ClpXP protease targets long-lived DNA translocation states of a helicase-like motor to cause restriction alleviation

We investigated how Escherichia coli ClpXP targets the helicase-nuclease (HsdR) subunit of the bacterial Type I restriction–modification enzyme EcoKI during restriction alleviation (RA). RA is a temporary reduction in endonuclease activity that occurs when Type I enzymes bind unmodified recognition sites on the host genome. These conditions arise upon acquisition of a new system by a naïve host, upon generation of new sites by genome rearrangement/mutation or during homologous recombination between hemimethylated DNA. Using recombinant DNA and proteins in vitro, we demonstrate that ClpXP targets EcoKI HsdR during dsDNA translocation on circular DNA but not on linear DNA. Protein roadblocks did not activate HsdR proteolysis. We suggest that DNA translocation lifetime, which is elevated on circular DNA relative to linear DNA, is important to RA. To identify the ClpX degradation tag (degron) in HsdR, we used bioinformatics and biochemical assays to design N- and C-terminal mutations that were analysed in vitro and in vivo. None of the mutants produced a phenotype consistent with loss of the degron, suggesting an as-yet-unidentified recognition pathway. We note that an EcoKI nuclease mutant still produces cell death in a clpx(−) strain, consistent with DNA damage induced by unregulated motor activity

Predictors and outcomes of sustained, intermittent or never achieving remission in patients with recent onset inflammatory polyarthritis:Results from the Norfolk Arthritis Register

Objectives: Early remission is the current treatment strategy for patients with inflammatory polyarthritis (IP) and RA. Our objective was to identify baseline factors associated with achieving remission: sustained (SR), intermittent (IR) or never (NR) over a 5-year period in patients with early IP.  Methods: Clinical and demographic data of patients with IP recruited to the Norfolk Arthritis Register (NOAR) were obtained at baseline and years 1, 2, 3 and 5. Remission was defined as no tender or swollen joints (out of 51). Patients were classified as NR or PR, respectively, if they were in remission at: no assessment or ⩾3 consecutive assessments after baseline, and IR otherwise. Ordinal regression and a random effects model, respectively, were used to examine the association between baseline factors, remission group and HAQ scores over time.  Results: A total of 868 patients (66% female) were included. Of these, 54%, 34% and 12% achieved NR, IR and SR, respectively. In multivariate analysis, female sex (odds ratio, OR 0.47, 95% CI: 0.35, 0.63), higher tender joint count (OR = 0.94, 95% CI: 0.93, 0.96), higher HAQ (OR = 0.59, 95% CI: 0.48, 0.74), being obese (OR = 0.70, 95% CI: 0.50, 0.99), hypertensive (OR = 0.67, 95% CI: 0.50, 0.90) or depressed (OR = 0.74, 95% CI: 0.55, 1.00) at baseline were independent predictors of being in a lower remission group. IR and SR were associated with lower HAQ scores over time and lower DAS28 at year 5.  Conclusion: Women with higher tender joint count and disability at baseline, depression, obesity and hypertension were less likely to achieve remission. This information could help when stratifying patients for more aggressive therapy

Continuation of Water Quality Monitoring of the Osage Creek above the Highway 112 Bridge Near Cave Springs, Arkansas

The City of Rogers is one of the fastest growing communities in Arkansas. Located in Northwest Arkansas, Rogers lies in two primary watersheds; the Illinois River Watershed and Beaver Lake Watershed. The Illinois River Watershed receives municipal wastewater discharge from most of the metropolitan communities in Northwest Arkansas, along with nonpoint source urban and agricultural runoff. The Illinois River originates in Arkansas and flows west into Oklahoma where it is classified as a scenic river. The river has been a source of interstate disputes for a number of years. A federal interstate compact commission was established to mediate these disputes, especially concerning high levels of phosphorus, which is perceived to degrade in-stream water quality as well as to accelerate eutrophication of an Oklahoma reservoir. Recent developments include establishment of in-stream phosphorus limits of 0.037 mg/l for Oklahoma scenic rivers (current base-flow only levels at the state line are around 0.1 mg/l) and an EPA mandate that the Spring Creek and Osage Creek be included on Arkansas’ 303d list for impacts by high levels of nutrients

Own identification of contributing factors for the success of toll roads in Australia under Public Private Partnerships

In Australia, Public Private Partnerships (PPPs) have been established as a common method for governments to deliver major road infrastructure projects. Success of PPPs has varied when measured against Government, Community, Market and Industry interests. Some projects have failed financially while still having a positive impact on the community. Other projects have failed to reach delivery stage as a result of community objections. The holistic success of PPP toll roads is ultimately determined by the needs of major project participants being satisfied in an unbiased equilibrium manner. PPP toll roads delivered in Sydney, Brisbane and Melbourne have had varying degrees of financial success, however there are other vitally important factors to be considered. Tollways directly contribute to travel time savings, vehicle operating cost savings, reduced accidents and vehicle emissions and can make a contribution to the overall economic performance of a city. Therefore these pieces of infrastructure contribute to society as a whole and not just the investors who provide capital for the projects. Even with recent financial failings of PPP toll roads, Governments within Australia are still actively pursuing the PPP model to deliver road infrastructure. Lessons must be learnt from past failures to ensure the successful delivery and operation of future projects. Overall success will be a result of finding a balance between the needs of Government, Private Sector and Society

Twenty-year outcome and association between early treatment and mortality and disability in an inception cohort of patients with rheumatoid arthritis: Results from the Norfolk Arthritis Register

Objective – To describe the outcome of patients with rheumatoid arthritis (RA) over 20 years from symptom onset; to assess the association between early treatment (DMARDs/steroids) and mortality and disability over follow-up. Methods – Patients from the Norfolk Arthritis Register recruited from 1990-94 who met the 2010 ACR/EULAR RA criteria at baseline were included in this analysis. Demographic/clinical variables were collected at baseline and years 1-3, 5, 7, 10, 15 and 20. Disease activity (swollen/tender joint counts (SJC/TJC)), disability (HAQ) and mortality over 20 years are described. Association between treatment group (early treatment (ET) = treatment ≤6 months after symptom onset; late treatment (LT) = treatment >6 months; never treatment (NT) = no treatment) and mortality and disability were assessed using weighted pooled logistic regression and weighted multilevel mixed-effects linear regression respectively. Inverse weights were used to account for indication/censoring confounding. Results – This study included 602 patients with RA (median (IQR) age = 56 (44, 68) years; 65.9% women). Median disease activity was low over follow-up (SJC 1-3, TJC 3-6). Median HAQ rose after year 1 but remained at low/moderate levels (median 1.25 after year 10). There was reduced mortality risk in the ET and LT compared to NT group. ET group had comparable HAQ to NT group over follow-up (β 0.03, 95% CI -0.06, 0.12); LT group had increased disability (LT vs NT β 0.10, 95% CI 0.02, 0.17). Conclusion – This study indicates the importance of early treatment regarding long-term outcomes of patients with RA. This article is protected by copyright. All rights reserved

CRISPR-Cas12a-mediated DNA clamping triggers target-strand cleavage

Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas12a is widely used for genome editing and diagnostics, so it is important to understand how RNA-guided DNA recognition activates the cleavage of the target strand (TS) following non-target-strand (NTS) cleavage. Here we used single-molecule magnetic tweezers, gel-based assays and nanopore sequencing to explore DNA unwinding and cleavage. In addition to dynamic and heterogenous R-loop formation, we also directly observed transient double-stranded DNA unwinding downstream of the 20-bp heteroduplex and, following NTS cleavage, formation of a hyperstable ‘clamped’ Cas12a–DNA intermediate necessary for TS cleavage. Annealing of a 4-nucleotide 3′ CRISPR RNA overhang to the unwound TS downstream of the heteroduplex inhibited clamping and slowed TS cleavage by ~16-fold. Alanine substitution of a conserved aromatic amino acid in the REC2 subdomain that normally caps the R-loop relieved this inhibition but favoured stabilisation of unwound states, suggesting that the REC2 subdomain regulates access of the 3′ CRISPR RNA to downstream DNA

ENDO-Pore:high-throughput linked-end mapping of single DNA cleavage events using nanopore sequencing

Mapping the precise position of DNA cleavage events plays a key role in determining the mechanism and function of endonucleases. ENDO-Pore is a high-throughput nanopore-based method that allows the time resolved mapping single molecule DNA cleavage events in vitro. Following linearisation of a circular DNA substrate by the endonuclease, a resistance cassette is ligated recording the position of the cleavage event. A library of single cleavage events is constructed and subjected to rolling circle amplification to generate concatemers. These are sequenced and used to produce accurate consensus sequences. To identify the cleavage site(s), we developed CSI (Cleavage Site Investigator). CSI recognizes the ends of the cassette ligated into the cleaved substrate and triangulates the position of the dsDNA break. We firstly benchmarked ENDO-Pore using Type II restriction endonucleases. Secondly, we analysed the effect of crRNA length on the cleavage pattern of CRISPR Cas12a. Finally, we mapped the time-resolved DNA cleavage by the Type ISP restriction endonuclease LlaGI that introduces random double-strand breaks into its DNA substrates

Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes

Endonucleolytic double-strand DNA break production requires separate strand cleavage events. Although catalytic mechanisms for simple dimeric endonucleases are available, there are many complex nuclease machines which are poorly understood in comparison. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide following convergent ATP-driven translocation. We report the 2.7 Angstroms resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are unexpectedly located upstream of the direction of translocation, inconsistent with simple nuclease domain-dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex where the nuclease domains are distal. Sequencing of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand nicking events combine to produce DNA scission

The usefulness and acceptability of a personal health record to children and young people living with a complex health condition : a realist review of the literature

BackgroundThere are a growing number of children and young people (CYP) with chronic health needs or complex disabilities. Increasingly CYP with life‐limiting or life‐threatening conditions are surviving into adulthood. Communication between CYP, their family and health professionals can be challenging. The use of a personal‐health record (PHR) is one potential strategy for improving communication by promoting CYP's health advocacy skills. However, PHR implementation has proved difficult due to technical, organisational and professional barriers. The aim of this realist review is to identify the factors which help or hinder the use of PHRs with CYP living with a complex health condition.MethodsSystematic realist review. Literature was sourced from six databases: Medline, Embase, CINAHL, PsychInfo, The Cochrane Library and Science Direct (from 1946 to August Week 3 2018). The web was searched to identify grey literature. Articles were sourced from reference lists of included studies. Data were extracted using a standardised data extraction form. Two reviewers completed data extraction and synthesis. Methodological rigor was assessed using the relevant Critical Appraisal Skills Programme (CASP) tool.ResultsNine articles were included. Contextual factors which helped implementation included the CYP having a high perception of need for a PHR, and a high level of desire for self‐management. Service providers and CYP need knowledge about the purpose and benefits of the PHR, and organisations need a dedicated person to facilitate PHR use. Mechanisms triggered by the PHR included improved understanding and knowledge of healthcare condition(s) for CYP, an increased feeling of control over condition(s), and more active engagement in their healthcare. Outcomes for CYP included improved self‐advocacy and communication.ConclusionClearer definitions of which young people would benefit from using a PHR must be established to inform which organisations and service providers would be best suited to PHR implementation

Multislice CT Virtual Intravascular Endoscopy for Assessing Pulmonary Embolisms: a Pictorial Review

Multislice CT has been widely used in clinical practice for diagnosing cardiovascular disease due to its reduced invasiveness and its high spatial and temporal resolution. As a reliable alternative to conventional pulmonary angiography, multislice CT angiography has been recognized as the first line technique for detecting and diagnosing pulmonary embolism. A pulmonary embolism located in the main pulmonary artery, as well as being located in the segmental branches, can be accurately detected with multislice CT imaging, and especially with the use of 16- and 64-slice CT scanners. Visualization of pulmonary embolisms has traditionally been limited to 2D, multiplanar reformation and the 3D external surface visualizations. In this pictorial review, we present our experience of using 3D virtual intravascular endoscopy to characterize and evaluate the intraluminal appearance of pulmonary embolisms in a group of patients who were suspected of having pulmonary embolism and who were undergoing multislice CT angiography. We expect that the research findings from this study will provide insight into the extent of disease and the luminal changes to the pulmonary arteries that are due to the presence of thrombus, and so monitoring of the progress of disease and predicting the treatment outcome can well be achieved