An effective method for extraction and polymerase chain reaction (PCR) amplification of DNA from formalin preserved tissue samples of snow leopard

Formalin-preserved biological samples obtained from endangered species are valuable in assessing genetic diversity. To make use of snow leopard samples preserved in formalin over a period of two to seven years, we optimized the method of extracting DNA from these samples. We used (a) phenol chloroform : isoamyl alcohol, (b) the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Germany), (c) the Qiagen DNeasy Blood and Tissue Kit after treating the samples with NaOH for three days and (d) the Qiagen DNeasy Blood and Tissue Kit after treating the samples with phosphate buffered saline (PBS) for three days. The usefulness of the extracted DNA was assessed on the basis of mitochondrial (150 to 550 bp) and nuclear (95 to 229 bp) markers. There was no PCR amplification with the first two methods. The PCR amplification with the NaOH and PBS treatment had a success rate of 30 to 100% for both mitochondrial and nuclear markers. The PBS method is the best method for extraction of DNA from formalin-preserved samples of longer period (two to seven years) because of higher success rate in amplifying mitochondrial gene of ca. 550 bp (60%) than the NaOH method (28%). The overall amplification of microsatellite markers in such samples was also higher in samples treated with PBS (43 to 100%) than NaOH (0 to 100%). The PCR products obtained were confirmed through DNA sequencing to be of snow leopard origin. The optimized protocol will enable genetic studies to be conducted on tissue samples of other species that have been preserved in formalin. The protocol will be particularly useful for species that are elusive and from which it is difficult to collect fresh tissue samples.Keywords: Formalin, polymerase chain reaction (PCR), mtDNA, microsatellites, snow leopardAfrican Journal of Biotechnology Vol. 12(22), pp. 3399-340

Genome-wide haplotype-based association analysis of major depressive disorder in Generation Scotland and UK Biobank

Generation Scotland received core funding from the Chief Scientist Office of the Scottish Government Health Directorate CZD/16/6 and the Scottish Funding Council HR03006. Genotyping of the GS:SFHS samples was carried out by the Genetics Core Laboratory at the Wellcome Trust Clinical Research Facility, Edinburgh, Scotland and was funded by the Medical Research Council UK and the Wellcome Trust (Wellcome Trust Strategic Award “STratifying Resilience and Depression Longitudinally” (STRADL) Reference 104036/Z/14/Z. YZ acknowledges support from China Scholarship Council. IJD is supported by the Centre for Cognitive Ageing and Cognitive Epidemiology which is funded by the Medical Research Council and the Biotechnology and Biological Sciences Research Council (MR/K026992/1). AMMcI and T-KC acknowledges support from the Dr Mortimer and Theresa Sackler Foundation. We are grateful to all the families who took part, the general practitioners and the Scottish School of Primary Care for their help in recruiting them, and the whole Generation Scotland team, which includes interviewers, computer and laboratory technicians, clerical workers, research scientists, volunteers, managers, receptionists, healthcare assistants and nurses. Ethics approval for the study was given by the NHS Tayside committee on research ethics (reference 05/S1401/8)Peer reviewedPublisher PD

A roadmap of strain in doped anatase TiO2

Anatase titanium oxide is important for its high chemical stability and photocatalytic properties, however, the latter are plagued by its large band gap that limits its activity to only a small percentage of the solar spectrum. In that respect, straining the material can reduce its band gap increasing the photocatalytic activity of titanium oxide. We apply density functional theory with the introduction of the Hubbard + U model, to investigate the impact of stress on the electronic structure of anatase in conjunction with defect engineering by intrinsic defects (oxygen/titanium vacancies and interstitials), metallic dopants (iron, chromium) and non-metallic dopants (carbon, nitrogen). Here we show that both biaxial and uniaxial strain can reduce the band gap of undoped anatase with the use of biaxial strain being marginally more beneficial reducing the band gap up to 2.96 eV at a tensile stress of 8 GPa. Biaxial tensile stress in parallel with doping results in reduction of the band gap but also in the introduction of states deep inside the band gap mainly for interstitially doped anatase. Dopants in substitutional positions show reduced deep level traps. Chromium-doped anatase at a tensile stress of 8 GPa shows the most significant reduction of the band gap as the band gap reaches 2.4 eV

LEADER 5: prevalence and cardiometabolic impact of obesity in cardiovascular high-risk patients with type 2 diabetes mellitus: baseline global data from the LEADER trial

Background: Epidemiological data on obesity are needed, particularly in patients with type 2 diabetes mellitus (T2DM) and high cardiovascular (CV) risk. We used the baseline data of liraglutide effect and action in diabetes: evaluation of CV outcome results—A long term Evaluation (LEADER) (a clinical trial to assess the CV safety of liraglutide) to investigate: (i) prevalence of overweight and obesity; (ii) relationship of the major cardiometabolic risk factors with anthropometric measures of adiposity [body mass index (BMI) and waist circumference (WC)]; and (iii) cardiometabolic treatment intensity in relation to BMI and WC. Methods: LEADER enrolled two distinct populations of high-risk patients with T2DM in 32 countries: (1) aged ≥50 years with prior CV disease; (2) aged ≥60 years with one or more CV risk factors. Associations of metabolic variables, demographic variables and treatment intensity with anthropometric measurements (BMI and WC) were explored using regression models (ClinicalTrials.gov identifier: NCT01179048). Results: Mean BMI was 32.5 ± 6.3 kg/m2 and only 9.1 % had BMI <25 kg/m2. The prevalence of healthy WC was also extremely low (6.4 % according to International Joint Interim Statement for the Harmonization of the Metabolic Syndrome criteria). Obesity was associated with being younger, female, previous smoker, Caucasian, American, with shorter diabetes duration, uncontrolled blood pressure (BP), antihypertensive agents, insulin plus oral antihyperglycaemic treatment, higher levels of triglycerides and lower levels of high-density lipoprotein cholesterol. Conclusions: Overweight and obesity are prevalent in high CV risk patients with T2DM. BMI and WC are related to the major cardiometabolic risk factors. Furthermore, treatment intensity, such as insulin, statins or oral antihypertensive drugs, is higher in those who are overweight or obese; while BP and lipid control in these patients are remarkably suboptimal. LEADER confers a unique opportunity to explore the longitudinal effect of weight on CV risk factors and hard endpoints

Patterns of glucose-lowering medication use in patients with type 2 diabetes and heart failure.: Insights from the Diabetes Collaborative Registry (DCR)

Background: Optimal glucose-lowering strategies in patients with both heart failure (HF) and type 2 diabetes mellitus (T2D) are not well defined, particularly as novel medication classes emerge.We sought to evaluate current patterns of glucose-lowering medication use in adults with T2D with and without HF. Methods: The DCR is a US-based outpatient registry of adults with diabetes; currently includes 3074 providers in 203 practices. We used hierarchical, modified Poisson regression models to examine the relationship between concomitant HF with use of each glucose-lowering medication class, adjusting for other factors that could impact selection of one medication class over another: age, chronic kidney disease (CKD), coronary artery disease (CAD), number of glucose-lowering medications, and insurance.Results: Among 456,106 adults with T2D, 125,161 (27%) had a diagnosis of HF (30% HFrEF, 15%HFmrEF, 55% HFpEF). Patients with T2D and HF were more likely to be older and male, and to have CAD, atrial fibrillation, and CKD. In the multivariable models, HF was associated with a greater use of insulin (RR 1.39, 95% CI 1.36–1.42) and lower use of thiazolidinediones (RR 0.79, 95% CI 0.74–0.83), SGLT2 inhibitors (RR 0.83, 95% CI 0.79–0.89), and metformin (RR 0.84, 95% CI 0.82–0.86). Among the subgroup of patients with HF, thiazolidinediones, GLP-1 receptor agonists, and SGLT2 inhibitors were used even less often in patients with lower ejection fraction, indicating that both the diagnosis of clinical HF and ejection fraction may influence the choice of glucose-lowering medications.Conclusion: In a large US-based outpatient registry, we found that a quarter of adults with T2D had a diagnosis of HF, which was predominantly HFpEF. Although certain T2D medication use in patients with HF appeared consistent with evidence (less use of thiazolidinediones), others appeared contrary to evidence (less use of metformin and SGLT2 inhibitors).</p

Identification of an N-terminal 27 kDa fragment of Mycoplasma pneumoniae P116 protein as specific immunogen in M. pneumoniae infections

<p>Abstract</p> <p>Background</p> <p><it>Mycoplasma pneumoniae </it>is an important cause of respiratory tract infection and is increasingly being associated with other diseases such as asthma and extra-pulmonary complications. Considerable cross-reactivity is known to exist between the whole cell antigens used in the commercial serological testing assays. Identification of specific antigens is important to eliminate the risk of cross-reactions among different related organisms. Adherence of <it>M. pneumoniae </it>to human epithelial cells is mediated through a well defined apical organelle to which a number of proteins such as P1, P30, P116 and HMW1-3 have been localized, and are being investigated for adhesion, gliding and immunodiagnostic purposes.</p> <p>Methods</p> <p>A 609 bp fragment P116_(N-27),corresponding to the N-terminal region of <it>M. pneumoniae </it>P116 gene was cloned and expressed. A C-terminal fragment P1_(C-40),of P1 protein of <it>M. pneumoniae </it>was also expressed. Three IgM ELISA assays based on P116_(N-27),P1_(C-40)and (P116 _(N-27)+ P1_(C-40)) proteins were optimized and a detailed analysis comparing the reactivity of these proteins with a commercial kit was carried out. Comparative statistical analysis of these assays was performed with the SPSS version 15.0.</p> <p>Results</p> <p>The expressed P116_(N-27)protein was well recognized by the patient sera and was immunogenic in rabbit. P1_(C-40)of <it>M. pneumoniae </it>was also immunogenic in rabbit. In comparison to the reference kit, which is reported to be 100% sensitive and 75% specific, ELISA assay based on purified P116_(N-27),P1_(C-40)and (P116_(N-27)+ P1_(C-40)) proteins showed 90.3%, 87.1% and 96.8% sensitivity and 87.0%, 87.1% and 90.3% specificity respectively. The p value for all the three assays was found to be < 0.001, and there was a good correlation and association between them.</p> <p>Conclusion</p> <p>This study shows that an N-terminal fragment of P116 protein holds a promise for serodiagnosis of <it>M. pneumoniae </it>infection. The IgM ELISA assays based on the recombinant proteins seem to be suitable for the use in serodiagnosis of acute <it>M. pneumoniae </it>infections. The use of short recombinant fragments of P116 and P1 proteins as specific antigens may eliminate the risk of cross-reactions and help to develop a specific and sensitive immunodiagnostic assay for <it>M. pneumoniae </it>detection.</p

Higher expression of human kallikrein 10 in breast cancer tissue predicts tamoxifen resistance

The human tissue kallikreins are secreted serine proteases, encoded by a group of homologous genes clustered in tandem on chromosome 19q13.3-4. Human kallikrein 6 and human kallikrein 10 are two new members of this family. Recently, we developed highly sensitive and specific immunofluorometric assays for human kallikrein 6 and human kallikrein 10, which allow for their quantification in tissue extracts and biological fluids. Both human kallikrein 6 and human kallikrein 10 are found to be down-regulated in breast cancer cell lines, suggesting that they may be involved in breast cancer pathogenesis and progression. In this study, we investigated the potential value of human kallikrein 6 and human kallikrein 10 as prognostic and predictive factors in breast cancer. We quantified human kallikrein 6 and human kallikrein 10 protein levels in 749 breast tumour cytosolic extracts and correlated this data with various clinicopathological variables and patient outcomes. Human kallikrein 6 and human kallikrein 10 are positively correlated with each other. Higher human kallikrein 6 and human kallikrein 10 protein levels are associated with younger age, pre-menopausal, status and tumours which are negative for oestrogen and progesterone receptors. No correlation was found between human kallikrein 6 and human kallikrein 10 levels and tumour size, grade, and nodal status. Survival analysis showed that neither human kallikrein 6 nor human kallikrein 10 are related to the rate of relapse-free and overall survival. In the analysis with respect to response to tamoxifen therapy, although human kallikrein 6 levels were not associated with tamoxifen responsiveness, higher levels of human kallikrein 10 were significantly associated with a poor response rate. This association remained significant in the multivariate analysis. Furthermore, higher human kallikrein 10 levels were significantly related with a short progression-free and post-relapse overall survival after start of tamoxifen treatment for advanced disease. Taken together, our results suggest that although human kallikrein 6 and human kallikrein 10 are not prognostic markers for breast cancer, human kallikrein 10 is an independent predictive marker for response of tamoxifen therapy