Enhanced annealing of mismatched oligonucleotides using a novel melting curve assay allows efficient in vitro discrimination and restriction of a single nucleotide polymorphism

<p>Abstract</p> <p>Background</p> <p>Many SNP discrimination strategies employ natural restriction endonucleases to discriminate between allelic states. However, SNPs are often not associated with a restriction site and therefore, a number of attempts have been made to generate sequence-adaptable restriction endonucleases. In this study, a simple, sequence-adaptable SNP discrimination mechanism between a 'wild-type' and 'mutant' template is demonstrated. This model differs from other artificial restriction endonuclease models as <it>cis- </it>rather than <it>trans-</it>orientated regions of single stranded DNA were generated and cleaved, and therefore, overcomes potential issues of either inefficient or non-specific binding when only a single variant is targeted.</p> <p>Results</p> <p>A series of mismatch 'bubbles' that spanned 0-5-bp surrounding a point mutation was generated and analysed for sensitivity to S1 nuclease. In this model, generation of oligonucleotide-mediated ssDNA mismatch 'bubbles' in the presence of S1 nuclease resulted in the selective degradation of the mutant template while maintaining wild-type template integrity. Increasing the size of the mismatch increased the rate of mutant sequence degradation, until a threshold above which discrimination was lost and the wild-type sequence was degraded. This level of fine discrimination was possible due to the development of a novel high-resolution melting curve assay to empirically determine changes in Tm (~5.0°C per base-pair mismatch) and to optimise annealing conditions (~18.38°C below Tm) of the mismatched oligonucleotide sets.</p> <p>Conclusions</p> <p>The <it>in vitro </it>'cleavage bubble' model presented is sequence-adaptable as determined by the binding oligonucleotide, and hence, has the potential to be tailored to discriminate between any two or more SNPs. Furthermore, the demonstrated fluorometric assay has broad application potential, offering a rapid, sensitive and high-throughput means to determine Tm and annealing rates as an alternative to conventional hybridisation detection strategies.</p

Concurrent acute illness and comorbid conditions poorly predict antibiotic use in upper respiratory tract infections: a cross-sectional analysis

<p>Abstract</p> <p>Background</p> <p>Inappropriate antibiotic use promotes resistance. Antibiotics are generally not indicated for upper respiratory infections (URIs). Our objectives were to describe patterns of URI treatment and to identify patient and provider factors associated with antibiotic use for URIs.</p> <p>Methods</p> <p>This study was a cross-sectional analysis of medical and pharmacy claims data from the Pennsylvania Medicaid fee-for-service program database. We identified Pennsylvania Medicaid recipients with a URI office visit over a one-year period. Our outcome variable was antibiotic use within seven days after the URI visit. Study variables included URI type and presence of concurrent acute illnesses and chronic conditions. We considered the associations of each study variable with antibiotic use in a logistic regression model, stratifying by age group and adjusting for confounders.</p> <p>Results</p> <p>Among 69,936 recipients with URI, 35,786 (51.2%) received an antibiotic. In all age groups, acute sinusitis, chronic sinusitis, otitis, URI type and season were associated with antibiotic use. Except for the oldest group, physician specialty and streptococcal pharyngitis were associated with antibiotic use. History of chronic conditions was not associated with antibiotic use in any age group. In all age groups, concurrent acute illnesses and history of chronic conditions had only had fair to poor ability to distinguish patients who received an antibiotic from patients who did not.</p> <p>Conclusion</p> <p>Antibiotic prevalence for URIs was high, indicating that potentially inappropriate antibiotic utilization is occurring. Our data suggest that demographic and clinical factors are associated with antibiotic use, but additional reasons remain unexplained. Insight regarding reasons for antibiotic prescribing is needed to develop interventions to address the growing problem of antibiotic resistance.</p

New targets for therapy in breast cancer: Farnesyltransferase inhibitors

Current systemic therapies for breast cancer are often limited by their nonspecific mechanism of action, unwanted toxicities on normal tissues, and short-term efficacy due to the emergence of drug resistance. However, identification of the molecular abnormalities in cancer, in particular the key proteins involved in abnormal cell growth, has resulted in development of various signal transduction inhibitor drugs as new treatment strategies against the disease. Protein farnesyltransferase inhibitors (FTIs) were originally designed to target the Ras signal transduction pathway, although it is now clear that several other intracellular proteins are dependent on post-translational farnesylation for their function. Preclinical data revealed that although FTIs inhibit the growth of ras-transformed cells, they are also potent inhibitors of a wide range of cancer cell lines that contain wild-type ras, including breast cancer cells. Additive or synergistic effects were observed when FTIs were combined with cytotoxic agents (in particular the taxanes) or endocrine therapies (tamoxifen). Phase I trials with FTIs have explored different schedules for prolonged administration, and dose-limiting toxicities included myelosuppression, gastrointestinal toxicity and neuropathy. Clinical efficacy against breast cancer was seen for the FTI tipifarnib in a phase II study. Based on promising preclinical data that suggest synergy with taxanes or endocrine therapy, combination clinical studies are now in progress to determine whether FTIs can add further to the efficacy of conventional breast cancer therapies

Joint Practice Guidelines for Radionuclide Lymphoscintigraphy for Sentinel Node Localization in Oral/Oropharyngeal Squamous Cell Carcinoma

Involvement of the cervical lymph nodes is the most important prognostic factor for patients with oral/oropharyngeal squamous cell carcinoma (OSCC), and the decision of whether to electively treat patients with clinically negative necks remains a controversial topic. Sentinel node biopsy (SNB) provides a minimally invasive method for determining the disease status of the cervical node basin, without the need for a formal neck dissection. This technique potentially improves the accuracy of histologic nodal staging and avoids overtreating three-quarters of this patient population, minimizing associated morbidity. The technique has been validated for patients with OSCC, and larger-scale studies are in progress to determine its exact role in the management of this patient population. This document is designed to outline the current best practice guidelines for the provision of SNB in patients with early-stage OSCC, and to provide a framework for the currently evolving recommendations for its use. Preparation of this guideline was carried out by a multidisciplinary surgical/nuclear medicine/pathology expert panel under the joint auspices of the European Association of Nuclear Medicine (EANM) Oncology Committee and the Sentinel European Node Trial (SENT) Committee

Joint practice guidelines for radionuclide lymphoscintigraphy for sentinel node localization in oral/oropharyngeal squamous cell carcinoma

Involvement of the cervical lymph nodes is the most important prognostic factor for patients with oral/oropharyngeal squamous cell carcinoma (OSCC), and the decision whether to electively treat patients with clinically negative necks remains a controversial topic. Sentinel node biopsy (SNB) provides a minimally invasive method of determining the disease status of the cervical node basin, without the need for a formal neck dissection. This technique potentially improves the accuracy of histological nodal staging and avoids over-treating three-quarters of this patient population, minimizing associated morbidity. The technique has been validated for patients with OSCC, and larger-scale studies are in progress to determine its exact role in the management of this patient population. This article was designed to outline the current best practice guidelines for the provision of SNB in patients with early-stage OSCC, and to provide a framework for the currently evolving recommendations for its use. These guidelines were prepared by a multidisciplinary surgical/nuclear medicine/pathology expert panel under the joint auspices of the European Association of Nuclear Medicine (EANM) Oncology Committee and the Sentinel European Node Trial Committee

Rural waste generation: a geographical survey at local scale

"The paper examines the per capita waste generation rates from from rural areas of Neamț County (Romania) using thematic cartography. Geographical approach of this issue is difficult because the lack of a geostatistic database at commune scale. Spatial analysis of waste indicators reveals several disparities between localities. Comparability of data between communes located in various geographical conditions must be carrefully made according to local waste management systems. Several dysfunctionalities are outlined in order to compare these results, on the one hand, between localities and on the one hand, between recent years. Geographical analysis of waste generation rates is imperative for a proper monitoring of this sector. Data from 2009, 2010 and 2012 shows that rural waste management is in a full process of change towards a more organized, stable and efficient system." (author's abstract

The impact of polyphenols on chondrocyte growth and survival: a preliminary report

Background: Imbalances in the functional binding of fibroblast growth factors (FGFs) to their receptors (FGFRs) have consequences for cell proliferation and differentiation that in chondrocytes may lead to degraded cartilage. The toxic, proinflammatory, and oxidative response of cytokines and FGFs can be mitigated by dietary polyphenols. Objective: We explored the possible effects of polyphenols in the management of osteoarticular diseases using a model based on the transduction of a mutated human FGFR3 (G380R) in murine chondrocytes. This mutation is present in most cases of skeletal dysplasia and is responsible for the overexpression of FGFR3 that, in the presence of its ligand, FGF9, results in toxic effects leading to altered cellular growth. Design: Different combinations of dietary polyphenols derived from plant extracts were assayed in FGFR3 (G380R) mutated murine chondrocytes, exploring cell survival, chloride efflux, extracellular matrix (ECM) generation, and grade of activation of mitogen-activated protein kinases. Results: Bioactive compounds from Hibiscus sabdariffa reversed the toxic effects of FGF9 and restored normal growth, suggesting a probable translation to clinical requests in humans. Indeed, these compounds activated the intracellular chloride efflux, increased ECM generation, and stimulated cell proliferation. The inhibition of mitogen-activated protein kinase phosphorylation was interpreted as the main mechanism governing these beneficial effects. Conclusions: These findings support the rationale behind the encouragement of the development of drugs that repress the overexpression of FGFRs and suggest the dietary incorporation of supplementary nutrients in the management of degraded cartilage.The authors are grateful for the constant support provided by the Hospital Universitari de Sant Joan and the Universitat Rovira i Virgili. Salvador Fernández-Arroyo is the recipient of a Sara Borrell grant (CD12/00672) from the Instituto de Salud Carlos III, Madrid, Spain. The authors also thank the Andalusian Regional Government Council of Innovation and Science for the Excellence Project P11-CTS-7625 and Generalitat Valenciana for the project PROMETEO/2012/007. This work was also supported by projects of the Fundación Areces and the Fundación MAGAR

IMPRoving Outcomes for children exposed to domestic ViolencE (IMPROVE): an evidence synthesis

BackgroundExposure to domestic violence and abuse (DVA) during childhood and adolescence increases the risk of negative outcomes across the lifespan.ObjectivesTo synthesise evidence on the clinical effectiveness, cost-effectiveness and acceptability of interventions for children exposed to DVA, with the aim of making recommendations for further research.Design(1) A systematic review of controlled trials of interventions; (2) a systematic review of qualitative studies of participant and professional experience of interventions; (3) a network meta-analysis (NMA) of controlled trials and cost-effectiveness analysis; (4) an overview of current UK provision of interventions; and (5) consultations with young people, parents, service providers and commissioners.SettingsNorth America (11), the Netherlands (1) and Israel (1) for the systematic review of controlled trials of interventions; the USA (4) and the UK (1) for the systematic review of qualitative studies of participant and professional experience of interventions; and the UK for the overview of current UK provision of interventions and consultations with young people, parents, service providers and commissioners.ParticipantsA total of 1345 children for the systematic review of controlled trials of interventions; 100 children, 202 parents and 39 professionals for the systematic review of qualitative studies of participant and professional experience of interventions; and 16 young people, six parents and 20 service providers and commissioners for the consultation with young people, parents, service providers and commissioners.InterventionsPsychotherapeutic, advocacy, parenting skills and advocacy, psychoeducation, psychoeducation and advocacy, guided self-help.Main outcome measuresInternalising symptoms and externalising behaviour, mood, depression symptoms and diagnosis, post-traumatic stress disorder symptoms and self-esteem for the systematic review of controlled trials of interventions and NMA; views about and experience of interventions for the systematic review of qualitative studies of participant and professional experience of interventions and consultations.Data sourcesMEDLINE, Cumulative Index to Nursing and Allied Health Literature, PsycINFO, EMBASE, Cochrane Central Register of Controlled Trials, Science Citation Index, Applied Social Sciences Index and Abstracts, International Bibliography of the Social Sciences, Social Services Abstracts, Social Care Online, Sociological Abstracts, Social Science Citation Index, World Health Organization trials portal and clinicaltrials.gov.Review methodsA narrative review; a NMA and incremental cost-effectiveness analysis; and a qualitative synthesis.ResultsThe evidence base on targeted interventions was small, with limited settings and types of interventions; children were mostly &lt; 14 years of age, and there was an absence of comparative studies. The interventions evaluated in trials were mostly psychotherapeutic and psychoeducational interventions delivered to the non-abusive parent and child, usually based on the child’s exposure to DVA (not specific clinical or broader social needs). Qualitative studies largely focused on psychoeducational interventions, some of which included the abusive parent. The evidence for clinical effectiveness was as follows: 11 trials reported improvements in behavioural or mental health outcomes, with modest effect sizes but significant heterogeneity and high or unclear risk of bias. Psychoeducational group-based interventions delivered to the child were found to be more effective for improving mental health outcomes than other types of intervention. Interventions delivered to (non-abusive) parents and to children were most likely to be effective for improving behavioural outcomes. However, there is a large degree of uncertainty around comparisons, particularly with regard to mental health outcomes. In terms of evidence of cost-effectiveness, there were no economic studies of interventions. Cost-effectiveness was modelled on the basis of the NMA, estimating differences between types of interventions. The outcomes measured in trials were largely confined to children’s mental health and behavioural symptoms and disorders, although stakeholders’ concepts of success were broader, suggesting that a broader range of outcomes should be measured in trials. Group-based psychoeducational interventions delivered to children and non-abusive parents in parallel were largely acceptable to all stakeholders. There is limited evidence for the acceptability of other types of intervention. In terms of the UK evidence base and service delivery landscape, there were no UK-based trials, few qualitative studies and little widespread service evaluation. Most programmes are group-based psychoeducational interventions. However, the funding crisis in the DVA sector is significantly undermining programme delivery.ConclusionsThe evidence base regarding the acceptability, clinical effectiveness and cost-effectiveness of interventions to improve outcomes for children exposed to DVA is underdeveloped. There is an urgent need for more high-quality studies, particularly trials, that are designed to produce actionable, generalisable findings that can be implemented in real-world settings and that can inform decisions about which interventions to commission and scale. We suggest that there is a need to pause the development of new interventions and to focus on the systematic evaluation of existing programmes. With regard to the UK, we have identified three types of programme that could be justifiably prioritised for further study: psycho-education delivered to mothers and children, or children alone; parent skills training in combination with advocacy: and interventions involving the abusive parent/caregiver. We also suggest that there is need for key stakeholders to come together to explicitly identify and address the structural, practical and cultural barriers that may have hampered the development of the UK evidence base to date.Future work recommendationsThere is a need for well-designed, well-conducted and well-reported UK-based randomised controlled trials with cost-effectiveness analyses and nested qualitative studies. Development of consensus in the field about core outcome data sets is required. There is a need for further exploration of the acceptability and effectiveness of interventions for specific groups of children and young people (i.e. based on ethnicity, age, trauma exposure and clinical profile). There is also a need for an investigation of the context in which interventions are delivered, including organisational setting and the broader community context, and the evaluation of qualities, qualifications and disciplines of personnel delivering interventions. We recommend prioritisation of psychoeducational interventions and parent skills training delivered in combination with advocacy in the next phase of trials, and exploratory trials of interventions that engage both the abusive and the non-abusive parent.Study registrationThis study is registered as PROSPERO CRD42013004348 and PROSPERO CRD420130043489.FundingThe National Institute for Health Research Public Health Research programme.</jats:sec