bluuebunny/crossref_metadata_2025 huggingface dataset:
Emotional Reconciliation
Authors: Hutchison Emma , Bleiker Roland
Published: August 2008

This article examines the public significance of emotions, most specifically their role in constituting identity and community in the wake of political violence and trauma. It offers a conceptual engagement with processes of healing and reconciliation, showing that emotions are central to how societies experience and work through the legacy of catastrophe. In many instances, political actors deal with the legacy of trauma in restorative ways, by re-imposing the order that has been violated. Emotions can in this way be directed by elites who are concerned with reinstating political stability and social control. Healing often becomes more about retribution and revenge, rather than a long-term project begetting peace, collaboration and emotional catharsis. The emotions triggered by trauma thus tend to perpetuate existing antagonisms, further entrenching the disingenuous perceptions of identity that may have created violence in the first place. Surveying this process, this article suggests that scholars of politics and reconciliation need to be more attentive to the role emotion plays in shaping particular forms of community. Doing so requires a systematic understanding not only of the feelings associated with first-hand experiences of trauma, but also of the manner in which these affective reactions can spread and generate collective emotions, thus producing new forms of antagonism. Addressing this challenge, the authors explore how a more conscious and active appreciation of the whole spectrum of emotions — not only anger and fear, for instance, but also empathy, compassion and wonder — may facilitate more lasting and ingenuous forms of social healing and reconciliation.
Does Television Crowd Out Spectators?
Authors: Allan Grant , Roy Graeme
Published: August 2008

This paper examines the impact of live television coverage on attendance at Scottish Premier League soccer matches during the 2002—2003 season. The authors exploit a rich data set which distinguishes match-day attendance into three groups: season ticket holders, pay-at-the-gate home team supporters, and pay-at-the-gate visiting team supporters. This examination of these categories is the first study of its kind. The results indicate matches broadcast live reduce pay-at-the-gate home team supporters by 30%. These results suggest that league administrators and club owners must consider the impact on clubs' traditional supporters when negotiating future broadcasting rights.
Effective dasatinib uptake may occur without human organic cation transporter 1 (hOCT1): implications for the treatment of imatinib-resistant chronic myeloid leukemia
Authors: Giannoudis Athina , Davies Andrea , Lucas Claire M. , Harris Robert J. , Pirmohamed Munir , Clark Richard E.
Published: August 2008

We have previously shown that imatinib uptake into chronic myeloid leukemia (CML) cells is dependent on human organic cation transporter 1 (hOCT1; SLC22A1), and that low hOCT1 expression is an important determinant of clinical outcome to imatinib treatment. We hypothesized that dasatinib might be transported differently than imatinib, possibly accounting for its favorable effects in imatinib-resistant patients. 14C-dasatinib uptake was greater in KCL22-transfected cells with pcDNA3-hOCT1 plasmid (high hOCT1-expressing cells) than in control cells (P = .02). However, hOCT inhibitors did not decrease dasatinib uptake into either control or primary cells, in contrast to their block on imatinib uptake. Dasa-tinib decreased the level of phosphorylated CrkL to 49.9% in control and 40.3% in high hOCT1-expressing cells. Dasa-tinib efflux was investigated in confluent ABCB1-transfected MDCKII cell monolayers. Both dasatinib and imatinib were transported from the basal to the apical layer, indicating that they were transported by ABCB1, which was confirmed using the ABCB1 inhibitor PSC833 (P = .001 and P < .001, respectively). Compared with imatinib, dasatinib achieved superior intracellular levels and BCR-ABL suppression even in cells with low or blocked hOCT1. Efflux of dasatinib and imatinib appear similar via ABCB1. Dasatinib may therefore offer an advantage over imatinib in patients with low hOCT1 expression.
The tetraspanin CD63 is involved in granule targeting of neutrophil elastase
Authors: Källquist Linda , Hansson Markus , Persson Ann-Maj , Janssen Hans , Calafat Jero , Tapper Hans , Olsson Inge
Published: August 2008

Targeting mechanisms of neutrophil elastase (NE) and other luminal proteins stored in myeloperoxidase (MPO)–positive secretory lysosomes/primary granules of neutrophils are unknown. These granules contain an integral membrane protein, CD63, with an adaptor protein-3–dependent granule delivery system. Therefore, we hypothesized that CD63 cooperates in granule delivery of the precursor of NE (proNE). Supporting this hypothesis, an association was demonstrated between CD63 and proNE upon coexpression in COS cells. This also involved augmented cellular retention of proNE requiring intact large extracellular loop of CD63. Furthermore, depletion of CD63 in promyelocytic HL-60 cells with RNA interference or a CD63 mutant caused reduction of cellular NE. However, the proNE steady-state level was similar to wild type in CD63-depleted clones, making it feasible to examine possible effects of CD63 on NE trafficking. Thus, depletion of CD63 led to reduced processing of proNE into mature NE and reduced constitutive secretion. Furthermore, CD63-depleted cells showed a lack of morphologically normal granules, but contained MPO-positive cytoplasmic vacuoles with a lack of proNE and NE. Collectively, our data suggest that granule proteins may cooperate in targeting; CD63 can be involved in ER or Golgi export, cellular retention, and granule targeting of proNE before storage as mature NE.
CD40: an upstream master switch for endothelial cell activation uncovered by RNAi-coupled transcriptional profiling
Authors: Pluvinet Raquel , Olivar Rut , Krupinski Jerzy , Herrero-Fresneda Inmaculada , Luque Anna , Torras Joan , Cruzado Josep M. , Grinyó Josep M. , Sumoy Lauro , Aran Josep M.
Published: August 2008

The CD40-CD154 dyad seems to play a prominent role fostering the immune-inflammatory response triggered by endothelial cell (EC)–T-cell communication. To delineate comprehensively the involvement of CD40 (TNFRSF5) in EC activation, we combined RNAi-mediated CD40 knockdown with comparative genome-wide transcriptional profiling of ECs interacting with (CD154+) T cells. We report the initiation of a profound stress response in ECs upon CD40-CD154 engagement through early up-regulation of, among others, the major proinflammatory NF-κB and MAPK/SAPK pathways and their associated transcription factors. Moreover, we have identified novel genes regulated through the CD40-CD154 interaction, and pathways previously unrecognized to be induced by CD40 signaling in ECs. Thus, we document a significant down-regulation of endothelial APLN by CD40-CD154 interaction, TNFα/IFNγ exposure, and in immune-inflammatory pathologies, which could lead to hemodynamic dysfunction. Conversely, CD40-mediated up-regulation of the viral immune surveillance system, notably TLR3, IFIH1, RIG-I, and RNASEL, establishes a reverse link from adaptive to innate immunity in ECs. Moreover, systematic enrichment analysis substantiates endothelial CD40 involvement in the transcriptional regulation of gene networks associated with adhesion and motility, immunity, cell fate control, hemostasis, and metabolism. Our study also highlights the anti-inflammatory potential of RNAi-mediated CD40 inhibition, and the relevance of CD40 signaling for therapeutic intervention.
Single cell cytometry of protein function in RNAi treated cells and in native populations
Authors: LaPan Peter , Zhang Jing , Pan Jing , Hill Andrew , Haney Steven A
Published: August 2008

Background

High Content Screening has been shown to improve results of RNAi and other perturbations, however significant intra-sample heterogeneity is common and can complicate some analyses. Single cell cytometry can extract important information from subpopulations within these samples. Such approaches are important for immune cells analyzed by flow cytometry, but have not been broadly available for adherent cells that are critical to the study of solid-tumor cancers and other disease models.

Results

We have directly quantitated the effect of resolving RNAi treatments at the single cell level in experimental systems for both exogenous and endogenous targets. Analyzing the effect of an siRNA that targets GFP at the single cell level permits a stronger measure of the absolute function of the siRNA by gating to eliminate background levels of GFP intensities. Extending these methods to endogenous proteins, we have shown that well-level results of the knockdown of PTEN results in an increase in phospho-S6 levels, but at the single cell level, the correlation reveals the role of other inputs into the pathway. In a third example, reduction of STAT3 levels by siRNA causes an accumulation of cells in the G1 phase of the cell cycle, but does not induce apoptosis or necrosis when compared to control cells that express the same levels of STAT3. In a final example, the effect of reduced p53 levels on increased adriamycin sensitivity for colon carcinoma cells was demonstrated at the whole-well level using siRNA knockdown and in control and untreated cells at the single cell level.

Conclusion

We find that single cell analysis methods are generally applicable to a wide range of experiments in adherent cells using technology that is becoming increasingly available to most laboratories. It is well-suited to emerging models of signaling dysfunction, such as oncogene addition and oncogenic shock. Single cell cytometry can demonstrate effects on cell function for protein levels that differ by as little as 20%. Biological differences that result from changes in protein level or pathway activation state can be modulated directly by RNAi treatment or extracted from the natural variability intrinsic to cells grown under normal culture conditions.
Differential control of Zap1-regulated genes in response to zinc deficiency in Saccharomyces cerevisiae
Authors: Wu Chang-Yi , Bird Amanda J , Chung Lisa M , Newton Michael A , Winge Dennis R , Eide David J
Published: August 2008

Background

The Zap1 transcription factor is a central player in the response of yeast to changes in zinc status. We previously used transcriptome profiling with DNA microarrays to identify 46 potential Zap1 target genes in the yeast genome. In this new study, we used complementary methods to identify additional Zap1 target genes.

Results

With alternative growth conditions for the microarray experiments and a more sensitive motif identification algorithm, we identified 31 new potential targets of Zap1 activation. Moreover, an analysis of the response of Zap1 target genes to a range of zinc concentrations and to zinc withdrawal over time demonstrated that these genes respond differently to zinc deficiency. Some genes are induced under mild zinc deficiency and act as a first line of defense against this stress. First-line defense genes serve to maintain zinc homeostasis by increasing zinc uptake, and by mobilizing and conserving intracellular zinc pools. Other genes respond only to severe zinc limitation and act as a second line of defense. These second-line defense genes allow cells to adapt to conditions of zinc deficiency and include genes involved in maintaining secretory pathway and cell wall function, and stress responses.

Conclusion

We have identified several new targets of Zap1-mediated regulation. Furthermore, our results indicate that through the differential regulation of its target genes, Zap1 prioritizes mechanisms of zinc homeostasis and adaptive responses to zinc deficiency.
Sequence conservation and combinatorial complexity of Drosophila neural precursor cell enhancers
Authors: Brody Thomas , Rasband Wayne , Baler Kevin , Kuzin Alexander , Kundu Mukta , Odenwald Ward F
Published: August 2008

Background

The presence of highly conserved sequences within cis-regulatory regions can serve as a valuable starting point for elucidating the basis of enhancer function. This study focuses on regulation of gene expression during the early events of Drosophila neural development. We describe the use of EvoPrinter and cis-Decoder, a suite of interrelated phylogenetic footprinting and alignment programs, to characterize highly conserved sequences that are shared among co-regulating enhancers.

Results

Analysis of in vivo characterized enhancers that drive neural precursor gene expression has revealed that they contain clusters of highly conserved sequence blocks (CSBs) made up of shorter shared sequence elements which are present in different combinations and orientations within the different co-regulating enhancers; these elements contain either known consensus transcription factor binding sites or consist of novel sequences that have not been functionally characterized. The CSBs of co-regulated enhancers share a large number of sequence elements, suggesting that a diverse repertoire of transcription factors may interact in a highly combinatorial fashion to coordinately regulate gene expression. We have used information gained from our comparative analysis to discover an enhancer that directs expression of the nervy gene in neural precursor cells of the CNS and PNS.

Conclusion

The combined use EvoPrinter and cis-Decoder has yielded important insights into the combinatorial appearance of fundamental sequence elements required for neural enhancer function. Each of the 30 enhancers examined conformed to a pattern of highly conserved blocks of sequences containing shared constituent elements. These data establish a basis for further analysis and understanding of neural enhancer function.
Temporal gene expression profiling reveals CEBPD as a candidate regulator of brain disease in prosaposin deficient mice
Authors: Sun Ying , Jia Li , Williams Michael T , Zamzow Matt , Ran Huimin , Quinn Brian , Aronow Bruce J , Vorhees Charles V , Witte David P , Grabowski Gregory A
Published: August 2008

Background

Prosaposin encodes, in tandem, four small acidic activator proteins (saposins) with specificities for glycosphingolipid (GSL) hydrolases in lysosomes. Extensive GSL storage occurs in various central nervous system regions in mammalian prosaposin deficiencies.

Results

Our hypomorphic prosaposin deficient mouse, PS-NA, exhibited 45% WT levels of brain saposins and showed neuropathology that included neuronal GSL storage and Purkinje cell loss. Impairment of neuronal function was observed as early as 6 wks as demonstrated by the narrow bridges tests. Temporal transcriptome microarray analyses of brain tissues were conducted with mRNA from three prosaposin deficient mouse models: PS-NA, prosaposin null (PS-/-) and a V394L/V394L glucocerebrosidase mutation combined with PS-NA (4L/PS-NA). Gene expression alterations in cerebrum and cerebellum were detectable at birth preceding the neuronal deficits. Differentially expressed genes encompassed a broad spectrum of cellular functions. The number of down-regulated genes was constant, but up-regulated gene numbers increased with age. CCAAT/enhancer-binding protein delta (CEBPD) was the only up-regulated transcription factor in these two brain regions of all three models. Network analyses revealed that CEBPD has functional relationships with genes in transcription, pro-inflammation, cell death, binding, myelin and transport.

Conclusion

These results show that: 1) Regionally specific gene expression abnormalities precede the brain histological and neuronal function changes, 2) Temporal gene expression profiles provide insights into the molecular mechanism during the GSL storage disease course, and 3) CEBPD is a candidate regulator of brain disease in prosaposin deficiency to participate in modulating disease acceleration or progression.
Epigenetic mechanisms involved in differential MDR1mRNA expression between gastric and colon cancer cell lines and rationales for clinical chemotherapy
Authors: Lee Tae-Bum , Park Jung-Hee , Min Young-Don , Kim Kyung-Jong , Choi Cheol-Hee
Published: August 2008

Background

The membrane transporters such as P-glycoprotein (Pgp), the MDR1 gene product, are one of causes of treatment failure in cancer patients. In this study, the epigenetic mechanisms involved in differential MDR1 mRNA expression were compared between 10 gastric and 9 colon cancer cell lines.

Methods

The MDR1 mRNA levels were determined using PCR and real-time PCR assays after reverse transcription. Cytotoxicity was performed using the MTT assay. Methylation status was explored by quantification PCR-based methylation and bisulfite DNA sequencing analyses.

Results

The MDR1 mRNA levels obtained by 35 cycles of RT-PCR in gastric cancer cells were just comparable to those obtained by 22 cycles of RT-PCR in colon cancer cells. Real-time RT-PCR analysis revealed that MDR1 mRNA was not detected in the 10 gastric cancer cell lines but variable MDR1 mRNA levels in 7 of 9 colon cancer cell lines except the SNU-C5 and HT-29 cells. MTT assay showed that Pgp inhibitors such as cyclosporine A, verapamil and PSC833 sensitized Colo320HSR (colon, highest MDR1 expression) but not SNU-668 (gastric, highest) and SNU-C5 (gastric, no expression) to paclitaxel. Quantification PCR-based methylation analysis revealed that 90% of gastric cancer cells, and 33% of colon cancer cells were methylated, which were completely matched with the results obtained by bisulfite DNA sequencing analysis. 5-aza-2'-deoxcytidine (5AC, a DNA methyltransferase inhibitor) increased the MDR1 mRNA levels in 60% of gastric cells, and in 11% of colon cancer cells. Trichostatin A (TSA, histone deacetylase inhibitor) increased the MDR1 mRNA levels in 70% of gastric cancer cells and 55% of colon cancer cells. The combined treatment of 5AC with TSA increased the MDR1 mRNA levels additively in 20% of gastric cancer cells, but synergistically in 40% of gastric and 11% of colon cancer cells.

Conclusion

These results indicate that the MDR1 mRNA levels in gastric cancer cells are significantly lower than those in colon cancer cells, which is at least in part due to different epigenetic regulations such as DNA methylation and/or histone deacetylation. These results can provide a better understanding of the efficacy of combined chemotherapy as well as their oral bioavailability.
Cytomegalovirus Infections among African-Americans
Authors: Wilms Isca R , Best Al M , Adler Stuart P
Published: August 2008

Background

Since African-Americans have twice the prevalence of cytomegalovirus (CMV) infections as age-matched Caucasians we sought to determine the ages and possible sources of infection of African-American children.

Methods

Subjects were 157 African-American healthy children and adolescents and their 113 household adults in Richmond VA. Families completed a questionnaire, provided saliva for antibody testing, and adolescents were interviewed regarding sexual activity.

Results

Regardless of age CMV seropositivity was not associated with gender, breast feeding, health insurance, sexual activity, or household income, education, or size. In the final regression model, prior CMV infection in adults was over two-fold higher than in children (chi-square = 18.8, p < 0.0001). At one year of age the CMV seropositivity rate was 11% (95%CI = 4% – 24%) and increased 1.8% each year until age 13 years. Between ages 13 and 20 years the CMV seropositivity rate remained between 22% and 33%. For adults, the CMV seropositivity rate was 84% in 21 year olds (95%CI = 69%–.92%). There was no association between CMV infections of the children and their mothers but CMV infections among siblings were associated.

Conclusion

We observed that African-American children had CMV seroprevalence rates by age 20 years at less than one-half of that of their adult mothers and caregivers. Sibling-to-sibling transmission was a likely source of CMV infections for the children. The next generation of African-American women may be highly susceptible to a primary CMV infection during pregnancy and may benefit from a CMV vaccine.
No effect of preterm birth on the risk of multiple sclerosis: a population based study
Authors: Ramagopalan Sreeram V , Valdar William , Dyment David A , DeLuca Gabriele C , Orton Sarah-Michelle , Yee Irene M , Criscuoli Maria , Ebers George C , Sadovnick A Dessa ,   for the Canadian Collaborative Study Group
Published: August 2008

Background

Genetic and environmental factors have important roles in multiple sclerosis (MS) susceptibility. A clear parent of origin effect has been shown in several populations, perhaps resulting from factors operating during gestation. Preterm birth (birth at less than 37 weeks gestational age) has been shown to result in long-term health problems, including impaired neurological development. Here, in a population-based cohort, we investigate whether preterm birth increases the risk to subsequently develop MS.

Methods

We identified 6585 MS index cases and 2509 spousal controls with preterm birth information from the Canadian Collaborative Project on Genetic Susceptibility to MS. Rates of individuals born preterm were compared for index cases and controls.

Results

There were no significant differences between cases and controls with respect to preterm births. 370 (5.6%) MS index cases and 130 (5.2%) spousal controls were born preterm, p = 0.41.

Conclusion

Preterm birth does not appear to contribute to MS aetiology. Other factors involved in foetal and early development need to be explored to elucidate the mechanism of the increased risk conferred by the apparent maternal effect.
Androgen regulation of the androgen receptor coregulators
Authors: Urbanucci Alfonso , Waltering Kati K , Suikki Hanna E , Helenius Merja A , Visakorpi Tapio
Published: August 2008

Background

The critical role of the androgen receptor (AR) in the development of prostate cancer is well recognized. The transcriptional activity of AR is partly regulated by coregulatory proteins. It has been suggested that these coregulators could also be important in the progression of prostate cancer. The aim of this study was to identify coregulators whose expression is regulated by either the androgens and/or by the expression level of AR.

Methods

We used empty vector and AR cDNA-transfected LNCaP cells (LNCaP-pcDNA3.1, and LNCaP-ARhi, respectively), and grew them for 4 and 24 hours in the presence of dihydrotestosterone (DHT) at various concentrations. The expression of 25 AR coregulators (SRC1, TIF2, PIAS1, PIASx, ARIP4, BRCA1, β-catenin, AIB3, AIB1, CBP, STAT1, NCoR1, AES, cyclin D1, p300, ARA24, LSD1, BAG1L, gelsolin, prohibitin, JMJD2C, JMJD1A, MAK, PAK6 and MAGE11) was then measured by using real-time quantitative RT-PCR (Q-RT-PCR).

Results

Five of the coregulators (AIB1, CBP, MAK, BRCA1 and β-catenin) showed more than 2-fold induction and 5 others (cyclin D1, gelsolin, prohibitin, JMJD1A, and JMJD2C) less than 2-fold induction. Overexpression of AR did not affect the expression of the coregulators alone. However, overexpression of AR enhanced the DHT-stimulated expression of MAK, BRCA1, AIB1 and CBP and reduced the level of expression of β-catenin, cyclinD1 and gelsolin.

Conclusion

In conclusion, we identified 5 coactivators whose expression was induced by androgens suggesting that they could potentiate AR signaling. Overexpression of AR seems to sensitize cells for low levels of androgens.
A quantitative risk assessment approach for mosquito-borne diseases: malaria re-emergence in southern France
Authors: Ponçon Nicolas , Tran Annelise , Toty Céline , Luty Adrian JF , Fontenille Didier
Published: August 2008

Background

The Camargue region is a former malaria endemic area, where potential Anopheles vectors are still abundant. Considering the importation of Plasmodium due to the high number of imported malaria cases in France, the aim of this article was to make some predictions regarding the risk of malaria re-emergence in the Camargue.

Methods

Receptivity (vectorial capacity) and infectivity (vector susceptibility) were inferred using an innovative probabilistic approach and considering both Plasmodium falciparum and Plasmodium vivax. Each parameter of receptivity (human biting rate, anthropophily, length of trophogonic cycle, survival rate, length of sporogonic cycle) and infectivity were estimated based on field survey, bibliographic data and expert knowledge and fitted with probability distributions taking into account the variability and the uncertainty of the estimation. Spatial and temporal variations of the parameters were determined using environmental factors derived from satellite imagery, meteorological data and entomological field data. The entomological risk (receptivity/infectivity) was calculated using 10,000 different randomly selected sets of values extracted from the probability distributions. The result was mapped in the Camargue area. Finally, vulnerability (number of malaria imported cases) was inferred using data collected in regional hospitals.

Results

The entomological risk presented large spatial, temporal and Plasmodium species-dependent variations. The sensitivity analysis showed that susceptibility, survival rate and human biting rate were the three most influential parameters for entomological risk. Assessment of vulnerability showed that among the imported cases in the region, only very few were imported in at-risk areas.

Conclusion

The current risk of malaria re-emergence seems negligible due to the very low number of imported Plasmodium. This model demonstrated its efficiency for mosquito-borne diseases risk assessment.
Immune modulation and increased neurotrophic factor production in multiple sclerosis patients treated with testosterone
Authors: Gold Stefan M , Chalifoux Sara , Giesser Barbara S , Voskuhl Rhonda R
Published: August 2008

Background

Multiple sclerosis is a chronic inflammatory disease of the central nervous system with a pronounced neurodegenerative component. It has been suggested that novel treatment options are needed that target both aspects of the disease. Evidence from basic and clinical studies suggests that testosterone has an immunomodulatory as well as a potential neuroprotective effect that could be beneficial in MS.

Methods

Ten male MS patients were treated with 10 g of gel containing 100 mg of testosterone in a cross-over design (6 month observation period followed by 12 months of treatment). Blood samples were obtained at three-month intervals during the observation and the treatment period. Isolated blood peripheral mononuclear cells (PBMCs) were used to examine lymphocyte subpopulation composition by flow cytometry andex vivoprotein production of cytokines (IL-2, IFNγ, TNFα, IL-17, IL-10, IL-12p40, TGFβ1) and growth factors (brain-derived neurotrophic factor BDNF, platelet-derived growth factor PDGF-BB, nerve growth factor NGF, and ciliary neurotrophic factor CNTF). Delayed type hypersensitivity (DTH) skin recall tests were obtained before and during treatment as anin vivofunctional immune measure.

Results

Testosterone treatment significantly reduced DTH recall responses and induced a shift in peripheral lymphocyte composition by decreasing CD4+ T cell percentage and increasing NK cells. In addition, PBMC production of IL-2 was significantly decreased while TGFβ1 production was increased. Furthermore, PBMCs obtained during the treatment period produced significantly more BDNF and PDGF-BB.

Conclusion

These results are consistent with an immunomodulatory effect of testosterone treatment in MS. In addition, increased production of BDNF and PDGF-BB suggests a potential neuroprotective effect.

Trial Registration

NCT00405353http://www.clinicaltrials.gov
Non-invasive cardiac assessment in high risk patients (The GROUND study): rationale, objectives and design of a multi-center randomized controlled clinical trial
Authors: de Vos Alexander M , Rutten Annemarieke , van de Zaag-Loonen Hester J , Bots Michiel L , Dikkers Riksta , Buiskool Robert A , Mali Willem P , Lubbers Daniel D , Mosterd Arend , Prokop Mathias , Rensing Benno J , Cramer Maarten J , van Es H Wouter , Moll Frans L , van de Pavoordt Eric D , Doevendans Pieter A , Velthuis Birgitta K , Mackaay Albert J , Zijlstra Felix , Oudkerk Matthijs
Published: August 2008

Background

Peripheral arterial disease (PAD) is a common disease associated with a considerably increased risk of future cardiovascular events and most of these patients will die from coronary artery disease (CAD). Screening for silent CAD has become an option with recent non-invasive developments in CT (computed tomography)-angiography and MR (magnetic resonance) stress testing. Screening in combination with more aggressive treatment may improve prognosis. Therefore we propose to study whether a cardiac imaging algorithm, using non-invasive imaging techniques followed by treatment will reduce the risk of cardiovascular disease in PAD patients free from cardiac symptoms.

Design

The GROUND study is designed as a prospective, multi-center, randomized clinical trial. Patients with peripheral arterial disease, but without symptomatic cardiac disease will be asked to participate. All patients receive a proper risk factor management before randomization. Half of the recruited patients will enter the 'control group' and only undergo CT calcium scoring. The other half of the recruited patients (index group) will undergo the non invasive cardiac imaging algorithm followed by evidence-based treatment. First, patients are submitted to CT calcium scoring and CT angiography. Patients with a left main (or equivalent) coronary artery stenosis of > 50% on CT will be referred to a cardiologist without further imaging. All other patients in this group will undergo dobutamine stress magnetic resonance (DSMR) testing. Patients with a DSMR positive for ischemia will also be referred to a cardiologist. These patients are candidates for conventional coronary angiography and cardiac interventions (coronary artery bypass grafting (CABG) or percutaneous cardiac interventions (PCI)), if indicated. All participants of the trial will enter a 5 year follow up period for the occurrence of cardiovascular events. Sequential interim analysis will take place. Based on sample size calculations about 1200 patients are needed to detect a 24% reduction in primary outcome.

Implications

The GROUND study will provide insight into the question whether non-invasive cardiac imaging reduces the risk of cardiovascular events in patients with peripheral arterial disease, but without symptoms of coronary artery disease.

Trial registration

Clinicaltrials.gov NCT00189111
Urinary interleukin-18 does not predict acute kidney injury after adult cardiac surgery: a prospective observational cohort study
Authors: Haase Michael , Bellomo Rinaldo , Story David , Davenport Piers , Haase-Fielitz Anja
Published: August 2008

Introduction

Urinary interleukin-18 (IL-18) measured during the immediate postoperative period could be a promising predictor of acute kidney injury following adult cardiac surgery.

Methods

In a single-centre prospective observational cohort study, we enrolled 100 adult cardiac surgical patients undergoing cardiopulmonary bypass at a tertiary hospital. We measured the urinary concentration of IL-18 and creatinine preoperatively, on arrival in the intensive care unit, and 24 hours postoperatively. We assessed urinary IL-18 concentration and urinary IL-18/urinary creatinine ratio in relation to the postoperative development of acute kidney injury defined as an increase in serum creatinine of greater than 50% from preoperative to postoperative peak value within 48 hours after surgery.

Results

Twenty patients developed acute kidney injury. On arrival in the intensive care unit and at 24 hours postoperatively, urinary IL-18 (median [interquartile range]) was not different in patients who subsequently developed acute kidney injury compared with those who did not: on arrival in the intensive care unit (168 [717] versus 104 [256] pg/mL; P = 0.70) and at 24 hours (195 [483] versus 165 [246] pg/mL; P = 0.47). On arrival in the intensive care unit (area under the curve for the receiver operating characteristic curve [AUC-ROCC] 0.53, 95% confidence interval [CI] 0.38 to 0.68; P = 0.70) and at 24 hours postoperatively (AUC-ROCC 0.55, 95% CI 0.40 to 0.71; P = 0.48), urinary IL-18 was not better than chance in predicting acute kidney injury. All findings were confirmed when urinary IL-18 was adjusted for urinary creatinine. Urinary IL-18 correlated with duration of cardiopulmonary bypass (P < 0.001).

Conclusion

In adults, early postoperative measurement of urinary IL-18 appears not to be valuable in identifying patients who develop acute kidney injury after cardiac surgery, but rather represents a nonspecific marker of cardiopulmonary bypass-associated systemic inflammation.
Persistent Hypomethylation in the Promoter of Nucleosomal Binding Protein 1 (Nsbp1) Correlates with Overexpression of Nsbp1 in Mouse Uteri Neonatally Exposed to Diethylstilbestrol or Genistein
Authors: Tang Wan-Yee , Newbold Retha , Mardilovich Katerina , Jefferson Wendy , Cheng Robert Y. S. , Medvedovic Mario , Ho Shuk-Mei
Published: August 2008

Neonatal exposure of CD-1 mice to diethylstilbestrol (DES) or genistein (GEN) induces uterine adenocarcinoma in aging animals. Uterine carcinogenesis in this model is ovarian dependent because its evolution is blocked by prepubertal ovariectomy. This study seeks to discover novel uterine genes whose expression is altered by such early endocrine disruption via an epigenetic mechanism. Neonatal mice were treated with 1 or 1000 μg/kg DES, 50 mg/kg GEN, or oil (control) on d 1–5. One group of treated mice was killed before puberty on d 19. Others were ovariectomized or left intact, and killed at 6 and 18 months of age. Methylation-sensitive restriction fingerprinting was performed to identify differentially methylated sequences associated with neonatal exposure to DES/GEN. Among 14 candidates, nucleosomal binding protein 1 (Nsbp1), the gene for a nucleosome-core-particle binding protein, was selected for further study because of its central role in chromatin remodeling. In uteri of immature control mice, Nsbp1 promoter CpG island (CGI) was minimally methylated. Once control mice reached puberty, the Nsbp1 CGI became hypermethylated, and gene expression declined further. In contrast, in neonatal DES/GEN-treated mice, the Nsbp1 CGI stayed anomalously hypomethylated, and the gene exhibited persistent overexpression throughout life. However, if neonatal DES/GEN-treated mice were ovariectomized before puberty, the CGI remained minimally to moderately methylated, and gene expression was subdued except in the group treated with 1000 μg/kg DES. Thus, the life reprogramming of uterine Nsbp1 expression by neonatal DES/GEN exposure appears to be mediated by an epigenetic mechanism that interacts with ovarian hormones in adulthood.
Identification of Melatonin-Regulated Genes in the Ovine Pituitary Pars Tuberalis, a Target Site for Seasonal Hormone Control
Authors: Dupré Sandrine M. , Burt Dave W. , Talbot Richard , Downing Alison , Mouzaki Daphne , Waddington David , Malpaux Benoit , Davis Julian R. E. , Lincoln Gerald A. , Loudon Andrew S. I.
Published: August 2008

The pars tuberalis (PT) of the pituitary gland expresses a high density of melatonin (MEL) receptors and is believed to regulate seasonal physiology by decoding changes in nocturnal melatonin secretion. Circadian clock genes are known to be expressed in the PT in response to the decline (Per1) and onset (Cry1) of MEL secretion, but to date little is known of other molecular changes in this key MEL target site. To identify transcriptional pathways that may be involved in the diurnal and photoperiod-transduction mechanism, we performed a whole genome transcriptome analysis using PT RNA isolated from sheep culled at three time points over the 24-h cycle under either long or short photoperiods. Our results reveal 153 transcripts where expression differs between photoperiods at the light-dark transition and 54 transcripts where expression level was more globally altered by photoperiod (all time points combined). Cry1 induction at night was associated with up-regulation of genes coding for NeuroD1 (neurogenic differentiation factor 1), Pbef / Nampt (nicotinamide phosphoribosyltransferase), Hif1α (hypoxia-inducible factor-1α), and Kcnq5 (K+ channel) and down-regulation of Rorβ, a key clock gene regulator. Using in situ hybridization, we confirmed day-night differences in expression for Pbef / Nampt, NeuroD1, and Rorβ in the PT. Treatment of sheep with MEL increased PT expression for Cry1, Pbef / Nampt, NeuroD1, and Hif1α, but not Kcnq5. Our data thus reveal a cluster of Cry1-associated genes that are acutely responsive to MEL and novel transcriptional pathways involved in MEL action in the PT.
Low Concentrations of the Soy Phytoestrogen Genistein Induce Proteinase Inhibitor 9 and Block Killing of Breast Cancer Cells by Immune Cells
Authors: Jiang Xinguo , Patterson Nicole M. , Ling Yan , Xie Jianwei , Helferich William G. , Shapiro David J.
Published: August 2008

The risks and benefits of diets and supplements containing the estrogenic soy isoflavone genistein are not well established. We report that 10 nm genistein potently induces the granzyme B inhibitor, proteinase inhibitor 9 (PI-9) in MCF-7 human breast cancer cells. By inducing PI-9, genistein inhibits the ability of human natural killer (NK) cells to lyse the target breast cancer cells. In ERαHA cells, stably transfected MCF-7 cells, which contain elevated levels of estrogen receptor-α (ERα), 100 pm genistein or 17β-estradiol potently induce PI-9 and prevent NK cells from killing the target breast cancer cells. The concentrations of genistein that fully induce PI-9 in MCF-7 cells, and in ERαHA cells, are far lower than those previously reported to elicit estrogenic responses through ERα. Because 4-hydroxytamoxifen, raloxifene, and ICI 182,780/Faslodex all block genistein induction of PI-9 and elevated levels of ERα enhance induction of PI-9, genistein acts via ERα to induce PI-9. Increasing levels of ERα in breast cancer cells results in a progressive increase in induction of PI-9 by genistein and in the cell’s ability to evade killing by NK cells. Moderate levels of dietary genistein and soy flour effectively induce PI-9 in human breast cancers grown in ovariectomized athymic mice. A significant population consumes levels of genistein in soy products that may be high enough to induce PI-9, perhaps potentiating the survival of some preexisting breast cancers by enabling them to evade immunosurveillance.
Minireview: How Peptide Hormone Vesicles Are Transported to the Secretion Site for Exocytosis
Authors: Park Joshua J. , Loh Y. Peng
Published: August 2008

Post-Golgi transport of peptide hormone-containing vesicles from the site of genesis at the trans-Golgi network to the release site at the plasma membrane is essential for activity-dependent hormone secretion to mediate various endocrinological functions. It is known that these vesicles are transported on microtubules to the proximity of the release site, and they are then loaded onto an actin/myosin system for distal transport through the actin cortex to just below the plasma membrane. The vesicles are then tethered to the plasma membrane, and a subpopulation of them are docked and primed to become the readily releasable pool. Cytoplasmic tails of vesicular transmembrane proteins, as well as many cytosolic proteins including adaptor proteins, motor proteins, and guanosine triphosphatases, are involved in vesicle budding, the anchoring of the vesicles, and the facilitation of movement along the transport systems. In addition, a set of cytosolic proteins is also necessary for tethering/docking of the vesicles to the plasma membrane. Many of these proteins have been identified from different types of (neuro)endocrine cells. Here, we summarize the proteins known to be involved in the mechanisms of sorting various cargo proteins into regulated secretory pathway hormone-containing vesicles, movement of these vesicles along microtubules and actin filaments, and their eventual tethering/docking to the plasma membrane for hormone secretion.
Screening for Two Selective Androgen Receptor Modulators Using Gas Chromatography-Mass Spectrometry in Doping Control Analysis
Authors: Thevis Mario , Kohler Maxie , Schlörer Nils , Fußhöller Gregor , Schänzer Wilhelm
Published: August 2008

Selective androgen receptor modulators (SARMs) have become a major field of clinical research enabling the tissue-selective stimulation of androgen receptors. The treatment of debilitating diseases, osteoporosis and frailty are primary goals and promising results have been obtained from clinical trials. However, the potential for misuse of SARMs in sport is great and drug testing methods based on liquid chromatography were established for different classes including arylpropionamide-, 2-quinolinone- and bicyclic hydantoin-derived compounds. As gas chromatography and mass spectrometry (GC-MS) are still important analytical tools in sports drug testing, a method to determine 2-quinolinone- and bicyclic hydantoin-derived SARMs established. Spiked urine samples were subjected to routine doping control protocols including enzymatic hydrolysis, liquid–liquid extraction, concentration and derivatisation to trimethylsilylated analogues followed by GC-MS analysis. The method was validated for the items specificity, lower limit of detection (0.2–10 ng mL−1), recovery (83–85%), intraday and interday precision (9–15% and 13–18%, respectively), which demonstrates the suitability of conventional GC-MS systems to determine representatives of an emerging class of compounds in doping control specimens.
Identification and Quantification of the Osmodiuretic Mannitol in Urine for Sports Drug Testing Using Gas Chromatography-Mass Spectrometry
Authors: Guddat Sven , Thevis Mario , Schänzer Wilhelm
Published: August 2008

The osmodiuretic mannitol can be potentially misused in sports, owing to its urine diluting effect and the possibility to decrease body-weight. To reveal a doping offence, resulting urinary mannitol concentrations after a prohibited intravenous application and a permitted oral intake have to be differentiated. Therefore, a reliable gas chromatography-mass spectrometry (GC-MS) method was established based on peracetyl derivatives of the analytes. All possible hexitols (allitol, galactitol, iditol, altritols, sorbitol and mannitol) that can occur in human urine were separated and identified on a phenyl-methylpolysiloxane column (HP-5MS) within 10.75 min, and the method demonstrated its capability for quantification purposes. The lower limit of detection and lower limit of quantification were estimated at 0.9 μg mL−1 and 2.4 μg mL−1, respectively, and the assay was validated for mannitol and sorbitol regarding the parameters specificity, linearity, intra- (<10%) and inter-day precision (<15%) and accuracy (92–102%). To investigate urinary mannitol concentrations after oral intake the method was applied to an excretion study, providing a mean urinary excretion of mannitol of 19.5%. Comparison of theoretically expected urinary levels after a common therapeutic dose of mannitol and preliminary results on physiological urinary mannitol levels were promising, regarding a threshold level for mannitol that can be utilised for doping control purposes.
Quantitative Analysis of Urinary Glycerol Levels for Doping Control Purposes Using Gas Chromatography-Mass Spectrometry
Authors: Thevis Mario , Guddat Sven , Flenker Ulrich , Schänzer Wilhelm
Published: August 2008

The administration of glycerol to endurance athletes results in an increased fluid retention and improved performance, particularly under hot and humid conditions. Consequently, glycerol is considered relevant for sports drug testing and methods for its detection in urine specimens are required. A major issue in this regard is the natural occurrence of trace amounts of glycerol in human urine, which necessitates a quantitative analysis and the determination of normal urinary glycerol levels under various sporting conditions. A quantitative method was established using a gas chromatography/isotope-dilution mass spectrometry-based approach that was validated with regard to lower limit of detection (0.3 μg mL−1), lower limit of quantification (0.9 μg mL−1), specificity, linearity (1.0–98.0 μg mL−1), intraday and interday precision (<20% at 2.4, 24.1 and 48.2 μg mL−1) as well as accuracy (92–110%). Sample aliquots of 20 μL were enriched with five-fold deuterated glycerol, dried and derivatised using N-methyl-trimethylsilyltrifluoroacetamide (MSTFA) before analysis. The established method was applied to a total of 1039 doping control samples covering various sport disciplines (349 endurance samples, 286 strength sport samples, 325 game sport samples and 79 other samples) in- and out-of-competition, which provided quantitative information about the glycerol content commonly observed in elite athletes' urine samples. About 85% of all specimens yielded glycerol concentrations < 20.0 μg mL−1 and few reached values up to 132.6 μg mL−1. One further sample, however, was found to contain 2690 μg mL−1, which might indicate the misuse of glycerol, but no threshold for urinary glycerol concentrations has been established yet due to the lack of substantial data. Based on the results obtained from the studied reference population, a threshold for glycerol levels in urine set at 200 μg mL−1 is suggested, which provides a tool to doping control laboratories to test for the misuse of this agent in elite and amateur sport.
High-Throughput and Sensitive Screening by Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry of Diuretics and other Doping Agents
Authors: Ventura Rosa , Roig Meritxell , Monfort Núria , Sáez Pilar , Bergés Rosa , Segura Jordi
Published: August 2008

The reliability of ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) for high throughput screening in anti-doping control has been tested. A method to screen for the presence of diuretics and other doping agents in urine has been optimised and validated. The extraction procedure consisted of an alkaline extraction (pH 9.5) with ethyl acetate and salting-out effect (sodium chloride). The extracts were analysed by UPLC-MS/MS. Analysis of 34 forbidden drugs and metabolites was achieved in a total run time of 5 min, using a C18 column (100 mm × 2.1 mm i.d., 1.7 μm particle size) and a mobile phase containing deionised water and acetonitrile with formic acid, with gradient elution at a flow-rate of 0.6 mL min−1. Identification of the compounds was performed by multiple reaction monitoring, using electrospray ionisation in positive- or negative-ion mode. Precursor and product ions were studied for each compound and cone voltage and collision energy were optimised. Due to the different chemical structure of the compounds under study, extraction recoveries varied from less than 10% to 100% depending on the analyte. The limits of detection ranged from 50 ng mL−1 to 200 ng mL−1, and all the compounds comply with the requirements of quality established by the World Anti-doping Agency. Intra-assay precision was evaluated at two concentrations for each compound and, in most cases, a relative standard deviation of the signal ratio lower than 20% was obtained. The method has demonstrated to be reliable when analysing routine samples and the short analysis time resulting from a simple sample preparation and a rapid instrumental analysis allow a fast turn-around time and makes it of great interest for routine anti-doping control purposes.