An atlas of human gene expression from massively parallel signature sequencing (MPSS).

We have used massively parallel signature sequencing (MPSS) to sample the transcriptomes of 32 normal human tissues to an unprecedented depth, thus documenting the patterns of expression of almost 20,000 genes with high sensitivity and specificity. The data confirm the widely held belief that differences in gene expression between cell and tissue types are largely determined by transcripts derived from a limited number of tissue-specific genes, rather than by combinations of more promiscuously expressed genes. Expression of a little more than half of all known human genes seems to account for both the common requirements and the specific functions of the tissues sampled. A classification of tissues based on patterns of gene expression largely reproduces classifications based on anatomical and biochemical properties. The unbiased sampling of the human transcriptome achieved by MPSS supports the idea that most human genes have been mapped, if not functionally characterized. This data set should prove useful for the identification of tissue-specific genes, for the study of global changes induced by pathological conditions, and for the definition of a minimal set of genes necessary for basic cell maintenance. The data are available on the Web at http://mpss.licr.org and http://sgb.lynxgen.com.

Remains of the day: Biliary complications related to single-port laparoscopic cholecystectomy.

AIM: To assesse the rate of bile duct injuries (BDI) and overall biliary complications during single-port laparoscopic cholecystectomy (SPLC) compared to conventional laparoscopic cholecystectomy (CLC). METHODS: SPLC has recently been proposed as an innovative surgical approach for gallbladder surgery. So far, its safety with respect to bile duct injuries has not been specifically evaluated. A systematic review of the literature published between January 1990 and November 2012 was performed. Randomized controlled trials (RCT) comparing SPLC versus CLC reporting BDI rate and overall biliary complications were included. The quality of RCT was assessed using the Jadad score. Analysis was made by performing a meta-analysis, using Review Manager 5.2. This study was based on the Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines. A retrospective study including all retrospective reports on SPLC was also performed alongside. RESULTS: From 496 publications, 11 RCT including 898 patients were selected for meta-analysis. No studies were rated as high quality (Jadad score ≥ 4). Operative indications included benign gallbladder disease operated in an elective setting in all studies, excluding all emergency cases and acute cholecystitis. The median follow-up was 1 mo (range 0.03-18 mo). The incidence of BDI was 0.4% for SPLC and 0% for CLC; the difference was not statistically different (P = 0.36). The incidence of overall biliary complication was 1.6% for SPLC and 0.5% for CLC, the difference did not reached statistically significance (P = 0.21, 95%CI: 0.66-15). Sixty non-randomized trials including 3599 patients were also analysed. The incidence of BDI reported then was 0.7%. CONCLUSION: The safety of SPLC cannot be assumed, based on the current evidence. Hence, this new technology cannot be recommended as standard technique for laparoscopic cholecystectomy.

Gemcitabine, oxaliplatin and 5-FU in advanced bile duct and gallbladder carcinoma: two parallel, multicentre phase-II trials.

BACKGROUND: Gemcitabine, oxaliplatin and 5-fluorouracil (5-FU) are active in biliary tract cancer and have a potentially synergistic mode of action and non-overlapping toxicity. The objective of these trials was to determine response, survival and toxicity separately in patients with bile duct cancer (BDC) and gallbladder cancer (GBC) treated with gemcitabine/oxaliplatin/5-FU chemotherapy. METHODS: Eligible patients with histologically proven, advanced or metastatic BDC (n=37) or GBC (n=35) were treated with gemcitabine (900 mg m(-2) over 30 min), oxaliplatin (65 mg m(-2)) and 5-FU (1500 mg m(-2) over 24 h) on days 1 and 8 of a 21-day cycle. Tumour response was the primary outcome measure. RESULTS: Response rates were 19% (95% CI: 6-32%) and 23% (95% CI: 9-37%) for BDC and GBC, respectively. Median survivals were 10.0 months (95% CI: 8.6-12.4) and 9.9 months (95% CI: 7.5-12.2) for BDC and GBC, respectively, and 1- and 2-year survival rates were 40 and 23% in BDC and 34 and 6% in GBC (intention-to-treat analysis). Major grade III and IV adverse events were neutropenia, thrombocytopenia, elevated bilirubin and anorexia. CONCLUSION: Triple-drug chemotherapy achieves comparable results for response and survival to previously reported regimens, but with more toxicity.

NOTES et trocart unique: révolution chirurgicale ou marketing [NOTES and single port access: surgical or marketing revolution?].

Promising new technologies are emerging in digestive surgery: Natural Orifice Transluminal Endoscopic Surgery (NOTES) and Single Port Access Surgery. They both aim to limit the surgical morbidity by decreasing the number of parietal accesses. The feasibility in human is obviously demonstrated, but numerous issues remain concerning the safety of these techniques. Furthermore, the expected advantages are not clearly demonstrated until now in the literature. In the future, it will be advisable to standardize techniques, in order to allow large clinical studies and to limit the potential complications of these approaches.

Unpacking the cognitive map: the parallel map theory of hippocampal function

In the parallel map theory, the hippocampus encodes space with 2 mapping systems. The bearing map is constructed primarily in the dentate gyrus from directional cues such as stimulus gradients. The sketch map is constructed within the hippocampus proper from positional cues. The integrated map emerges when data from the bearing and sketch maps are combined. Because the component maps work in parallel, the impairment of one can reveal residual learning by the other. Such parallel function may explain paradoxes of spatial learning, such as learning after partial hippocampal lesions, taxonomic and sex differences in spatial learning, and the function of hippocampal neurogenesis. By integrating evidence from physiology to phylogeny, the parallel map theory offers a unified explanation for hippocampal function.

Parallel changes in genetic diversity and species diversity following a natural disturbance.

We examined the spatial and temporal variation of species diversity and genetic diversity in a metacommunity comprising 16 species of freshwater gastropods. We monitored species abundance at five localities of the Ain river floodplain in southeastern France, over a period of four years. Using 190 AFLP loci, we monitored the genetic diversity of Radix balthica, one of the most abundant gastropod species of the metacommunity, twice during that period. An exceptionally intense drought occurred during the last two years and differentially affected the study sites. This allowed us to test the effect of natural disturbances on changes in both genetic and species diversity. Overall, local (alpha) diversity declined as reflected by lower values of gene diversity H(S) and evenness. In parallel, the among-sites (beta) diversity increased at both the genetic (F(ST)) and species (F(STC)) levels. These results suggest that disturbances can lead to similar changes in genetic and community structure through the combined effects of selective and neutral processes.

Antibiotic lock technique combined with systemic antimicrobial therapy for management of port-related bloodstream infections in onco-haematological patients without catheter removal

Background : Port-related bloodstream infection (PRBSI) is a common complication associated with long-term use of ports systems. Systemic antimicrobial therapy (ST) and removal of the device is the standard management of PRBSI. However, a conservative management combining ST with antibiotic lock therapy (ALT) without port removal has been suggested as an alternative management option for infections due to gram-positive skin colonizers with low virulence.¦Objectives : i) to assess the frequency of management of PRBSI in onco-hematological patients by combining the ALT with ST, without catheter removal and ii) to analyze the efficacy of such an approach.¦Methods : Retrospective observational study over a 6-year period between 2005 and 2010, including patients who where diagnosed with PRBSI and who were treated with ST and ALT. PRBSI diagnosis consisted in clinical signs of bacteremia with blood cultures positive for gram-positive skin colonizers. The primary endpoint was failure to cure the PRBSI.¦Results : 61 port infections were analysed, of which 23 PRBSI met the inclusion criteria. All the patients were suffering from haematological conditions and 75% were neutropenic at the time of PRBSI diagnosis. S. epidermidis was responsible for 91% of PRBSI (21/23). The median duration of ST was 14 days (range 7-35) and the median duration of ALT was 15 days (range 8-41). Failure to cure the PRBSI requiring port removal was observed in 4 patients, but was not associated with severe infectious complications. Kaplan-Meier analysis showed a success rate in port salvage at day 180 (6 months) of 78% (95%CI 59-97%).¦Conclusion : The success rate observed in the present study suggests that combining ST and ALT is an effective option to conservatively treat PRBSI caused by pathogens of low virulence such as S. epidermidis.

Stick insect genomes reveal natural selection's role in parallel speciation.

Natural selection can drive the repeated evolution of reproductive isolation, but the genomic basis of parallel speciation remains poorly understood. We analyzed whole-genome divergence between replicate pairs of stick insect populations that are adapted to different host plants and undergoing parallel speciation. We found thousands of modest-sized genomic regions of accentuated divergence between populations, most of which are unique to individual population pairs. We also detected parallel genomic divergence across population pairs involving an excess of coding genes with specific molecular functions. Regions of parallel genomic divergence in nature exhibited exceptional allele frequency changes between hosts in a field transplant experiment. The results advance understanding of biological diversification by providing convergent observational and experimental evidence for selection's role in driving repeatable genomic divergence.

Highly parallel genetic approaches in Mendelian and complex diseases

The recent advance in high-throughput sequencing and genotyping protocols allows rapid investigation of Mendelian and complex diseases on a scale not previously been possible. In my thesis research I took advantage of these modern techniques to study retinitis pigmentosa (RP), a rare inherited disease characterized by progressive loss of photoreceptors and leading to blindness; and hypertension, a common condition affecting 30% of the adult population. Firstly, I compared the performance of different next generation sequencing (NGS) platforms in the sequencing of the RP-linked gene PRPF31. The gene contained a mutation in an intronic repetitive element, which presented difficulties for both classic sequencing methods and NGS. We showed that all NGS platforms are powerful tools to identify rare and common DNA variants, also in case of more complex sequences. Moreover, we evaluated the features of different NGS platforms that are important in re-sequencing projects. The main focus of my thesis was then to investigate the involvement of pre-mRNA splicing factors in autosomal dominant RP (adRP). I screened 5 candidate genes in a large cohort of patients by using long-range PCR as enrichment step, followed by NGS. We tested two different approaches: in one, all target PCRs from all patients were pooled and sequenced as a single DNA library; in the other, PCRs from each patient were separated within the pool by DNA barcodes. The first solution was more cost-effective, while the second one allowed obtaining faster and more accurate results, but overall they both proved to be effective strategies for gene screenings in many samples. We could in fact identify novel missense mutations in the SNRNP200 gene, encoding an essential RNA helicase for splicing catalysis. Interestingly, one of these mutations showed incomplete penetrance in one family with adRP. Thus, we started to study the possible molecular causes underlying phenotypic differences between asymptomatic and affected members of this family. For the study of hypertension, I joined a European consortium to perform genome-wide association studies (GWAS). Thanks to the use of very informative genotyping arrays and of phenotipically well-characterized cohorts, we could identify a novel susceptibility locus for hypertension in the promoter region of the endothelial nitric oxide synthase gene (NOS3). Moreover, we have proven the direct causality of the associated SNP using three different methods: 1) targeted resequencing, 2) luciferase assay, and 3) population study. - Le récent progrès dans le Séquençage à haut Débit et les protocoles de génotypage a permis une plus vaste et rapide étude des maladies mendéliennes et multifactorielles à une échelle encore jamais atteinte. Durant ma thèse de recherche, j'ai utilisé ces nouvelles techniques de séquençage afin d'étudier la retinite pigmentale (RP), une maladie héréditaire rare caractérisée par une perte progressive des photorécepteurs de l'oeil qui entraine la cécité; et l'hypertension, une maladie commune touchant 30% de la population adulte. Tout d'abord, j'ai effectué une comparaison des performances de différentes plateformes de séquençage NGS (Next Generation Sequencing) lors du séquençage de PRPF31, un gène lié à RP. Ce gène contenait une mutation dans un élément répétable intronique, qui présentait des difficultés de séquençage avec la méthode classique et les NGS. Nous avons montré que les plateformes de NGS analysées sont des outils très puissants pour identifier des variations de l'ADN rares ou communes et aussi dans le cas de séquences complexes. De plus, nous avons exploré les caractéristiques des différentes plateformes NGS qui sont importantes dans les projets de re-séquençage. L'objectif principal de ma thèse a été ensuite d'examiner l'effet des facteurs d'épissage de pre-ARNm dans une forme autosomale dominante de RP (adRP). Un screening de 5 gènes candidats issus d'une large cohorte de patients a été effectué en utilisant la long-range PCR comme étape d'enrichissement, suivie par séquençage avec NGS. Nous avons testé deux approches différentes : dans la première, toutes les cibles PCRs de tous les patients ont été regroupées et séquencées comme une bibliothèque d'ADN unique; dans la seconde, les PCRs de chaque patient ont été séparées par code barres d'ADN. La première solution a été la plus économique, tandis que la seconde a permis d'obtenir des résultats plus rapides et précis. Dans l'ensemble, ces deux stratégies se sont démontrées efficaces pour le screening de gènes issus de divers échantillons. Nous avons pu identifier des nouvelles mutations faux-sens dans le gène SNRNP200, une hélicase ayant une fonction essentielle dans l'épissage. Il est intéressant de noter qu'une des ces mutations montre une pénétrance incomplète dans une famille atteinte d'adRP. Ainsi, nous avons commencé une étude sur les causes moléculaires entrainant des différences phénotypiques entre membres affectés et asymptomatiques de cette famille. Lors de l'étude de l'hypertension, j'ai rejoint un consortium européen pour réaliser une étude d'association Pangénomique ou genome-wide association study Grâce à l'utilisation de tableaux de génotypage très informatifs et de cohortes extrêmement bien caractérisées au niveau phénotypique, un nouveau locus lié à l'hypertension a été identifié dans la région promotrice du gène endothélial nitric oxide sinthase (NOS3). Par ailleurs, nous avons prouvé la cause directe du SNP associé au moyen de trois méthodes différentes: i) en reséquençant la cible avec NGS, ii) avec des essais à la luciférase et iii) une étude de population.

Sleep deprivation effects on circadian clock gene expression in the cerebral cortex parallel electroencephalographic differences among mouse strains.

Sleep deprivation (SD) results in increased electroencephalographic (EEG) delta power during subsequent non-rapid eye movement sleep (NREMS) and is associated with changes in the expression of circadian clock-related genes in the cerebral cortex. The increase of NREMS delta power as a function of previous wake duration varies among inbred mouse strains. We sought to determine whether SD-dependent changes in circadian clock gene expression parallel this strain difference described previously at the EEG level. The effects of enforced wakefulness of incremental durations of up to 6 h on the expression of circadian clock genes (bmal1, clock, cry1, cry2, csnk1epsilon, npas2, per1, and per2) were assessed in AKR/J, C57BL/6J, and DBA/2J mice, three strains that exhibit distinct EEG responses to SD. Cortical expression of clock genes subsequent to SD was proportional to the increase in delta power that occurs in inbred strains: the strain that exhibits the most robust EEG response to SD (AKR/J) exhibited dramatic increases in expression of bmal1, clock, cry2, csnkIepsilon, and npas2, whereas the strain with the least robust response to SD (DBA/2) exhibited either no change or a decrease in expression of these genes and cry1. The effect of SD on circadian clock gene expression was maintained in mice in which both of the cryptochrome genes were genetically inactivated. cry1 and cry2 appear to be redundant in sleep regulation as elimination of either of these genes did not result in a significant deficit in sleep homeostasis. These data demonstrate transcriptional regulatory correlates to previously described strain differences at the EEG level and raise the possibility that genetic differences underlying circadian clock gene expression may drive the EEG differences among these strains.

Bloodstream infections related to totally implantable venous access port: What is the situation in our hospital?

Port-a-Cath© (PAC) are totally implantable devices that offer an easy and long term access to venous circulation. They have been extensively used for intravenous therapy administration and are particularly well suited for chemotherapy in oncologic patients. Previous comparative studies have shown that these devices have the lowest catheter-related bloodstream infection rates among all intravascular access systems. However, bloodstream infection (BSI) still remains a major issue of port use and epidemiology data for PAC-associated BSI (PABSI) rates differ strongly depending on studies. Also, current literature about PABSI risk factors is scarce and sometimes controversial. Such heterogeneity may depend on type of studied population and local factors. Therefore, the aim of this study was to describe local epidemiology and risk factors for PABSI in adult patients in our tertiary- care university hospital. We conducted a retrospective cohort study in order to describe local epidemiology. We also performed a nested case-control study to identify local risk factors of PABSI. We analyzed medical files of adult patients who had a PAC implanted between January 1st, 2008 and December 31st, 2009 and looked for PABSI occurrence before May 1st, 2011 to define cases. Thirty nine PABSI occurred in this population with an attack rate of 5.8%. We estimated an incidence rate of 0.08/1000 PAC-days using the case-control study. PABSI causative agents were mainly Gram positive cocci (62%). We identified three predictive factors of PABSI by multivariate statistical analysis: neutropenia on outcome date (Odds Ratio [OR]: 4.05; 95% confidence interval [CI]:1.05- 15.66; p=0.042), diabetes (OR: 11.53; 95% CI: 1.07-124.70; p=0.044) and having another infection than PABSI on outcome date (OR: 6.35; 95% CI: 1.50-26.86; p=0.012). Patients suffering from acute or renal failure (OR: 4.26; 95% CI: 0.94-19.21; p=0.059) or wearing another invasive device (OR: 2.99; 95%CI:0.96-9.31; p=0.059) did not have a statistically increased risk for developing a PABSI according to classical threshold (p<0.05) but nevertheless remained close to significance. Our study demonstrated that local epidemiology and microbiology of PABSI in our institution was similar to previous reports. A larger prospective study is required to confirm our results or to test preventive measures.

Parallel imaging with phase scrambling.

PURPOSE: Most existing methods for accelerated parallel imaging in MRI require additional data, which are used to derive information about the sensitivity profile of each radiofrequency (RF) channel. In this work, a method is presented to avoid the acquisition of separate coil calibration data for accelerated Cartesian trajectories. METHODS: Quadratic phase is imparted to the image to spread the signals in k-space (aka phase scrambling). By rewriting the Fourier transform as a convolution operation, a window can be introduced to the convolved chirp function, allowing a low-resolution image to be reconstructed from phase-scrambled data without prominent aliasing. This image (for each RF channel) can be used to derive coil sensitivities to drive existing parallel imaging techniques. As a proof of concept, the quadratic phase was applied by introducing an offset to the x(2) - y(2) shim and the data were reconstructed using adapted versions of the image space-based sensitivity encoding and GeneRalized Autocalibrating Partially Parallel Acquisitions algorithms. RESULTS: The method is demonstrated in a phantom (1 × 2, 1 × 3, and 2 × 2 acceleration) and in vivo (2 × 2 acceleration) using a 3D gradient echo acquisition. CONCLUSION: Phase scrambling can be used to perform parallel imaging acceleration without acquisition of separate coil calibration data, demonstrated here for a 3D-Cartesian trajectory. Further research is required to prove the applicability to other 2D and 3D sampling schemes. Magn Reson Med, 2014. © 2014 Wiley Periodicals, Inc.

Inherently self-calibrating non-Cartesian parallel imaging.

The use of self-calibrating techniques in parallel magnetic resonance imaging eliminates the need for coil sensitivity calibration scans and avoids potential mismatches between calibration scans and subsequent accelerated acquisitions (e.g., as a result of patient motion). Most examples of self-calibrating Cartesian parallel imaging techniques have required the use of modified k-space trajectories that are densely sampled at the center and more sparsely sampled in the periphery. However, spiral and radial trajectories offer inherent self-calibrating characteristics because of their densely sampled center. At no additional cost in acquisition time and with no modification in scanning protocols, in vivo coil sensitivity maps may be extracted from the densely sampled central region of k-space. This work demonstrates the feasibility of self-calibrated spiral and radial parallel imaging using a previously described iterative non-Cartesian sensitivity encoding algorithm.