Optimization of Non-Natural Nucleotides For Selective Incorporation Opposite Damaged DNA

The promutagenic process known as translesion DNA synthesis reflects the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template. To study the underlying mechanism of nucleotide selection during this process, we quantified the incorporation of various non-natural nucleotide analogs opposite an abasic site, a non-templating DNA lesion. Our kinetic studies using the bacteriophage T4 DNA polymerase reveal that the π-electron surface area of the incoming nucleotide substantially contributes to the efficiency of incorporation opposite an abasic site. A remaining question is whether the selective insertion of these non-hydrogen-bonding analogs can be achieved through optimization of shape and π-electron density. In this report, we describe the synthesis and kinetic characterization of four novel nucleotide analogs, 5-cyanoindolyl-2′-deoxyriboside 5′-triphosphate (5-CyITP), 5-ethyleneindolyl-2′-deoxyriboside 5′-triphosphate (5-EyITP), 5-methylindolyl-2′-deoxyriboside 5′-triphosphate (5-MeITP), and 5-ethylindolyl-2′-deoxyriboside 5′-triphosphate (5-EtITP). Kinetic analyses indicate that the overall catalytic efficiencies of all four nucleotides are related to their base-stacking properties. In fact, the catalytic efficiency for nucleotide incorporation opposite an abasic site displays a parabolic trend in the overall π-electron surface area of the non-natural nucleotide. In addition, each non-natural nucleotide is incorporated opposite templating DNA ∼100-fold worse than opposite an abasic site. These data indicate that selectivity for incorporation opposite damaged DNA can be achieved through optimization of the base-stacking properties of the incoming nucleotide

Non-invasive airway health assessment: Synchrotron imaging reveals effects of rehydrating treatments on mucociliary transit in-vivo

To determine the efficacy of potential cystic fibrosis (CF) therapies we have developed a novel mucociliary transit (MCT) measurement that uses synchrotron phase contrast X-ray imaging (PCXI) to non-invasively measure the transit rate of individual micron-sized particles deposited into the airways of live mice. The aim of this study was to image changes in MCT produced by a rehydrating treatment based on hypertonic saline (HS), a current CF clinical treatment. Live mice received HS containing a long acting epithelial sodium channel blocker (P308); isotonic saline; or no treatment, using a nebuliser integrated within a small-animal ventilator circuit. Marker particle motion was tracked for 20 minutes using PCXI. There were statistically significant increases in MCT in the isotonic and HS-P308 groups. The ability to quantify in vivo changes in MCT may have utility in pre-clinical research studies designed to bring new genetic and pharmaceutical treatments for respiratory diseases into clinical trials.Martin Donnelley, Kaye S. Morgan, Karen K. W. Siu, Nigel R. Farrow, Charlene S. Stahr, Richard C. Boucher, Andreas Fouras & David W. Parson

The disappearance of the "revolving door" patient in Scottish general practice: successful policies

<b>Background</b> We describe the health of "revolving door" patients in general practice in Scotland, estimate changes in their number over the timescale of the study, and explore reasons for changes, particularly related to NHS and government policy.<p></p> <b>Methods</b> A mixed methods predominantly qualitative study, using a grounded theory approach, set in Scottish general practice. Semi-structured interviews were conducted with professional key informants, 6 Practitioner Services staff who administer the GP registration system and 6 GPs with managerial or clinical experience of working with "revolving door" patients. Descriptive statistical analysis and qualitative analysis of patient removal episodes linked with routine hospital admissions, outpatient appointments, drug misuse treatment episodes and deaths were carried out with cohorts of "revolving door" patients identified from 1999 to 2005 in Scotland.<p></p> <b>Results</b> A "revolving door" patient is removed 4 or more times from GP lists in 7 years. Patients had complex health issues including substance misuse, psychiatric and physical health problems and were at high risk of dying. There was a dramatic reduction in the number of "revolving door" patients during the course of the study.<p></p> <b>Conclusions</b> "Revolving door" patients in general practice had significant health problems. Their numbers have reduced dramatically since 2004 and this probably resulted from improved drug treatment services, pressure from professional bodies to reduce patient removals and the positive ethical regulatory and financial climate of the 2004 GMS GP contract. This is a positive development for the NHS

Assessment of Lentiviral Vector Mediated CFTR Correction in Mice Using an Improved Rapid in vivo Nasal Potential Difference Measurement Protocol

Cystic Fibrosis (CF) is caused by a defect in the CF transmembrane conductance regulator (CFTR) gene responsible for epithelial ion transport. Nasal potential difference (PD) measurement is a well established diagnostic technique for assessing the efficacy of therapies in CF patients and animal models. The aim was to establish a rapid nasal PD protocol in mice and quantify the efficacy of lentiviral (LV) vector-based CFTR gene therapy. Anaesthetised wild-type (WT) and CF mice were non-surgically intubated and nasal PD measurements were made using a range of buffer flow rates. Addition of the cAMP agonist, isoproterenol, to the buffer sequence was then examined. The optimised rapid PD technique was then used to assess CFTR function produced by second and third generation LV-CFTR vectors. V5 epitope tagged-CFTR in nasal tissue was identified by immunohistochemistry. When intubated, mice tolerated higher flow rates. Isoproterenol could discriminate between WT and CF mice. Improved chloride transport was observed for the second and third generation LV-CFTR vectors, with up to 60% correction of the cAMP-driven chloride response towards WT. V5-CFTR was located in ciliated epithelial cells. The rapid PD technique enables improved functional assessment of the bioelectrical ion transport defect for both current and potential CF therapies.P. Cmielewski, J. Delhove, M. Donnelley, and D. Parson

Absence of "Ghost Images" Excludes Large Values of the Cosmological Constant

We used the 1.4 GHz NRAO NVSS survey to search for ghost images of radio sources, expected in cosmologies with a positive cosmological constant and positive space curvature. No statistically significant evidence for ghost images was found, placing constraints on the values of L, the space curvature or the duration of the radio-luminous phase of extragalactic radio sources.Comment: 11 pages 2 figure

The Initial Economic Burden of Femur Fractures on Informal Caregivers in Dar es Salaam, Tanzania

BackgroundFemur fracture patients require significant in-hospital care. The burden incurred by caregivers of such patients amplifies the direct costs of these injuries and remains unquantified. Aim Here we aim to establish the in-hospital economic burden faced by informal caregivers of femur fracture patients. Methods 70 unique caregivers for 46 femoral shaft fracture patients were interviewed. Incurred economic burden was determined by the Human Capital Approach, using standardized income data to quantify productivity loss (in $USD). Linear regression assessed the relationship between caregiver burden and patient time-in-hospital.ResultsThe average economic burden incurred was$149, 9% of a caregiver’s annual income and positively correlated with patient time in hospital (p<0.01). Conclusion Caregivers of patients treated operatively for femur fractures lost a large portion of their annual income, and this loss increased with patient time in hospital. These indirect costs of femur fracture treatment constitute an important component of the total injury burden

Single-dose lentiviral mediated gene therapy recovers CFTR function in cystic fibrosis knockout rats

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective ion transport in the airways. Addition of a functioning CFTR gene into affected airway cells has the potential to be an effective treatment for lung disease. The therapeutic efficacy of airway gene transfer can be quantified in animal models by assessing ion transport in the treated nasal epithelium using the nasal potential difference (PD) measurement technique. The nasal PD technique is routinely used in CF mice, however when applied to a recently developed CF rat model those animals did not tolerate the initial nasal PD assessment, therefore the procedure was firstly optimised in rats. This study evaluated the effect of lentiviral (LV)-mediated CFTR airway gene delivery on nasal PD in a CFTR knockout rat model. LV gene vector containing the CFTR gene tagged with a V5 epitope tag (LV-V5-CFTR) was delivered to the nasal epithelium of CF rats, and one week later nasal PD was analysed. This study demonstrated for the first time that LV-V5-CFTR treatment produced a mean correction of 46% towards wild-type chloride response in treated CF rats. Transduced cells were subsequently identifiable using V5 immunohistochemical staining. These findings in the nose validate the use of airway gene therapy for future lung based experiments.Nicole Reyne, Patricia Cmielewski, Alexandra McCarron, Juliette Delhove, David Parsons and Martin Donnelle

Cost utility analysis of intramedullary nailing and skeletal traction treatment for patients with femoral shaft fractures in Malawi

Background and purpose — In Malawi, both skeletal traction (ST) and intramedullary nailing (IMN) are used in the treatment of femoral shaft fractures, ST being the mainstay treatment. Previous studies have found that IMN has improved outcomes and is less expensive than ST. However, no cost-effectiveness analyses have yet compared IMN and ST in Malawi. We report the results of a cost-utility analysis (CUA) comparing treatment using either IMN or ST. Patients and methods — This was an economic evaluation study, where a CUA was done using a decision-tree model from the government healthcare payer and societal perspectives with an 1-year time horizon. We obtained EQ-5D-3L utility scores and probabilities from a prospective observational study assessing quality of life and function in 187 adult patients with femoral shaft fractures treated with either IMN or ST. The patients were followed up at 6 weeks, and 3, 6, and 12 months post-injury. Quality adjusted life years (QALYs) were calculated from utility scores using the area under the curve method. Direct treatment costs were obtained from a prospective micro costing study. Indirect costs included patient lost productivity, patient transportation, meals, and childcare costs associated with hospital stay and follow-up visits. Multiple sensitivity analyses assessed model uncertainty. Results — Total treatment costs were higher for ST ($1,349) compared with IMN ($1,122). QALYs were lower for ST than IMN, 0.71 (95% confidence interval [CI] 0.66–0.76) and 0.77 (CI 0.71–0.82) respectively. Based on lower cost and higher utility, IMN was the dominant strategy. IMN remained dominant in 94% of simulations. IMN would be less cost-effective than ST at a total procedure cost exceeding $880 from the payer’s perspective, or$1,035 from the societal perspective. Interpretation — IMN was cost saving and more effective than ST in the treatment of adult femoral shaft fractures in Malawi, and may be an efficient use of limited healthcare resources.publishedVersio

A Scalable Middleware Solution for Advanced Wide Area Web Services

To alleviate scalability problems in the Web, many researchers concentrate on how to incorporate advanced caching and replication techniques. Many solutions incorporate object-based techniques. In particular, Web resources are considered as distributed objects offering a well-defined interface. We argue that most proposals ignore two important aspects. First, there is little discussion on what kind of coherence should be provided. Proposing specific caching or replication solutions makes sense only if we know what coherence model they should implement. Second, most proposals treat all Web resources alike. Such a one-size-fits-all approach will never work in a wide-area system. We propose a solution in which Web resources are encapsulated in physically distributed shared objects. Each object should encapsulate not only state and operations, but also the policy by which its state is distributed, cached, replicated, migrated, etc