Relationships between CYP2D6 phenotype, breast cancer and hot flushes in women at high risk of breast cancer receiving prophylactic tamoxifen: results from the IBIS-I trial

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A mutate-and-map protocol for inferring base pairs in structured RNA

Chemical mapping is a widespread technique for structural analysis of nucleic acids in which a molecule's reactivity to different probes is quantified at single-nucleotide resolution and used to constrain structural modeling. This experimental framework has been extensively revisited in the past decade with new strategies for high-throughput read-outs, chemical modification, and rapid data analysis. Recently, we have coupled the technique to high-throughput mutagenesis. Point mutations of a base-paired nucleotide can lead to exposure of not only that nucleotide but also its interaction partner. Carrying out the mutation and mapping for the entire system gives an experimental approximation of the molecules contact map. Here, we give our in-house protocol for this mutate-and-map strategy, based on 96-well capillary electrophoresis, and we provide practical tips on interpreting the data to infer nucleic acid structure.Comment: 22 pages, 5 figure

Massively Parallel RNA Chemical Mapping with a Reduced Bias MAP-seq Protocol

Chemical mapping methods probe RNA structure by revealing and leveraging correlations of a nucleotide's structural accessibility or flexibility with its reactivity to various chemical probes. Pioneering work by Lucks and colleagues has expanded this method to probe hundreds of molecules at once on an Illumina sequencing platform, obviating the use of slab gels or capillary electrophoresis on one molecule at a time. Here, we describe optimizations to this method from our lab, resulting in the MAP-seq protocol (Multiplexed Accessibility Probing read out through sequencing), version 1.0. The protocol permits the quantitative probing of thousands of RNAs at once, by several chemical modification reagents, on the time scale of a day using a table-top Illumina machine. This method and a software package MAPseeker (http://simtk.org/home/map_seeker) address several potential sources of bias, by eliminating PCR steps, improving ligation efficiencies of ssDNA adapters, and avoiding problematic heuristics in prior algorithms. We hope that the step-by-step description of MAP-seq 1.0 will help other RNA mapping laboratories to transition from electrophoretic to next-generation sequencing methods and to further reduce the turnaround time and any remaining biases of the protocol.Comment: 22 pages, 5 figure

Upper extremity impairments in women with or without lymphedema following breast cancer treatment

Breast-cancer-related lymphedema affects ∼25% of breast cancer (BC) survivors and may impact use of the upper limb during activity. The purpose of this study is to compare upper extremity (UE) impairment and activity between women with and without lymphedema after BC treatment. 144 women post BC treatment completed demographic, symptom, and Disability of Arm-Shoulder-Hand (DASH) questionnaires. Objective measures included Purdue pegboard, finger-tapper, Semmes-Weinstein monofilaments, vibration perception threshold, strength, range of motion (ROM), and volume. Women with lymphedema had more lymph nodes removed (p < .001), more UE symptoms (p < .001), higher BMI (p = .041), and higher DASH scores (greater limitation) (p < .001). For all participants there was less strength (elbow flexion, wrist flexion, grip), less shoulder ROM, and decreased sensation at the medial upper arm (p < .05) in the affected UE. These differences were greater in women with lymphedema, particularly in shoulder abduction ROM (p < .05). Women with lymphedema had bilaterally less elbow flexion strength and shoulder ROM (p < .05). Past diagnosis of lymphedema, grip strength, shoulder abduction ROM, and number of comorbidities contributed to the variance in DASH scores (R 2 of 0.463, p < .001). UE impairments are found in women following treatment for BC. Women with lymphedema have greater UE impairment and limitation in activities than women without. Many of these impairments are amenable to prevention measures or treatment, so early detection by health care providers is essential

The Evolution of Gene Expression QTL in Saccharomyces cerevisiae

Understanding the evolutionary forces that influence patterns of gene expression variation will provide insights into the mechanisms of evolutionary change and the molecular basis of phenotypic diversity. To date, studies of gene expression evolution have primarily been made by analyzing how gene expression levels vary within and between species. However, the fundamental unit of heritable variation in transcript abundance is the underlying regulatory allele, and as a result it is necessary to understand gene expression evolution at the level of DNA sequence variation. Here we describe the evolutionary forces shaping patterns of genetic variation for 1206 cis-regulatory QTL identified in a cross between two divergent strains of Saccharomyces cerevisiae. We demonstrate that purifying selection against mildly deleterious alleles is the dominant force governing cis-regulatory evolution in S. cerevisiae and estimate the strength of selection. We also find that essential genes and genes with larger codon bias are subject to slightly stronger cis-regulatory constraint and that positive selection has played a role in the evolution of major trans-acting QTL

Impact of seasonal variation, age and smoking status on human semen parameters: The Massachusetts General Hospital experience

BACKGROUND: To investigate the relationship of human semen parameters with season, age and smoking status. METHODS: The present study used data from subjects recruited into an ongoing cross-sectional study on the relationship between environmental agents and semen characteristics. Our population consisted of 306 patients who presented to the Vincent Memorial Andrology Laboratory of Massachusetts General Hospital for semen evaluation. Sperm concentration and motility were measured with computer aided sperm analysis (CASA). Sperm morphology was scored using Tygerberg Kruger strict criteria. Regression analyses were used to investigate the relationships between semen parameters and season, age and smoking status, adjusting for abstinence interval. RESULTS: Sperm concentration in the spring was significantly higher than in winter, fall and summer (p < 0.05). There was suggestive evidence of higher sperm motility and percent of sperm with normal morphology in the spring than in the other seasons. There were no statistically significant relationships between semen parameters and smoking status, though current smokers tended to have lower sperm concentration. We also did not find a statistically significant relationship between age and semen parameters. CONCLUSIONS: We found seasonal variations in sperm concentration and suggestive evidence of seasonal variation in sperm motility and percent sperm with normal morphology. Although smoking status was not a significant predictor of semen parameters, this may have been due to the small number of current smokers in the study

Choice of Bacterial Growth Medium Alters the Transcriptome and Phenotype of Salmonella enterica Serovar Typhimurium

The type of bacterial culture medium is an important consideration during design of any experimental protocol. The aim of this study was to understand the impact of medium choice on bacterial gene expression and physiology by comparing the transcriptome of Salmonella enterica SL1344 after growth in the widely used LB broth or the rationally designed MOPS minimal medium. Transcriptomics showed that after growth in MOPS minimal media, compared to LB, there was increased expression of 42 genes involved in amino acid synthesis and 23 genes coding for ABC transporters. Seven flagellar genes had decreased expression after growth in MOPS minimal medium and this correlated with a decreased motility. In both MOPS minimal medium and MEM expression of genes from SPI-2 was increased and the adhesion of S. Typhimurium to intestinal epithelial cells was higher compared to the levels after growth in LB. However, SL1344 invasion was not significantly altered by growth in either MOPs minimal media or MEM. Expression of SPI-2 was also measured using chromosomal GFP reporter fusions followed by flow cytometry which showed, for the first time, that the reduction in SPI-2 transcript after growth in different media related to a reduction in the proportion of the bacterial population expressing SPI-2. These data highlight the profound differences in the global transcriptome after in vitro growth in different media and show that choice of medium should be considered carefully during experimental design, particularly when virulence related phenotypes are being measured

Energy input and dissipation in a temperate lake during the spring transition

ADCP and temperature chain measurements have been used to estimate the rate of energy input by wind stress to the water surface in the south basin of Windermere. The energy input from the atmosphere was found to increase markedly as the lake stratified in spring. The efficiency of energy transfer (Eff), defined as the ratio of the rate of working in near-surface waters (RW) to that above the lake surface (P10), increased from ∼0.0013 in vertically homogenous conditions to ∼0.0064 in the first 40 days of the stratified regime. A maximum value of Eff∼0.01 was observed when, with increasing stratification, the first mode internal seiche period decreased to match the diurnal wind period of 24 h. The increase in energy input, following the onset of stratification was reflected in enhancement of the mean depth-varying kinetic energy without a corresponding increase in wind forcing. Parallel estimates of energy dissipation in the bottom boundary layer, based on determination of the structure function show that it accounts for ∼15% of RW in stratified conditions. The evolution of stratification in the lake conforms to a heating stirring model which indicates that mixing accounts for ∼21% of RW. Taken together, these estimates of key energetic parameters point the way to the development of full energy budgets for lakes and shallow seas

Alzheimer's disease: new diagnostic and therapeutic tools

On March 19, 2008 a Symposium on Pathophysiology of Ageing and Age-Related diseases was held in Palermo, Italy. Here, the lectures of M. Racchi on History and future perspectives of Alzheimer Biomarkers and of G. Scapagnini on Cellular Stress Response and Brain Ageing are summarized. Alzheimer's disease (AD) is a heterogeneous and progressive neurodegenerative disease, which in Western society mainly accounts for clinica dementia. AD prevention is an important goal of ongoing research. Two objectives must be accomplished to make prevention feasible: i) individuals at high risk of AD need to be identified before the earliest symptoms become evident, by which time extensive neurodegeneration has already occurred and intervention to prevent the disease is likely to be less successful and ii) safe and effective interventions need to be developed that lead to a decrease in expression of this pathology. On the whole, data here reviewed strongly suggest that the measurement of conformationally altered p53 in blood cells has a high ability to discriminate AD cases from normal ageing, Parkinson's disease and other dementias. On the other hand, available data on the involvement of curcumin in restoring cellular homeostasis and rebalancing redox equilibrium, suggest that curcumin might be a useful adjunct in the treatment of neurodegenerative illnesses characterized by inflammation, such as AD