Sorting of chromosomes by magnetic separation

Chromosomes were isolated from Chinese hamster x human hybrid cell lines containing four and nine human chromosomes. Human genomic DNA was biotinylated by nick translation and used to label the human chromosomes by in situ hybridization in suspension. Streptavidin was covalently coupled to the surface of magnetic beads and these were incubated with the hybridized chromosomes. The human chromosomes were bound to the magnetic beads through the strong biotin-streptavidin complex and then rapidly separated from nonlabeled Chinese hamster chromosomes by a simple permanent magnet. The hybridization was visualized by additional binding of avidin-FITC (fluorescein) to the unoccupied biotinylated human DNA bound to the human chromosomes. After magnetic separation, up to 98% of the individual chromosomes attached to magnetic beads were classified as human chromosomes by fluorescence microscopy

Thermographic imaging in sports and exercise medicine: A Delphi study and consensus statement on the measurement of human skin temperature

This is an accepted manuscript of an article published by Elsevier in Journal of Thermal Biology on 18/07/2017, available online: https://doi.org/10.1016/j.jtherbio.2017.07.006 The accepted version of the publication may differ from the final published version.© 2017 Elsevier Ltd The importance of using infrared thermography (IRT) to assess skin temperature (tsk) is increasing in clinical settings. Recently, its use has been increasing in sports and exercise medicine; however, no consensus guideline exists to address the methods for collecting data in such situations. The aim of this study was to develop a checklist for the collection of tsk using IRT in sports and exercise medicine. We carried out a Delphi study to set a checklist based on consensus agreement from leading experts in the field. Panelists (n  =  24) representing the areas of sport science (n = 8; 33%), physiology (n = 7; 29%), physiotherapy (n = 3; 13%) and medicine (n = 6; 25%), from 13 different countries completed the Delphi process. An initial list of 16 points was proposed which was rated and commented on by panelists in three rounds of anonymous surveys following a standard Delphi procedure. The panel reached consensus on 15 items which encompassed the participants’ demographic information, camera/room or environment setup and recording/analysis of tsk using IRT. The results of the Delphi produced the checklist entitled “Thermographic Imaging in Sports and Exercise Medicine (TISEM)” which is a proposal to standardize the collection and analysis of tsk data using IRT. It is intended that the TISEM can also be applied to evaluate bias in thermographic studies and to guide practitioners in the use of this technique.Published versio

Predicted risks of radiogenic cardiac toxicity in two pediatric patients undergoing photon or proton radiotherapy

BACKGROUND: Hodgkin disease (HD) and medulloblastoma (MB) are common malignancies found in children and young adults, and radiotherapy is part of the standard treatment. It was reported that these patients who received radiation therapy have an increased risk of cardiovascular late effects. We compared the predicted risk of developing radiogenic cardiac toxicity after photon versus proton radiotherapies for a pediatric patient with HD and a pediatric patient with MB. METHODS: In the treatment plans, each patient’s heart was contoured in fine detail, including substructures of the pericardium and myocardium. Risk calculations took into account both therapeutic and stray radiation doses. We calculated the relative risk (RR) of cardiac toxicity using a linear risk model and the normal tissue complication probability (NTCP) values using relative seriality and Lyman models. Uncertainty analyses were also performed. RESULTS: The RR values of cardiac toxicity for the HD patient were 7.27 (proton) and 8.37 (photon), respectively; the RR values for the MB patient were 1.28 (proton) and 8.39 (photon), respectively. The predicted NTCP values for the HD patient were 2.17% (proton) and 2.67% (photon) for the myocardium, and were 2.11% (proton) and 1.92% (photon) for the whole heart. The predicted ratios of NTCP values (proton/photon) for the MB patient were much less than unity. Uncertainty analyses revealed that the predicted ratio of risk between proton and photon therapies was sensitive to uncertainties in the NTCP model parameters and the mean radiation weighting factor for neutrons, but was not sensitive to heart structure contours. The qualitative findings of the study were not sensitive to uncertainties in these factors. CONCLUSIONS: We conclude that proton and photon radiotherapies confer similar predicted risks of cardiac toxicity for the HD patient in this study, and that proton therapy reduced the predicted risk for the MB patient in this study

Genetically Programmed Differences in Epidermal Host Defense between Psoriasis and Atopic Dermatitis Patients

In the past decades, chronic inflammatory diseases such as psoriasis, atopic dermatitis, asthma, Crohn’s disease and celiac disease were generally regarded as immune-mediated conditions involving activated T-cells and proinflammatory cytokines produced by these cells. This paradigm has recently been challenged by the finding that mutations and polymorphisms in epithelium-expressed genes involved in physical barrier function or innate immunity, are risk factors of these conditions. We used a functional genomics approach to analyze cultured keratinocytes from patients with psoriasis or atopic dermatitis and healthy controls. First passage primary cells derived from non-lesional skin were stimulated with pro-inflammatory cytokines, and expression of a panel of 55 genes associated with epidermal differentiation and cutaneous inflammation was measured by quantitative PCR. A subset of these genes was analyzed at the protein level. Using cluster analysis and multivariate analysis of variance we identified groups of genes that were differentially expressed, and could, depending on the stimulus, provide a disease-specific gene expression signature. We found particularly large differences in expression levels of innate immunity genes between keratinocytes from psoriasis patients and atopic dermatitis patients. Our findings indicate that cell-autonomous differences exist between cultured keratinocytes of psoriasis and atopic dermatitis patients, which we interpret to be genetically determined. We hypothesize that polymorphisms of innate immunity genes both with signaling and effector functions are coadapted, each with balancing advantages and disadvantages. In the case of psoriasis, high expression levels of antimicrobial proteins genes putatively confer increased protection against microbial infection, but the biological cost could be a beneficial system gone awry, leading to overt inflammatory disease

Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis

The mitotic checkpoint prevents mitotic exit until all chromosomes are attached to spindle microtubules. Aurora B kinase indirectly invokes this checkpoint by destabilizing incorrect attachments; however, a more direct role remains controversial. In contrast, activity of the kinase Mps1 is indispensible for the mitotic checkpoint. Here we show that Aurora B and Hec1 are needed for efficient Mps1 recruitment to unattached kinetochores, allowing rapid Mps1 activation at the onset of mitosis. Live monitoring of cyclin B degradation reveals that this is essential to establish the mitotic checkpoint quickly at the start of mitosis. Delayed Mps1 activation and checkpoint establishment upon Aurora B inhibition or Hec1 depletion are rescued by tethering Mps1 to kinetochores, demonstrating that Mps1 recruitment is the primary role of Aurora B and Hec1 in mitotic checkpoint signalling. These data demonstrate a direct role for Aurora B in initiating the mitotic checkpoint rapidly at the onset of mitosis

An empirical Bayes model for gene expression and methylation profiles in antiestrogen resistant breast cancer

<p>Abstract</p> <p>Background</p> <p>The nuclear transcription factor estrogen receptor alpha (ER-alpha) is the target of several antiestrogen therapeutic agents for breast cancer. However, many ER-alpha positive patients do not respond to these treatments from the beginning, or stop responding after being treated for a period of time. Because of the association of gene transcription alteration and drug resistance and the emerging evidence on the role of DNA methylation on transcription regulation, understanding of these relationships can facilitate development of approaches to re-sensitize breast cancer cells to treatment by restoring DNA methylation patterns.</p> <p>Methods</p> <p>We constructed a hierarchical empirical Bayes model to investigate the simultaneous change of gene expression and promoter DNA methylation profiles among wild type (WT) and OHT/ICI resistant MCF7 breast cancer cell lines.</p> <p>Results</p> <p>We found that compared with the WT cell lines, almost all of the genes in OHT or ICI resistant cell lines either do not show methylation change or hypomethylated. Moreover, the correlations between gene expression and methylation are quite heterogeneous across genes, suggesting the involvement of other factors in regulating transcription. Analysis of our results in combination with H3K4me2 data on OHT resistant cell lines suggests a clear interplay between DNA methylation and H3K4me2 in the regulation of gene expression. For hypomethylated genes with alteration of gene expression, most (~80%) are up-regulated, consistent with current view on the relationship between promoter methylation and gene expression.</p> <p>Conclusions</p> <p>We developed an empirical Bayes model to study the association between DNA methylation in the promoter region and gene expression. Our approach generates both global (across all genes) and local (individual gene) views of the interplay. It provides important insight on future effort to develop therapeutic agent to re-sensitize breast cancer cells to treatment.</p

Why Um Helps Auditory Word Recognition: The Temporal Delay Hypothesis

Several studies suggest that speech understanding can sometimes benefit from the presence of filled pauses (uh, um, and the like), and that words following such filled pauses are recognised more quickly. Three experiments examined whether this is because filled pauses serve to delay the onset of upcoming words and these delays facilitate auditory word recognition, or whether the fillers themselves serve to signal upcoming delays in a way which informs listeners' reactions. Participants viewed pairs of images on a computer screen, and followed recorded instructions to press buttons corresponding to either an easy (unmanipulated, with a high-frequency name) or a difficult (visually blurred, low-frequency) image. In all three experiments, participants were faster to respond to easy images. In 50% of trials in each experiment, the name of the image was directly preceded by a delay; in the remaining trials an equivalent delay was included earlier in the instruction. Participants were quicker to respond when a name was directly preceded by a delay, regardless of whether this delay was filled with a spoken um, was silent, or contained an artificial tone. This effect did not interact with the effect of image difficulty, nor did it change over the course of each experiment. Taken together, our consistent finding that delays of any kind help word recognition indicates that natural delays such as fillers need not be seen as ‘signals’ to explain the benefits they have to listeners' ability to recognise and respond to the words which follow them

The Temporal Pattern of Mating Behavior of the Fruit Fly, Anastrepha zenildae in the Laboratory

The state of Rio Grande do Norte is an important fruit-producing and exporting area in northeastern Brazil. The success of this industry depends on fruit fly population control, especially in fly-free exporting zones. However, many fruits are not exported because of quarantine restrictions imposed by importing countries. A survey in the state has detected a considerable increase of the fruit fly, Anastrepha zenildae Zucchi (Diptera: Tephritidae), probably a result of the introduction of irrigated guava orchards that make fruit available all year. Knowledge of the sexual behavior of Tephritidae has great importance to pest control programs, particularly those that employ the Sterile Insect Technique. In order to characterize the reproductive behavior of A. zenildae, 32 individuals (16 males; 16 females) in each of six generations were submitted to an artificial 12:12 L:D cycle (750: < 1 lux, lights on 07:00–19:00) and observed over their lifetimes. The courtship and copulation occurred in leks and the episodes varied with the time of day, courtship being most frequent between Zeitgeber time (ZT) 3 and ZT 7, peaking at ZT 5–6. Copulations occurred between ZT 2 and ZT 8, with a higher frequency between ZT 5–7 and a peak at ZT 6. Mean duration was 0.28 ± 0.03 min/male (range: 5–163 min). Males in the leks attempted to copulate mainly between ZT 3 and ZT 7 with a peak at ZT 6, and males outside leks peaked at ZT 7. The different timing of sexual behaviors among related sympatric species, including A. zenildae, may contribute to species isolation