bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic Manipulation of the Toxoplasma gondii Genome by Fosmid Recombineering
Authors: Vinayak Sumiti , Brooks Carrie F. , Naumov Anatoli , Suvorova Elena S. , White Michael W. , Striepen Boris 
Published: December 2014

ABSTRACT

Apicomplexa are obligate intracellular parasites that cause important diseases in humans and animals. Manipulating the pathogen genome is the most direct way to understand the functions of specific genes in parasite development and pathogenesis. In
Toxoplasma gondii
, nonhomologous recombination is typically highly favored over homologous recombination, a process required for precise gene targeting. Several approaches, including the use of targeting vectors that feature large flanks to drive site-specific recombination, have been developed to overcome this problem. We have generated a new large-insert repository of
T. gondii
genomic DNA that is arrayed and sequenced and covers 95% of all of the parasite’s genes. Clones from this fosmid library are maintained at single copy, which provides a high level of stability and enhances our ability to modify the organism dramatically. We establish a robust recombineering pipeline and show that our fosmid clones can be easily converted into gene knockout constructs in a 4-day protocol that does not require plate-based cloning but can be performed in multiwell plates. We validated this approach to understand gene function in
T. gondii
and produced a conditional null mutant for a nucleolar protein belonging to the NOL1/NOP2/SUN family, and we show that this
gene is essential for parasite growth. We also demonstrate a powerful complementation strategy in the context of chemical mutagenesis and whole-genome sequencing. This repository is an important new resource that will accelerate both forward and reverse genetic analysis of this important pathogen.

IMPORTANCE
Toxoplasma gondii
is an important genetic model to understand intracellular parasitism. We show here that large-insert genomic clones are effective tools that enhance homologous recombination and allow us to engineer conditional mutants to understand gene function. We have generated, arrayed, and sequenced a fosmid library of
T. gondii
genomic DNA in a copy control vector that provides excellent coverage of the genome. The fosmids are maintained in a single-copy state that dramatically improves their stability and allows modification by means of a simple and highly scalable protocol. We show here that modified and unmodified fosmid clones are powerful tools for forward and reverse genetics.
Is clopidogrel better than aspirin following breakthrough strokes while on aspirin? A retrospective cohort study
Authors: Lee Meng , Wu Yi-Ling , Saver Jeffrey L , Lee Hsuei-Chen , Lee Jiann-Der , Chang Ku-Chou , Wu Chih-Ying , Lee Tsong-Hai , Wang Hui-Hsuan , Rao Neal M , Ovbiagele Bruce 
Published: December 2014

ObjectiveThere is insufficient evidence on which to base a recommendation for optimal antiplatelet therapy following a stroke while on aspirin. The objective was to compare clopidogrel initiation vs aspirin reinitiation for vascular risk reduction among patients with ischaemic stroke on aspirin at the time of their index stroke.DesignRetrospective.SettingWe conducted a nationwide cohort study by retrieving all hospitalised patients (≥18 years) with a primary diagnosis of ischaemic stroke between 2003 and 2009 from Taiwan National Health Insurance Research Database.ParticipantsAmong 3862 patients receiving aspirin before the index ischaemic stroke and receiving either aspirin or clopidogrel after index stroke during follow-up period, 1623 were excluded due to a medication possession ratio <80%. Also, 355 were excluded due to history of atrial fibrillation, valvular heart disease or coagulopathy. Therefore, 1884 patients were included in our final analysis.InterventionsPatients were categorised into two groups based on whether clopidogrel or aspirin was prescribed during the follow-up period. Follow-up was from time of the index stroke to admission for recurrent stroke or myocardial infarction, death or the end of 2010.Primary and secondary outcome measuresThe primary end point was hospitalisation due to a new-onset major adverse cardiovascular event (MACE: composite of any stroke or myocardial infarction). The leading secondary end point was any recurrent stroke.ResultsCompared to aspirin, clopidogrel was associated with a lower occurrence of future MACE (HR=0.54, 95% CI 0.43 to 0.68, p<0.001, number needed to treat: 8) and recurrent stroke (HR=0.54, 95% CI 0.42 to 0.69, p<0.001, number needed to treat: 9) after adjustment of relevant covariates.ConclusionsAmong patients with an ischaemic stroke while taking aspirin, clopidogrel initiation was associated with fewer recurrent vascular events than aspirin reinitiation.
Unplanned, urgent and emergency care: what are the roles that EMS plays in providing for older people with dementia? An integrative review of policy, professional recommendations and evidence
Authors: Buswell Marina , Lumbard Philip , Prothero Larissa , Lee Caroline , Martin Steven , Fleming Jane , Goodman Claire 
Published: December 2014

ObjectiveTo synthesise the existing literature on the roles that emergency medical services (EMS) play in unplanned, urgent and emergency care for older people with dementia (OPWD), to define these roles, understand the strength of current research and to identify where the focus of future research should lie.DesignAn integrative review of the synthesised reports, briefings, professional recommendations and evidence. English-language articles were included if they made any reference to the role of EMS in the urgent or emergency care of OPWD. Preparatory scoping and qualitative work with frontline ambulance and primary care staff and carers of OPWD informed our review question and subsequent synthesis.ResultsSeventeen literature sources were included. Over half were from the grey literature. There was no research that directly addressed the review question. There was evidence in reports, briefings and professional recommendations of EMS addressing some of the issues they face in caring for OPWD. Three roles of EMS could be drawn out of the literature: emergency transport, assess and manage and a ‘last resort’ or safety net role.ConclusionsThe use of EMS by OPWD is not well understood, although the literature reviewed demonstrated a concern for this group and awareness that services are not optimum. Research in dementia care should consider the role that EMS plays, particularly if considering crises, urgent care responses and transitions between care settings. EMS research into new ways of working, training or extended paramedical roles should consider specific needs and challenges of responding to people with dementia.
Improving Power to Detect Changes in Blood miRNA Expression by Accounting for Sources of Variability in Experimental Designs
Authors: Daniels Sarah I. , Sillé Fenna C.M. , Goldbaum Audrey , Yee Brenda , Key Ellen F. , Zhang Luoping , Smith Martyn T. , Thomas Reuben 
Published: December 2014

Abstract
Background: Blood miRNAs are a new promising area of disease research, but variability in miRNA measurements may limit detection of true-positive findings. Here, we measured sources of miRNA variability and determine whether repeated measures can improve power to detect fold-change differences between comparison groups.
Methods: Blood from healthy volunteers (N = 12) was collected at three time points. The miRNAs were extracted by a method predetermined to give the highest miRNA yield. Nine different miRNAs were quantified using different qPCR assays and analyzed using mixed models to identify sources of variability. A larger number of miRNAs from a publicly available blood miRNA microarray dataset with repeated measures were used for a bootstrapping procedure to investigate effects of repeated measures on power to detect fold changes in miRNA expression for a theoretical case–control study.
Results: Technical variability in qPCR replicates was identified as a significant source of variability (P &lt; 0.05) for all nine miRNAs tested. Variability was larger in the TaqMan qPCR assays (SD = 0.15–0.61) versus the qScript qPCR assays (SD = 0.08–0.14). Inter- and intraindividual and extraction variability also contributed significantly for two miRNAs. The bootstrapping procedure demonstrated that repeated measures (20%–50% of N) increased detection of a 2-fold change for approximately 10% to 45% more miRNAs.
Conclusion: Statistical power to detect small fold changes in blood miRNAs can be improved by accounting for sources of variability using repeated measures and choosing appropriate methods to minimize variability in miRNA quantification.
Impact: This study demonstrates the importance of including repeated measures in experimental designs for blood miRNA research.
See all the articles in this CEBP Focus section, “Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.”
Cancer Epidemiol Biomarkers Prev; 23(12); 2658–66. ©2014 AACR.
A Phase I Study of Cixutumumab (IMC-A12) in Combination with Temsirolimus (CCI-779) in Children with Recurrent Solid Tumors: A Children's Oncology Group Phase I Consortium Report
Authors: Fouladi Maryam , Perentesis John P. , Wagner Lars M. , Vinks Alexander A. , Reid Joel M. , Ahern Charlotte , Thomas George , Mercer Carol A. , Krueger Darcy A. , Houghton Peter J. , Doyle L. Austin , Chen Helen , Weigel Brenda , Blaney Susan M. 
Published: December 2014

Abstract
Purpose: To determine the MTD, dose-limiting toxicities (DLT), pharmacokinetics, and biologic effects of cixutumumab administered in combination with temsirolimus to children with refractory solid tumors.
Experimental Design: Cixutumumab and temsirolimus were administered intravenously once every 7 days in 28-day cycles. Pharmacokinetic and biology studies, including assessment of mTOR downstream targets in peripheral blood mononuclear cells, were performed during the first cycle.
Results: Thirty-nine patients, median age 11.8 years (range, 1–21.5), with recurrent solid or central nervous system tumors were enrolled, of whom 33 were fully assessable for toxicity. There were four dose levels, which included two dose reductions and a subsequent intermediated dose escalation: (i) IMC-A12 6 mg/kg, temsirolimus 15 mg/m2; (ii) IMC-A12 6 mg/kg, temsirolimus 10 mg/m2; (iii) IMC-A12 4 mg/kg, temsirolimus 8 mg/m2; and (iv) IMC-A12 6 mg/kg, temsirolimus 8 mg/m2. Mucositis was the predominant DLT. Other DLTs included hypercholesterolemia, fatigue, thrombocytopenia, and increased alanine aminotransferase. Target inhibition (decreased S6K1 and PAkt) in peripheral blood mononuclear cells was noted at all dose levels. Marked interpatient variability in temsirolimus pharmacokinetic parameters was noted. At 8 mg/m2, the median temsirolimus AUC was 2,946 ng • h/mL (range, 937–5,536) with a median sirolimus AUC of 767 ng • h/mL (range, 245–3,675).
Conclusions: The recommended pediatric phase II doses for the combination of cixutumumab and temsirolimus are 6 mg/kg and 8 mg/m2, respectively. Clin Cancer Res; 21(7); 1558–65. ©2014 AACR.
A Phase I Study of DMS612, a Novel Bifunctional Alkylating Agent
Authors: Appleman Leonard J. , Balasubramaniam Sanjeeve , Parise Robert A. , Bryla Christine , Redon Christophe E. , Nakamura Asako J. , Bonner William M. , Wright John J. , Piekarz Richard , Kohler David R. , Jiang Yixing , Belani Chandra P. , Eiseman Julie , Chu Edward , Beumer Jan H. , Bates Susan E. 
Published: December 2014

Abstract
Purpose: DMS612 is a dimethane sulfonate analog with bifunctional alkylating activity and preferential cytotoxicity to human renal cell carcinoma (RCC) in the NCI-60 cell panel. This first-in-human phase I study aimed to determine dose-limiting toxicity (DLT), maximum tolerated dose (MTD), pharmacokinetics (PK), and pharmacodynamics (PD) of DMS612 administered by 10-minute intravenous infusion on days 1, 8, and 15 of an every-28-day schedule.
Experimental Design: Patients with advanced solid malignancies were eligible. Enrollment followed a 3+3 design. PKs of DMS612 and metabolites were assessed by mass spectroscopy and PD by γ-H2AX immunofluorescence.
Results: A total of 31 patients, including those with colorectal (11), RCC (4), cervical (2), and urothelial (1) cancers, were enrolled. Six dose levels were studied, from 1.5 mg/m2 to 12 mg/m2. DLTs of grade 4 neutropenia and prolonged grade 3 thrombocytopenia were observed at 12 mg/m2. The MTD was determined to be 9 mg/m2 with a single DLT of grade 4 thrombocytopenia in 1 of 12 patients. Two patients had a confirmed partial response at the 9 mg/m2 dose level, in renal (1) and cervical (1) cancer. DMS612 was rapidly converted into active metabolites. γ-H2AX immunofluorescence revealed dose-dependent DNA damage in both peripheral blood lymphocytes and scalp hairs.
Conclusions: The MTD of DMS12 on days 1, 8, and 15 every 28 days was 9 mg/m2. DMS612 appears to be an alkylating agent with unique tissue specificities. Dose-dependent PD signals and two partial responses at the MTD support further evaluation of DMS612 in phase II trials. Clin Cancer Res; 21(4); 721–9. ©2014 AACR.
High HER2 Expression Correlates with Response to the Combination of Lapatinib and Trastuzumab
Authors: Scaltriti Maurizio , Nuciforo Paolo , Bradbury Ian , Sperinde Jeff , Agbor-Tarh Dominique , Campbell Christine , Chenna Ahmed , Winslow John , Serra Violeta , Parra Josep Lluis , Prudkin Ludmila , Jimenez José , Aura Claudia , Harbeck Nadia , Pusztai Lajos , Ellis Catherine , Eidtmann Holger , Arribas Joaquin , Cortes Javier , de Azambuja Evandro , Piccart Martine , Baselga José 
Published: December 2014

Abstract
Purpose: Expression of p95HER2 has been associated with resistance to trastuzumab-based therapy in patients with metastatic breast cancer. Conversely, high levels of HER2 have been linked with increased clinical benefit from anti-HER2 therapy. In this work, we aimed to investigate whether the levels of p95HER2 and HER2 can predict response to anti-HER2 therapy in patients with breast cancer.
Experimental Design: We measured p95HER2 and HER2 by VeraTag and HERmark, respectively, in primary tumors of patients enrolled in the neoadjuvant phase III study NeoALTTO and correlated these variables with pathologic complete response (pCR) and progression-free survival (PFS) following lapatinib (L), trastuzumab (T), or the combination of both agents (L+T).
Results: A positive correlation between p95HER2 and HER2 levels was found in the 274 cases (60%) in which quantification of both markers was possible. High levels of these markers were predictive for pCR, especially in the hormone receptor (HR)–positive subset of patients. High HER2 expression was associated with increased pCR rate upon L+T irrespective of the HR status. To examine whether the levels of either p95HER2 or HER2 could predict for PFS in patients treated with lapatinib, trastuzumab or L+T, we fit to the PFS data in Cox models containing log2(p95HER2) or log2(HER2). Both variables correlated with longer PFS.
Conclusions: Increasing HER2 protein expression correlated with increased benefit of adding lapatinib to trastuzumab. HER2 expression is a stronger predictor of pCR and PFS than p95HER2 for response to lapatinib, trastuzumab and, more significantly, L+T. Clin Cancer Res; 21(3); 569–76. ©2014 AACR.
Anti‐Inflammatory Peptides From Cardiac Progenitors Ameliorate Dysfunction After Myocardial Infarction
Authors: Liu Mei‐Lan , Nagai Toshio , Tokunaga Masakuni , Iwanaga Koji , Matsuura Katsuhisa , Takahashi Toshinao , Kanda Masato , Kondo Naomichi , Naito Atsuhiko T. , Komuro Issei , Kobayashi Yoshio 
Published: December 2014

Background
Cardiac cell therapy has been proposed as one of the new strategies against myocardial infarction. Although several reports showed improvement of the function of ischemic heart, the effects of cell therapy vary among the studies and the mechanisms of the beneficial effects are still unknown. Previously, we reported that clonal stem cell antigen‐1–positive cardiac progenitor cells exerted a therapeutic effect when transplanted into the ischemic heart. Our aims were to identify the cardiac progenitor‐specific paracrine factor and to elucidate the mechanism of its beneficial effect.

Methods and Results

By using an antibody array, we found that soluble junctional adhesion molecule‐A (
JAM
‐A) was abundantly secreted from cardiac progenitor cells. Pretreatment of neutrophils with conditioned medium from cultured cardiac progenitor cells or soluble
JAM
‐A inhibited transendothelial migration and reduced motility of neutrophils. These inhibitory effects were attenuated by anti–
JAM
‐A neutralizing antibody. Injection of cardiac progenitor cells into infarct heart attenuated neutrophil infiltration and expression of inflammatory cytokines. Injection of soluble
JAM
‐A–expressing, but not of
JAM
‐A si
RNA
–expressing, cardiac progenitor cells into the infarct heart prevented cardiac remodeling and reduced fibrosis area.

Conclusions

Soluble
JAM
‐A secreted from cardiac progenitor cells reduces infiltration of neutrophils after myocardial infarction and ameliorates tissue damage through prevention of excess inflammation. Our finding may lead to a new therapy for cardiovascular disease by using the anti‐inflammatory effect of
JAM
‐A.
Dietary Salt Restriction Improves Cardiac and Adipose Tissue Pathology Independently of Obesity in a Rat Model of Metabolic Syndrome
Authors: Hattori Takuya , Murase Tamayo , Takatsu Miwa , Nagasawa Kai , Matsuura Natsumi , Watanabe Shogo , Murohara Toyoaki , Nagata Kohzo 
Published: December 2014

Background
Metabolic syndrome (MetS) enhances salt sensitivity of blood pressure and is an important risk factor for cardiovascular disease. The effects of dietary salt restriction on cardiac pathology associated with metabolic syndrome remain unclear.

Methods and Results

We investigated whether dietary salt restriction might ameliorate cardiac injury in DahlS.Z‐
Lepr

fa

/
Lepr

fa

(
DS
/obese) rats, which are derived from a cross between Dahl salt‐sensitive and Zucker rats and represent a model of metabolic syndrome.
DS
/obese rats were fed a normal‐salt (0.36% NaCl in chow) or low‐salt (0.0466% NaCl in chow) diet from 9 weeks of age and were compared with similarly treated homozygous lean littermates (DahlS.Z‐
Lepr

+

/
Lepr

+

, or
DS
/lean rats).
DS
/obese rats fed the normal‐salt diet progressively developed hypertension and showed left ventricular hypertrophy, fibrosis, and diastolic dysfunction at 15 weeks. Dietary salt restriction attenuated all of these changes in
DS
/obese rats. The levels of cardiac oxidative stress and inflammation and the expression of cardiac renin–angiotensin–aldosterone system genes were increased in
DS
/obese rats fed the normal‐salt diet, and dietary salt restriction downregulated these parameters in both
DS
/obese and
DS
/lean rats. In addition, dietary salt restriction attenuated the increase in visceral adipose tissue inflammation and the decrease in insulin signaling apparent in
DS
/obese rats without reducing body weight or visceral adipocyte size. Dietary salt restriction did not alter fasting serum glucose levels but it markedly decreased the fasting serum insulin concentration in
DS
/obese rats.

Conclusions
Dietary salt restriction not only prevents hypertension and cardiac injury but also ameliorates insulin resistance, without reducing obesity, in this model of metabolic syndrome.
Hypertension Is Associated With Preamyloid Oligomers in Human Atrium: A Missing Link in Atrial Pathophysiology?
Authors: Sidorova Tatiana N. , Mace Lisa C. , Wells K. Sam , Yermalitskaya Liudmila V. , Su Pei‐Fang , Shyr Yu , Atkinson James B. , Fogo Agnes B. , Prinsen Joseph K. , Byrne John G. , Petracek Michael R. , Greelish James P. , Hoff Steven J. , Ball Stephen K. , Glabe Charles G. , Brown Nancy J. , Barnett Joey V. , Murray Katherine T. 
Published: December 2014

Background

Increasing evidence indicates that proteotoxicity plays a pathophysiologic role in experimental and human cardiomyopathy. In organ‐specific amyloidoses, soluble protein oligomers are the primary cytotoxic species in the process of protein aggregation. While isolated atrial amyloidosis can develop with aging, the presence of preamyloid oligomers (
PAO
s) in atrial tissue has not been previously investigated.

Methods and Results

Atrial samples were collected during elective cardiac surgery in patients without a history of atrial arrhythmias, congestive heart failure, cardiomyopathy, or amyloidosis. Immunohistochemistry was performed for
PAO
s using a conformation‐specific antibody, as well as for candidate proteins identified previously in isolated atrial amyloidosis. Using a myocardium‐specific marker, the fraction of myocardium colocalizing with
PAO
s (
PAO
burden) was quantified (green/red ratio). Atrial samples were obtained from 92 patients, with a mean age of 61.7±13.8 years. Most patients (62%) were male, 23% had diabetes, 72% had hypertension, and 42% had coronary artery disease. A majority (n=62) underwent aortic valve replacement, with fewer undergoing coronary artery bypass grafting (n=34) or mitral valve replacement/repair (n=24). Immunostaining detected intracellular
PAO
s in a majority of atrial samples, with a heterogeneous distribution throughout the myocardium. Mean green/red ratio value for the samples was 0.11±0.1 (range 0.03 to 0.77), with a value ≥0.05 in 74 patients. Atrial natriuretic peptide colocalized with
PAO
s in myocardium, whereas transthyretin was located in the interstitium. Adjusting for multiple covariates,
PAO
burden was independently associated with the presence of hypertension.

Conclusion
PAOs are frequently detected in human atrium, where their presence is associated with clinical hypertension.
Habitual Sleep Duration and Predicted 10‐Year Cardiovascular Risk Using the Pooled Cohort Risk Equations Among US Adults
Authors: Ford Earl S. 
Published: December 2014

Background

The association between sleep duration and predicted cardiovascular risk has been poorly characterized. The objective of this study was to examine the association between self‐reported sleep duration and predicted 10‐year cardiovascular risk among
US
adults.

Methods and Results

Data from 7690 men and nonpregnant women who were aged 40 to 79 years, who were free of self‐reported heart disease and stroke, and who participated in a National Health and Nutrition Examination Survey from 2005 to 2012 were analyzed. Sleep duration was self‐reported. Predicted 10‐year cardiovascular risk was calculated using the pooled cohort equations. Among the included participants, 13.1% reported sleeping ≤5 hours, 24.4% reported sleeping 6 hours, 31.9% reported sleeping 7 hours, 25.2% reported sleeping 8 hours, 4.0% reported sleeping 9 hours, and 1.3% reported sleeping ≥10 hours. After adjustment for covariates, geometric mean–predicted 10‐year cardiovascular risk was 4.0%, 3.6%, 3.4%, 3.5%, 3.7%, and 3.7% among participants who reported sleeping ≤5, 6, 7, 8, 9, and ≥10 hours per night, respectively (
P
Wald chi‐square
<0.001). The age‐adjusted percentages of predicted cardiovascular risk ≥20% for the 6 intervals of sleep duration were 14.5%, 11.9%, 11.0%, 11.4%, 11.8%, and 16.3% (
P
Wald chi‐square
=0.022). After maximal adjustment, however, sleep duration was not significantly associated with cardiovascular risk ≥20% (
P
Wald chi‐square
=0.698).

Conclusions
Mean‐predicted 10‐year cardiovascular risk was lowest among adults who reported sleeping 7 hours per night and increased as participants reported sleeping fewer and more hours.