The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. The protein Fused in Sarcoma (FUS) undergoes LLPS and mutations in FUS have been causally linked to the motor neuron disease Amyotrophic Lateral Sclerosis (ALS-FUS). LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. However, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. Hence, we developed a method allowing for the purification of LLPS FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome, depending on its biophysical state. While non-LLPS FUS interacts mainly with factors involved in pre-mRNA processing, LLPS FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, also mitochondrial factors are strongly enriched with LLPS FUS, providing a potential explanation for the observed changes in mitochondrial gene expression in mouse models of ALS-FUS. In summary, we present a methodology to investigate the interactomes of phase separating proteins and provide evidence that LLPS shapes the FUS interactome with implications for function and disease.

General practitioner and nurse practitioner attitudes towards electronic reminders in primary care: a qualitative analysis.

OBJECTIVES: Reminders in primary care administrative systems aim to help clinicians provide evidence-based care, prescribe safely and save money. However, increased use of reminders can lead to alert fatigue. Our study aimed to assess general practitioners' (GPs) and nurse practitioners' (NPs) views on electronic reminders in primary care. DESIGN: A qualitative analysis using semistructured interviews. SETTING AND PARTICIPANTS: Fifteen GPs and NP based in general practices located in North-West London and Yorkshire, England. METHODS: We collected data on participants' views on: (1) perceptions of the value of information provided; (2) reminder-related behaviours and (3) how to improve reminders. We carried out a thematic analysis. RESULTS: Participants were familiar with reminders in their clinical systems and felt many were important to support their clinical work. However, participants reported, on average, 70% of reminders were ignored. Four major themes emerged: (1) reaction to a reminder, which was mixed and varied by situation. (2) Factors influencing the decision to act on reminders, often related to experience, consultation styles and interests of participants. Time constraints, alert design, inappropriate presentation and litigation were also factors. (3) Negative consequences of using reminders were increased workload or costs and compromising GP and NPs behaviour. (4) Factors relating to improving users' engagement with reminders were prevention of unnecessary reminders through data linkage across healthcare administrative systems or the development of more intelligent algorithms. Participants felt training was vital to effectively manage reminders. CONCLUSIONS: GPs and NPs believe reminders are useful in supporting the provision of good quality patient care. Improving GPs and NPs' engagement with reminders centres on further developing their relevance to their clinical practice, which is personalised, considers cognitive workflow and suppresses inappropriate presentation.

Assessing the utility of long-read nanopore sequencing for rapid and efficient characterization of mobile element insertions.

Short-read next generation sequencing (NGS) has become the predominant first-line technique used to diagnose patients with rare genetic conditions. Inherent limitations of short-read technology, notably for the detection and characterization of complex insertion-containing variants, are offset by the ability to concurrently screen many disease genes. "Third-generation" long-read sequencers are increasingly being deployed as an orthogonal adjunct technology, but their full potential for molecular genetic diagnosis has yet to be exploited. Here, we describe three diagnostic cases in which pathogenic mobile element insertions were refractory to characterization by short-read sequencing. To validate the accuracy of the long-read technology, we first used Sanger sequencing to confirm the integration sites and derive curated benchmark sequences of the variant-containing alleles. Long-read nanopore sequencing was then performed on locus-specific amplicons. Pairwise comparison between these data and the previously determined benchmark alleles revealed 100% identity of the variant-containing sequences. We demonstrate a number of technical advantages over existing wet-laboratory approaches, including in silico size selection of a mixed pool of amplification products, and the relative ease with which an automated informatics workflow can be established. Our findings add to a growing body of literature describing the diagnostic utility of long-read sequencing.

Epidural Versus Transabdominal Wall Catheters: A Comparative Study of Outcomes After Pancreatic Resection.

BACKGROUND: This study compared epidural analgesia with local anesthetic administration via transabdominal wall catheters (TAWC), to determine the effect on perioperative outcomes in pancreatic surgery. MATERIALS AND METHODS: A retrospective review of patients undergoing open pancreatic surgery at Auckland City Hospital from 2015 to 2018 was undertaken. Data collected included patient demographics, type of perioperative analgesia, intravenous fluid and vasopressor use, length of high dependency unit stay, postoperative complications, and length of hospital stay. RESULTS: Seventy-two patients underwent pancreatic surgery, of which 47 had epidural analgesia and 25 TAWC. The median age was 64 y (range 29-85). Failure of analgesia method occurred in 45% of epidural patients and 28% of TAWC patients (P = 0.209). There was no significant difference in volume of intravenous fluid given or need for vasopressors in the first 3 postoperative days, length of high dependency unit stay (median 1 d, P = 0.2836), rates of postoperative pancreatic fistula (32% versus 40%, P = 0.6046), postoperative complications (38% versus 20%, P = 0.183), or mortality (0.04% versus 0.04%, P = 1.0). CONCLUSIONS: Epidural analgesia and TAWC may have comparable perioperative outcomes in patients undergoing pancreatic surgery. Further randomized studies with a larger cohort of patients are warranted to identify the best postoperative analgesic method in patients undergoing pancreatic resection.

Developing and validating subjective and objective risk-assessment measures for predicting mortality after major surgery: An international prospective cohort study.

BACKGROUND: Preoperative risk prediction is important for guiding clinical decision-making and resource allocation. Clinicians frequently rely solely on their own clinical judgement for risk prediction rather than objective measures. We aimed to compare the accuracy of freely available objective surgical risk tools with subjective clinical assessment in predicting 30-day mortality. METHODS AND FINDINGS: We conducted a prospective observational study in 274 hospitals in the United Kingdom (UK), Australia, and New Zealand. For 1 week in 2017, prospective risk, surgical, and outcome data were collected on all adults aged 18 years and over undergoing surgery requiring at least a 1-night stay in hospital. Recruitment bias was avoided through an ethical waiver to patient consent; a mixture of rural, urban, district, and university hospitals participated. We compared subjective assessment with 3 previously published, open-access objective risk tools for predicting 30-day mortality: the Portsmouth-Physiology and Operative Severity Score for the enUmeration of Mortality (P-POSSUM), Surgical Risk Scale (SRS), and Surgical Outcome Risk Tool (SORT). We then developed a logistic regression model combining subjective assessment and the best objective tool and compared its performance to each constituent method alone. We included 22,631 patients in the study: 52.8% were female, median age was 62 years (interquartile range [IQR] 46 to 73 years), median postoperative length of stay was 3 days (IQR 1 to 6), and inpatient 30-day mortality was 1.4%. Clinicians used subjective assessment alone in 88.7% of cases. All methods overpredicted risk, but visual inspection of plots showed the SORT to have the best calibration. The SORT demonstrated the best discrimination of the objective tools (SORT Area Under Receiver Operating Characteristic curve [AUROC] = 0.90, 95% confidence interval [CI]: 0.88-0.92; P-POSSUM = 0.89, 95% CI 0.88-0.91; SRS = 0.85, 95% CI 0.82-0.87). Subjective assessment demonstrated good discrimination (AUROC = 0.89, 95% CI: 0.86-0.91) that was not different from the SORT (p = 0.309). Combining subjective assessment and the SORT improved discrimination (bootstrap optimism-corrected AUROC = 0.92, 95% CI: 0.90-0.94) and demonstrated continuous Net Reclassification Improvement (NRI = 0.13, 95% CI: 0.06-0.20, p < 0.001) compared with subjective assessment alone. Decision-curve analysis (DCA) confirmed the superiority of the SORT over other previously published models, and the SORT-clinical judgement model again performed best overall. Our study is limited by the low mortality rate, by the lack of blinding in the 'subjective' risk assessments, and because we only compared the performance of clinical risk scores as opposed to other prediction tools such as exercise testing or frailty assessment. CONCLUSIONS: In this study, we observed that the combination of subjective assessment with a parsimonious risk model improved perioperative risk estimation. This may be of value in helping clinicians allocate finite resources such as critical care and to support patient involvement in clinical decision-making.

Patient safety in inpatient mental health settings: a systematic review.

OBJECTIVES: Patients in inpatient mental health settings face similar risks (eg, medication errors) to those in other areas of healthcare. In addition, some unsafe behaviours associated with serious mental health problems (eg, self-harm), and the measures taken to address these (eg, restraint), may result in further risks to patient safety. The objective of this review is to identify and synthesise the literature on patient safety within inpatient mental health settings using robust systematic methodology. DESIGN: Systematic review and meta-synthesis. Embase, Cumulative Index to Nursing and Allied Health Literature, Health Management Information Consortium, MEDLINE, PsycINFO and Web of Science were systematically searched from 1999 to 2019. Search terms were related to 'mental health', 'patient safety', 'inpatient setting' and 'research'. Study quality was assessed using the Hawker checklist. Data were extracted and grouped based on study focus and outcome. Safety incidents were meta-analysed where possible using a random-effects model. RESULTS: Of the 57 637 article titles and abstracts, 364 met inclusion criteria. Included publications came from 31 countries and included data from over 150 000 participants. Study quality varied and statistical heterogeneity was high. Ten research categories were identified: interpersonal violence, coercive interventions, safety culture, harm to self, safety of the physical environment, medication safety, unauthorised leave, clinical decision making, falls and infection prevention and control. CONCLUSIONS: Patient safety in inpatient mental health settings is under-researched in comparison to other non-mental health inpatient settings. Findings demonstrate that inpatient mental health settings pose unique challenges for patient safety, which require investment in research, policy development, and translation into clinical practice. PROSPERO REGISTRATION NUMBER: CRD42016034057.

A conceptual framework for capacity strengthening of health research in conflict: the case of the Middle East and North Africa region.

BACKGROUND: In conflict settings, research capacities have often been de-prioritized as resources are diverted to emergency needs, such as addressing elevated morbidity, mortality and health system challenges directly and/or indirectly associated to war. This has had an adverse long-term impact in such protracted conflicts such as those found in the Middle East and North Africa region (MENA), where research knowledge and skills have often been compromised. In this paper, we propose a conceptual framework for health research capacity strengthening that adapts existing models and frameworks in low- and middle-income countries and uses our knowledge of the MENA context to contextualise them for conflict settings. METHODS: The framework was synthesized using "best fit" framework synthesis methodology. Relevant literature, available in English and Arabic, was collected through PubMed, Google Scholar and Google using the keywords: capacity building; capacity strengthening; health research; framework and conflict. Grey literature was also assessed. RESULTS: The framework is composed of eight principal themes: "structural levels", "the influence of the external environment", "funding, community needs and policy environment", "assessing existing capacity and needs", "infrastructure and communication", "training, leadership and partnership", "adaptability and sustainability", and "monitoring and evaluation"; with each theme being supported by examples from the MENA region. Our proposed framework takes into consideration safety, infrastructure, communication and adaptability as key factors that affect research capacity strengthening in conflict. As it is the case more generally, funding, permissible political environments and sustainability are major determinants of success for capacity strengthening for health research programmes, though these are significantly more challenging in conflict settings. Nonetheless, health research capacity strengthening should remain a priority. CONCLUSION: The model presented is the first framework that focuses on strengthening health research capacity in conflict with a focus on the MENA region. It should be viewed as a non-prescriptive reference tool for health researchers and practitioners, from various disciplines, involved in research capacity strengthening to evaluate, use, adapt and improve. It can be further extended to include representative indicators and can be later evaluated by assessing its efficacy for interventions in conflict settings.

Postoperative critical care and high-acuity care provision in the United Kingdom, Australia, and New Zealand.

BACKGROUND: Decisions to admit high-risk postoperative patients to critical care may be affected by resource availability. We aimed to quantify adult ICU/high-dependency unit (ICU/HDU) capacity in hospitals from the UK, Australia, and New Zealand (NZ), and to identify and describe additional 'high-acuity' beds capable of managing high-risk patients outside the ICU/HDU environment. METHODS: We used a modified Delphi consensus method to design a survey that was disseminated via investigator networks in the UK, Australia, and NZ. Hospital- and ward-level data were collected, including bed numbers, tertiary services offered, presence of an emergency department, ward staffing levels, and the availability of critical care facilities. RESULTS: We received responses from 257 UK (response rate: 97.7%), 35 Australian (response rate: 32.7%), and 17 NZ (response rate: 94.4%) hospitals (total 309). Of these hospitals, 91.6% reported on-site ICU or HDU facilities. UK hospitals reported fewer critical care beds per 100 hospital beds (median=2.7) compared with Australia (median=3.7) and NZ (median=3.5). Additionally, 31.1% of hospitals reported having high-acuity beds to which high-risk patients were admitted for postoperative management, in addition to standard ICU/HDU facilities. The estimated numbers of critical care beds per 100 000 population were 9.3, 14.1, and 9.1 in the UK, Australia, and NZ, respectively. The estimated per capita high-acuity bed capacities per 100 000 population were 1.2, 3.8, and 6.4 in the UK, Australia, and NZ, respectively. CONCLUSIONS: Postoperative critical care resources differ in the UK, Australia, and NZ. High-acuity beds may have developed to augment the capacity to deliver postoperative critical care.

Mutational Signature Analysis Reveals NTHL1 Deficiency to Cause a Multi-tumor Phenotype.

Biallelic germline mutations affecting NTHL1 predispose carriers to adenomatous polyposis and colorectal cancer, but the complete phenotype is unknown. We describe 29 individuals carrying biallelic germline NTHL1 mutations from 17 families, of which 26 developed one (n = 10) or multiple (n = 16) malignancies in 14 different tissues. An unexpected high breast cancer incidence was observed in female carriers (60%). Mutational signature analysis of 14 tumors from 7 organs revealed that NTHL1 deficiency underlies the main mutational process in all but one of the tumors (93%). These results reveal NTHL1 as a multi-tumor predisposition gene with a high lifetime risk for extracolonic cancers and a typical mutational signature observed across tumor types, which can assist in the recognition of this syndrome.

Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep.

The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in ~ 50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.

Treatment of a metabolic liver disease by in vivo genome base editing in adult mice.

CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)enu2 mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)1, using recently developed CRISPR-Cas-associated base editors2-4. These systems enable conversion of C∙G to T∙A base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an intein-split base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pahenu2 gene correction rates that restored physiological blood phenylalanine (L-Phe) levels below 120 µmol/l [5]. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pahenu2 mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.

Cancer modeling by Transgene Electroporation in Adult Zebrafish (TEAZ).

Transgenic animals are invaluable for modeling cancer genomics, but often require complex crosses of multiple germline alleles to obtain the desired combinations. Zebrafish models have advantages in that transgenes can be rapidly tested by mosaic expression, but typically lack spatial and temporal control of tumor onset, which limits their utility for the study of tumor progression and metastasis. To overcome these limitations, we have developed a method referred to as Transgene Electroporation in Adult Zebrafish (TEAZ). TEAZ can deliver DNA constructs with promoter elements of interest to drive fluorophores, oncogenes or CRISPR-Cas9-based mutagenic cassettes in specific cell types. Using TEAZ, we created a highly aggressive melanoma model via Cas9-mediated inactivation of Rb1 in the context of BRAFV600E in spatially constrained melanocytes. Unlike prior models that take ∼4 months to develop, we found that TEAZ leads to tumor onset in ∼7 weeks, and these tumors develop in fully immunocompetent animals. As the resulting tumors initiated at highly defined locations, we could track their progression via fluorescence, and documented deep invasion into tissues and metastatic deposits. TEAZ can be deployed to other tissues and cell types, such as the heart, with the use of suitable transgenic promoters. The versatility of TEAZ makes it widely accessible for rapid modeling of somatic gene alterations and cancer progression at a scale not achievable in other in vivo systems.

Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair.

The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, however, leads to relatively low rates of precise editing from donor DNA. Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold, and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus. Enhanced correction rates were observed in multiple cell types and on different genomic loci, suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired.

Large-scale image-based profiling of single-cell phenotypes in arrayed CRISPR-Cas9 gene perturbation screens.

High-content imaging using automated microscopy and computer vision allows multivariate profiling of single-cell phenotypes. Here, we present methods for the application of the CISPR-Cas9 system in large-scale, image-based, gene perturbation experiments. We show that CRISPR-Cas9-mediated gene perturbation can be achieved in human tissue culture cells in a timeframe that is compatible with image-based phenotyping. We developed a pipeline to construct a large-scale arrayed library of 2,281 sequence-verified CRISPR-Cas9 targeting plasmids and profiled this library for genes affecting cellular morphology and the subcellular localization of components of the nuclear pore complex (NPC). We conceived a machine-learning method that harnesses genetic heterogeneity to score gene perturbations and identify phenotypically perturbed cells for in-depth characterization of gene perturbation effects. This approach enables genome-scale image-based multivariate gene perturbation profiling using CRISPR-Cas9.

Genomic Subtypes of Non-invasive Bladder Cancer with Distinct Metabolic Profile and Female Gender Bias in KDM6A Mutation Frequency.

Bladder cancer incurs a higher lifetime treatment cost than other cancers due to frequent recurrence of non-invasive disease. Improved prognostic biomarkers and localized therapy are needed for this large patient group. We defined two major genomic subtypes of primary stage Ta tumors. One of these was characterized by loss of 9q including TSC1, increased KI67 labeling index, upregulated glycolysis, DNA repair, mTORC1 signaling, features of the unfolded protein response, and altered cholesterol homeostasis. Comparison with muscle-invasive bladder cancer mutation profiles revealed lower overall mutation rates and more frequent mutations in RHOB and chromatin modifier genes. More mutations in the histone lysine demethylase KDM6A were present in non-invasive tumors from females than males.

Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes.

The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economic impact and has become an emerging model for developing new genetic control strategies as an alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific single guide RNAs (sgRNA) into early embryos. When targeting the eye pigmentation gene white eye (we), a high rate of somatic mosaicism in surviving G0 adults was observed. Germline transmission rate of mutated we alleles by G0 animals was on average above 52%, with individual cases achieving nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end-joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in C. capitata will significantly advance the design and development of new effective strategies for pest control management.

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription.

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes.

Neuroprotection during aortic surgery.

Neurological injury is a major limitation of aortic surgery, whether it involves spinal cord injury following intervention to the thoracoabdominal aorta, or stroke following surgery on the arch and ascending aorta. Despite an extensive body of literature and various proposals, a completely effective strategy to prevent or treat neurological injury remains elusive. In this article, we summarise the evidence for established and emerging strategies, and review current concepts in pathophysiology and risk assessment as they relate to neurological injury.