Prominin-1 controls stem cell activation by orchestrating ciliary dynamics.

Proper temporal and spatial activation of stem cells relies on highly coordinated cell signaling. The primary cilium is the sensory organelle that is responsible for transmitting extracellular signals into a cell. Primary cilium size, architecture, and assembly-disassembly dynamics are under rigid cell cycle-dependent control. Using mouse incisor tooth epithelia as a model, we show that ciliary dynamics in stem cells require the proper functions of a cholesterol-binding membrane glycoprotein, Prominin-1 (Prom1/CD133), which controls sequential recruitment of ciliary membrane components, histone deacetylase, and transcription factors. Nuclear translocation of Prom1 and these molecules is particularly evident in transit amplifying cells, the immediate derivatives of stem cells. The absence of Prom1 impairs ciliary dynamics and abolishes the growth stimulation effects of sonic hedgehog (SHH) treatment, resulting in the disruption of stem cell quiescence maintenance and activation. We propose that Prom1 is a key regulator ensuring appropriate response of stem cells to extracellular signals, with important implications for development, regeneration, and diseases.

Methods and sensors for functional genomic studies of cell-cycle transitions in single cells.

Much of our understanding of the regulatory mechanisms governing the cell cycle in mammals has relied heavily on methods that measure the aggregate state of a population of cells. While instrumental in shaping our current understanding of cell proliferation, these approaches mask the genetic signatures of rare subpopulations such as quiescent (G0) and very slowly dividing (SD) cells. Results described in this study and those of others using single-cell analysis reveal that even in clonally derived immortalized cancer cells, ∼1-5% of cells can exhibit G0 and SD phenotypes. Therefore to enable the study of these rare cell phenotypes we established an integrated molecular, computational, and imaging approach to track, isolate, and genetically perturb single cells as they proliferate. A genetically encoded cell-cycle reporter (K67p-FUCCI) was used to track single cells as they traversed the cell cycle. A set of R-scripts were written to quantify K67p-FUCCI over time. To enable the further study G0 and SD phenotypes, we retrofitted a live cell imaging system with a micromanipulator to enable single-cell targeting for functional validation studies. Single-cell analysis revealed HT1080 and MCF7 cells had a doubling time of ∼24 and ∼48 h, respectively, with high duration variability in G1 and G2 phases. Direct single-cell microinjection of mRNA encoding (GFP) achieves detectable GFP fluorescence within ∼5 h in both cell types. These findings coupled with the possibility of targeting several hundreds of single cells improves throughput and sensitivity over conventional methods to study rare cell subpopulations.

The European Association for Clinical Pharmacology and Therapeutics-25 years' young and going strong.

Clinical pharmacology as a scientific discipline and medical specialty was unarguably born in the twentieth century. Whilst pharmacology-the science behind the treatment of disease-had been in evolution since at least medieval times, the clinical discipline of pharmacology has had a more recent genesis and rather insidious evolution. During the 1900s, there were some clear father (parent) figures of clinical pharmacology in Europe that emerged and were responsible for the development of the specialty in this continent. This was a time when there were parallel developments in geographically dispersed academic departments (around the globe), during an age of excitement in drug discovery and clinical application of new therapeutic agents. It was the meeting of minds of some of these progenitors of the specialty that led to the development of the European Association for Clinical Pharmacology and Therapeutics (EACPT) 25 years ago arising from a working party supported by the World Health Organization in Europe. The EACPT now includes all major national organizations for clinical pharmacology in Europe, representing over 4000 individual professionals interested in clinical pharmacology and therapeutics. The EACPT has a major interest in promoting the safe use of medicines across Europe and internationally and has supported these aims since 1995, through biennial international scientific congresses and summer schools with delegates and presenters from around the world as well as various working group activities. In this article, the current executive committee members of EACPT recall this history, describe the evolution of the association over the last quarter of a century, and provide an update on the activities and ambitions of the association today.

Osler Centenary Papers: Osler as medical leader.

The Canadian physician Sir William Osler is a key figure in the history of modern medicine. He encouraged lifelong learning for doctors, starting with bedside teaching. Contemporary with Old World figures such as Pasteur in Paris and Virchow in Berlin, he played a major role in raising awareness among clinicians of the importance of the scientific basis for the practice of medicine. He championed a rational approach to treatment and did much to encourage avoidance of 'unnecessary drugging' by prescribers. He is credited with playing a key role in improving education of medical students and postgraduate education of doctors, with important benefits for the care of hospital patients. He also had a major influence on his medical colleagues through founding and leading medical societies. A century on from his death in December 1919, his specific contributions and how he achieved them are not well known. The aim of this article is to consider the evidence that Osler was an influential medical leader and to reflect on the extent to which the achievements which resulted from his leadership are relevant to modern clinical medicine. Questions of interest include his leadership style, what made for his success as a leader, his medical achievements both in North America and in England, his own insight into leadership and how he was viewed by his peers.

A randomised controlled trial of the impact of structured written and verbal advice by community pharmacists on improving hypertension education and control in patients with high blood pressure.

PURPOSE: This study was aimed to determine whether structured written and verbal education provided to patients by community pharmacists about high blood pressure (BP) and its treatment would be (a) better retained and (b) be associated with improved BP control as compared to patients receiving verbal advice only. METHODS: The study was designed as a randomised controlled trial and was conducted in the West Midlands, UK, between January 2014 and June 2014. The primary outcome measures were differences in systolic and diastolic BP from baseline and retention of information about high BP assessed with a questionnaire at 2-, 4- and 26-week follow-up points. RESULTS: A total of 64 adults were included in the study. At the week 26 follow-up, compared to participants in the control group, there was a significant improvement in the knowledge of intervention participants about the risks associated with high BP (p < 0.001) and awareness about potential adverse effects of the new BP medicine (p < 0.001). Similarly, there was a greater and more significant reduction in systolic BP in favour of the intervention group 8 mmHg (95% CI 2.1-13.3 p = 0.009) compared to 6 mmHg (95% CI 0.6-11.7 p = 0.02) in the control group at the week 4 follow-up. However, this greater effect of an intervention on BP was not sustained at the 26-week follow-up. For diastolic BP, there was no added effect of the intervention. CONCLUSION: This randomised controlled trial suggests that although written advice provided by community pharmacists in comparison to verbal advice was more effective in improving knowledge and understanding of patients about hypertension and its treatment, it did not lead to better blood pressure control.

Registry report of structural and functional cardiac abnormalities diagnosed by echocardiography in an asymptomatic population.

BACKGROUND: The epidemiology of heart disease is changing, with rheumatic heart disease becoming less common but degenerative valve disorders, heart failure and atrial fibrillation (AF) increasing. OBJECTIVE: We sought to determine the prevalence of structural cardiac abnormalities in the apparently symptom-free adult population within our prospective echocardiography (echo) registry. METHODS: Our echo registry comprised echo studies and associated demographic and clinical data obtained prospectively from 362 consecutive asymptomatic subjects aged 50-74 years and without known heart disease referred between 2011 and 2012 from general practices in the South East of England. RESULTS: 221 echo abnormalities were detected in 178 (49%) subjects (46% men; mean (±SD) age 63.9±9.2 years; 98% Caucasian). A major abnormality was detected in seven subjects: four had a large secundum atrial septal defect, one had critical aortic stenosis, one severe mitral regurgitation and one features of hypertrophic cardiomyopathy. Twelve subjects had left ventricular systolic dysfunction with an ejection fraction (EF) <50% (of whom 10 had EF <40%). Four subjects had AF. Minor echo abnormalities were evident in the remaining 171 (47%) subjects. Abnormalities were commoner in patients with cardiovascular risk factors or a history of cardiac disease than in those without (53% vs 38%). In multivariate analyses stratified by gender, for women, increased age (F=33.3, p<0.001) and systolic blood pressure (F=9.2, p=0.003) were associated with abnormal echo findings; for men, increased age (F=12.0, p<0.001) and lower cholesterol (F=4.2, p=0.042) predicted an increase in abnormal findings on echo. CONCLUSIONS: Unrecognised cardiac abnormalities are very common in middle-aged men and women with no overt symptoms. Echo offers the potential to identify the need for early intervention and treatment to improve cardiovascular outcomes.

The impact of interventions by pharmacists in community pharmacies on control of hypertension: a systematic review and meta-analysis of randomized controlled trials.

AIMS: To undertake a systematic review and meta-analysis of randomized controlled trials concerned with the impact of community pharmacist-led interventions on blood pressure control in patients with hypertension. METHODS: Eight electronic databases were searched up to 30 November 2013, with no start date (Web of Science, Embase, The Cochrane Library, Medline Ovid, Biomed Central, Biosis Citation Index, CINAHL, PsycINFO). All studies included were randomized controlled trials involving patients with hypertension, with or without cardiovascular-related co-morbidities, with difference in blood pressure as an outcome. Data collected included the study design, baseline characteristics of study populations, types of interventions and outcomes. The Cochrane tool was used to assess risk of bias. RESULTS: From 340 articles identified on initial searching, 16 randomized controlled trials (3032 patients) were included. Pharmacist-led interventions were patient education on hypertension, management of prescribing and safety problems associated with medication, and advice on lifestyle. These interventions were associated with significant reductions in systolic [11 studies (2240 patients); -6.1 mmHg (95% confidence interval, -3.8 to -8.4 mmHg); P < 0.00001] and diastolic blood pressure [11 studies (2246 patients); -2.5 mmHg (95% confidence interval, -1.5 to -3.4 mmHg); P < 0.00001]. CONCLUSIONS: Community pharmacist-led interventions can significantly reduce systolic and diastolic blood pressure. These interventions could be useful for improving clinical management of hypertension.

Risk-based approach to setting sterile filtration microbial bioburden limits - Focus on biotech-derived products.

Holistic concepts should be applied that reduce risks prior to final bioburden testing and sterile filtration, based on enhanced process and product attribute understanding, which could be key to successful bioburden risk management. Key findings of this paper include.

Registry report on kinetics of rescue antiplatelet treatment to abolish cerebral microemboli after carotid endarterectomy.

BACKGROUND AND PURPOSE: Cerebral microemboli signals (MES) are associated with increased risk of acute stroke syndromes. We compared the effects on cerebral microemboli after carotid endarterectomy of tirofiban with dextran-40. METHODS: We used transcranial Doppler ultrasound to study transient MES acutely after carotid endarterectomy between August 2000 and December 2010 in 128 subjects refractory to preoperative antiplatelet treatment. Antithrombotic treatment was given for MES ≥50 hour(-1) (tirofiban: 40 patients [age 74 ± 1 {SEM}, males 27, and white 38]; dextran-40: 34 patients [age 69 ± 2, males 22, white 30]). In 54 patients with MES <50 hour(-1) (age 71 ± 1, male 36, white 52), MES were monitored during their spontaneous resolution (controls). Data are median (interquartile range). RESULTS: The time to 50% reduction in MES (tirofiban 23 minutes [15-28]; dextran-56 [43-83]; controls 30 [22-38]; P<0.001, Kruskal-Wallis analysis) and for complete MES resolution (tirofiban 68 minutes [53-94]; dextran-113 [79-146]; controls 53 [49-68]; P<0.001, Kruskal-Wallis analysis) were shorter with tirofiban. The early cardiovascular event rate was similar with tirofiban compared with controls but increased in patients who received dextran. CONCLUSIONS: These findings suggest that transcranial Doppler-directed tirofiban therapy is more effective than dextran-40 in suppression of cerebral microemboli after carotid endarterectomy.

Risk Assessment Approach to Microbiological Controls of Cell Therapies.

This technology review, written by a small group of pharmaceutical microbiologists experienced in cell therapies, discussed a risk-based approach to microbiological contamination detection and control during gene and cell therapy production. Topics discussed include a brief overview of cell therapies, a risk analysis related to donor selection, cell collection and infectious agent testing, cell transformation and expansion, packaging, storage, and administration, and cell therapy microbial contamination testing and release.

Effects of cyclic strain on endothelial cell apoptosis and tubulogenesis are dependent on ROS production via NAD(P)H subunit p22phox.

OBJECTIVE: Vascular endothelial cells (ECs) are constantly exposed to blood flow associated forces such as cyclic strain due to blood pressure, which affects ECs survival and angiogenesis by producing ROS via NAD(P)H oxidase. NAD(P)H oxidase subunit p22phox is reported to be related to the development of atherosclerosis and increased levels of p22phox mRNA are correlated to ECs proliferation. However, the importance and signaling mechanism of p22phox on ECs survival and angiogenesis under cyclic strain are unclear. METHODS: 5%-20% cyclic strain were applied by the Flexercell system to simulate in vivo environment of human ECs; the effect of p22phox on mechanical ECs survival mechanism and tubulogenesis was determined by western blot and 3-D tissue culture by knocking down p22phox expression via shRNA plasmid. RESULTS: Knockdown of p22phox induced expression of cleaved caspase-3 and decreased cell viability ratio (CVR). 5% strain increased and 20% strain decreased CVR of shp22phox cells. There were complex biphasic effects of cyclic strain on ECs survival signaling. 5% strain continuously increased Akt phosphorylation; 20% strain increased after 10min stimulation and decreased Akt phosphorylation lately. 5% strain increased and 20% strain decreased eNOS phosphorylation. Knockdown of p22phox decreased Akt and eNOS phosphorylation with or without cyclic strain. ROS production was increasingly stimulated progressively by strain via the p22phox pathway. 5% strain increased and 20% strain decreased total NO production and vascular tubulogenesis via p22phox pathway. CONCLUSION: ROS production is pivotal to responses to physiological or pathological strain. Physiological strain increases but pathological strain decreases ECs survival and tubulogenesis, and these effects occur via the NAD(P)H subunit p22phox pathway.

Grossly elevated plasma BNP does not exclude the diagnosis of constrictive pericarditis.

Pericardial effusion with constrictive physiology describes a condition in which the pericardial fluid and thickened and dense pericardium limit left ventricular (LV) diastolic filling and prevent ventricular stretch. This leads to equalization of the end-diastolic pressure in cardiac chambers and poor ventricular filling. We report two patients, who presented with symptoms and signs of severe heart failure and with significantly raised BNP levels who were subsequently diagnosed to have pericardial effusion with constrictive physiology. When VATS pericardial window procedure was performed, the BNP values transiently increased even more in both patients, and returned to pre-operative levels at 5 days post-op. We therefore propose that in contrast to current evidence, grossly elevated BNP levels can coexist with a diagnosis of constrictive pathology. Further studies into constrictive pericarditis should take into account the transient changes in BNP observed in our study that may reveal more regarding the pathophysiology of constrictive pericarditis.

Transit amplifying cells coordinate mouse incisor mesenchymal stem cell activation.

Stem cells (SCs) receive inductive cues from the surrounding microenvironment and cells. Limited molecular evidence has connected tissue-specific mesenchymal stem cells (MSCs) with mesenchymal transit amplifying cells (MTACs). Using mouse incisor as the model, we discover a population of MSCs neibouring to the MTACs and epithelial SCs. With Notch signaling as the key regulator, we disclose molecular proof and lineage tracing evidence showing the distinct MSCs contribute to incisor MTACs and the other mesenchymal cell lineages. MTACs can feedback and regulate the homeostasis and activation of CL-MSCs through Delta-like 1 homolog (Dlk1), which balances MSCs-MTACs number and the lineage differentiation. Dlk1's function on SCs priming and self-renewal depends on its biological forms and its gene expression is under dynamic epigenetic control. Our findings can be validated in clinical samples and applied to accelerate tooth wound healing, providing an intriguing insight of how to direct SCs towards tissue regeneration.

Registry report on prediction by Pocock cardiovascular score of cerebral microemboli acutely following carotid endarterectomy.

BACKGROUND: Cerebral microemboli may lead to ischaemic neurological complications after carotid endarterectomy (CEA). The association between classical cardiovascular risk factors and acute cerebral microemboli following carotid surgery has not been studied. The aim of this study was to explore whether an established cardiovascular risk score (Pocock score) predicts the presence of cerebral microemboli acutely after CEA. SUBJECTS AND METHODS: Pocock scores were assessed for the 670 patients from the Carotid Surgery Registry (age 71±1 (SEM) years, 474 (71%) male, 652 (97%) Caucasian) managed from January 2002 to December 2012 in the Regional Vascular Centre at University Hospitals Coventry and Warwickshire NHS Trust, which serves a population of 950 000. CEA was undertaken in 474 (71%) patients for symptomatic carotid stenosis and in 196 (25%) asymptomatic patients during the same period. 74% of patients were hypertensive, 71% were smokers and 49% had hypercholesterolaemia. RESULTS: A high Pocock score (≥2.3%) was significantly associated with evidence of cerebral microemboli acutely following CEA (P=0.039, Mann-Whitney (MW) test). A Pocock score (≥2.3%) did not predict patients who required additional antiplatelet therapy (microemboli signal (MES) rate >50 hour-1: P=0.164, MW test). Receiver operating characteristic analysis also showed that the Pocock score predicts acute postoperative microemboli (area under the curve (AUC) 0.546, 95% CI 0.502 to 0.590, P=0.039) but not a high rate of postoperative microemboli (MES >50 hour-1: AUC 0.546, 95% CI 0.482 to 0.610, P=0.164). A Pocock score ≥2.3% showed a sensitivity of 74% for the presence of acute postoperative cerebral microemboli. A Pocock score ≥2.3% also showed a sensitivity of 77% and a negative predictive value of 90% for patients who developed a high microembolic rate >50 hour-1 after carotid surgery. CONCLUSION: These findings demonstrate that the Pocock score could be used as a clinical tool to identify patients at high risk of developing acute postoperative microemboli.