A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites

Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice, however until recently a major limitation to performing screens on this scale has been the cost effective isolation and sequencing of insertion sites. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA isolation, DNA digestion, linker or splinkerette ligation, primary and secondary PCR amplification, and sequencing. This method, which takes about 1 week to perform, has allowed us to isolate hundreds of thousands of insertion sites from mouse tumours and, unlike other methods, has been specifically optimised for the isolation of insertion sites generated with the murine leukaemia virus (MuLV), and can easily be performed in 96 well plate format for the efficient multiplex isolation of insertion sites

The Dutch Healthcare System in International Perspective

In this address, important aspects of the Dutch system of managed competition are discussed from the economic perspective, highlighting both its merits and the major challenges posed by the development of this system. Reasons for government intervention in healthcare markets are provided, and the outline the different types of healthcare systems are sketched. Followed by a description of the Dutch healthcare system and healthcare outcomes in different countries are compared. The inaugural address concludes with some suggestions for improving the Dutch system

Determination of association constants between 5 '-guanosine monophosphate gel and aromatic compounds by capillary electrophoresis

Hydro gel formed by 5'-guanosine monophosphate (GMP) in the presence of a potassium ion is expected to exhibit interesting selectivity in capillary electrophoretic separations. Here, we estimated the conditional association constants between the hydro gel (G-gel) and aromatic compounds by capillary electrophoresis in order to investigate the separation selectivity that is induced by the G-gel. Several aromatic compounds were separated in a solution containing GMP and potassium ion at different concentrations. The association constants were calculated by correlating the electrophoretic mobilities of the analytes obtained experimentally using a concentration of G-gel. During semi-quantitative estimation, naphthalene derivatives had larger association constants (K-ass = 10.3-16.8) compared with those of benzene derivatives (K-ass = 3.91-5.31), which means that the binding sites of G-gel match better to a naphthalene ring than to a benzene ring. A hydrophobic interaction was also found when the association constants for alkyl resorcinol were compared with those of different hydrocarbon chains. The association constants of nucleobases and tryptophan ranged from 6.05 to 12.6, which approximated the intermediate values between benzene and naphthalene derivatives. Consequently, the selective interaction between G-gel and aromatic compounds was classified as one of three types: (1) an intercalation into stacked planar GMP tetramers; (2) a hydrophobic interaction with a long alkyl chain; or, (3) a small contribution of steric hindrance and/or hydrogen bonding with functional groups such as amino and hydroxyl groups

Beyond the Clock:Exploring the Causal Relationship between General Practitioner Time Allocation and Hospital Referrals

This paper presents an analysis of an experiment conducted in the Netherlands to evaluate the impact of a new way of working for General Practitioners (GPs) with respect to hospital referrals and healthcare spending. The new way of working gave GPs more time to spend on each patient, and we employed a Bayesian difference-in-differences approach to estimate the effect of this intervention. To assess the impact of the new way of working, we created a control group consisting of regional GP practices that did not implement the new approach and analyzed data on hospital referrals and healthcare spending in both the practice that implemented the new approach (the treatment practice) and the control group, both before and after the intervention. Our analysis shows that the new approach led to a substantial reduction in the total number of hospital referrals. We also observed a decrease in overall healthcare spending on combined GP and hospital care for the treatment practice relative to the control group

Novel monoclonal antibodies detect Smad-Interacting Protein 1 (SIP1) in the cytoplasm of human cells from multiple tumor tissue arrays

Cataloged from PDF version of article.Smad-interacting protein 1 (SIP1, also known as ZEB2) represses the transcription of E-cadherin and mediates epithelial-mesenchymal transition in development and tumor metastasis. Due to the lack of human SIP1-specific antibodies, its expression in human tumor tissues has not been studied in detail by immunohistochemistry. Hence, we generated two anti-SIP1 monoclonal antibodies, clones 1C6 and 6E5, with IgG1 and IgG2a isotypes, respectively. The specificity of these antibodies was shown by Western blotting studies using siRNA mediated downregulation of SIP1 and ZEB1 in a human osteosarcoma cell line. In the same context, we also compared them with 5 commercially available SIP1 antibodies. Antibody specificity was further verified in an inducible cell line system by immunofluorescence. By using both antibodies, we evaluated the tissue expression of SIP1 in paraffin-embedded tissue microarrays consisting of 22 normal and 101 tumoral tissues of kidney, colon, stomach, lung, esophagus, uterus, rectum, breast and liver. Interestingly, SIP1 predominantly displayed a cytoplasmic expression, while the nuclear localization of SIP1 was observed in only 6 cases. Strong expression of SIP1 was found in distal tubules of kidney, glandular epithelial cells of stomach and hepatocytes, implicating a co-expression of SIP1 and E-cadherin. Squamous epithelium of the esophagus and surface epithelium of colon and rectum were stained with moderate to weak intensity. Normal uterus, breast and lung tissues remained completely negative. By comparison with their normal tissues, we observed SIP1 overexpression in cancers of the kidney, breast, lung and uterus. However, SIP1 expression was found to be downregulated in tumors from colon, rectum, esophagus, liver and stomach tissues. Finally we did nuclear/cytoplasmic fractionation in 3 carcinoma cell lines and detected SIP1 in both fractions, nucleus being the dominant one. To our best knowledge, this is the first comprehensive immunohistochemical study of the expression of SIP1 in a series of human cancers. Our finding that SIP1 is not exclusively localized to nucleus suggests that the subcellular localization of SIP1 is regulated in normal and tumor tissues. These novel monoclonal antibodies may help elucidate the role of SIP1 in tumor development. © 2010 Elsevier Inc

Advancing District Nursing Care Through a Learning Healthcare System:A Viewpoint on Key Requirements

The increasing complexity of healthcare needs driven by an ageing population places pressure on district nursing care. Many vulnerable older adults prefer to remain at home, requiring care coordinated with general practitioners and other professionals. This demand for integrated care is further challenged by a shortage of nursing professionals and the lack of standardised approaches to measure care quality. This article identifies the key requirements for implementing a learning healthcare system in district nursing care, using patient outcome data to foster continuous improvement and create a more adaptive, evidence-based, and patient-centred approach. This paper synthesises findings from multiple studies conducted as part of a PhD thesis, utilising a multi-method approach. These methods include examining patient outcomes in district nursing care and evaluating necessary cultural, organisational, and financial changes. Four key requirements were identified: (1) standardising patient outcome measures; (2) fostering a data-driven culture and strengthening professional autonomy; (3) enhancing organisational support and integrated care; and (4) adopting financing models that incentivise continuous learning and quality improvement. Implementing a learning healthcare system with patient outcome data in district nursing care requires a transformative shift. Standardising outcome measures, investing in information systems, and promoting continuous learning are crucial. Aligning financial incentives with patient outcomes, strengthening professional autonomy, and enhancing organisational support can make district nursing more responsive and capable of meeting complex needs. The described requirements are essential for advancing district nursing care through a more adaptive, evidence-based, and patient-centred approach.</p