The Utility of High-Resolution Melting Analysis of SNP Nucleated PCR Amplicons—An MLST Based Staphylococcus aureus Typing Scheme

High resolution melting (HRM) analysis is gaining prominence as a method for discriminating DNA sequence variants. Its advantage is that it is performed in a real-time PCR device, and the PCR amplification and HRM analysis are closed tube, and effectively single step. We have developed an HRM-based method for Staphylococcus aureus genotyping. Eight single nucleotide polymorphisms (SNPs) were derived from the S. aureus multi-locus sequence typing (MLST) database on the basis of maximized Simpson's Index of Diversity. Only G↔A, G↔T, C↔A, C↔T SNPs were considered for inclusion, to facilitate allele discrimination by HRM. In silico experiments revealed that DNA fragments incorporating the SNPs give much higher resolving power than randomly selected fragments. It was shown that the predicted optimum fragment size for HRM analysis was 200 bp, and that other SNPs within the fragments contribute to the resolving power. Six DNA fragments ranging from 83 bp to 219 bp, incorporating the resolution optimized SNPs were designed. HRM analysis of these fragments using 94 diverse S. aureus isolates of known sequence type or clonal complex (CC) revealed that sequence variants are resolved largely in accordance with G+C content. A combination of experimental results and in silico prediction indicates that HRM analysis resolves S. aureus into 268 “melt types” (MelTs), and provides a Simpson's Index of Diversity of 0.978 with respect to MLST. There is a high concordance between HRM analysis and the MLST defined CCs. We have generated a Microsoft Excel key which facilitates data interpretation and translation between MelT and MLST data. The potential of this approach for genotyping other bacterial pathogens was investigated using a computerized approach to estimate the densities of SNPs with unlinked allelic states. The MLST databases for all species tested contained abundant unlinked SNPs, thus suggesting that high resolving power is not dependent upon large numbers of SNPs

Natural images from the birthplace of the human eye

Here we introduce a database of calibrated natural images publicly available through an easy-to-use web interface. Using a Nikon D70 digital SLR camera, we acquired about 5000 six-megapixel images of Okavango Delta of Botswana, a tropical savanna habitat similar to where the human eye is thought to have evolved. Some sequences of images were captured unsystematically while following a baboon troop, while others were designed to vary a single parameter such as aperture, object distance, time of day or position on the horizon. Images are available in the raw RGB format and in grayscale. Images are also available in units relevant to the physiology of human cone photoreceptors, where pixel values represent the expected number of photoisomerizations per second for cones sensitive to long (L), medium (M) and short (S) wavelengths. This database is distributed under a Creative Commons Attribution-Noncommercial Unported license to facilitate research in computer vision, psychophysics of perception, and visual neuroscience.Comment: Submitted to PLoS ON

Early-life adversity selectively impairs α2-GABAA receptor expression in the mouse nucleus accumbens and influences the behavioral effects of cocaine

Haplotypes of the Gabra2 gene encoding the α2-subunit of the GABAA receptor (GABAAR) are associated with drug abuse, suggesting that α2-GABAARs may play an important role in the circuitry underlying drug misuse. The genetic association of Gabra2 haplotypes with cocaine addiction appears to be evident primarily in individuals who had experienced childhood trauma. Given this association of childhood trauma, cocaine abuse and the Gabra2 haplotypes, we have explored in a mouse model of early life adversity (ELA) whether such events influence the behavioral effects of cocaine and if, as suggested by the human studies, α2-GABAARs in the nucleus accumbens (NAc) are involved in these perturbed behaviors. In adult mice prior ELA caused a selective decrease of accumbal α2-subunit mRNA, resulting in a selective decrease in the number and size of the α2-subunit (but not the α1-subunit) immunoreactive clusters in NAc core medium spiny neurons (MSNs). Functionally, in adult MSNs ELA decreased the amplitude and frequency of GABAAR-mediated miniature inhibitory postsynaptic currents (mIPSCs), a profile similar to that of α2 “knock-out” (α2−/−) mice. Behaviourally, adult male ELA and α2−/− mice exhibited an enhanced locomotor response to acute cocaine and blunted sensitisation upon repeated cocaine administration, when compared to their appropriate controls. Collectively, these findings reveal a neurobiological mechanism which may relate to the clinical observation that early trauma increases the risk for substance abuse disorder (SAD) in individuals harbouring haplotypic variations in the Gabra2 gene

The Prevalence of Natural Health Product Use in Patients with Acute Cardiovascular Disease

Background: Natural health products (NHP) use may have implications with respect to adverse effects, drug interactions and adherence yet the prevalence of NHP use by patients with acute cardiovascular disease and the best method to ascertain this information is unknown. Objective: To identify the best method to ascertain information on NHP, and the prevalence of use in a population with acute cardiovascular disease. Methods: Structured interviews were conducted with a convenience sample of consecutive patients admitted with acute cardiovascular disease to the University of Alberta Hospital during January 2009. NHP use was explored using structured and open-ended questions based on Health Canada’s definition of NHP. The medical record was reviewed, and documentation of NHP use by physicians, nurses, and pharmacists, compared against the gold-standard structured interview. Results: 88 patients were interviewed (mean age 62 years, standard deviation [SD 14]; 80 % male; 41 % admitted for acute coronary syndromes). Common co-morbidities included hypertension (59%), diabetes (26%) and renal impairment (19%). NHP use was common (78 % of patients) and 75 % of NHP users reported daily use. The category of NHP most commonly used was vitamins and minerals (73%) followed by herbal products (20%), traditional medicines including Chines

Dopamine D2 receptor polymorphisms and susceptibility to alcohol dependence in Indian males: a preliminary study

<p>Abstract</p> <p>Background</p> <p>Dopamine is an important neurotransmitter involved in reward mechanism in the brain and thereby influences development and relapse of alcohol dependence. The dopamine D2 receptor (<it>DRD2</it>) gene on chromosome 11 (q22-q23) has been found to be associated with increased alcohol consumption through mechanisms involving incentive salience attributions and craving in alcoholic patients. Therefore, we investigated the association of three single nucleotide polymorphisms (SNP) in <it>DRD2 </it>gene with alcohol dependence in the north Indian subjects.</p> <p>Methods</p> <p>In a retrospective analysis, genetic association of three polymorphisms from <it>DRD2 </it>gene with alcohol dependence was investigated using a case-control approach. Alcohol dependence was determined by DSM-IV criteria and a total of 90 alcoholics and 60 healthy unrelated age-matched control subjects were recruited. Odds ratio and confidence interval was calculated to determine risk conferred by a predisposing allele/genotype/haplotype. Logistic regression analysis was carried out to correlate various clinical parameters with genotypes, and to study pair-wise interactions between SNPs.</p> <p>Results</p> <p>The study showed a significant association of -141C Ins allele and a trend of association of TaqI A1 allele of <it>DRD2 </it>with alcohol dependence. Haplotype with the predisposing -141C Ins and TaqI A1 alleles (-141C Ins-A-A1) seems to confer ≈ 2.5 times more risk to develop alcohol dependence.</p> <p>Conclusions</p> <p>The study provides preliminary insight into genetic risk to alcohol dependence in Indian males. Two polymorphisms namely, -141C Ins/Del and TaqI A in <it>DRD2 </it>gene may have clinical implications among Indian alcoholic subjects.</p

Trends and variation in the management of oesophagogastric cancer patients: a population-based survey

<p>Abstract</p> <p>Background</p> <p>Previous evidence indicates potential variation in the quality of care of cancer patients. We aimed to examine whether recent changes in the treatment of oesophagogastric cancers have been distributed equally among different patient subgroups.</p> <p>Methods</p> <p>We analysed population-based cancer registry data about the treatment patterning of oesophagogastric cancer (other than oesophageal squamous cell carcinoma) during 1995-2006.</p> <p>Results</p> <p>There were 14,077 patients aged ≥40 years (69% men). There was only limited information on stage, and no information on co-morbidity status. During successive triennia, curative surgery use decreased from 28% to 20% (p < 0.001) whilst chemotherapy use increased from 9% to 30% (p < 0.001). Use of palliative surgery and of radiotherapy increased significantly but modestly (7% to 10%, and 9% to 11%, respectively). In multivariable logistic regression adjusting for age group, gender, diagnosis period and tumour type, curative surgery and chemotherapy were used less frequently in more deprived patients [per increasing deprivation group Odds Ratio (OR) = 0.96, 95% Confidence Interval (CI) 0.93-0.99, and OR = 0.90, 95%CI 0.87-0.93, respectively, p < 0.001 for both)]. Chemotherapy was also used less frequently in women (OR = 0.76, p < 0.001).</p> <p>Conclusions</p> <p>During the study period, curative surgery decreased by a third and chemotherapy use increased by more than three-fold, reflecting improvements in the appropriateness and quality of management, but chemotherapy use, in particular, was unequal, both by socioeconomic status and gender.</p

Minim Typing – A Rapid and Low Cost MLST Based Typing Tool for Klebsiella pneumoniae

Here we report a single nucleotide polymorphism (SNP) based genotyping method for Klebsiella pneumoniae utilising high-resolution melting (HRM) analysis of fragments within the multilocus sequence typing (MLST) loci. The approach is termed mini-MLST or Minim typing and it has previously been applied to Streptococcus pyogenes, Staphylococcus aureus and Enterococcus faecium. Six SNPs were derived from concatenated MLST sequences on the basis of maximisation of the Simpsons Index of Diversity (D). DNA fragments incorporating these SNPs and predicted to be suitable for HRM analysis were designed. Using the assumption that HRM alleles are defined by G+C content, Minim typing using six fragments was predicted to provide a D = 0.979 against known STs. The method was tested against 202 K. pneumoniae using a blinded approach in which the MLST analyses were performed after the HRM analyses. The HRM-based alleles were indeed in accordance with G+C content, and the Minim typing identified known STs and flagged new STs. The tonB MLST locus was determined to be very diverse, and the two Minim fragments located herein contribute greatly to the resolving power. However these fragments are refractory to amplification in a minority of isolates. Therefore, we assessed the performance of two additional formats: one using only the four fragments located outside the tonB gene (D = 0.929), and the other using HRM data from these four fragments in conjunction with sequencing of the tonB MLST fragment (D = 0.995). The HRM assays were developed on the Rotorgene 6000, and the method was shown to also be robust on the LightCycler 480, allowing a 384-well high through-put format. The assay provides rapid, robust and low-cost typing with fully portable results that can directly be related to current MLST data. Minim typing in combination with molecular screening for antibiotic resistance markers can be a powerful surveillance tool kit