A high-efficiency HeLa cell nuclear transcription extract.

A HeLa cell nuclear transcription extract that is approximately 20 times more efficient than standard HeLa cell transcription extracts was developed. Transcription of the strong adenovirus II major late promoter by this extract results in the synthesis of 1.5-4 molecules of product RNA per molecule of template, indicating that the extract is capable of multiple rounds of initiation. Standard HeLa cell nuclear extracts transcribe closed circular and linear adenovirus major late promoter templates with equal efficiency. In contrast, the new extract exhibits an increase of approximately twofold on transcription of a closed circular, as opposed to a linear, major late promoter template.

Ultraweak photon emission in assessing bone growth factor efficiency using fibroblastic differentiation.

Photons participate in many atomic and molecular interactions and changes. Recent biophysical research has shown the induction of ultraweak photons in biological tissue. It is now established that plants, animal and human cells emit a very weak radiation which can be readily detected with an appropriate photomultiplier system. Although the emission is extremely low in mammalian cells, it can be efficiently induced by ultraviolet light. In our studies, we used the differentiation system of human skin fibroblasts from a patient with Xeroderma Pigmentosum of complementation group A in order to test the growth stimulation efficiency of various bone growth factors at concentrations as low as 5 ng/ml of cell culture medium. In additional experiments, the cells were irradiated with a moderate fluence of ultraviolet A. The different batches of growth factors showed various proliferation of skin fibroblasts in culture which could be correlated with the ultraweak photon emission. The growth factors reduced the acceleration of the fibroblast differentiation induced by mitomycin C by a factor of 10-30%. In view that fibroblasts play an essential role in skin aging and wound healing, the fibroblast differentiation system is a very useful tool in order to elucidate the efficacy of growth factors.

Rationale and design of a randomized, double-blind, parallel-group study of terutroban 30 mg/day versus aspirin 100 mg/day in stroke patients: the prevention of cerebrovascular and cardiovascular events of ischemic origin with terutroban in patients with a history of ischemic stroke or transient ischemic attack (PERFORM) study.

BACKGROUND: Ischemic stroke is the leading cause of mortality worldwide and a major contributor to neurological disability and dementia. Terutroban is a specific TP receptor antagonist with antithrombotic, antivasoconstrictive, and antiatherosclerotic properties, which may be of interest for the secondary prevention of ischemic stroke. This article describes the rationale and design of the Prevention of cerebrovascular and cardiovascular Events of ischemic origin with teRutroban in patients with a history oF ischemic strOke or tRansient ischeMic Attack (PERFORM) Study, which aims to demonstrate the superiority of the efficacy of terutroban versus aspirin in secondary prevention of cerebrovascular and cardiovascular events. METHODS AND RESULTS: The PERFORM Study is a multicenter, randomized, double-blind, parallel-group study being carried out in 802 centers in 46 countries. The study population includes patients aged > or =55 years, having suffered an ischemic stroke (< or =3 months) or a transient ischemic attack (< or =8 days). Participants are randomly allocated to terutroban (30 mg/day) or aspirin (100 mg/day). The primary efficacy endpoint is a composite of ischemic stroke (fatal or nonfatal), myocardial infarction (fatal or nonfatal), or other vascular death (excluding hemorrhagic death of any origin). Safety is being evaluated by assessing hemorrhagic events. Follow-up is expected to last for 2-4 years. Assuming a relative risk reduction of 13%, the expected number of primary events is 2,340. To obtain statistical power of 90%, this requires inclusion of at least 18,000 patients in this event-driven trial. The first patient was randomized in February 2006. CONCLUSIONS: The PERFORM Study will explore the benefits and safety of terutroban in secondary cardiovascular prevention after a cerebral ischemic event.

Fish oil supplementation does not alter energy efficiency in healthy males

BACKGROUND AND AIMS: Fish oil (FO) supplementation prevents the development of obesity and insulin resistance, and upregulate the expression of UCP3 in skeletal muscle in rodents. This may represent indirect evidence that FO promotes fat oxidation and/or alter energy efficiency. The aim of this study was to evaluate whether such effects can be observed in humans. The metabolic effects of FO were assessed during exercise in order to obtain a direct measurement of energy efficiency. METHODS: Eight healthy male volunteers were studied with and without supplementation with 7.2 g/day FO (including 1.1 g/day eicosopentaenoic acid and 0.7 g/day decosahexaenoic acid) during 14 days. Their VO(2 max) was measured on cycle ergometer. Thereafter, energy metabolism (substrate oxidation, energy expenditure and energy efficiency) was assessed during a 30 min cycling exercise at 50% VO(2 max) performed 2 h 30 after a standardized, high carbohydrate breakfast. RESULTS: VO(2 max) was 38.6+/-2.2 after FO and 38.4+/-2.0 (mL x kg(-1) x min(-1)) in control conditions (NS). Basal plasma glucose, insulin and NEFA concentrations, and energy metabolism were similar with FO and in controls. During exercise, the increases in plasma NEFA concentrations, energy expenditure, glucose and lipid oxidation, and the decreases in glycaemia and insulinemia were not altered by FO intake. Energy efficiency was 22.4+/-0.6% after FO vs 21.8+/-0.7% in controls. In order to ascertain that the absence of effects of FO was not due to consumption of a carbohydrate meal immediately before exercise, 4 of the 8 subjects were re-studied in fasting conditions, FO also failed to alter energy efficiency in this subset of studies. CONCLUSION: FO supplementation did not significantly alter energy metabolism and energy efficiency during exercise in healthy humans.

Identification of cancer/testis-antigen genes by massively parallel signature sequencing.

Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer/testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 intron-containing genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family.

A random linear functional approach to efficiency bounds

This paper suggests a method for obtaining efficiency bounds in models containing either only infinite-dimensional parameters or both finite- and infinite-dimensional parameters (semiparametric models). The method is based on a theory of random linear functionals applied to the gradient of the log-likelihood functional and is illustrated by computing the lower bound for Cox's regression model

C4 Photosynthesis evolved in grasses via parallel adaptive genetic changes.

Phenotypic convergence is a widespread and well-recognized evolutionary phenomenon. However, the responsible molecular mechanisms remain often unknown mainly because the genes involved are not identified. A well-known example of physiological convergence is the C4 photosynthetic pathway, which evolved independently more than 45 times [1]. Here, we address the question of the molecular bases of the C4 convergent phenotypes in grasses (Poaceae) by reconstructing the evolutionary history of genes encoding a C4 key enzyme, the phosphoenolpyruvate carboxylase (PEPC). PEPC genes belong to a multigene family encoding distinct isoforms of which only one is involved in C4 photosynthesis [2]. By using phylogenetic analyses, we showed that grass C4 PEPCs appeared at least eight times independently from the same non-C4 PEPC. Twenty-one amino acids evolved under positive selection and converged to similar or identical amino acids in most of the grass C4 PEPC lineages. This is the first record of such a high level of molecular convergent evolution, illustrating the repeatability of evolution. These amino acids were responsible for a strong phylogenetic bias grouping all C4 PEPCs together. The C4-specific amino acids detected must be essential for C4 PEPC enzymatic characteristics, and their identification opens new avenues for the engineering of the C4 pathway in crops.

Efficiency and significance of multiple vocal signals in sibling competition

Animals can compete for resources by displaying various acoustic signals that may differentially affect the outcome of competition. We propose the hypothesis that the most efficient signal to deter opponents should be the one that most honestly reveals motivation to compete. We tested this hypothesis in the barn owl (Tyto alba) in which nestlings produce more calls of longer duration than siblings to compete for priority access to the indivisible prey item their parents will deliver next. Because nestlings increase call rate to a larger extent than call duration when they become hungrier, call rate would signal more accurately hunger level. This leads us to propose three predictions. First, a high number of calls should be more efficient in deterring siblings to compete than long calls. Second, the rate at which an individual calls should be more sensitive to variation in the intensity of the sibling vocal competition than the duration of its calls. Third, call rate should influence competitors' vocalization for a longer period of time than call duration. To test these three predictions we performed playback experiments by broadcasting to singleton nestlings calls of varying durations and at different rates. According to the first prediction, singleton nestlings became less vocal to a larger extent when we broadcasted more calls compared to longer calls. In line with the second prediction, nestlings reduced vocalization rate to a larger extent than call duration when we broadcasted more or longer calls. Finally, call rate had a longer influence on opponent's vocal behavior than call duration. Young animals thus actively and differentially use multiple signaling components to compete with their siblings over parental resources.

The potential effects of social interactions on reproductive efficiency of stallions

The reproductive efficiency of stabled domestic stallions is often lower than what could be expected from observations in feral herds. In the wild, stallions typically live with mares in harem bands, with other stallions in bachelor bands, or occasionally in mixed-sex transitional bands. We, therefore, argue that permanent contact with mares may increase reproductive efficiency of stallions suffering from low libido and/or fertility. We also provide a summary of our present knowledge of natural conditions, management, and husbandry of domestic stallions, and of intra- and intersexual behavioral interactions in horses.

Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin

Variation in cellular gene expression levels has been shown to be inherited. Expression is controlled at transcriptional and post-transcriptional levels. Internal ribosome entry sites (IRES) are used by viruses to bypass inhibition of cap-dependent translation, and by eukaryotic cells to control translation under conditions when protein synthesis is inhibited. We aimed at identifying genomic determinants of variability in IRES-mediated translation of viral [Encephalomyocarditis virus (EMCV)] and cellular IRES [X-linked inhibitor-of-apoptosis (XIAP) and c-myc]. Bicistronic lentiviral constructs expressing two fluorescent reporters were used to transduce laboratory and B lymphoblastoid cell lines [15 CEPH pedigrees (n = 205) and 50 unrelated individuals]. IRES efficiency varied according to cell type and among individuals. Control of IRES activity has a significant genetic component (h(2) of 0.47 and 0.36 for EMCV and XIAP, respectively). Quantitative linkage analysis identified a suggestive locus (LOD 2.35) on chromosome 18q21.2, and genome-wide association analysis revealed of a cluster of SNPs on chromosome 3, intronic to the FHIT gene, marginally associated (P = 5.9E-7) with XIAP IRES function. This study illustrates the in vitro generation of intermediate phenotypes by using cell lines for the evaluation of genetic determinants of control of elements such as IRES.

Guideline adaptation: an approach to enhance efficiency in guideline development and improve utilisation.

BACKGROUND: Developing and updating high-quality guidelines requires substantial time and resources. To reduce duplication of effort and enhance efficiency, we developed a process for guideline adaptation and assessed initial perceptions of its feasibility and usefulness. METHODS: Based on preliminary developments and empirical studies, a series of meetings with guideline experts were organised to define a process for guideline adaptation (ADAPTE) and to develop a manual and a toolkit made available on a website (http://www.adapte.org). Potential users, guideline developers and implementers, were invited to register and to complete a questionnaire evaluating their perception about the proposed process. RESULTS: The ADAPTE process consists of three phases (set-up, adaptation, finalisation), 9 modules and 24 steps. The adaptation phase involves identifying specific clinical questions, searching for, retrieving and assessing available guidelines, and preparing the draft adapted guideline. Among 330 registered individuals (46 countries), 144 completed the questionnaire. A majority found the ADAPTE process clear (78%), comprehensive (69%) and feasible (60%), and the manual useful (79%). However, 21% found the ADAPTE process complex. 44% feared that they will not find appropriate and high-quality source guidelines. DISCUSSION: A comprehensive framework for guideline adaptation has been developed to meet the challenges of timely guideline development and implementation. The ADAPTE process generated important interest among guideline developers and implementers. The majority perceived the ADAPTE process to be feasible, useful and leading to improved methodological rigour and guideline quality. However, some de novo development might be needed if no high quality guideline exists for a given topic.

Bunch frequency multiplication by RF injection into an isochronous ring

La collaboration CLIC (Compact LInear Collider, collisionneur linéaire compact) étudie la possibilité de réaliser un collisionneur électron-positon linéaire à haute énergie (3 TeV dans le centre de masse) et haute luminosité (1034 cm-2s-1), pour la recherche en physique des particules. Le projet CLIC se fonde sur l'utilisation de cavités accélératrices à haute fréquence (30 GHz). La puissance nécessaire à ces cavités est fournie par un faisceau d'électrons de basse énergie et de haute intensité, appelé faisceau de puissance, circulant parallèlement à l'accélérateur linéaire principal (procédé appelé « Accélération à Double Faisceau »). Dans ce schéma, un des principaux défis est la réalisation du faisceau de puissance, qui est d'abord généré dans un complexe accélérateur à basse fréquence, puis transformé pour obtenir une structure temporelle à haute fréquence nécessaire à l'alimentation des cavités accélératrices de l'accélérateur linéaire principal. La structure temporelle à haute fréquence des paquets d'électrons est obtenue par le procédé de multiplication de fréquence, dont la manipulation principale consiste à faire circuler le faisceau d'électrons dans un anneau isochrone en utilisant des déflecteurs radio-fréquence (déflecteurs RF) pour injecter et combiner les paquets d'électrons. Cependant, ce type de manipulation n'a jamais été réalisé auparavant et la première phase de la troisième installation de test pour CLIC (CLIC Test Facility 3 ou CTF3) a pour but la démonstration à faible charge du procédé de multiplication de fréquence par injection RF dans un anneau isochrone. Cette expérience, qui a été réalisée avec succès au CERN au cours de l'année 2002 en utilisant une version modifiée du pré-injecteur du grand collisionneur électron-positon LEP (Large Electron Positron), est le sujet central de ce rapport. L'expérience de combinaison des paquets d'électrons consiste à accélérer cinq impulsions dont les paquets d'électrons sont espacés de 10 cm, puis à les combiner dans un anneau isochrone pour obtenir une seule impulsion dont les paquets d'électrons sont espacés de 2 cm, multipliant ainsi la fréquence des paquets d'électrons, ainsi que la charge par impulsion, par cinq. Cette combinaison est réalisée au moyen de structures RF résonnantes sur un mode déflecteur, qui créent dans l'anneau une déformation locale et dépendante du temps de l'orbite du faisceau. Ce mécanisme impose plusieurs contraintes de dynamique de faisceau comme l'isochronicité, ainsi que des tolérances spécifiques sur les paquets d'électrons, qui sont définies dans ce rapport. Les études pour la conception de la Phase Préliminaire du CTF3 sont détaillées, en particulier le nouveau procédé d'injection avec les déflecteurs RF. Les tests de haute puissance réalisés sur ces cavités déflectrices avant leur installation dans l'anneau sont également décrits. L'activité de mise en fonctionnement de l'expérience est présentée en comparant les mesures faites avec le faisceau aux simulations et calculs théoriques. Finalement, les expériences de multiplication de fréquence des paquets d'électrons sont décrites et analysées. On montre qu'une très bonne efficacité de combinaison est possible après optimisation des paramètres de l'injection et des déflecteurs RF. En plus de l'expérience acquise sur l'utilisation de ces déflecteurs, des conclusions importantes pour les futures activités CTF3 et CLIC sont tirées de cette première démonstration de la multiplication de fréquence des paquets d'électrons par injection RF dans un anneau isochrone.<br/><br/>The Compact LInear Collider (CLIC) collaboration studies the possibility of building a multi-TeV (3 TeV centre-of-mass), high-luminosity (1034 cm-2s-1) electron-positron collider for particle physics. The CLIC scheme is based on high-frequency (30 GHz) linear accelerators powered by a low-energy, high-intensity drive beam running parallel to the main linear accelerators (Two-Beam Acceleration concept). One of the main challenges to realize this scheme is to generate the drive beam in a low-frequency accelerator and to achieve the required high-frequency bunch structure needed for the final acceleration. In order to provide bunch frequency multiplication, the main manipulation consists in sending the beam through an isochronous combiner ring using radio-frequency (RF) deflectors to inject and combine electron bunches. However, such a scheme has never been used before, and the first stage of the CLIC Test Facility 3 (CTF3) project aims at a low-charge demonstration of the bunch frequency multiplication by RF injection into an isochronous ring. This proof-of-principle experiment, which was successfully performed at CERN in 2002 using a modified version of the LEP (Large Electron Positron) pre-injector complex, is the central subject of this report. The bunch combination experiment consists in accelerating in a linear accelerator five pulses in which the electron bunches are spaced by 10 cm, and combining them in an isochronous ring to obtain one pulse in which the electron bunches are spaced by 2 cm, thus achieving a bunch frequency multiplication of a factor five, and increasing the charge per pulse by a factor five. The combination is done by means of RF deflecting cavities that create a time-dependent bump inside the ring, thus allowing the interleaving of the bunches of the five pulses. This process imposes several beam dynamics constraints, such as isochronicity, and specific tolerances on the electron bunches that are defined in this report. The design studies of the CTF3 Preliminary Phase are detailed, with emphasis on the novel injection process using RF deflectors. The high power tests performed on the RF deflectors prior to their installation in the ring are also reported. The commissioning activity is presented by comparing beam measurements to model simulations and theoretical expectations. Eventually, the bunch frequency multiplication experiments are described and analysed. It is shown that the process of bunch frequency multiplication is feasible with a very good efficiency after a careful optimisation of the injection and RF deflector parameters. In addition to the experience acquired in the operation of these RF deflectors, important conclusions for future CTF3 and CLIC activities are drawn from this first demonstration of the bunch frequency multiplication by RF injection into an isochronous ring.<br/><br/>La collaboration CLIC (Compact LInear Collider, collisionneur linéaire compact) étudie la possibilité de réaliser un collisionneur électron-positon linéaire à haute énergie (3 TeV) pour la recherche en physique des particules. Le projet CLIC se fonde sur l'utilisation de cavités accélératrices à haute fréquence (30 GHz). La puissance nécessaire à ces cavités est fournie par un faisceau d'électrons de basse énergie et de haut courant, appelé faisceau de puissance, circulant parallèlement à l'accélérateur linéaire principal (procédé appelé « Accélération à Double Faisceau »). Dans ce schéma, un des principaux défis est la réalisation du faisceau de puissance, qui est d'abord généré dans un complexe accélérateur à basse fréquence, puis transformé pour obtenir une structure temporelle à haute fréquence nécessaire à l'alimentation des cavités accélératrices de l'accélérateur linéaire principal. La structure temporelle à haute fréquence des paquets d'électrons est obtenue par le procédé de multiplication de fréquence, dont la manipulation principale consiste à faire circuler le faisceau d'électrons dans un anneau isochrone en utilisant des déflecteurs radio-fréquence (déflecteurs RF) pour injecter et combiner les paquets d'électrons. Cependant, ce type de manipulation n'a jamais été réalisé auparavant et la première phase de la troisième installation de test pour CLIC (CLIC Test Facility 3 ou CTF3) a pour but la démonstration à faible charge du procédé de multiplication de fréquence par injection RF dans un anneau isochrone. L'expérience consiste à accélérer cinq impulsions, puis à les combiner dans un anneau isochrone pour obtenir une seule impulsion dans laquelle la fréquence des paquets d'électrons et le courant sont multipliés par cinq. Cette combinaison est réalisée au moyen de structures déflectrices RF qui créent dans l'anneau une déformation locale et dépendante du temps de la trajectoire du faisceau. Les résultats de cette expérience, qui a été réalisée avec succès au CERN au cours de l?année 2002 en utilisant une version modifiée du pré-injecteur du grand collisionneur électron-positon LEP (Large Electron Positon), sont présentés en détail.

Efficiency of coronary artery disease screening guidelines in asymptomatic and atypical chest pain type 2 diabetic patients

RESUME Objectifs. Évaluer la prévalence de maladie coronarienne chez les patients diabétiques de type 2 asymptomatiques ou avec angor atypique selon les recommandations américaines de l'American Diabetes Association et de l'American College of Cardiology. Méthodes. Cent cinquante-quatre patients diabétiques de type 2 asymptomatiques ou avec angor atypique et présentant au minimum 2 facteurs de risque cardio-vasculaires additionnels ont été dépistés par échocardiographie de stress (71%, n=109), scintigraphie myocardique de perfusion (26%, n=40) ou l'association des 2 examens (3%, n=5). Résultats. L'échocardiographie de stress s'est révélée positive chez 16 patients (14%) et 14 ont eu une coronarographie révélant des sténoses significatives chez 12 (86%). La scintigraphie myocardique de perfusion était positive chez 16 patients (36%). Huit patients ont eu une coronarographie et 4 (50%) présentaient des sténoses significatives. Au total, 31 patients (20%) ont montré des signes d'ischémie lors de l'examen non-invasif et 15 (10%) ont présenté des sténoses significatives à la coronarographie. Les facteurs prédictifs indépendants de la maladie coronarienne étaient le tabagisme (OR 6.5, p=0.05), la microalbuminurie (OR 3.9, p=0.03), ainsi que les souffles fémoraux (OR 17.1, p=0.008). Conclusions. En suivant les recommandations américaines, un patient sur cinq présentait une ischémie lors des examens non-invasifs, tandis que 1 sur 10 avait des sténoses significatives à la coronarographie. L'analyse multivariée suggère que des marqueurs des complications micro- et macro-vasculaires en combinaison avec des facteurs de risque cardio-vasculaire classiques pourraient améliorer le pouvoir diagnostic de ces recommandations. SUMMARY Aims. We evaluated the prevalence of coronary artery disease in asymptomatic and atypical chest pain type 2 diabetic patients according to the American Diabetes Association and American College of Cardiology guidelines. Methods. Asymptomatic or atypical chest pain type 2 diabetic patients (n=154), with at least two additional cardiovascular risk factors, were screened for coronary artery disease using stress echocardiography (71%, n=109), myocardial perfusion imaging (26%, n=40) or both (3%, n=5). Results. Stress echocardiography was positive in 16 patients (14%) and 14 had a coronary angiography, revealing significant stenoses in 12 (86%). Myocardial perfusion imaging was positive in 16 patients (36%). Eight patients underwent angiography and 4 (50%) presented significant stenoses. Overall, 31 patients (20%) demonstrated signs of ischemia on non-invasive tests and 15 (10%) presented significant stenoses on coronary angiography. Independent predictors of coronary artery disease were smoking (OR 6.5, p=0.05), microalbuminuria (OR 3.9, p=0.03) and femoral murmur (OR 17.1, p=0.008). Conclusions. Following the guidelines, one in five diabetic patient presented ischemia on noninvasive tests, while one in ten presented significant coronary stenoses. Multivariate analysis suggests that adding markers of micro- and macro-vascular complications to classical cardiovascular risk factors may enhance the diagnostic efficiency of the guidelines.