/2 proteins from organisms belonging to all three domains of life, we propose that RNases HII/2's dual roles in ribonucleotide excision repair and RNA/DNA hydrolysis result in limited acceptance of modified rNMPs embedded in DNA.

RNase H2-initiated ribonucleotide excision repair.

Ribonucleotides are incorporated into DNA by the replicative DNA polymerases at frequencies of about 2 per kb, which makes them by far the most abundant form of potential DNA damage in the cell. Their removal is essential for restoring a stable intact chromosome. Here, we present a complete biochemical reconstitution of the ribonucleotide excision repair (RER) pathway with enzymes purified from Saccharomyces cerevisiae. RER is most efficient when the ribonucleotide is incised by RNase H2, and further excised by the flap endonuclease FEN1 with strand displacement synthesis carried out by DNA polymerase δ, the PCNA clamp, its loader RFC, and completed by DNA ligase I. We observed partial redundancy for several of the enzymes in this pathway. Exo1 substitutes for FEN1 and Pol ε for Pol δ with reasonable efficiency. However, RNase H1 fails to substitute for RNase H2 in the incision step of RER.

Limited pharmaceuticalisation: a qualitative case study of physiotherapist prescribing practices in an NHS Trust in England following the expansion of non-medical prescribing in the UK.

Over the last quarter century, non-medical prescribing in the UK has grown significantly; eight non-medical professional groups now have authority to prescribe a wide range of medicines, suggesting it could be a potent driver of pharmaceuticalisation. In this article, we present data from a case study of physiotherapists' prescribing practices. UK physiotherapists have had legal rights to prescribe medicines since 2005, but relatively little is known about the contribution they make to expanding patient access to medicines. We approached our study through a lens of governmentality to capture the mentalities and micro-practices governing physiotherapist non-medical prescribing. Ethnographic methods were used to gather data from an outpatient orthopaedic service in an NHS Trust in England employing physiotherapist prescribers. From the data, we identified a grid of intelligibility - an organising framework formulated by powerful discourses and technologies of government through which physiotherapist prescribing was acted into being. A primary effect of this grid was the constitution of new physiotherapist subjectivities, mostly as non-prescribers of medicines contrary to policy intentions, underpinned by a familiar and enduring template of medical professionalism.

Abasic and oxidized ribonucleotides embedded in DNA are processed by human APE1 and not by RNase H2.

Ribonucleoside 5'-monophosphates (rNMPs) are the most common non-standard nucleotides found in DNA of eukaryotic cells, with over 100 million rNMPs transiently incorporated in the mammalian genome per cell cycle. Human ribonuclease (RNase) H2 is the principal enzyme able to cleave rNMPs in DNA. Whether RNase H2 may process abasic or oxidized rNMPs incorporated in DNA is unknown. The base excision repair (BER) pathway is mainly responsible for repairing oxidized and abasic sites into DNA. Here we show that human RNase H2 is unable to process an abasic rNMP (rAP site) or a ribose 8oxoG (r8oxoG) site embedded in DNA. On the contrary, we found that recombinant purified human apurinic/apyrimidinic endonuclease-1 (APE1) and APE1 from human cell extracts efficiently process an rAP site in DNA and have weak endoribonuclease and 3'-exonuclease activities on r8oxoG substrate. Using biochemical assays, our results provide evidence of a human enzyme able to recognize and process abasic and oxidized ribonucleotides embedded in DNA.

Pathological ribonuclease H1 causes R-loop depletion and aberrant DNA segregation in mitochondria.

The genetic information in mammalian mitochondrial DNA is densely packed; there are no introns and only one sizeable noncoding, or control, region containing key cis-elements for its replication and expression. Many molecules of mitochondrial DNA bear a third strand of DNA, known as "7S DNA," which forms a displacement (D-) loop in the control region. Here we show that many other molecules contain RNA as a third strand. The RNA of these R-loops maps to the control region of the mitochondrial DNA and is complementary to 7S DNA. Ribonuclease H1 is essential for mitochondrial DNA replication; it degrades RNA hybridized to DNA, so the R-loop is a potential substrate. In cells with a pathological variant of ribonuclease H1 associated with mitochondrial disease, R-loops are of low abundance, and there is mitochondrial DNA aggregation. These findings implicate ribonuclease H1 and RNA in the physical segregation of mitochondrial DNA, perturbation of which represents a previously unidentified disease mechanism.

Crystal structures of RNase H2 in complex with nucleic acid reveal the mechanism of RNA-DNA junction recognition and cleavage.

Two classes of RNase H hydrolyze RNA of RNA/DNA hybrids. In contrast to RNase H1 that requires four ribonucleotides for cleavage, RNase H2 can nick duplex DNAs containing a single ribonucleotide, suggesting different in vivo substrates. We report here the crystal structures of a type 2 RNase H in complex with substrates containing a (5')RNA-DNA(3') junction. They revealed a unique mechanism of recognition and substrate-assisted cleavage. A conserved tyrosine residue distorts the nucleic acid at the junction, allowing the substrate to function in catalysis by participating in coordination of the active site metal ion. The biochemical and structural properties of RNase H2 explain the preference of the enzyme for junction substrates and establish the structural and mechanistic differences with RNase H1. Junction recognition is important for the removal of RNA embedded in DNA and may play an important role in DNA replication and repair.

Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication.

Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in mitochondrial DNA replication. Analysis of replicating mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in mitochondrial DNA. However, the retained primers present an obstacle to the mitochondrial DNA polymerase γ in subsequent rounds of replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in mitochondrial DNA replication is the removal of primers at the origin of replication.

RNase H2 roles in genome integrity revealed by unlinking its activities.

Ribonuclease H2 (RNase H2) protects genome integrity by its dual roles of resolving transcription-related R-loops and ribonucleotides incorporated in DNA during replication. To unlink these two functions, we generated a Saccharomyces cerevisiae RNase H2 mutant that can resolve R-loops but cannot cleave single ribonucleotides in DNA. This mutant definitively correlates the 2-5 bp deletions observed in rnh201Δ strains with single rNMPs in DNA. It also establishes a connection between R-loops and Sgs1-mediated replication reinitiation at stalled forks and identifies R-loops uniquely processed by RNase H2. In mouse, deletion of any of the genes coding for RNase H2 results in embryonic lethality, and in humans, RNase H2 hypomorphic mutations cause Aicardi-Goutières syndrome (AGS), a neuroinflammatory disorder. To determine the contribution of R-loops and rNMP in DNA to the defects observed in AGS, we characterized in yeast an AGS-related mutation, which is impaired in processing both substrates, but has sufficient R-loop degradation activity to complement the defects of rnh201Δ sgs1Δ strains. However, this AGS-related mutation accumulates 2-5 bp deletions at a very similar rate as the deletion strain.

What is important to patients in wound management.

Traumatic wounds are a common reason for patients to attend emergency departments. There are many ways of managing these wounds from glue to suturing. The authors conducted a patient survey to identify the outcome measures most important to patients after closure of traumatic wounds. The results showed that having the least chance of infection was the most important outcome, followed by being looked after by caring staff and a quick recovery. These finding were consistent regardless of the anatomical location of the wound or age of the patient. This information is being used to guide the authors in the most appropriate outcome measures for further research.

Clinical handovers between prehospital and hospital staff: literature review.

BACKGROUND: Clinical handover plays a vital role in patient care and has been investigated in hospital settings, but less attention has been paid to the interface between prehospital and hospital settings. This paper reviews the published research on these handovers. METHODS: A computerised literature search was conducted for papers published between 2000 and 2013 using combinations of terms: 'handover', 'handoff', 'prehospital', 'ambulance', 'paramedic' and 'emergency' and citation searching. Papers were assessed and included if determined to be at least moderate quality with a primary focus on prehospital to hospital handover. FINDINGS: 401 studies were identified, of which 21 met our inclusion criteria. These revealed concerns about communication and information transfer, and themes concerning context, environment and interprofessional relationships. It is clear that handover exchanges are complicated by chaotic and noisy environments, lack of time and resources. Poor communication is linked to behaviours such as not listening, mistrust and misunderstandings between staff. While standardisation is offered as a solution, notably in terms of the use of mnemonics (alphabetical memory aids), evidence for benefit appears inconclusive. CONCLUSIONS: This review raises concerns about handovers at the interface between prehospital and hospital settings. The quality of existing research in this area is relatively poor and further high-quality research is required to understand this important part of emergency care. We need to understand the complexity of handover better to grasp the challenges of context and interprofessional relationships before we reach for tools and techniques to standardise part of the handover process.

Safe staffing for nursing in emergency departments: evidence review.

OBJECTIVE: Getting staffing levels wrong in hospitals is linked to excess mortality and poor patient experiences but establishing the safe nurse staffing levels in the emergency department (ED) is challenging because patient demand is so variable. This paper reports a review conducted for the National Institute for Health and Care Excellence (NICE) which sought to identify the research evidence to inform UK nursing workforce planning. DESIGN: We searched 10 electronic databases and relevant websites for English language studies published from 1994. Studies included reported a direct measure of nurse staffing relative to an activity measure (eg, attendances, patient throughput) or an estimate of nurse staffing requirements. Randomised or non-randomised trials, prospective or retrospective observational, cross-sectional or correlational studies, interrupted time-series, and controlled before and after studies were considered. RESULTS: We identified 16,132 items via databases and 2193 items through manual and other searching. After title/abstract screening (by one reviewer, checked by a second) 55 studies underwent full assessment by the review team. 18 studies met the inclusion criteria for the NICE review, however 3 simulation studies that reported simulated rather than measured outcomes are not reported here. CONCLUSIONS: The evidence is weak but indicates that levels of nurse staffing in the ED are associated with patients leaving without being seen, ED care time and patient satisfaction. Lower staffing is associated with worse outcomes. There remain significant gaps and in particular a lack of evidence on the impact of staffing on direct patient outcomes and adequate economic analyses to inform decisions about nurse staffing. Given that an association between nurse staffing levels and patient outcomes on inpatient wards has been demonstrated, this gap in the evidence about nurse staffing in EDs needs to be addressed.

Dynamic nurse leadership in high-pressure situations.

Traditionally, healthcare professionals have been expected to acquire technical skills while minimal attention has been paid to the non-technical skills (NTS) they require to work in complex health environments, such as resuscitation rooms. This article explains the importance of NTS in improving patient outcomes and why a model of dynamic nurse leadership is useful in resuscitative care.

Extending access to specialist services: the impact of an onsite helipad and analysis of the first 100 flights.

BACKGROUND: In November 2011, University Hospital Southampton (UHS), now a major trauma centre, opened its onsite helipad, allowing patients to be brought to the emergency department (ED) directly by air ambulance. Prior to this, helicopters were required to land at a local playing field and the patient had to be transferred by land ambulance. This study aims to investigate the impact this change in practice has had on the flow of patients to the ED. METHODS: The authors completed a retrospective case analysis of the first 100 patients brought directly to UHS by helicopter. Data were obtained from ED notes and helicopter provider databases. Analysis was conducted on the type of incident and appropriateness of referral. Incident locations were plotted geographically. RESULTS: 100 patients arrived at UHS ED by helicopter between 17 November 2011 and 31 March 2012. Of these, 79 were primary helicopter emergency medical service (HEMS) missions and 21 were secondary transfers from other hospitals. Of the HEMS patients, 38 were likely to have been transported to another hospital, had there not been an onsite helipad at UHS. 29 passed another suitable receiving hospital en route and therefore may have come to UHS for speciality services. CONCLUSIONS: The provision of an onsite, 24 h helipad facility at UHS has resulted in a significant number of patients being transported to the hospital by helicopter who might otherwise have attended an alternative hospital.