A novel FRET pair for detection of parallel DNA triplexes by the LightCycler

<p>Abstract</p> <p>Background</p> <p>Melting temperature of DNA structures can be determined on the LightCycler using quenching of FAM. This method is very suitable for pH independent melting point (Tm) determination performed at basic or neutral pH, as a high throughput alternative to UV absorbance measurements. At acidic pH quenching of FAM is not very suitable, since the fluorescence of FAM is strongly pH dependent and drops with acidic pH.</p> <p>Hoogsteen based parallel triplex helix formation requires protonation of cytosines in the triplex forming strand. Therefore, nucleic acid triplexes show strong pH dependence and are stable only at acidic pH. This led us to establish a new pH independent fluorophore based measuring system on the LightCycler for thermal stability studies of parallel triplexes.</p> <p>Results</p> <p>A novel LightCycler FRET pair labelled with ATTO495 and ATTO647N was established for parallel triplex detection with antiparallel duplex as a control for the general applicability of these fluorophores for Tm determination. The ATTO fluorophores were pH stable from pH 4.5 to 7.5. Melting of triplex and duplex structures were accompanied by a large decrease in fluorescence intensity leading to well defined Tm and high reproducibility. Validation of Tm showed low intra- and inter-assay coefficient of variation; 0.11% and 0.14% for parallel triplex and 0.19% and 0.12% for antiparallel duplex. Measurements of Tm and fluorescence intensity over time and multiple runs showed great time and light stability of the ATTO fluorophores. The variance on Tm determinations was significant lower on the LightCycler platform compared to UV absorbance measurements, which enable discrimination of DNA structures with very similar Tm. Labelling of DNA probes with ATTO fluorophore increased Tm of antiparallel duplexes significantly, but not Tm of parallel triplexes.</p> <p>Conclusions</p> <p>We have established a novel pH independent FRET pair with high fluorescence signals on the LightCycler platform for both antiparallel duplex and parallel triplex formation. The method has been thoroughly validated, and is characterized by an excellent accuracy and reproducibility. This FRET pair is especially suitable for ΔTm and Tm<it/> determinations of pH dependent parallel triplex formation.</p

Association of smoking and cancer with the risk of venous thromboembolism: the Scandinavian Thrombosis and Cancer cohort

Smoking is a well-established risk factor for cancer, and cancer patients have a high risk of venous thromboembolism (VTE). Conflicting results have been reported on the association between smoking and risk of VTE, and the effect of smoking on VTE-risk in subjects with cancer is scarcely studied. We aimed to investigate the association between smoking and VTE in subjects with and without cancer in a large population-based cohort. The Scandinavian Thrombosis and Cancer (STAC) cohort included 144,952 participants followed from 1993–1997 to 2008–2012. Information on smoking habits was derived from self-administered questionnaires. Active cancer was defined as the first two years following the date of cancer diagnosis. Former smokers (n = 35,890) and those with missing information on smoking status (n = 3680) at baseline were excluded. During a mean follow up of 11 years, 10,181 participants were diagnosed with cancer, and 1611 developed incident VTE, of which 214 were cancer-related. Smoking was associated with a 50% increased risk of VTE (HR 1.49, 95% CI 1.12–1.98) in cancer patients, whereas no association was found in cancer-free subjects (HR 1.07, 95% CI 0.96–1.20). In cancer patients, the risk of VTE among smokers remained unchanged after adjustment for cancer site and metastasis. Stratified analyses showed that smoking was a risk factor for VTE among those with smoking-related and advanced cancers. In conclusion, smoking was associated with increased VTE risk in subjects with active cancer, but not in those without cancer. Our findings imply a biological interaction between cancer and smoking on the risk of VTE

Major TCR Repertoire Perturbation by Immunodominant HLA-B*44:03-Restricted CMV-Specific T Cells

Lack of disease during chronic human cytomegalovirus (CMV) infection depends on the maintenance of a high-frequency CMV-specific T cell response. The composition of the T cell receptor (TCR) repertoire underlying this response remains poorly characterised, especially within African populations in which CMV is endemic from infancy. Here we focus on the immunodominant CD8+ T cell response to the immediate-early 2 (IE-2)-derived epitope NEGVKAAW (NW8) restricted by HLA-B*44:03, a highly prevalent response in African populations, which in some subjects represents &gt;10% of the circulating CD8+ T cells. Using pMHC multimer staining and sorting of NW8-specific T cells, the TCR repertoire raised against NW8 was characterised here using high-throughput sequencing in 20 HLA-B*44:03 subjects. We found that the CD8+ T cell repertoire raised in response to NW8 was highly skewed and featured preferential use of a restricted set of V and J gene segments. Furthermore, as often seen in immunity against ancient viruses like CMV and Epstein-Barr virus (EBV), the response was strongly dominated by identical TCR sequences shared by multiple individuals, or “public” TCRs. Finally, we describe a pair “superdominant” TCR clonotypes, which were germline or nearly germline-encoded and produced at remarkably high frequencies in certain individuals, with a single CMV-specific clonotype representing up to 17% of all CD8+ T cells. Given the magnitude of the NW8 response, we propose that this major skewing of CMV-specific immunity leads to massive perturbations in the overall TCR repertoire in HLA-B*44:03 individuals

Stringent or nonstringent complete remission and prognosis in acute myeloid leukemia:a Danish population-based study

Key Points sCR and non-sCR in acute myeloid leukemia are associated with different prognoses. Three lineage blood cell recoveries have independent impacts on prognosis.</jats:p

Reliability of measuring abductor hallucis muscle parameters using two different diagnostic ultrasound machines

<p>Abstract</p> <p>Background</p> <p>Diagnostic ultrasound provides a method of analysing soft tissue structures of the musculoskeletal system effectively and reliably. The aim of this study was to evaluate within and between session reliability of measuring muscle dorso-plantar thickness, medio-lateral length and cross-sectional area, of the abductor hallucis muscle using two different ultrasound machines, a higher end Philips HD11 Ultrasound machine and clinically orientated Chison 8300 Deluxe Digital Portable Ultrasound System.</p> <p>Methods</p> <p>The abductor hallucis muscle of both the left and right feet of thirty asymptomatic participants was imaged and then measured using both ultrasound machines. Interclass correlation coefficients (ICC) with 95% confidence intervals (CI) were used to calculate both within and between session intra-tester reliability. Standard error of the measurement (SEM) calculations were undertaken to assess difference between the actual measured score across trials and the smallest real difference (SRD) was calculated from the SEM to indicate the degree of change that would exceed the expected trial to trial variability.</p> <p>Results</p> <p>The ICCs, SEM and SRD for dorso-plantar thickness and medial-lateral length were shown to have excellent to high within and between-session reliability for both ultrasound machines. The between-session reliability indices for cross-sectional area were acceptable for both ultrasound machines.</p> <p>Conclusion</p> <p>The results of the current study suggest that regardless of the type ultrasound machine, intra-tester reliability for the measurement the abductor hallucis muscle parameters is very high.</p

Ultrasound evaluation of the abductor hallucis muscle: Reliability study

© 2008 Cameron et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Reversal of Obesity and Insulin Resistance by a Non-Peptidic Glucagon-Like Peptide-1 Receptor Agonist in Diet-Induced Obese Mice

BACKGROUND: Glucagon-like peptide-1 (GLP-1) is recognized as an important regulator of glucose homeostasis. Efforts to utilize GLP-1 mimetics in the treatment of diabetes have yielded clinical benefits. A major hurdle for an effective oral therapy has been the difficulty of finding a non-peptidic GLP-1 receptor (GLP-1R) agonist. While its oral bioavailability still poses significant challenges, Boc5, one of the first such compounds, has demonstrated the attainment of GLP-1R agonism in diabetic mice. The present work was to investigate whether subchronic Boc5 treatment can restore glycemic control and induce sustainable weight loss in diet-induced obese (DIO) mice, an animal model of human obesity and insulin resistance. METHODOLOGY/PRINCIPAL FINDINGS: DIO mice were treated three times a week with Boc5 (0.3, 1 and 3 mg) for 12 weeks. Body weight, body mass index (BMI), food intake, fasting glucose, intraperitoneal glucose tolerance and insulin induced glucose clearance were monitored regularly throughout the treatment. Glucose-stimulated insulin secretion, β-cell mass, islet size, body composition, serum metabolic profiles, lipogenesis, lipolysis, adipose hypertrophy and lipid deposition in the liver and muscle were also measured after 12 weeks of dosing. Boc5 dose-dependently reduced body weight, BMI and food intake in DIO mice. These changes were associated with significant decreases in fat mass, adipocyte hypertrophy and peripheral tissue lipid accumulation. Boc5 treatment also restored glycemic control through marked improvement of insulin sensitivity and normalization of β-cell mass. Administration of Boc5 (3 mg) reduced basal but enhanced insulin-mediated glucose incorporation and noradrenaline-stimulated lipolysis in isolated adipocytes from obese mice. Furthermore, circulating leptin, adiponectin, triglyceride, total cholesterol, nonesterified fatty acid and high-density lipoprotein/low-density lipoprotein ratio were normalized to various extents by Boc5 treatment. CONCLUSIONS/SIGNIFICANCE: Boc5 may produce metabolic benefits via multiple synergistic mechanisms and may represent an attractive tool for therapeutic intervention of obesity and diabetes, by means of non-peptidic GLP-1R agonism