A Method for Quantitative Analysis of Standard and High-Throughput qPCR Expression Data Based on Input Sample Quantity

Over the past decade rapid advances have occurred in the understanding of RNA expression and its regulation. Quantitative polymerase chain reactions (qPCR) have become the gold standard for quantifying gene expression. Microfluidic next generation, high throughput qPCR now permits the detection of transcript copy number in thousands of reactions simultaneously, dramatically increasing the sensitivity over standard qPCR. Here we present a gene expression analysis method applicable to both standard polymerase chain reactions (qPCR) and high throughput qPCR. This technique is adjusted to the input sample quantity (e.g., the number of cells) and is independent of control gene expression. It is efficiency-corrected and with the use of a universal reference sample (commercial complementary DNA (cDNA)) permits the normalization of results between different batches and between different instruments – regardless of potential differences in transcript amplification efficiency. Modifications of the input quantity method include (1) the achievement of absolute quantification and (2) a non-efficiency corrected analysis. When compared to other commonly used algorithms the input quantity method proved to be valid. This method is of particular value for clinical studies of whole blood and circulating leukocytes where cell counts are readily available

Metabolism of ticagrelor in patients with acute coronary syndromes.

© The Author(s) 2018Ticagrelor is a state-of-the-art antiplatelet agent used for the treatment of patients with acute coronary syndromes (ACS). Unlike remaining oral P2Y12 receptor inhibitors ticagrelor does not require metabolic activation to exert its antiplatelet action. Still, ticagrelor is extensively metabolized by hepatic CYP3A enzymes, and AR-C124910XX is its only active metabolite. A post hoc analysis of patient-level (n = 117) pharmacokinetic data pooled from two prospective studies was performed to identify clinical characteristics affecting the degree of AR-C124910XX formation during the first six hours after 180 mg ticagrelor loading dose in the setting of ACS. Both linear and multiple regression analyses indicated that ACS patients presenting with ST-elevation myocardial infarction or suffering from diabetes mellitus are more likely to have decreased rate of ticagrelor metabolism during the acute phase of ACS. Administration of morphine during ACS was found to negatively influence transformation of ticagrelor into AR-C124910XX when assessed with linear regression analysis, but not with multiple regression analysis. On the other hand, smoking appears to increase the degree of ticagrelor transformation in ACS patients. Mechanisms underlying our findings and their clinical significance warrant further research.Peer reviewedFinal Published versio

Meat quality traits and fatty acid composition of breast muscles from ducks fed with yellow lupin

Abstract The protein sources in feed have a huge impact on good-quality and -quantity meat traits. Yellow lupin (YL) seeds have a similar level of protein as soybean meal (SBM). The most popular is SBM that is genetically modified (GMO). During this age, the consumer market requires non-GMO products. Yellow lupin used as a high-protein substitute for SBM in feed has an effect on the quality of meat from broiler ducks. The aim of the study was to analyse and compare meat quality traits in breast and leg muscles as well as fatty acid (FA) composition in breast muscles from ducks fed mixtures containing YL as an alternative to SBM. Two hundred 1-day-old Cherry Valley ducks were kept in pens on litter in two equal dietary groups, four replications with 25 birds per group. The control group (1) received balanced feed containing SBM. The treatment group (2) received balanced feed containing YL. The feed provided to both groups contained 55% of concentrate and 45% of wheat. Birds received feed and water ad libitum and were reared for 8 weeks. After that, 16 ducks (eight from each group) of BW close to the mean for the whole group were slaughtered. Plucked and gutted carcasses were analysed in a laboratory for quality parameters. Meat was analysed for pH, colour, water-holding capacity and drip loss. Samples of breast muscles were analysed for the content of cholesterol, collagen, intramuscular fat and FA composition. The proposed feed mixture containing YL had no impact on meat traits, content of muscles or fat in duck carcasses (P > 0.05). The values of lightness (L*) and yellowness (b*) and collagen content in breast muscles were significantly higher (P < 0.05) in group 2 (YL). A lower ability to retain water, that is, higher water-holding capacity (percentage of water lost from meat) (P < 0.05), was found for leg muscles from group 2 (YL). The content of C16:0, C18:0, C20:4 n-6, C22:4 n-6, C22:5 n-3, total content of saturated fatty acids (SFA), values of atherogenic index and thrombogenic index were significantly lower (P < 0.05) in group 2 (YL) than in group 1 (SBM). The content of C18:2 n-6 and the polyunsaturated fatty acids-to-SFA ratio (P/S) were significantly higher (P < 0.05) in ducks fed the diet with the inclusion of YL. Diets with YL could be proposed as a partial substitute for SBM in duck-rearing

Combining SNP discovery from next-generation sequencing data with bulked segregant analysis (BSA) to fine-map genes in polyploid wheat

<p>Abstract</p> <p>Background</p> <p>Next generation sequencing (NGS) technologies are providing new ways to accelerate fine-mapping and gene isolation in many species. To date, the majority of these efforts have focused on diploid organisms with readily available whole genome sequence information. In this study, as a proof of concept, we tested the use of NGS for SNP discovery in tetraploid wheat lines differing for the previously cloned grain protein content (GPC) gene <it>GPC-B1</it>. Bulked segregant analysis (BSA) was used to define a subset of putative SNPs within the candidate gene region, which were then used to fine-map <it>GPC-B1</it>.</p> <p>Results</p> <p>We used Illumina paired end technology to sequence mRNA (RNAseq) from near isogenic lines differing across a ~30-cM interval including the <it>GPC-B1 </it>locus. After discriminating for SNPs between the two homoeologous wheat genomes and additional quality filtering, we identified inter-varietal SNPs in wheat unigenes between the parental lines. The relative frequency of these SNPs was examined by RNAseq in two bulked samples made up of homozygous recombinant lines differing for their GPC phenotype. SNPs that were enriched at least 3-fold in the corresponding pool (6.5% of all SNPs) were further evaluated. Marker assays were designed for a subset of the enriched SNPs and mapped using DNA from individuals of each bulk. Thirty nine new SNP markers, corresponding to 67% of the validated SNPs, mapped across a 12.2-cM interval including <it>GPC-B1</it>. This translated to 1 SNP marker per 0.31 cM defining the <it>GPC-B1 </it>gene to within 13-18 genes in syntenic cereal genomes and to a 0.4 cM interval in wheat.</p> <p>Conclusions</p> <p>This study exemplifies the use of RNAseq for SNP discovery in polyploid species and supports the use of BSA as an effective way to target SNPs to specific genetic intervals to fine-map genes in unsequenced genomes.</p

Extracellular citrate and metabolic adaptations of cancer cells

It is well established that cancer cells acquire energy via the Warburg effect and oxidative phosphorylation. Citrate is considered to play a crucial role in cancer metabolism by virtue of its production in the reverse Krebs cycle from glutamine. Here, we review the evidence that extracellular citrate is one of the key metabolites of the metabolic pathways present in cancer cells. We review the different mechanisms by which pathways involved in keeping redox balance respond to the need of intracellular citrate synthesis under different extracellular metabolic conditions. In this context, we further discuss the hypothesis that extracellular citrate plays a role in switching between oxidative phosphorylation and the Warburg effect while citrate uptake enhances metastatic activities and therapy resistance. We also present the possibility that organs rich in citrate such as the liver, brain and bones might form a perfect niche for the secondary tumour growth and improve survival of colonising cancer cells. Consistently, metabolic support provided by cancer-associated and senescent cells is also discussed. Finally, we highlight evidence on the role of citrate on immune cells and its potential to modulate the biological functions of pro- and anti-tumour immune cells in the tumour microenvironment. Collectively, we review intriguing evidence supporting the potential role of extracellular citrate in the regulation of the overall cancer metabolism and metastatic activity

Montessori's mediation of meaning: a social semiotic perspective

The distinctive objects designed by Dr Maria Montessori as the centrepiece of her approach to pedagogy are the topic of this study. The Montessori approach to pedagogy, celebrating its centenary in 2007, continues to be used in classrooms throughout the world. Despite such widespread and enduring use, there has been little analysis of the Montessori objects to evaluate or understand their pedagogic impact. This study begins by outlining the provenance of the Montessori objects, reaching the conclusion that the tendency to interpret them from the perspective of the progressive education movement of the early twentieth century fails to provide insights into the developmental potential embodied in the objects. In order to appreciate that potential more fully, the study explores the design of the objects, specifically, the way in which the semiotic qualities embodied in their design orient children to the meanings of educational knowledge. A meta-analytic framework comprising three components is used to analyse the semiotic potential of the Montessori objects as educational artefacts. First, Vygotsky’s model of development is used to analyse the objects as external mediational means and to recognise the objects as complexes of signs materialising educational knowledge. In order to understand how the objects capture, in the form of concrete analogues, the linguistic meanings which construe educational knowledge, systemic functional linguistics, the second component of the framework, is used to achieve a rich and detailed social semiotic analysis of these relations, in particular, material and linguistic representations of abstract educational meanings. Finally, the pedagogic device, a central feature of Bernstein’s sociology of pedagogy, is used to analyse how the Montessori objects re-contextualise educational knowledge as developmental pedagogy. Particular attention is paid to the Montessori literacy pedagogy, in which the study of grammar plays a central role. The study reveals a central design principle which distinguishes the Montessori objects. This principle is the redundant representation of educational knowledge across multiple semiotic modes. Each representation holds constant the underlying meaning relations which construe quanta of educational knowledge, giving children the freedom to engage with this knowledge playfully, independently and successfully. The conclusion drawn from this study is that the design of the Montessori objects represents valuable educational potential which deserves continued investigation, as well as wider recognition and application. To initiate this process, the findings in this study may provide insights which can be used to develop tools for evaluating and enhancing the implementation of Montessori pedagogy in Montessori schools. The findings may also be used to adapt Montessori design principles for the benefit of educators working in non-Montessori contexts, in particular, those educators concerned with developing pedagogies which promote equitable access to educational knowledge

The AGTR1 gene A1166C polymorphism as a risk factor and outcome predictor of primary intracerebral and aneurysmal subarachnoid hemorrhages

Associations between the angiotensin II type 1 receptor (AGTR1) gene A1166C polymorphism and hypertension, aortic abdominal aneurysms (as a risk factor) as well as cardiovascular disorders (as a risk factor and an outcome predictor) have been demonstrated. We aimed to investigate the role of this polymorphism as risk factors and outcome predictors in primary intracerebral hemorrhage (PICH) and aneurysmal subarachnoid hemorrhage (aSAH). We have prospectively recruited 1078 Polish participants to the study: 261 PICH patients, 392 aSAH patients, and 425 unrelated control subjects. The A1166C AGTR1 gene polymorphism was studied using the tetra-primer ARMS-PCR method. Allele and genotype frequencies were compared with other ethnically different populations. The A1166C polymorphism was not associated with the risk of PICH or aSAH. Among the aSAH patients the AA genotype was associated with a good outcome, defined by a Glasgow Outcome Scale of 4 or 5 (p&lt;0.02). The distribution of A1166C genotypes in our cohort did not differ from other white or other populations of European descent. In conclusion, we found an association between the A1166C AGTR1 polymorphism and outcome of aSAH patients, but not with the risk of PICH or aSAH