Evaluation of Dynamic Cell Processes and Behavior Using Video Bioinformatics Tools

Just as body language can reveal a person’s state of well-being, dynamic changes in cell behavior and morphology can be used to monitor processes in cultured cells. This chapter discusses how CL-Quant software, a commercially available video bioinformatics tool, can be used to extract quantitative data on: (1) growth/proliferation, (2) cell and colony migration, (3) reactive oxygen species (ROS) production, and (4) neural differentiation. Protocols created using CL-Quant were used to analyze both single cells and colonies. Time-lapse experiments in which different cell types were subjected to various chemical exposures were done using Nikon BioStations. Proliferation rate was measured in human embryonic stem cell colonies by quantifying colony area (pixels) and in single cells by measuring confluency (pixels). Colony and single cell migration were studied by measuring total displacement (distance between the starting and ending points) and total distance traveled by the colonies/cells. To quantify ROS production, cells were pre-loaded with MitoSOX Red™, a mitochondrial ROS (superoxide) indicator, treated with various chemicals, then total intensity of the red fluorescence was measured in each frame. Lastly, neural stem cells were incubated in differentiation medium for 12 days, and time lapse images were collected daily. Differentiation of neural stem cells was quantified using a protocol that detects young neurons. CLQuant software can be used to evaluate biological processes in living cells, and the protocols developed in this project can be applied to basic research and toxicological studies, or to monitor quality control in culture facilities

Effect of P2X4 and P2X7 receptor antagonism on the pressure diuresis relationship in rats.

Reduced glomerular filtration, hypertension and renal microvascular injury are hallmarks of chronic kidney disease, which has a global prevalence of ~10%. We have shown previously that the Fischer (F344) rat has lower GFR than the Lewis rat, and is more susceptible to renal injury induced by hypertension. In the early stages this injury is limited to the pre-glomerular vasculature. We hypothesized that poor renal hemodynamic function and vulnerability to vascular injury are causally linked and genetically determined. In the present study, normotensive F344 rats had a blunted pressure diuresis relationship, compared with Lewis rats. A kidney microarray was then interrogated using the Endeavour enrichment tool to rank candidate genes for impaired blood pressure control. Two novel candidate genes, P2rx7 and P2rx4, were identified, having a 7- and 3- fold increased expression in F344 rats. Immunohistochemistry localized P2X4 and P2X7 receptor expression to the endothelium of the pre-glomerular vasculature. Expression of both receptors was also found in the renal tubule; however there was no difference in expression profile between strains. Brilliant Blue G (BBG), a relatively selective P2X7 antagonist suitable for use in vivo, was administered to both rat strains. In Lewis rats, BBG had no effect on blood pressure, but increased renal vascular resistance, consistent with inhibition of some basal vasodilatory tone. In F344 rats BBG caused a significant reduction in blood pressure and a decrease in renal vascular resistance, suggesting that P2X7 receptor activation may enhance vasoconstrictor tone in this rat strain. BBG also reduced the pressure diuresis threshold in F344 rats, but did not alter its slope. These preliminary findings suggest a physiological and potential pathophysiological role for P2X7 in controlling renal and/or systemic vascular function, which could in turn affect susceptibility to hypertension-related kidney damage

A Prospective Study of the Association of Metacognitive Beliefs and Processes with Persistent Emotional Distress After Diagnosis of Cancer

Two hundred and six patients, diagnosed with primary breast or prostate cancer completed self-report questionnaires on two occasions: before treatment (T1) and 12 months later (T2). The questionnaires included: the Hospital Anxiety and Depression Scale; Impact of Events Scale; the Metacognitions Questionnaire-30 (MCQ-30) and the Illness Perceptions Questionnaire-revised. A series of regression analyses indicated that metacognitive beliefs at T1 predicted between 14 and 19 % of the variance in symptoms of anxiety, depression and trauma at T2 after controlling for age and gender. For all three outcomes, the MCQ-30 subscale ‘negative beliefs about worry’ made the largest individual contribution with ‘cognitive confidence’ also contributing in each case. For anxiety, a third metacognitive variable, ‘positive beliefs about worry’ also predicted variance in T2 symptoms. In addition, hierarchical analyses indicated that metacognitive beliefs explained a small but significant amount of variance in T2 anxiety (2 %) and T2 depression (4 %) over and above that explained by demographic variables, T1 symptoms and T1 illness perceptions. The findings suggest that modifying metacognitive beliefs and processes has the potential to alleviate distress associated with cancer

Exergetic performance and comparative assessment of bottoming power cycles operating with carbon dioxide–based binary mixture as working fluid

This paper presents CO2-toluene (CO2-C7H8) binary mixture as working fluid to enhance the energetic and exergetic performance of CO2 bottoming power cycles in warm ambient conditions. A criterion for selection of CO2-based binary mixture is defined, and 0.9 CO2/0.1 C7H8 composition is decided based on the required minimum cycle temperature compatible with ambient conditions. Bottoming simple regenerative cycle (BSRC) and bottoming preheating cycle (BPHC) configurations are selected, and their realistic operating conditions are determined based on sensitivity analysis. The performance of bottoming cycles using CO2-C7H8 binary mixture is compared with the bottoming cycles using pure CO2 as working fluid at different ambient temperatures. It is observed that the cycles operating with pure CO2 can only perform better at lower ambient temperature conditions, whereas, at the increased ambient temperatures, bottoming cycles with CO2-C7H8 binary mixture outperform and produce significant gains in exergetic and energetic performance compared with pure CO2 bottoming cycles. A maximum gain of exergetic efficiency for BSRC and BPHC observed is 26.83% and 18.71%, respectively, at an operating ambient temperature of 313 K, whereas an overall gain in energetic efficiencies for BSRC and BPHC observed is 28.92% and 10.12%, respectively. Taking into consideration thermodynamic performance, overall UA (product of overall heat transfer coefficient and heat transfer area for the heat exchanger) and specific investment cost, BPHC configuration is suggested as reasonable choice for higher ambient temperature conditions

Dilation of the ascending aorta in Turner syndrome - a prospective cardiovascular magnetic resonance study

<p>Abstract</p> <p>Background</p> <p>The risk of aortic dissection is 100-fold increased in Turner syndrome (TS). Unfortunately, risk stratification is inadequate due to a lack of insight into the natural course of the syndrome-associated aortopathy. Therefore, this study aimed to prospectively assess aortic dimensions in TS.</p> <p>Methods</p> <p>Eighty adult TS patients were examined twice with a mean follow-up of 2.4 ± 0.4 years, and 67 healthy age and gender-matched controls were examined once. Aortic dimensions were measured at nine predefined positions using 3D, non-contrast and free-breathing cardiovascular magnetic resonance. Transthoracic echocardiography and 24-hour ambulatory blood pressure were also performed.</p> <p>Results</p> <p>At baseline, aortic diameters (body surface area indexed) were larger at all positions in TS. Aortic dilation was more prevalent at all positions excluding the distal transverse aortic arch. Aortic diameter increased in the aortic sinus, at the sinotubular junction and in the mid-ascending aorta with growth rates of 0.1 - 0.4 mm/year. Aortic diameters at all other positions were unchanged. The bicuspid aortic valve conferred higher aortic sinus growth rates (p < 0.05). No other predictors of aortic growth were identified.</p> <p>Conclusion</p> <p>A general aortopathy is present in TS with enlargement of the ascending aorta, which is accelerated in the presence of a bicuspid aortic valve.</p

The pairwise disconnectivity index as a new metric for the topological analysis of regulatory networks

<p>Abstract</p> <p>Background</p> <p>Currently, there is a gap between purely theoretical studies of the topology of large bioregulatory networks and the practical traditions and interests of experimentalists. While the theoretical approaches emphasize the global characterization of regulatory systems, the practical approaches focus on the role of distinct molecules and genes in regulation. To bridge the gap between these opposite approaches, one needs to combine 'general' with 'particular' properties and translate abstract topological features of large systems into testable functional characteristics of individual components. Here, we propose a new topological parameter – the pairwise disconnectivity index of a network's element – that is capable of such bridging.</p> <p>Results</p> <p>The pairwise disconnectivity index quantifies how crucial an individual element is for sustaining the communication ability between connected pairs of vertices in a network that is displayed as a directed graph. Such an element might be a vertex (i.e., molecules, genes), an edge (i.e., reactions, interactions), as well as a group of vertices and/or edges. The index can be viewed as a measure of topological redundancy of regulatory paths which connect different parts of a given network and as a measure of sensitivity (robustness) of this network to the presence (absence) of each individual element. Accordingly, we introduce the notion of a path-degree of a vertex in terms of its corresponding incoming, outgoing and mediated paths, respectively. The pairwise disconnectivity index has been applied to the analysis of several regulatory networks from various organisms. The importance of an individual vertex or edge for the coherence of the network is determined by the particular position of the given element in the whole network.</p> <p>Conclusion</p> <p>Our approach enables to evaluate the effect of removing each element (i.e., vertex, edge, or their combinations) from a network. The greatest potential value of this approach is its ability to systematically analyze the role of every element, as well as groups of elements, in a regulatory network.</p

Genetic risk factors for body mass index and obesity in an ethnically diverse population: results from the Population Architecture using Genomics and Epidemiology (PAGE) Study

Several genome-wide association studies (GWAS) have demonstrated that common genetic variants contribute to obesity. However, studies of this complex trait have focused on ancestrally European populations, despite the high prevalence of obesity in some minority groups. As part of the ‘Population Architecture using Genomics and Epidemiology (PAGE)’ Consortium, we investigated the association between thirteen GWAS-identified SNPs and BMI and obesity in 69,775 subjects, including 6,149 American Indians, 15,415 African-Americans, 2,438 East Asians, 7,346 Hispanics, 604 Pacific Islanders, and 37,823 European Americans. For the BMI-increasing allele of each SNP, we calculated beta coefficients using linear regression (for BMI) and risk estimates using logistic regression (for obesity defined as BMI ≥ 30) followed by fixed-effects meta-analysis to combine results across PAGE sites. Analyses stratified by racial/ethnic group assumed an additive genetic model and adjusted for age, sex, and current smoking. We defined “replicating SNPs” (in European Americans) and “generalizing SNPs” (in other racial/ethnic groups) as those associated with an allele frequency-specific increase in BMI. By this definition, we replicated 9/13 SNP associations (5 out of 8 loci) in European Americans. We also generalized 8/13 SNP associations (5/8 loci) in East Asians, 7/13 (5/8 loci) in African Americans, 6/13 (4/8 loci) in Hispanics, 5/8 in Pacific Islanders (5/8 loci), and 5/9 (4/8 loci) in American Indians. Linkage disequilibrium patterns suggest that tagSNPs selected for European Americans may not adequately tag causal variants in other ancestry groups. Accordingly, fine-mapping in large samples is needed to comprehensively explore these loci in diverse populations