An ATM distributed simulator for network management research

Due to the high cost of a large ATM network working up to full strength to apply our ideas about network management, i.e., dynamic virtual path (VP) management and fault restoration, we developed a distributed simulation platform for performing our experiments. This platform also had to be capable of other sorts of tests, such as connection admission control (CAC) algorithms, routing algorithms, and accounting and charging methods. The platform was posed as a very simple, event-oriented and scalable simulation. The main goal was the simulation of a working ATM backbone network with a potentially large number of nodes (hundreds). As research into control algorithms and low-level, or rather cell-level methods, was beyond the scope of this study, the simulation took place at a connection level, i.e., there was no real traffic of cells. The simulated network behaved like a real network accepting and rejecting SNMP ones, or experimental tools using the API node

Network-based scoring system for genome-scale metabolic reconstructions

Background: Network reconstructions at the cell level are a major development in Systems Biology. However, we are far from fully exploiting its potentialities. Often, the incremental complexity of the pursued systems overrides experimental capabilities, or increasingly sophisticated protocols are underutilized to merely refine confidence levels of already established interactions. For metabolic networks, the currently employed confidence scoring system rates reactions discretely according to nested categories of experimental evidence or model-based likelihood. Results: Here, we propose a complementary network-based scoring system that exploits the statistical regularities of a metabolic network as a bipartite graph. As an illustration, we apply it to the metabolism of Escherichia coli. The model is adjusted to the observations to derive connection probabilities between individual metabolite-reaction pairs and, after validation, to assess the reliability of each reaction in probabilistic terms. This network-based scoring system uncovers very specific reactions that could be functionally or evolutionary important, identifies prominent experimental targets, and enables further confirmation of modeling results. Conclusions: We foresee a wide range of potential applications at different sub-cellular or supra-cellular levels of biological interactions given the natural bipartivity of many biological networks.

Inclusion of soil erosion impacts in life cycle assessment on a global scale: application to energy crops in Spain

Purpose: Despite the fundamental role of ecosystem goods and services in sustaining human activities, there is no harmonized and internationally agreed method for including them in life cycle assessment (LCA). The main goal of this study was to develop a globally applicable and spatially resolved method for assessing land-use impacts on the erosion regulation ecosystem service.Methods: Soil erosion depends much on location. Thus, unlike conventional LCA, the endpoint method was regionalized at the grid-cell level (5 arc-minutes, approximately 10×10 km2) to reflect the spatial conditions of the site. Spatially explicit characterization factors were not further aggregated at broader spatial scales. Results and discussion: Life cycle inventory data of topsoil and topsoil organic carbon (SOC) losses were interpreted at the endpoint level in terms of the ultimate damage to soil resources and ecosystem quality. Human health damages were excluded from the assessment. The method was tested on a case study of five three-year agricultural rotations, two of them with energy crops, grown in several locations in Spain. A large variation in soil and SOC losses was recorded in the inventory step, depending on climatic and edaphic conditions. The importance of using a spatially explicit model and characterization factors is shown in the case study.Conclusions and outlook: The regionalized assessment takes into account the differences in soil erosion-related environmental impacts caused by the great variability of soils. Taking this regionalized framework as the starting point, further research should focus on testing the applicability of the method trough the complete life cycle of a product and on determining an appropriate spatial scale at which to aggregate characterization factors, in order to deal with data gaps on location of processes, especially in the background system. Additional research should also focus on improving reliability of the method by quantifying and, insofar as it is possible, reducing uncertainty.

Ultrastructural characterisation of the cell wall of Arabidopsis thaliana transgenic plants

The plant cell wall is a strong fibrillar network that gives each cell its stable shape. It is constituted by a network of cellulose microfibrils embedded in a matrix of polysaccharides, such as xyloglucans. To enlarge, cells selectively loosen this network. Moreover, there is a pectin-rich intercellular material, the middle lamella, cementing together the walls of adjacent plant cells. Xyloglucan endotransglucosylase/hydrolases (XTHs) are a group of enzymes involved in the reorganisation of the cellulose-xyloglucan framework by catalysing cleavage and re-ligation of the xyloglucan chains in the plant cell wall, and are considered cell wall loosening agents. In the laboratory, it has been isolated and characterised a XTH gene, ZmXTH1, from an elongation root cDNA library of maize. To address the cellular function of ZmXTH1, transgenic Arabidopsis thaliana plants over-expressing ZmXTH1 (under the control of the CaMV35S promoter) were generated. The aim of the work performed was therefore the characterisation of these transgenic plants at the ultrastructural level, by transmission electron microscopy (TEM).The detailed cellular phenotype of transgenic plants was investigated by comparing ultra-thin transverse sections of basal stem of 5-weeks old plants of wild type (Col 0) and 35S-ZmXTH1 Arabidopsis plants. Transgenic plants show modifications in the cell walls, particularly a thicker middle lamella layer with respect the wild type plants, supporting the idea that the overexpression of ZmXTH1 could imply a pronounced wall-loosening. In sum, the work carried out reinforces the idea that ZmXTH1 is involved in the cell wall loosening process in maize.  

Interfacial instabilities of a fluid annulus in a rotating Hele-Shaw cell

We have studied the interfacial instabilities experienced by a liquid annulus as it moves radially in a circular Hele-Shaw cell rotating with angular velocity Omega. The instability of the leading interface (oil displacing air) is driven by the density difference in the presence of centrifugal forcing, while the instability of the trailing interface (air displacing oil) is driven by the large viscosity contrast. A linear stability analysis shows that the stability of the two interfaces is coupled through the pressure field already at a linear level. We have performed experiments in a dry cell and in a cell coated with a thin fluid layer on each plate, and found that the stability depends substantially on the wetting conditions at the leading interface. Our experimental results of the number of fingers resulting from the instability compare well with the predictions obtained through a numerical integration of the coupled equations derived from a linear stability analysis. Deep in the nonlinear regime we observe the emission of liquid droplets through the formation of thin filaments at the tip of outgrowing fingers.

Semiquantitative RT-PCR measurement of gene expression in rat tissues including a correction for varying cell size and number

Background: Current methodology of gene expression analysis limits the possibilities of comparison between cells/tissues of organs in which cell size and/or number changes as a consequence of the study (e.g. starvation). A method relating the abundance of specific mRNA copies per cell may allow direct comparison or different organs and/or changing physiological conditions. Methods: With a number of selected genes, we analysed the relationship of the number of bases and the fluorescence recorded at a present level using cDNA standards. A lineal relationship was found between the final number of bases and the length of the transcript. The constants of this equation and those of the relationship between fluorescence and number of bases in cDNA were determined and a general equation linking the length of the transcript and the initial number of copies of mRNA was deduced for a given pre-established fluorescence setting. This allowed the calculation of the concentration of the corresponding mRNAs per g of tissue. The inclusion of tissue RNA and the DNA content per cell, allowed the calculation of the mRNA copies per cell. Results: The application of this procedure to six genes: Arbp, cyclophilin, ChREBP, T4 deiodinase 2, acetyl-CoA carboxylase 1 and IRS-1, in liver and retroperitoneal adipose tissue of food-restricted rats allowed precise measures of their changes irrespective of the shrinking of the tissue, the loss of cells or changes in cell size, factors that deeply complicate the comparison between changing tissue conditions. The percentage results obtained with the present methods were essentially the same obtained with the delta-delta procedure and with individual cDNA standard curve quantitative RT-PCR estimation. Conclusion: The method presented allows the comparison (i.e. as copies of mRNA per cell) between different genes and tissues, establishing the degree of abundance of the different molecular species tested.

Fish Glucose Transporter (GLUT)-4 Differs from Rat GLUT4 in Its Traffic Characteristics but Can Translocate to the Cell Surface in Response to Insulin in Skeletal Muscle Cells

In mammals, glucose transporter (GLUT)-4 plays an important role in glucose homeostasis mediating insulin action to increase glucose uptake in insulin-responsive tissues. In the basal state, GLUT4 is located in intracellular compartments and upon insulin stimulation is recruited to the plasma membrane, allowing glucose entry into the cell. Compared with mammals, fish are less efficient restoring plasma glucose after dietary or exogenous glucose administration. Recently our group cloned a GLUT4-homolog in skeletal muscle from brown trout (btGLUT4) that differs in protein motifs believed to be important for endocytosis and sorting of mammalian GLUT4. To study the traffic of btGLUT4, we generated a stable L6 muscle cell line overexpressing myc-tagged btGLUT4 (btGLUT4myc). Insulin stimulated btGLUT4myc recruitment to the cell surface, although to a lesser extent than rat-GLUT4myc, and enhanced glucose uptake. Interestingly, btGLUT4myc showed a higher steady-state level at the cell surface under basal conditions than rat-GLUT4myc due to a higher rate of recycling of btGLUT4myc and not to a slower endocytic rate, compared with rat-GLUT4myc. Furthermore, unlike rat-GLUT4myc, btGLUT4myc had a diffuse distribution throughout the cytoplasm of L6 myoblasts. In primary brown trout skeletal muscle cells, insulin also promoted the translocation of endogenous btGLUT4 to the plasma membrane and enhanced glucose transport. Moreover, btGLUT4 exhibited a diffuse intracellular localization in unstimulated trout myocytes. Our data suggest that btGLUT4 is subjected to a different intracellular traffic from rat-GLUT4 and may explain the relative glucose intolerance observed in fish.

A system-level, molecular evolutionary analysis of mammalian phototransduction

Visual perception is initiated in the photoreceptor cells of the retina via the phototransduction system.This system has shown marked evolution during mammalian divergence in such complex attributes as activation time and recovery time. We have performed a molecular evolutionary analysis of proteins involved in mammalianphototransduction in order to unravel how the action of natural selection has been distributed throughout thesystem to evolve such traits. We found selective pressures to be non-randomly distributed according to both a simple protein classification scheme and a protein-interaction network representation of the signaling pathway. Proteins which are topologically central in the signaling pathway, such as the G proteins, as well as retinoid cycle chaperones and proteins involved in photoreceptor cell-type determination, were found to be more constrained in their evolution. Proteins peripheral to the pathway, such as ion channels and exchangers, as well as the retinoid cycle enzymes, have experienced a relaxation of selective pressures. Furthermore, signals of positive selection were detected in two genes: the short-wave (blue) opsin (OPN1SW) in hominids and the rod-specific Na+/Ca2+,K+ ion exchanger (SLC24A1) in rodents. The functions of the proteins involved in phototransduction and the topology of the interactions between them have imposed non-random constraints on their evolution. Thus, in shaping or conserving system-level phototransduction traits, natural selection has targeted the underlying proteins in a concerted manner.

Combined intermittent hypoxia and surface muscle electrostimulation as a method to increase peripheral blood progenitor cell concentration.

Background: Our goal was to determine whether short-term intermittent hypoxia exposure, at a level well tolerated by healthy humans and previously shown by our group to increase EPO and erythropoiesis, could mobilizehematopoietic stem cells (HSC) and increase their presence in peripheral circulation. Methods: Four healthy male subjects were subjected to three different protocols: one with only a hypoxic stimulus (OH), another with a hypoxic stimulus plus muscle electrostimulation (HME) and the third with only muscle electrostimulation (OME). Intermittent hypobaric hypoxia exposureconsisted of only three sessions of three hours at barometric pressure 540 hPa (equivalent to an altitude of 5000 m) for three consecutive days, whereas muscular electrostimulation was performed in two separate periods of 25 min in each session. Blood samples were obtained from an antecubital vein on three consecutive days immediately before the experiment and 24 h, 48 h, 4 days and 7 days after the last day of hypoxic exposure. Results: There was a clear increase in the number of circulating CD34+ cells after combined hypobaric hypoxia and muscular electrostimulation. This response was not observed after the isolated application of the same stimuli. Conclusion: Our results open a new application field for hypobaric systems as a way to increase efficiency in peripheral HSC collection.

Combined intermittent hypoxia and surface muscle electrostimulation as a method to increase peripheral blood progenitor cell concentration.

Background: Our goal was to determine whether short-term intermittent hypoxia exposure, at a level well tolerated by healthy humans and previously shown by our group to increase EPO and erythropoiesis, could mobilizehematopoietic stem cells (HSC) and increase their presence in peripheral circulation. Methods: Four healthy male subjects were subjected to three different protocols: one with only a hypoxic stimulus (OH), another with a hypoxic stimulus plus muscle electrostimulation (HME) and the third with only muscle electrostimulation (OME). Intermittent hypobaric hypoxia exposureconsisted of only three sessions of three hours at barometric pressure 540 hPa (equivalent to an altitude of 5000 m) for three consecutive days, whereas muscular electrostimulation was performed in two separate periods of 25 min in each session. Blood samples were obtained from an antecubital vein on three consecutive days immediately before the experiment and 24 h, 48 h, 4 days and 7 days after the last day of hypoxic exposure. Results: There was a clear increase in the number of circulating CD34+ cells after combined hypobaric hypoxia and muscular electrostimulation. This response was not observed after the isolated application of the same stimuli. Conclusion: Our results open a new application field for hypobaric systems as a way to increase efficiency in peripheral HSC collection.

The planarian flatworm: an in vivo model for stem cell biology and nervous system regeneration

Planarian flatworms are an exception among bilaterians in that they possess a large pool of adult stem cells that enables them to promptly regenerate any part of their body, including the brain. Although known for two centuries for their remarkable regenerative capabilities, planarians have only recently emerged as an attractive model for studying regeneration and stem cell biology. This revival is due in part to the availability of a sequenced genome and the development of new technologies, such as RNA interference and next-generation sequencing, which facilitate studies of planarian regeneration at the molecular level. Here, we highlight why planarians are an exciting tool in the study of regeneration and its underlying stem cell biology in vivo, and discuss the potential promises and current limitations of this model organism for stem cell research and regenerative medicine.

Newly described human polyomaviruses Merkel Cell, KI and WU are present in urban sewage and may represent potential environmental contaminants

Recently, three new polyomaviruses (KI, WU and Merkel cell polyomavirus) have been reported to infect humans. It has also been suggested that lymphotropic polyomavirus, a virus of simian origin, infects humans. KI and WU polyomaviruses have been detected mainly in specimens from the respiratory tract while Merkel cell polyomavirus has been described in a very high percentage of Merkel cell carcinomas. The distribution, excretion level and transmission routes of these viruses remain unknown. Here we analyzed the presence and characteristics of newly described human polyomaviruses in urban sewage and river water in order to assess the excretion level and the potential role of water as a route of transmission of these viruses. Nested-PCR assays were designed for the sensitive detection of the viruses studied and the amplicons obtained were confirmed by sequencing analysis. The viruses were concentrated following a methodology previously developed for the detection of JC and BK human polyomaviruses in environmental samples. JC polyomavirus and human adenoviruses were used as markers of human contamination in the samples. Merkel cell polyomavirus was detected in 7/8 urban sewage samples collected and in 2/7 river water samples. Also one urine sample from a pregnant woman, out of 4 samples analyzed, was positive for this virus. KI and WU polyomaviruses were identified in 1/8 and 2/8 sewage samples respectively. The viral strains detected were highly homologous with other strains reported from several other geographical areas. Lymphotropic polyomavirus was not detected in any of the 13 sewage neither in 9 biosolid/sludge samples analyzed. This is the first description of a virus isolated from sewage and river water with a strong association with cancer. Our data indicate that the Merkel cell polyomavirus is prevalent in the population and that it may be disseminated through the fecal/urine contamination of water. The procedure developed may constitute a useful tool for studying the excreted strains, prevalence and transmission of these recently described polyomaviruses.

Cell fusion reprogramming leads to a specific hepatic expression pattern during mouse bone marrow derived hepatocyte formation In Vivo

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-β1 (TGF-β1) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation.

Progressive Purkinje Cell Degeneration in tambaleante Mutant Mice Is a Consequence of a Missense Mutation in HERC1 E3 Ubiquitin Ligase

The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domain shave been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a GuA transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N- terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology.

Estimation of lung cancer diagnosis and treatment costs based on a patient-level analysis in Catalonia (Spain)

Background: Assessing of the costs of treating disease is necessary to demonstrate cost-effectiveness and to estimate the budget impact of new interventions and therapeutic innovations. However, there are few comprehensive studies on resource use and costs associated with lung cancer patients in clinical practice in Spain or internationally. The aim of this paper was to assess the hospital cost associated with lung cancer diagnosis and treatment by histology, type of cost and stage at diagnosis in the Spanish National Health Service. Methods: A retrospective, descriptive analysis on resource use and a direct medical cost analysis were performed. Resource utilisation data were collected by means of patient files from nine teaching hospitals. From a hospital budget impact perspective, the aggregate and mean costs per patient were calculated over the first three years following diagnosis or up to death. Both aggregate and mean costs per patient were analysed by histology, stage at diagnosis and cost type. Results: A total of 232 cases of lung cancer were analysed, of which 74.1% corresponded to non-small cell lung cancer (NSCLC) and 11.2% to small cell lung cancer (SCLC); 14.7% had no cytohistologic confirmation. The mean cost per patient in NSCLC ranged from 13,218 Euros in Stage III to 16,120 Euros in Stage II. The main cost components were chemotherapy (29.5%) and surgery (22.8%). Advanced disease stages were associated with a decrease in the relative weight of surgical and inpatient care costs but an increase in chemotherapy costs. In SCLC patients, the mean cost per patient was 15,418 Euros for limited disease and 12,482 Euros for extensive disease. The main cost components were chemotherapy (36.1%) and other inpatient costs (28.7%). In both groups, the Kruskall-Wallis test did not show statistically significant differences in mean cost per patient between stages. Conclusions: This study provides the costs of lung cancer treatment based on patient file reviews, with chemotherapy and surgery accounting for the major components of costs. This cost analysis is a baseline study that will provide a useful source of information for future studies on cost-effectiveness and on the budget impact of different therapeutic innovations in Spain.