In vitro self-replication and multicistronic expression of large synthetic genomes.

The generation of a chemical system capable of replication and evolution is a key objective of synthetic biology. This could be achieved by in vitro reconstitution of a minimal self-sustaining central dogma consisting of DNA replication, transcription and translation. Here, we present an in vitro translation system, which enables self-encoded replication and expression of large DNA genomes under well-defined, cell-free conditions. In particular, we demonstrate self-replication of a multipartite genome of more than 116 kb encompassing the full set of Escherichia coli translation factors, all three ribosomal RNAs, an energy regeneration system, as well as RNA and DNA polymerases. Parallel to DNA replication, our system enables synthesis of at least 30 encoded translation factors, half of which are expressed in amounts equal to or greater than their respective input levels. Our optimized cell-free expression platform could provide a chassis for the generation of a partially self-replicating in vitro translation system.

Configurable NOR gate arrays from Belousov-Zhabotinsky micro-droplets.

We investigate the Belousov-Zhabotinsky (BZ) reaction in an attempt to establish a basis for computation using chemical oscillators coupled via inhibition. The system consists of BZ droplets suspended in oil. Interdrop coupling is governed by the non-polar communicator of inhibition, Br2. We consider a linear arrangement of three droplets to be a NOR gate, where the center droplet is the output and the other two are inputs. Oxidation spikes in the inputs, which we define to be TRUE, cause a delay in the next spike of the output, which we read to be FALSE. Conversely, when the inputs do not spike (FALSE) there is no delay in the output (TRUE), thus producing the behavior of a NOR gate. We are able to reliably produce NOR gates with this behavior in microfluidic experiment.

Bone Like Arterial Calcification in Femoral Atherosclerotic Lesions: Prevalence and Role of Osteoprotegerin and Pericytes.

OBJECTIVE/BACKGROUND: Arterial calcification, a process that mimics bone formation, is an independent risk factor of cardiovascular morbidity and mortality, and has a significant impact on surgical and endovascular procedures and outcomes. Research efforts have focused mainly on the coronary arteries, while data regarding the femoral territory remain scarce. METHODS: Femoral endarterectomy specimens, clinical data, and plasma from a cohort of patients were collected prospectively. Histological analysis was performed to characterize the cellular populations present in the atherosclerotic lesions, and that were potentially involved in the formation of bone like arterial calcification known as osteoid metaplasia (OM). Enzyme linked immunosorbent assays and cell culture assays were conducted in order to understand the cellular and molecular mechanisms underlying the formation of OM in the lesions. RESULTS: Twenty-eight of the 43 femoral plaques (65%) displayed OM. OM included osteoblast and osteoclast like cells, but very few of the latter exhibited the functional ability to resorb mineral tissue. As in bone, osteoprotegerin (OPG) was significantly associated with the presence of OM (p = .04). Likewise, a high plasma OPG/receptor activator for the nuclear factor kappa B ligand (RANKL) ratio was significantly associated with the presence of OM (p = .03). At the cellular level, there was a greater presence of pericytes in OM+ compared with OM- lesions (5.59 ± 1.09 vs. 2.42 ± 0.58, percentage of area staining [region of interest]; p = .04); in vitro, pericytes were able to inhibit the osteoblastic differentiation of human mesenchymal stem cells, suggesting that they are involved in regulating arterial calcification. CONCLUSION: These results suggest that bone like arterial calcification (OM) is highly prevalent at femoral level. Pericyte cells and the OPG/RANK/RANKL triad seem to be critical to the formation of this ectopic osteoid tissue and represent interesting potential therapeutic targets to reduce the clinical impact of arterial calcification.

Ceramic micro-injection molded nozzles for serial femtosecond crystallography sample delivery.

Serial femtosecond crystallography (SFX) using X-ray Free-Electron Lasers (XFELs) allows for room temperature protein structure determination without evidence of conventional radiation damage. In this method, a liquid suspension of protein microcrystals can be delivered to the X-ray beam in vacuum as a micro-jet, which replenishes the crystals at a rate that exceeds the current XFEL pulse repetition rate. Gas dynamic virtual nozzles produce the required micrometer-sized streams by the focusing action of a coaxial sheath gas and have been shown to be effective for SFX experiments. Here, we describe the design and characterization of such nozzles assembled from ceramic micro-injection molded outer gas-focusing capillaries. Trends of the emitted jet diameter and jet length as a function of supplied liquid and gas flow rates are measured by a fast imaging system. The observed trends are explained by derived relationships considering choked gas flow and liquid flow conservation. Finally, the performance of these nozzles in a SFX experiment is presented, including an analysis of the observed background.

Simple convergent-nozzle aerosol injector for single-particle diffractive imaging with X-ray free-electron lasers.

A major challenge in high-resolution x-ray free-electron laser-based coherent diffractive imaging is the development of aerosol injectors that can efficiently deliver particles to the peak intensity of the focused X-ray beam. Here, we consider the use of a simple convergent-orifice nozzle for producing tightly focused beams of particles. Through optical imaging we show that 0.5 µm particles can be focused to a full-width at half maximum diameter of 4.2 µm, and we demonstrate the use of such a nozzle for injecting viruses into a micro-focused soft-X-ray FEL beam.

Patient information ahead of thyroid surgery. Guidelines of the French Society of Oto-Rhino-Laryngology and Head and Neck Surgery (SFORL).

OBJECTIVE: The authors present the guidelines of the French Society of Oto-Rhino-Laryngology and Head and Neck Surgery (SFORL) on patient information ahead of thyroid surgery. METHODS: A multidisciplinary medical team was tasked with a scientific literature review on this topic. The texts retrieved were analyzed by an independent committee. A joint meeting drew up the final guidelines. The strength of the recommendations (grade A, B or C) was based on levels of evidence. RESULTS: It is recommended that the results of preoperative exploration and the indications for surgery should be explained to the patient. Patients should be informed as to the type of surgery, surgical objectives, risks and consequences. It is mandatory to obtain the patient's written consent before surgery. CONCLUSION: Appropriate medical information is a critical step in patient management.

[Arterial heterogeneity]. / Artériopathie et hétérogénéité artérielle.

More and more clinical observations and trials support the concept of heterogeneity of atheroma according to the arterial bed. In a pilot study named "Étude Comparative des Lésions Athéromateuses" (ECLA), we have shown that carotid and femoral plaques possess different characteristics. Carotid arteries display increased lipid content compared to femoral arteries whereas femoral arteries are more prone to calcify and to develop osteoid metaplasia. These observations should lead the researcher and the clinician to look at the cellular and molecular mechanisms governing the heterogeneity of atheromas. At last, a better understanding of the characteristics of plaques should help us to determine plaque stability, to prevent cardiovascular events and to choose the best medical, endovascular or surgical option.

Interleukin 34 expression is associated with synovitis severity in rheumatoid arthritis patients.

OBJECTIVES: Interleukin (IL) 34 is a new cytokine implicated in macrophage differentiation and osteoclastogenesis. This study assessed IL-34 expression in the tissue of patients with rheumatoid arthritis (RA). METHODS: Immunohistochemistry was performed in synovial biopsies from patients with RA (n=20), osteoarthritis (n=3) or other inflammatory arthritis (n=4). IL-34 was detected in the synovial fluid by ELISA and its messenger RNA expression was studied by quantitative PCR in rheumatoid synovial fibroblasts after stimulation by tumour necrosis factor α (TNFα) and IL-1ß. Wild-type, jnk1(-/-)-jnk2(-/-) and nemo(-/-) murine fibroblasts and pharmacological inhibition were used to determine the involvement of nuclear factor kappa B (NF-κB) and JNK in that effect. RESULTS: IL-34 was expressed in 24/27 biopsies, with three samples from RA patients being negative. A significant association was found between IL-34 expression and synovitis severity. Levels of IL-34 and the total leucocyte count in synovial fluid were correlated. TNFα and IL-1ß stimulated IL-34 expression by synovial fibroblasts in a dose/time-dependent manner through the NF-κB and JNK pathway. CONCLUSION: This work for the first time identifies IL-34 expression in the synovial tissue of patients with arthritis. This cytokine, as a downstream effector of TNFα and IL-1ß, may contribute to inflammation and bone erosions in RA.

The Bone Niche of Chondrosarcoma: A Sanctuary for Drug Resistance, Tumour Growth and also a Source of New Therapeutic Targets.

Chondrosarcomas are malignant cartilage-forming tumours representing around 20% of malignant primary tumours of bone and affect mainly adults in the third to sixth decade of life. Unfortunately, the molecular pathways controlling the genesis and the growth of chondrosarcoma cells are still not fully defined. It is well admitted that the invasion of bone by tumour cells affects the balance between early bone resorption and formation and induces an "inflammatory-like" environment which establishes a dialogue between tumour cells and their environment. The bone tumour microenvironment is then described as a sanctuary that contributes to the drug resistance patterns and may control at least in part the tumour growth. The concept of "niche" defined as a specialized microenvironment that can promote the emergence of tumour stem cells and provide all the required factors for their development recently emerges in the literature. The present paper aims to summarize the main evidence sustaining the existence of a specific bone niche in the pathogenesis of chondrosarcomas.

Décrypthon grid - grid resources dedicated to neuromuscular disorders.

Thanks to the availability of computational grids and their middleware, a seamless access to computation and storage resources is provided to application developers and scientists. The Décrypthon project is one example of such a high performance platform. In this paper, we present the architecture of the platform, the middleware developed to facilitate access to several servers deployed in France, and the data center for integrating large biological datasets over multiple sites, supported by a new query language and integration of various tools. The SM2PH project represents an example of a biological application that exploits the capacities of the Décrypthon grid. The goal of SM2PH is a better understanding of mutations involved in human monogenic diseases, their impact on the 3D structure of the protein and the subsequent consequences for the pathological phenotypes.

Endoscopic ultrasound-guided fine-needle aspiration for the diagnosis of solid pancreatic masses: the impact on patient-management strategy.

BACKGROUND: Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is a safe and accurate technique for diagnosing pancreatic cancer. However, its impact for management of these patients is poorly investigated. AIMS: To investigate the diagnostic yield and the therapeutic impact of EUS-FNA in the management of solid pancreatic masses. METHODS: One hundred consecutive patients who underwent EUS-FNA for a solid pancreatic mass were included. Aspirates were placed onto glass slides for cytological examination and microbiopsies were fixed in formaldehyde for histology. The impact on clinical management was analysed retrospectively according to different endpoints, such as its impact on indications for chemotherapy, surgery or appropriate follow-up modality. RESULTS: Eight procedures were considered failures and two patients were lost to follow-up. A final diagnosis was obtained in 90 patients. The sensitivity, specificity and accuracy of combined cytology and histology for the diagnosis of malignant or potentially-malignant tumours were 78%, 75%, and 78% respectively. The sensitivity and accuracy of cytology alone were significantly higher than those of histology alone (P = 0.0003). By intention-to-diagnose analysis, EUS-FNA directly influenced the management strategy in 62 of 100 patients. CONCLUSIONS: In patients with pancreatic mass and suspected malignancy, EUS-FNA provides an accurate diagnosis in approximately 80% of cases. EUS-FNA directly influences the management in two-thirds of patients.

[Hemoperitoneum due to rupture of an omental arterial aneurysm]. / Hémopéritoine par rupture spontanée d'un anévrisme de l'arcade vasculaire omentale.

We report a case of spontaneous hemoperitoneum due to rupture of an omental arterial aneurysm. This source of bleeding is unusual (2 cases published); the diagnosis was made preoperatively by doppler ultrasound and CT scan with IV contrast. Omental resection was performed and histological analysis confirmed the diagnosis. A literature review of the rare cases of hemoperitoneum due to rupture of a digestive arterial aneurysm is done.

A new experimental rat model of osteosarcoma established by intrafemoral tumor cell inoculation, useful for biology and therapy investigations.

Satisfactory experimental models for preclinical cancer studies must follow several criteria: (1) reproducibility of the method used to induce the tumor and (2) clinical, pathological and kinetic similarity with the corresponding human tumors. We developed a model of osteosarcoma locally induced by the intrafemoral injection of osteosarcoma (OSR) cells in Sprague-Dawley rats. This method yields nearly 80% of bone tumors at the injection site. These tumors double their volume fairly slowly (in approximately 20 days) and lung metastases occur in 96% of the animals. The OSR cell-induced tumor is characterized by a direct production of mineralized matrix by the tumor cells themselves, as revealed by histochemical analysis. The microarchitectural parameters which were quantified by a microscanner show an increased trabecular bone volume (+238%) when OSR cells were injected in the femur, as compared to controls injected with vehicle. Osteoblastic markers such as alkaline phosphatase, osteopontin, osteocalcin and bone sialoprotein were expressed by the tumor in vivo, whereas the initially injected OSR cells did not express some of these markers, suggesting that OSR cells reacquired an osteoblastic phenotype in a favorable environment. The clinical, radiological and histological data show that this model shares high similarities with the osteocondensing forms of osteosarcoma in humans.

Enhanced tumor regression and tissue repair when zoledronic acid is combined with ifosfamide in rat osteosarcoma.

The efficacy of zoledronic acid (ZOL), with or without the anticancer drug ifosfamide (IFO), was tested on primary bone tumor growth using a rat-transplantable model of osteosarcoma. The effects on bone remodeling and tumor growth were analyzed by radiography, micro-computed tomography (micro-CT), and histological staining. The in vitro effects of ZOL were studied by proliferation, apoptosis, and cell cycle analyses on the osteosarcoma cells OSRGA compared to rat primary osteoblasts. Treatment with ZOL was effective in preventing the formation of osteolytic lesions that developed in bone sites and in reducing the local tumor growth, as compared to the untreated rats. The combination of ZOL and IFO was more effective than each agent alone in preventing tumor recurrence, improving tissue repair, and increasing bone formation as revealed by the analysis of trabecular architecture. In vitro studies demonstrated that ZOL was more potent against the OSRGA cell line than osteoblasts (with a half-maximal inhibitory effect on proliferation seen at 0.2 and 20 microM, respectively), the ZOL-induced inhibition of OSRGA proliferation being due to cell cycle arrest in S-phase. No effect on OSRGA apoptosis could be observed in vitro, as assessed by Hoechst staining and caspase-1 and -3 activation. In situ cell death was determined by TUNEL staining on tumor tissue sections. No significant difference in TUNEL-positive cells could be observed between ZOL-treated and -untreated rats. This is the first report of the anti-bone resorption and antitumoral activities of zoledronic acid in a rat model of osteosarcoma, and its beneficial association with an antitumoral chemotherapeutic drug in preventing tumor recurrence.

Endocrine tumors of the duodenum. A study of 55 cases relative to clinicopathological features and hormone content.

BACKGROUND/AIMS: Study of prognosis of duodenal endocrine tumors. METHODOLOGY: Retrospective study concerned 55 duodenal endocrine tumors discovered in biopsy or surgical specimens. Follow-up records available for 49 patients indicated that inconspicuous associated clinical manifestations were often found subsequently. Seven patients were classified as Zollinger-Ellison syndrome and seven as multiple endocrine neoplasia (6 MEN I and 1 MEN II). RESULTS: Tumors were small (mean 1.28cm) and located preferentially in the first and second part of the duodenum. Fifty-four were well-differentiated and one poorly differentiated. Immunochemistry revealed 30 G-cell tumors (54.6%), 15 D-cell (27.3%), two plurihormonal (EC cell and G cell), and one GRH-cell, whereas seven could not be classified. Fifteen patients died (five in relation to their disease). Twenty-one had metastases (liver, nodes, lung), eight of whom are still alive. CONCLUSIONS: Eighty-eight percent of duodenal endocrine tumors were gastrinomas, small plurifocal tumors and somatostatinomas preferentially located in the ampullar region and diagnosed because of hematemesis or icterus. Size is an important prognostic factor in determining whether surgery is required. The prognosis is better for D- and G-cell tumors than pancreatic endocrine tumors. Duodenal endocrine tumors in multiple endocrine neoplasia have a good prognosis, but can be associated with pancreatic plurihormonal tumors and metastases.

Neointimal hyperplasia after stenting in a human mammary artery organ culture.

Although the use of stents has limited the incidence of restenosis, in-stent restenosis remains an important problem. In-stent restenosis is the result of a healing process that induced neointimal hyperplasia through mechanisms that are still not understood. The aim of this study was to analyze the histological consequences of the healing process following stent implantation. Internal mammary arteries from atheroslerotic patients undergoing coronary artery bypass surgery were stented and maintained in culture for 0-28 days. Stent implantation after predilatation induced an extensive loss of endothelial cells whereas direct stenting preserved endothelium between the struts. Morphometric analysis shows that stent placement induced neointimal thickening. Smooth muscle alpha-actin labeling indicates that neo-intimal formation was mainly due to proliferation and migration of smooth muscle cells. Smooth muscle cell proliferation, assessed by MIB-1 staining, was maximal at day 14 after stent insertion. Human mammary artery organ culture thus provides valuable information on histological consequences of stent implantation with or without predilatation regarding endothelial cell disappearance and neointimal hyperplasia. These data also demonstrate that neointimal thickening induced by stent implantation comprises an intrinsic component resulting from the vessel wall response to stent insertion and suggest that blood factors could play an amplifying but not necessary role.

[A rare cause of myocardial infarction: papillary fibroelastoma of the aortic valve]. / Cause rare d'infarctus du myocarde: le fibro-élastome papillaire de la valve aortique.

Papillary fibroelastoma is a rare, benign endocardial tumour usually located on the cardiac valves. Before echocardiography, these tumours were chance findings either at surgery or at autopsy. With the advent of echocardiography, the diagnosis has become commoner and they are often the cause of systemic embolism justifying surgical ablation. In this case, an aortic valve papillary fibroelastoma presented with myocardial infarction in a 78 year old woman with normal coronary angiography. The diagnosis was strongly suspected at echocardiography and confirmed by histological analysis of the surgically excised tumour.

Molecular mechanisms involved in the antiproliferative effect of two COX-2 inhibitors, nimesulide and NS-398, on colorectal cancer cell lines.

BACKGROUND: Cyclooxygenase (COX)-2 is up-regulated in most colorectal cancers. Chronic use of non-steroidal anti-inflammatory drugs, which target cyclooxygenases, have been shown to reduce the risk of these cancers. However, the mechanisms underlying this protective effect remain unclear. AIMS: The aim of our study was to characterize the effects of two COX-2 selective inhibitors, NS-398 and nimesulide, on colorectal cancer cell proliferation, and to describe the molecular mechanisms involved. MATERIALS AND METHODS: HT-29 and SW-1116 cell lines were cultured with either NS-398 or nimesulide. Cell proliferation was assessed by staining DNA with crystal violet. Cell cycle repartition and apoptosis were analysed by flow cytometry. The expression of COX-1 and COX-2. and of two cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1, was analysed by Western blotting and RT-PCR. RESULTS: Both drugs dose-dependently inhibited cell proliferation and induced G1 cell cycle blockade. HT-29 cells were more sensitive to both drugs than SW-1116 cells. p21Cip1 and p27Kip1 were induced on both cell lines. Concomitant induction of p21Cip1 mRNA indicates transcriptional modulation, whereas induction of p27Kip1 only at the protein level suggests post-translational modulation. CONCLUSION: NS-398 and nimesulide inhibit colorectal cell proliferation through induction of p21Cip1 and p27Kip1.

Cyclo-oxygenase-2 over-expression in sporadic colorectal carcinoma without lymph node involvement.

BACKGROUND: Cyclo-oxygenase-2 over-expression has been reported in most advanced human colorectal cancers. AIMS: To assess the prevalence of cyclo-oxygenase-2 over-expression in non-advanced colorectal cancers, to investigate the correlation between cyclo-oxygenase-2 status and tumour clinicopathological features and molecular phenotype, and to determine the impact of cyclo-oxygenase-2 status on long-term clinical outcome. METHODS: Sixty-one patients who had undergone surgery for colorectal cancer without lymph node involvement were evaluated retrospectively. Cyclo-oxygenase-2 expression was determined by immunohistochemistry. The tumour replication error phenotype was assessed by amplification of the two microsatellites, BAT-25 and BAT-26. RESULTS: Thirty-six tumours were classified as cyclo-oxygenase-2 positive and 25 as cyclo-oxygenase-2 negative. No correlation was found between cyclo-oxygenase-2 over-expression and clinicopathological features or molecular phenotype. Cyclo-oxygenase-2 over-expression was an independent predictor of a poor prognosis. Indeed, the relative risk of tumour recurrence or death for patients with cyclo-oxygenase-2-positive tumours was 2.13 times that of patients with cyclo-oxygenase-2-negative tumours (P=0.008; 95% confidence interval, 1.22-3.73). This difference remained significant when post-operative deaths were censored in the multivariate analysis (P=0.014). CONCLUSION: Cyclo-oxygenase-2 over-expression is not associated with tumour phenotype, but is indicative of a poorer clinical outcome in patients with non-advanced colorectal carcinoma.

[Parathyroid cyst. Report of ten cases]. / Les kystes parathyroïdiens. A propos de dix observations.

AIM OF THE STUDY: To evaluate the characteristics of the parathyroid cysts (PC). PATIENTS AND METHOD: Ten patients with PC were included in this retrospective study. The PC were discovered as follows: cervical mass (n = 3), hyperparathyroidism (n = 3), incidentally during thyroid surgery (n = 3) and screening for obesity (n = 1). Intracystic parathormone determination was performed after fine needle aspiration in 2 cases. RESULTS: Mean cyst measurements were 27 mm (ext: 5-70 mm) to 22 mm (5-45 mm). Nine cysts were cervical (resection by cervicotomy), and one was mediastinal (resection by sternotomy). In addition to the resection of the PC, 3 adenomas, 1 hyperplasia of the parathyroid glands and 3 benign thyroid diseases were recognized and treated during the cervicotomies. CONCLUSION: The diagnosis of PC is not common and must be based primarily on the study of the cyst liquid obtained by percutaneous puncture (intracystic parathormone measurement).