Using extraction techniques for ancient DNA, we obtained high-quality sequences for two of these loci from gut contents (rbcL and ITS2). Sequences were then compared to a comprehensive DNA barcode library of the Zingiberales. The rbcL locus identified host plants to family (success/sequence = 58.8%) and genus (success/sequence = 47%). For all Zingiberales except Heliconiaceae, ITS2 successfully identified host plants to genus (success/sequence = 67.1%) and species (success/sequence = 61.6%). Kindt’s sampling estimates suggest that by collecting ca. four individuals representing each plant-herbivore interaction, 99% of all host associations included in this study can be identified to genus. For plants that amplified ITS2, 99% of the hosts can be identified to species after collecting at least four individuals representing each interaction. Our study demonstrates that host plant identifications at the species-level using DNA barcodes are feasible, cost-effective, and reliable, and that reconstructing plant-herbivore networks with these methods will become the standard for a detailed understanding of these interactions.Plants and their associated insect herbivores, represent more than 50% of all known species on earth. The first step in understanding the mechanisms generating and maintaining this important component of biodiversity is to identify plant-herbivore associations. In this study we determined insect-host plant associations for an entire guild of insect herbivores using plant DNA extracted from insect gut contents. Over two years, in a tropical rain forest in Costa Rica , we recorded the full diet breadth of rolled-leaf beetles, a group of herbivores that feed on plants in the order Zingiberales. Field observations were used to determine the accuracy of diet identifications using a three-locus DNA barcode ( Plants, together with their associated insect herbivores, represent more than 50% of all known species on earth Developing and implementing cost effective and reliable methods to identify insect herbivore diets has been a challenge. The most common strategies have included: 1) direct observations of herbivory in the field; 2) laboratory feeding trials An alternative to identify insect diets is to use plant DNA sequences extracted from the guts of insects . This meCephaloleia and Chelobasis (Cassidinae: Chrysomelidae). We performed this study at La Selva Biological Station (hereafter La Selva) in a tropical rain forest habitat in Costa Rica, Central America To assess the efficacy and accuracy of using DNA barcodes as molecular markers to determine host species in whole communities of insect herbivores, we performed a field and laboratory experiment on a well-studied plant-insect interaction: plant hosts in the order Zingiberales and their rolled-leaf beetle herbivores in the genera i.e., rbcL, ITS2 and trnH-psbA) to accurately identify insect diets see Methods section). Finally, we estimated the sampling effort required to identify all plant-herbivore interactions in a community of rolled-leaf beetles using DNA barcodes.This well-studied plant-herbivore system allowed us to determine the ability of three plant DNA barcode loci . A potentially useful marker to identify insect diets is the trnL-intron UAA (P6 loop). This short molecular marker (<100 bp) can be easily amplified from degraded DNA samples such as feces. However, its taxonomic resolution is limited and should be used in studies aimed at determining insect diets to the plant family taxonomic levels only Our results show that each of these three plant DNA barcode loci is not as universal as desired in such studies and that more than one locus must be used when reconstructing a network of plant-herbivore interactions. For example, long A-T repeats in the rbcL sequences were assigned to the correct plant family or genus , diet identified as C. lasiostachia (Marantaceae), sequences 112858035, 112858396, All or genus . When usCephaloleia placida, a specialist that feeds on plants in the genus Renealmia (Zingiberaceae) was assigned to an incorrect host, Calathea lutea  and the probability of identification of equences , we succrbcL barcode, it was possible to identify 99% of the host plants to family by collecting 500 rolled-leaf beetles. In addition, 99% of the interactions can be identified to genus by collecting ca. 700 rolled-leaf beetles  technologies Although NGS has an enormous potential for the study of plant-herbivore interactions, this novel technique is affected by the same limitations that we described for traditional Sanger sequencing methods In conclusion, we tested in a community of rolled-leaf beetles the reliability of multiple DNA barcode loci for the identification of insect herbivore diets. DNA barcode techniques can identify insect host plants to the taxonomic level of family, genus and species. DNA barcodes are also successful at differentiating closely related host plant species allowing the successful reconstructing of multiple associations in plant-herbivore networks. As better DNA extraction methods are developed for degraded DNA sampled from insect guts and as more cost-effective and rapid next-generation DNA sequencing protocols are developed, we expect that reconstructing plant-herbivore networks with these methods will become the standard for a detailed understanding of these interactions.Fieldwork was conducted from October 2008 to March 2011 at La Selva Biological Station, a tropical rain forest site in Costa Rica, Central America . La Selva is classified as an aseasonal tropical wet forest Cephaloleia and Chelobasis  and Neotropical plants in the order Zingiberales  Cephaloleia and Chelobasis are also known as the “rolled-leaf beetles” because their lifecycle occurs inside the scrolls formed by the young rolled leaves of their host plants This study focuses on insect herbivores in the genera At La Selva 20 species of rolled-leaf beetles feed on 33 species of Zingiberales  [37]. ThDuring two years we surveyed the diets of all rolled-leaf beetles present in our study area. Surveys were performed during the rainy and dry seasons of each year. . Surveys consisted in carefully unrolling each young leaf and counting the number of individuals of each rolled-leaf beetle species. The minimum distance between plants surveyed was 5 m .We collected vouchers of at least three individuals of each species in the order Zingiberales at La Selva  buffer at 54°C. The lysate was then spun to pellet debris, and the cleared lysate mixed with GUSCN based binding buffer, then DNA was captured through binding on a 96 well glass-filter plate through centrifugation, washed twice and finally eluted in 200 µl of 10 µM Tris-HCl. All DNA extractions were conducted in a PCR free laboratory. Aliquots of DNA were prepared, with 30 µl material separated as a working stock for the PCR laboratory, and remaining DNA extract archived with 2-D barcode matrix tubes with numerical GUIDs  used to track the samples throughout the process of PCR, sequencing To identify insect herbivore diets using DNA barcodes, we collected 1 to 8 rolled-leaf beetle individuals for each of the insect-host plant associations included in this study . High resolution 3D images were obtained for each specimen using an Auto-Montage® system . Images are available upon request to the corresponding author. Each beetle was placed into a separate screw-cap tub with an O-ring and DNA extraction was conducted in a PCR free laboratory, where no plant DNA extractions had been implemented. Each beetle sample had the thorax or entire body used in the extraction depending on the size of the individual. The remnant parts of each sample were deposited in the National Museum of Natural History Biorepository - Smithsonian Institution. A mixture of disposable silica and zircon beads (0.1–2.3 mm diameter) were added to each tube containing a beetle and disrupted with a single tube bead-mill.We obtained high-quality DNA from insect gut contents by modifying DNA extraction techniques originally developed to obtain ancient DNA studies (Guanadinium-thyocyanate lysis buffer with glass fiber membrane recovery) rbcL, nuclear ribosomal ITS2 and plastid trnH-psbA intergenic spacer used in routine DNA barcoding To generate the reference plant DNA barcode library, three loci were sequenced for each of the vouchered samples: plastid rbcL, we used a pair of mini-barcodes which divided the normal rbcL barcode region into two segments, one 230 bases long and a second 320 bases long. We used two internal primers that annealed to the same position to create the mini-barcode partitions in conjunction with the standard barcode primers. The rbcLa-230 was a reverse primer used with rbcL_F, and rbcL 260-F was used with the standard reverse DNA barcode primer rbcLa_Rev. The sequence of these rbcL-mini-barcodes is given in For the insect herbivore gut content samples, we attempted PCR recovery for each of the three loci in our reference database. For rbcL mini-barcode, we used primers for ITS2 that produced the smallest possible amplicon size, to increase the chance that we could recover intact sequences from potentially highly degraded plant DNA from insect gut contents. We used the same ITS2-2-FOR primer used in our reference library construction and the standard ITS4-Rev primer. ITS4_Rev produced a 360bp amplicon that was approximately 90bp shorter than the reference library sequences. We were unable to identify a primer that would generate a mini-barcode for the trnH-psbA region that would work for the entire order of Zingiberales, and thus we attempted recovery of forensic sequences with the full-length barcode region using standard primers.Similar to the use of the see a summary of primers and PCR conditions in PCR was conducted under standard conditions for each gene, with the exception that we increased the number of PCR cycles to 40 for all samples  and ITS2 DNA sequences (for identifications to the genus and species taxonomic resolutions). We did not perform this analysis for the trnH-psbA as we failed to amplify high-quality DNA from Zingiberales or insect gut contents (see results). Because insect collections were modeled at random, our sampling assumes equivalent occurrence for all plant-herbivore associations.We estimated for the interactions included in this study the sampling effort required for reconstructing plant-herbivore associations using DNA barcodes. First we simulated random collections with replacement of 1000 rolled-leaf beetles from known host plants. For each collection event, we estimated the probability of successful host plant identification to a given taxonomic level using a given DNA barcode  by multiplying the probability of success during DNA extractions and the probability of successful host plant identification to a given taxonomic level using a molecular marker . We perfŜ represents the average number of plant-herbivore interactions. Stot represents the total of interactions recorded in the survey.We generated accumulation models of plant-herbivore interactions using the Kindt’s exact accumulator  [26]. WhWhere:N indicates the numbers of collections for which the average number of interaction is calculated, if represents the frequency of interaction i, and totF is the total number of rolled-leaf beetles surveyed.In our application of the Kindt’s exact accumulator, We estimated the variance for the accumulation curves:ijr is the correlation coefficient between interaction i and interaction j. Simulations and accumulation curves were estimated using Program R (package vegan) Where Table S1Data matrix to generate .(DOCX)Click here for additional data file.Table S2Primers and PCR conditions used in this study to obtain DNA barcode libraries and sequences from insect gut contents.(DOCX)Click here for additional data file.Supplement S1DNA sequences (rbcL and ITS2) obtained from insect herbivore gut contents.(TXT)Click here for additional data file.


Few attempts have been made to model learning of sensory-motor control using spiking neural units. We trained a 2-degree-of-freedom virtual arm to reach for a target using a spiking-neuron model of motor cortex that maps proprioceptive representations of limb position to motor commands and undergoes learning based on reinforcement mechanisms suggested by the dopaminergic reward system. A 2-layer model of layer 5 motor cortex (M1) passed motor commands to the virtual arm and received proprioceptive position information from it. The reinforcement algorithm trained synapses of M1 using reward (punishment) signals based on visual perception of decreasing (increasing) distance of the virtual hand from the target. Output M1 units were partially driven by noise, creating stochastic movements that were shaped to achieve desired outcomes.The virtual arm consisted of a shoulder joint, upper arm, elbow joint, and forearm. The upper- and forearm were each controlled by a pair of flexor/extensor muscles. These muscles received rotational commands from 192 output cells of the M1 model, while the M1 model received input from muscle-specific groups of sensory cells, each of which were tuned to fire over a range of muscle lengths. The M1 model had 384 excitatory and 192 inhibitory event-based integrate-and-fire neurons, with AMPA/NMDA and GABA synapses. Excitatory and inhibitory units were interconnected probabilistically. Plasticity was enabled in the feedforward connections between input and output excitatory units. Poisson noise was added to the output units for driving stochastic movements. The reinforcement learning (RL) algorithm used eligibility traces for synaptic credit/blame assignment, and a global signal  corresponding to dopaminergic bursting/dipping. Eligibility traces were spike-timing-dependent, with pre-before-post spiking required. Reward (punishment) was delivered when the distance between the hand and target decreased (increased) [RL learning occurred over 100 training sessions with the arm starting at 15 different initial positions. Each sub-session consisted of 15 s of RL training from a specific starting position. After training, the network was tested for its ability to reach the arm to target from each starting position, over the course of a 15 s trial. Compared to the naive network, the network post-training was able to reach the target from all starting positions. This was most clearly pronounced when the arm started at a large distance from the target. After reaching the target, the hand tended to oscillate around the target. Learning was most effective when recurrent connectivity in the output units was turned off or at low levels. Best overall performance was achieved with no recurrent connectivity and moderate maximal weights. Although learning typically increased average synaptic weight gains in the input-to-output M1 connections, there were frequent reductions in weights as well. Our model predicts that optimal motor performance is sensitive to perturbations in both strength and density of recurrent connectivity within motor cortex and that therefore the wiring of recurrent connectivity during development might be carefully regulated.


These developments are aiming for in vitro generation of organ equivalents such as liver, lymph nodes and lung or even multi-organ systems for substance testing, research on organ regeneration or transplant manufacturing. Initially technical perfusion systems based on membranes, hollow fibers or networks of micro-channels were used for these purposes. However, none of the currently available systems ensures long-term homeostasis of the respective tissue over months. This is caused by a lack of in vivo-like vasculature which leads to continuous accumulation of protein sediments and cell debris in the systems. Here, we demonstrate a closed and self-contained circulation system emulating the natural blood perfusion environment of vertebrates at tissue level.Enormous efforts have been made to develop circulation systems for physiological nutrient supply and waste removal of The Multi-Organ-Chip (MOC) device accommodates two microvascular circuits Figure . Each ci7 cells ml-1 into the channels, a homogeneous distribution of cells throughout all channels was achieved (Figure A miniaturized circulation system has been established over a period of 14 days by fully covering all channels and surfaces of the MOC with human microvascular endothelial cells. By injecting 2 × 10d Figure . During d Figure . During d Figure . After 1d Figure  and exprd Figure .A robust procedure applying pulsatile shear stress has been established to cover all fluid contact surfaces of the system with a functional, tightly closed layer of HDMEC.in vivo-like crosstalk between endothelial cells and tissues and prevents clumping inside the channels. Compared with conventional cell culture techniques, a microfluidic-based cell culture may mimic more accurate in vivo-like extracellular conditions, as the culture of cells and organ models in perfused microfluidic systems can improve their oxygen and nutrient supply. This makes it suitable for long-term cultivation and more efficient drug studies. In future, such endothelialized bioreactors might be used for testing vasoactive substances. Finally, the described system can now be used for the establishment of organ-specific capillary networks. Here, we will adhere to our recently published roadmap toward vascularized ''human-on-a-chip'' models to generate systemic data fully replacing the animals or human beings currently used [Long-term cultivation of elongated and flow-aligned HDMEC inside the chip-based microcirculation was demonstrated over a period of 14 days. For such endothelialized microfluidic devices to be useful for substance testing, it is essential to show long-term viability and function in the presence of physiological flow rates as shown here. These artificial vessels are an important approach for systemic substance testing in Multi-Organ-Chips. The miniaturized circulation system creates the conditions for circulation of nutrients through the organoid culture chamber, allows for tly used .


Salmo trutta) as a study species. We manipulated densities of large and small individuals, and observed effects on survival, migration, condition and individual growth rate in a target group of intermediate-sized individuals. The generality of the response was investigated by reducing population densities below and increasing above the natural levels . Reducing the density (relaxing the intensity of competition) had no influence on the response variables, suggesting that stream productivity was not a limiting factor at natural population density. Addition of large individuals resulted in a negative density-dependent response, while no effect was detected when adding small individuals or when maintaining the natural population structure. We found that the density-dependent response was revealed as reduced growth rate rather than increased mortality and movement, an effect that may arise from exclusion to suboptimal habitats or increased stress levels among inferior individuals. Our findings confirm the notion of interference competition as the primary mode of competition in juvenile salmonids, and also show that the feedback-mechanisms of density-dependence are primarily acting when increasing densities above their natural levels.While the prevalence of density-dependence is well-established in population ecology, few field studies have investigated its underlying mechanisms and their relative population-level importance. Here, we address these issues, and more specifically, how differences in body-size influence population regulation. For this purpose, two experiments were performed in a small coastal stream on the Swedish west coast, using juvenile brown trout ( Although negative density-dependence, through reduced survival and/or fecundity, is a well-established process for hampering population growth as resources become limited, the underlying mechanisms and their relative importance are less well known Among juvenile stream-living salmonids, interference through territoriality and dominance hierarchies is generally assumed to be the most prevalent mode of competition with strong density-dependent effects on mortality and migration, especially noticeable at the early stages after emergence Coregonus albula) have competitive advantage over older (and larger) conspecifics, through lower metabolic requirements, with subsequent negative effects on growth rate and ultimately also on fecundity of the latter 2) of salmon  fry had negative effects on the growth of older cohorts. Suggested explanations for this response include shadow (filtering) competition where fry either depletes the downstream drift of invertebrates or intercepts the territories of older fish For example, young  individuals of planktivorous vendace . The competitive environments were altered by reducing (hereafter referred to as the Reduction experiment) and increasing (hereafter referred to as the Addition experiment) the number of large and small individuals and the density-dependent responses on growth, condition, survival and migration were evaluated in a target group of intermediate-sized individuals. Furthermore, based on suggestions in previous literature To better understand how body-size influence density-dependent population regulation, two field experiments were conducted on juvenile (1-yr old) sea-migratory brown trout . Electro-fishing permits were obtained from the County Administrative Board Västra Götaland and all field work was approved by the private land owners. No protected species were collected during the field experiment.2 and runs through deciduous forests with much vegetative overhang −2 for young-of-the-year and yearling trout). Larger trout typically migrate sooner than smaller and size appears to be the main determinant of migration. After 1–2 years in the sea, adults return to their natal stream to breed. There is also a varying, but small, proportion of resident (non-migratory) trout that mature in the stream and they typically occupy the deeper pools. Predators are heron (Ardea cinerea), eel (Anguilla anguilla), larger resident brown trout, and mink (Mustela vison).The experiments were performed during 2006 (Reduction) and 2007 (Addition) in Jörlandaån , a small stream 40 km north of Gothenburg on the Swedish west coast. Jörlandaån has a catchment area of 39 kmThe experimental area was a stream stretch of approximately 1 km, within which three or four blocks  were distributed. Each block (replicate) consisted of three treatment units (stream sections) that were randomly distributed within the block and individually labelled on the stream bank:Large individuals removed (Reduction experiment) or added (Addition experiment)Small individuals removed (Reduction experiment) or added (Addition experiment)Control, natural density and size-structureThe length of the stream sections ranged between 40 and 60 m, determined by the number of target fish . Each treatment section was isolated with a buffer zone of approximately 20 m in length with natural density and population size-structure.The population-level response of altered competition conditions was investigated by removing one third of the total biomass in two out of three experimental sections per block, either many small (lower third) or few large individuals (upper third), while keeping the natural density intact in the remaining control section  beginning at the downstream end of the experimental site. All fish were captured and electro-fishing was paused after having collected approximately 30 individuals of intermediate size (target fish) . Collected fish were held in section-specific containers with aerated and continuously replaced stream water. The lower/middle/upper thirds of the biomass in each treatment section were estimated using a randomly selected sample of approximately 35 individuals from each section. Fish in the subsample were measured  and their individual body-weight was estimated using a length-weight relationship from previous population surveys at the same site and at the same time of year, in order to avoid anaesthetizing the fish and hence to reduce handling stress. The accumulated biomass of the subsample and the lower/middle/upper thirds were calculated in an Excel spread-sheet and the corresponding size-limits of the small group, the target group and the group of large fish were generated after sorting the subsample by body-length , measured (to the nearest mm), weighed (to the nearest g) and placed in three separate containers according to the size-groups estimated from the subsample. Target fish  were equipped with passive integrated transponders  for individual identification .After recuperating, the target group and a group of small, large or both (depending on treatment) were transferred back into the same stream section where they were captured, while removed small or large fish were released downstream of the experimental area. The release site was separated from the experimental area by a stream stretch of approximately 1.5 km consisting of low water level and poor habitat quality, in order to reduce the risk of homing.Estimated body-weights and size-limits of the three groups gave a relatively good fit when applied to the total data set (the biomass of the size-groups did not deviate greatly from 33% (one third) of the total section biomass). Biomass of removed large individuals was 36±3%  , while 29±1.1% of the biomass was removed as small individuals  . As expeRecaptures were performed on two occasions; one month after the start of the experiment  and in the autumn . A two-pass electro-fishing was performed at each recapture, starting 100 m downstream the first section and continuing 150 m upstream the uppermost section to include potential strayers from the experimental area. The capture location (block and section) was noted for each target individual and data on PIT-ID, length and weight was collected after anesthetising the fish. All individuals were released at the place of capture after recovering from the procedure.During the second year, different competitive environments were created by adding few large or many small individuals  at a biomass equal to that of the target group in each experimental section, while keeping the natural densities intact in the remaining control section. The nine experimental sections of the preceding experiment were re-used during the Addition experiment and three additional sections were included to increase statistical power , measured  and individuals between 60–110 mm (the target group) were equipped with PIT-tags. This target group generally comprised a majority of the fish in the treatment sections . The totThe supplementation fish were collected approximately one month after the experimental start-up (17 May 2007) at a site 3.5 km downstream the experimental area using electro-fishing. Immediately after capture, the fish were anaesthetized, measured , weighed (to the nearest gram), transported to the experimental area in aerated tanks and released in respective stream section.In total, 220 small  and 118 large fish (37.3±5.7%) were added to the treatment sections . The corRecaptures were performed on three occasions, two months after the start of the experiment , in the autumn  and after approximately one year . A two-pass electro-fishing was performed at each recapture, starting 100 m downstream the first section and continuing 150 m upstream the uppermost section to include potential strayers from the experimental area. The capture location (block and section) was noted for each target individual and data on PIT-ID, length and weight was collected after anesthetising the fish. All individuals were released at the place of capture after recovering from the procedure.w), difference in condition (Cdiff) from start to recapture, recapture rate  and movement (proportion of target fish recaptured within their home-section and home-block (section and block where they were released)). Specific growth rate was calculated as0W is initial body-size and tW is body-size t days later. Cdiff is the difference in residual value of the length-weight regression at the start and recapture, movement was analysed by assigning each recaptured individual a value of 1 (recaptured inside home-section or -block) and 0 (recaptured outside home-section or -block) and recapture rate was similarly analysed by assigning recaptured individuals a value of 1 and not recaptured individuals a value of 0.Response variables were specific growth rate in weight  and final recapture  (133-147 days). In the Addition experiment, response variables were calculated for the period between the start of the experiment (April) and final recapture  (354–358 days) but due to low recapture rates  these estimates were complemented by data for the period between start of experiment and the recapture in September (R2) (136–141 days).Mean per section of the response variables (n = 9 (Reduction experiment) and n = 12 (Addition experiment)) were tested in an ANCOVA according to the following model:Response variable  =  Treatment (fixed) + Block (random) + Initial length (covariate) + Proportion of total section biomass that was removed (Reduction experiment) or added (Addition experiment) (covariate)with a step-wise removal of factors at P>0.1. Initial length refers to the mean length of target individuals in each section at the start of the experiment. Proportions were arcsine(sqrt)-transformed prior to analyses. SPSS (v.19) was used for all statistical analyses.A total of 175 individuals (54%) were recaptured at the final sampling in September-October (R2)  were recaptured during the final sampling , with no effect of treatment or added biomass  final d. Due to Most target fish moved only short distances, with 75% being recaptured within their home-section and 89% within their home-block before winter  while no target individuals were recaptured in their home-section after winter  but 57% within their home block. There was no difference in movement rate between treatments at either of the occasions .All recaptured target individuals maintained positive growth throughout the sampling period. Growth rate was significantly affected by block and treatment at both recapture occasions (R2 and R3) , with taTo our knowledge, these are the first field studies to explore the mechanisms of density-dependence in a stream-living salmonid population using a fully experimental approach. We tested for differential effects of adding and removing large and small individuals on a group of intermediate-sized individuals. Most field evidence of density-dependence in salmonids is derived from populations that are experimentally increased by the addition of supplementation fish, either hatchery-reared alone e.g.  or togetReducing the population density below the natural level by removing few large and several small individuals had no feedback-effects on the performance of intermediate-sized target fish. This finding contrasts the response of recent studies using a similar experimental approach but investigating competition effects between, rather than within, cohorts The high productivity of the stream used in this experiment may be one explanation for the lack of response. If resource availability was in excess at the natural population density, i.e. if the natural population density was below carrying capacity, we would indeed expect the effect of density reduction to be less apparent. Such a scenario would also appear if the population density is pushed below carrying capacity through high mortality at the early critical phase after emergence, as suggested in the extensive work by Elliott Increasing the natural population density by adding few large and several small individuals gave rise to a negative feedback-effect. Target individuals in treatment sections, to which large fish were added, experienced a significantly slower growth rate whereas no such effect was detected in control sections  or in sections with an increased number of small fish. The reduced growth rate was revealed at both tested time periods, from the experimental start-up to the autumn sample as well as to the spring sample, one year later. This finding suggests that the body-size of the introduced fish relative to that of the target group was more important for the performance of intermediate-sized target individuals, than population density per se. Such a response would be expected if feeding territories, rather than food itself, is the main limiting resource, with few large individuals being able to monopolize high-quality areas of the stream habitat The observed reduction of individual growth rate disagrees with the prediction that larger individuals (through interference competition) affect primarily mortality and movement. Several studies, especially the extensive observational data by Elliott Although not specifically investigated here, several mechanisms may explain the observed reduction in growth rate among the target fish. Size-dependent habitat use is frequently reported within e.g.  as well In accordance with the findings of previous studies e.g. , we predIn conclusion, our results show that addition of few large individuals results in a negative feedback-response on the growth rate of intermediate-sized target individuals. We argue that this is likely an effect of interference competition, where the larger  supplementation fish exclude target individuals from favourable feeding habitats. For organisms with indeterminate growth, such as fishes, a reduction in growth rate can have substantial life-history consequences, ultimately by affecting fecundity Figure S13.1100) with an estimation based on data from previous years of electro-fishing surveys (red dots) (y = 6E−06x3.1258).Length-weight relationships. Comparisons of the actual length-weight relationship  (blue dots) (y = 6E−06x(DOCX)Click here for additional data file.Sheet S1Procedure for establishing size-limits during the Reduction experiment. A subsample of 35 individuals from each experimental stream section was collected to estimate the size-limits of the three size-groups  and large) and applied to the total stock of each section.(XLSX)Click here for additional data file.Table S1Distribution of treatment sections and blocks within the experimental site. The reduction experiment comprised three blocks (replicates) while four blocks were used during the addition experiment.(DOCX)Click here for additional data file.Table S2−2) of tagged and untagged (1 yr and older) fish at the final recapture of the Reduction experiment (September-October 2006) and the Addition experiment (April 2008), respectively.Final densities. Average densities Click here for additional data file.


Dose adjustments of low molecular weight heparin (LMWH) based on daily anti-Xa measurement by chromogenic assay remain controversial in daily clinical practice. One of the major obstacles is the cost of such a test. An affordable and reliable bedside test could change practice to an individual tailored dosing of LMWH. The aim of our study was to evaluate whether a prophylactic dose regimen of 40 mg enoxaparine in cardiac surgical patients increases the anti-Xa activity to the level necessary for efficient prevention of a thromboembolic event . Seconda®) and the bedside assay (Hemochron® Jr).This was an open, single-centre, prospective, nonrandomized clinical trial at a university hospital. All patients that needed prophylactic dosing of enoxaparine after cardiac surgery were duly informed and after giving written consent we included 44 patients with a mean Euroscore of 1.66. The demographic specifications, medical and surgical history of all patients were collected. Anti-Xa activity was measured at three different points in time. We determined baseline, peak and trough anti-Xa activity: preoperatively, and respectively 4 hours after the third dose of enoxaparine and 30 minutes before the fourth dose. Each measurement was done with both techniques, the two-stage chromogenic assay at the laboratory (Biophen2. Comparison of the bedside assay with the two-stage chromogenic assay by means of the Pearson's correlation coefficient showed correlation of the two tests if no variables were taken into account. In the Bland-Altman analysis we could not confirm this correlation.Our dose regimen of enoxaparine achieved in one-half of the included patients a sufficient anti-Xa activity for prevention of thromboembolic events. One-half of the patients with insufficient anti-Xa activity had a body mass index over 30 kg/mThe bedside anti-Xa activity assay with a Hemochron device tends to show some correlation with the two-stage chromogenic assay, but insufficient to be used as an alternative, in this small but uniform patient population. Use of a standard dosing protocol for enoxaparine administration is prone for underdosage in post-cardiac surgery patients and may increase postoperative morbidity.


Chronic tinnitus is a brain network disorder with involvement of auditory and non-auditory areas. Repetitive transcranial magnetic stimulation (rTMS) over the temporal cortex has been investigated for the treatment of tinnitus. Several small studies suggest that motor cortex excitability is altered in people with tinnitus. We retrospectively analysed data from 231 patients with chronic tinnitus and 120 healthy controls by pooling data from different studies. Variables of interest were resting motor threshold (RMT), short-interval intra-cortical inhibition (SICI), intra-cortical facilitation (ICF), and cortical silent period (CSP). 118 patients were tested twice - before and after ten rTMS treatment sessions over the left temporal cortex. In tinnitus patients SICI and ICF were increased and CSP was shortened as compared to healthy controls. There was no group difference in RMT. Treatment related amelioration of tinnitus symptoms were correlated with normalisations in SICI. These findings confirm earlier studies of abnormal motor cortex excitability in tinnitus patients. Moreover our longitudinal data suggest that altered SICI may reflect a state parameter, whereas CSP and ICF may rather mirror a trait-like predisposing factor of tinnitus. These findings are new and innovative as they enlarge the knowledge about basic physiologic and neuroplastic processes in tinnitus. Tinnitus is associated with neural changes in both the auditory pathway , and in non-auditory brain areas In addition TMS can also be used as a diagnostic tool for the assessment of motor cortex excitability by quantifying contractions of peripheral muscles induced by stimulation of the corresponding motor cortex representation. Assessment of motor cortex excitability in a longitudinal study design before and after a specific therapeutic intervention (e.g. multiple sessions of rTMS) enables to describe treatment-related neuroplastic changes.Several pilot studies suggest that tinnitus is related to alterations in motor cortex excitability. ICF was found to be enhanced in 19 patients with tinnitus in contrast to 19 controls Moreover it has been shown, that rTMS over the temporal cortex can induce changes in motor cortex excitability. Five days of active low-frequency rTMS over auditory cortex lead to prolongation of the CSP in 18 healthy subjects Here, we aimed to investigate in a retrospective analysis whether the observed alterations of cortical excitability can be replicated in a large sample of patients with tinnitus and controls. In order to address the open question whether such changes represent state or trait markers, we analysed longitudinal data before and after a therapeutic intervention.All participants gave written informed consent after a comprehensive explanation of the procedures. All studies whose data contributed to this analysis were approved by the Ethics Committee at the University of Regensburg. All experiments were conducted in accordance with the last revision of the Declaration of Helsinki.Patients presenting with a phantom sound of at least two month duration were recruited and investigated at the Tinnitus center Regensburg (Germany) which is a collaboration of the Department of Otolaryngology and the Department of Psychiatry and Psychotherapy of the University Hospital of Regensburg (Germany). Measurements of cortical excitability were performed in 231 patients (172 (74.5%) males; 49.5±12.2 (20–83) years) with chronic tinnitus (duration 93±95 (2–476) months). 44 (19%) out of 208 patients reported a purely left-sided, 37 (16%) a purely right-sided tinnitus, and 127 (55%) patients described their tinnitus as bilateral or originating within the head. Tinnitus distress was assessed by the German version 2 = 25.3; df = 1; p<0.001).Healthy volunteers  males; age: 28.2±7.5 (19–55) years) were recruited through advertisements and by requesting the hospital staff. All healthy participants were free of medication and free of any mental disorder as verified during an interview by board-certified psychiatrists . Controls did significantly differ from patients with respect to age  and gender  connected via a Bistim module to a figure-of-eight coil (double-circular-70-mm coil). The coil was held tangential to the skull and with the handle pointing backwards and about 45° away from the midline. The optimal coil position for stimulation was defined as the position above the left motor cortex for eliciting MEPs of maximal amplitude in abductor digiti minimi muscle with a slightly supra-threshold stimulus. Once this position was found, it was marked on a scientific head cap and the coil was held in this position by the investigator.MEPs of the abductor digiti minimi of the right hand were recorded with surface electrodes. The analogue signal was registered, band-pass filtered between 20 Hz and 10 kHz, then digitised at a frequency of 5 kHz and analysed off-line. The employed hardware setup required the analogue signal to be acquired with a band-pass filter between 20 Hz and 10 kHz and a digitization frequency of 5 kHz. Whereas these suboptimal filter settings yield the possibility of aliasing effects , the small signal amplitudes for high frequencies render it unlikely that the recording setup may have biased or altered the present findings Resting and active motor threshold were determined according to Rossini and colleagues Short-interval intra-cortical inhibition and intracortical facilitation were measured with a paired-pulse TMS protocol Cortical silent period was measured in 10 trials  in the moderately active abductor digiti minimi muscle on the non-rectified recording of every individual sweep and then averaged . Treatment effects were evaluated by change in TQ between the first and the last day of treatment. Motor cortex excitability was examined on these days, too. We analyzed longitudinal data of 118 patients participating in three different treatments The therapeutic intervention consisted of 10 rTMS sessions on consecutive weekdaysFirstly, we were interested in differences in motor cortex excitability between patients with tinnitus and healthy controls. We contrasted both groups with two-sided Student t-tests. As groups were not comparable for age and gender, we also contrasted both groups with an analysis of variance (ANOVA) with group as between-subjects factor and age and gender as covariate (ANCOVA). As we included data from former studies Secondly, we were interested if rTMS induced clinical amelioration was associated with changes in motor cortex excitability. Thus, we correlated changes/differences in the TQ with changes/differences in excitability parameters. This analysis would shed light on the question if tinnitus-related changes in motor cortex excitability represent state or trait markers of tinnitus. As three different treatments were included we were interested in the interrelationship of kind of treatment with correlations. Thus, we repeated the correlation analysis with partial correlations using kind of treatment as control variable.As we had four variables of interest , only p-values below a Bonferroni corrected significance level of 0.0125 (0.05/4 = 0.0125) were presented as significant. Statistical analyses were performed with SPSS 18.0.0 .Firstly, group contrasts of two big samples of patients with tinnitus (n = 231) and healthy controls (n = 120) revealed significant differences in short-interval intra-cortical inhibition (SICI), intra-cortical facilitation , and cortical silent period (CSP). Patients with tinnitus showed increased SICI , increased intra-cortical facilitation, and shortened cortical silent period . As grouSecondly, for a sub-group of patients (n = 118) data of motor cortex excitability were available at two different time points before and after a therapeutic intervention. Here we found that a change in tinnitus severity correlated significantly with changes in SICI, i.e., the higher the improvement the higher the decrease in SICI . A scatter plot  indicateMotor cortex excitability as measured by short-interval intra-cortical inhibition (SICI), intra-cortical facilitation (ICF), and cortical silent period (CSP) appeared to be altered in tinnitus patients . SICI anSICI and CSP are measures for inhibitory and ICF of facilitatory processes in the motor system. Tinnitus is known to be related to neural changes along the auditory pathway emerging from a dysbalance of inhibitory and excitatory neurons Several confounding factors may potentially influence motor cortex excitability in tinnitus patients. First, the influence of hearing loss has to be considered as a confounding factor. Hearing loss results in sensory deprivation and is considered to be the most relevant risk factor for tinnitus. At the same time sensory deprivation may have a direct influence on motor cortex excitability. Increased ICF has been reported after experimental somatosensory deafferentiation Alterations of motor cortex excitability in a cross-sectional group comparison between tinnitus patients and controls could be either a consequence or a pre-condition of tinnitus or both. This could be evaluated by large-sampled prospective studies in non-tinnitus sufferers. Another approach for disentangling state and trait markers is to evaluate the influence of treatment effects. Here the cross-sectional comparison between tinnitus patients and controls was complemented by a longitudinal data analysis in order to identify to which extent the observed alterations of motor cortex excitability in tinnitus patients reflect an alteration of inhibitory-excitatory cortical processes predisposing for tinnitus, or a consequence of tinnitus.By analysing longitudinal data before and after rTMS treatment we found a significant association of treatment-related change in TQ with change in SICI, i.e., the higher amelioration of symptoms the higher the decrease in inhibition . PendingAlternatively, the increased SICI may reflect a direct consequence of tinnitus. Neural changes in the auditory system could induce changes in the motor system via mechanisms of cross-modal plasticity. Functional imaging and transcranial magnetic stimulation studies suggest multisensory association areas as neural basis for cross-modal or multisensory integration. However, recent findings in synaesthesia - an example for the incorporation of one modality in two modalities - changed the focus of research from a single core region to network properties and connectivity N-methyl-d-aspartate (NMDA) increased ICF and shortened CSP reflect globally increased glutamatergic and reduced GABA-B transmission, respectively. Support for this theory comes from a recent genetic study providing preliminary evidence for a role of genetic variations of the GABA-B receptor in the pathophysiology of tinnitus Our findings fit well to concepts and findings in tinnitus with respect to trans-synaptic chemical signalling. Inhibitory-excitatory dysbalance in tinnitus is considered to be related to altered activity of inhibitory-acting γ-aminobutyric acid (GABA) and excitatory-acting glutamate or In conclusion, present data show that tinnitus is accompanied by excitability and plasticity changes in the motor cortex. This finding sheds light on neglected areas in tinnitus research and highlights the inter-connectedness of the brain.


Therefore we sought to investigate whether NRP-1 is expressed in EEC and how NRP-1 and EEC respond to butyrate and other short-chain fatty acids . Additionally we sought to assess whether there is a field effect around adenomas.Previous reports have suggested that the VEGF receptor neuropilin-1 (NRP-1) is expressed in a singly dispersed subpopulation of cells in the normal colonic epithelium, but that expression becomes dysregulated during colorectal carcinogenesis, with higher levels in tumour suggestive of a poor prognosis. We noted that the spatial distribution and morphology if NRP-1 expressing cells resembles that of enteroendocrine cells (EEC) which are altered in response to disease state including cancer and irritable bowel syndrome (IBS). We have shown that NRP-1 is down-regulated by butyrate in colon cancer cell lines Biopsies were collected at the mid-sigmoid, at the adenoma and at the contralateral wall (field) of 28 subjects during endoscopy. Samples were fixed for IHC and stained for either NRP-1 or for chromogranin A (CgA), a marker of EEC. Stool sampling was undertaken to assess individuals' butyrate, acetate and propionate levels.+ cell number was also inversely related to SCFA at the landmark, but was lower and unresponsive at the field. Crypt cellularity was unaltered by field effect. A colocalisation analysis showed only a small subset of NRP-1 localised with CgA. Adenomas showed extensive, weaker staining for NRP-1 which contrastingly correlated positively with butyrate level. Field effects cause this relationship to be lost. Adenoma tissue shows dissociation of the co-regulation of NRP-1 and EEC.NRP-1 expression was inversely related to SCFA concentration at the colon landmark (mid-sigmoid), but expression was lower and not related to SCFA concentration at the field. Likewise CgAin vivo and is expressed in a subset of CgA expressing cells. EEC number is related to butyrate level in the same way.NRP-1 is inversely associated with levels of butyrate and other SCFA  The incidence of colorectal cancer has been shown to be decreased in populations with a high dietary fibre intake ,2. This Our recent data show that the transmembrane glycoprotein neuropilin-1 (NRP-1) is downregulated by butyrate in several colon cancer cell lines . NRP-1 wEnteroendocrine cells (EEC) are hormone-producing intestinal epithelial cells that are individually dispersed throughout the epithelium where they have a critical role in regulating gastrointestinal physiology . The numin vitro [in vivo systems and the same relationship will occur in vivo. In the present study we have investigated the relationship between faecal butyrate, acetate and propionate concentration and NRP-1 expression in human colonic mucosa. Furthermore, as NRP-1 expression is limited in the normal mucosa and is widespread in cancer tissue, we sought to investigate the expression profile of NRP-1 in adenoma and in fields around adenoma to map the onset of NRP-1 dysregulation. We have undertaken the same analyses on EEC and sought to establish whether EEC are the NRP-1 expressing compartment of the colon mucosa.Our recent data show an inverse causal relationship between butyrate concentration and NRP-1 expression at both the mRNA and protein level in vitro . We hypo2. The concentration range of faecal butyrate was 0.64-16.4 mM.A total of 28 subjects with adenoma were recruited for whom biopsies and faecal butyrate data were available. All subjects were included. Subjects had a mean age of 68.1 ± 10.1 yr and a mean BMI of 25.5 ± 3.4 kg/mIn order to investigate any baseline associations between butyrate concentration and field effect on crypt cell number and to contextualize the main assessment of the impacts of these two factors on the proportion of NRP-1 and CgA positive cells, the total number of cells per hemi-crypt across 10 hemi-crypts per sample was determined on samples from 23 field and 28 mid-sigmoid specimens. The correlation between crypt cellularity and butyrate level was assessed by Spearman's rho Figure  and usinin vitro [p < 0.001; Figure p = 0.235; Figure p = 0.013). A post hoc analysis revealed that the NRP-1 positive cell count in the low butyrate group (1.69%) was significantly higher than in medium  and high  butyrate groups  in mid-sigmoid sections  . The cels Figure . There ws Figure . These d+ and fewer than 20% of the NRP-1 positive cells expressed CgA+  and a near-significant correlation was seen between the percentage of positive cells and butyrate  depresses EEC cell number in the intestine [Although CgA is at present considered to be the broadest EEC marker, some EEC sub-populations are CgA negative . The ant the rat . As withntestine , suggestNRP-1 has been linked to cancer progression and aggressiveness in colon tumours . Our datin vitro studies using butyrate treatment of cell lines has shown that it induces apoptosis and cell cycle arrest at physiologically achievable concentrations (circa 1-10mM). Given that loss of regulation of both cell cycle and resistance to apoptosis are hallmarks of the cancer cell [A plethora of cer cell , these ecer cell , leadingcer cell . The impcer cell ,29 and ocer cell  have asscer cell ,32, butycer cell . These dOne limitation of this study is the use of faecal SCFA as a proxy measure of luminal SCFA. There is a gradation of level of SCFA within the different regions of the colon lumen  but sampTaken together our data provide evidence for progressive field effects in the vicinity of colon adenoma and in adenoma. Both NRP-1 and EEC number decreased in relation to increasing SCFA concentrations at sites distant to the adenoma. In the immediate field  this apparent relationship to SCFA level is lost and in the neoplasia the effect is reversed with neuropilin showing positive association with butyrate and dissociated from EEC-like expression. Our data therefore suggest a progressive and complete reversal of the response to butyrate as a hallmark of field effects. Despite widespread acceptance of the field effect hypothesis -37 in coin vitro data. NRP-1 expression is related to SCFA expression, but this association is lost in fields and expression becomes unlinked from EEC-like patterns in adenomatous tissue, implying an early and potential alternative role for NRP-1 in neoplasia. Our data showed for the first time that EEC number is also related to butyrate concentration. Future studies will now address whether there is a difference in EEC number in normal subjects by comparison with those carrying an adenoma, to examine whether there are pan-colon field effects in addition to local effects. Studies must also establish what the role and interactions of NRP-1 are in the normal colon epithelium in order to establish a clear role for butyrate in the regulation of function as well as homeostasis.In summary our data show that NRP-1 is expressed in the normal colon epithelium in a pattern redolent of EEC, and this expression appears related to butyrate levels, in agreement with the hypothesis raised from our The study protocol through which samples were acquired has been described elsewhere in detail . Briefly+ /NRP+, CGA+ /NRP-, GCA- /NRP+, and CGA- /NRP-,. Adenomas were scored for the intensity and percentage of positive NRP-1 and positive or negative of CgA stained cells per each section. All staining was scored by an assessor (DY) blinded to the cases and trained by the project histopathologist (JPB), and second scored by the project histopathologist. The co-localisation analysis was double scored by two assessors (DY & JT), under the supervision of the project pathologist.NRP-1 and CgA were stained in serial sections to enable analysis of co-localisation of the two factors. Antigen retrieval was performed using heat induced epitope retrieval using a microwave oven, with citrate buffer (pH6) for NRP-1 and DAKO Target Retrieval Solution (DAKO) for CgA. For NRP-1 staining a polyclonal rabbit anti-human NRP-1 antibody (Santa Cruz) and for CgA staining a monoclonal mouse anti-CgA antibody (DAKO) were used. A standard horse-radish peroxidise staining procedure was performed for both antibodies, using biotinylated antibodies  followed by the elite ABC kit  and DAB as the chromogen substrate (Vector laboratories) for visualisation. Sections of normal mucosa from the landmark site and adenoma field  were scored as the percentage of positively stained cells per hemi-crypt for each marker. Only well-orientated hemi-crypts were scored, up to a maximum of 10/section. To assess the colocalisation of NRP-1 and CGA, staining was performed in serial sections and 400 cells classified as GCAp < 0.05.Statistical analysis into the relationship between NRP-1 or CgA staining and faecal butyrate was conducted using SPSS v18 software . As the continuous data were not normally distributed the correlation between faecal butyrate levels and NRP-1 or CgA expression was analysed using Spearman's correlation statistics. A further analysis grouped the samples into tertiles by faecal butyrate and used the nonparametric Jonckheere-Terpstra test for ordinal categorical groupings. Data were considered statistically significant at the level of The authors declare that they have no competing interests.DCWY undertook all laboratory work, preliminary statistical analysis and wrote the first draft of the paper; JPB directed the development of IHC methods and directed statistical analysis, co-wrote and edited the manuscript; JT undertook the colocalisation analysis; JSW supervised laboratory work and co-conceived the study; CAS co-conceived the study, undertook statistical analyses, co-wrote and edited the manuscript; BMC conceived the study, directed the work and co-wrote and edited the manuscript. All authors read and approved the final manuscript.


We are introducing a novel, refined MR-compatible, explanted, blood-perfused and free-beating pig heart model for translational research within a state of the art 3T XMR environment.An isolated perfused pig heart model has recently been proposed for the development of novel methods in standard clinical magnetic resonance (MR) scanners. The original set-up required the electrical system to be within the safe part of the MR-room, which introduced significant background noise. The purpose of the current work was to refine the system to overcome this limitation so that all electrical parts are completely outside the scanner room.Four pig hearts were explanted under terminal anaesthesia from large white cross landrace pigs. All hearts underwent cardiovascular magnetic resonance (CMR) scanning in the MR part of a novel 3T XMR suite. CMR scanning included real-time k-t SENSE functional imaging, k-t SENSE accelerated perfusion imaging and late gadolinium enhancement imaging. Interference with image quality was assessed by spurious echo imaging and compared to noise levels, acquired while operating the electrical parts within the scanner room.Imaging was performed successfully in all hearts. The system proved suitable for isolated heart perfusion in a novel 3T XMR suite. No significant additional noise was introduced into the scanner room by our set-up.We have substantially improved a previous version of an isolated perfused pig heart model and made it applicable for MR imaging in a state of the art clinical 3T XMR imaging suite. The use of this system should aid novel CMR sequence development and translation into clinical practice.This work was supported by the BHF , the BRC  a European Union Grant  and the Wellcome Trust and EPSRC Medical Engineering Centre .


MicroRNAs (miRNAs) have emerged as fundamental regulators that silence gene expression at the post-transcriptional and translational levels. The identification of their targets is a major challenge to elucidate the regulated biological processes. The overall effect of miRNA is reflected on target mRNA expression, suggesting the design of new investigative methods based on high-throughput experimental data such as miRNA and transcriptome profiles. We propose a novel statistical measure of non-linear dependence between miRNA and mRNA expression, in order to infer miRNA-target interactions. This approach, which we name antagonism pattern detection, is based on the statistical recognition of a triangular-shaped pattern in miRNA-target expression profiles. This pattern is observed in miRNA-target expression measurements since their simultaneously elevated expression is statistically under-represented in the case of miRNA silencing effect. The proposed method enables miRNA target prediction to strongly rely on cellular context and physiological conditions reflected by expression data. The procedure has been assessed on synthetic datasets and tested on a set of real positive controls. Then it has been applied to analyze expression data from Ewing’s sarcoma patients. The antagonism relationship is evaluated as a good indicator of real miRNA-target biological interaction. The predicted targets are consistently enriched for miRNA binding site motifs in their 3′UTR. Moreover, we reveal sets of predicted targets for each miRNA sharing important biological function. The procedure allows us to infer crucial miRNA regulators and their potential targets in Ewing’s sarcoma disease. It can be considered as a valid statistical approach to discover new insights in the miRNA regulatory mechanisms. MicroRNAs (miRNAs) are single-stranded RNA molecules of Multicellular eukaryotes use miRNAs to regulate many biological processes. In animals, examples of documented miRNA functions include regulation of signaling pathways, apoptosis, metabolism, cardiogenesis and brain development The molecular mechanisms of miRNA action remain intensely debated. There are evidences for multiple modes of miRNA-mediated regulation, including translational inhibition, increased mRNA de-adenylation and degradation, and/or mRNA sequestration Since the discovery of miRNAs, the identification of genuine targets is a key issue to decipher their role in different biological processes. To date, the experimentally validated miRNA interactions are little more than 3500 in all species We propose here a novel measure of dependence between miRNA and mRNA expression to infer miRNA-target interactions. We assume that miRNAs and target transcripts can create a non-linear expression pattern due to the effect of cancer specific genomic alterations, additional regulatory factors and external noise. Two different relationships can be distinguished between a given miRNA and their targets, as depicted in hsa-miR-20b and its target gene MYLIP is illustrated in The antagonsim pattern detection described herein is based on large-scale analysis of paired miRNA and mRNA expression profiles. The procedure requires a set of coupled miRNA and mRNA expression measures from the same samples. The expression profile of each miRNA-mRNA pair across samples is scored for the presence of antagonism pattern. A representative example of antagonism pattern observed in Ewing’s sarcoma data between The measure of antagonism is a particularly suitable method to elucidate the role of miRNA regulation in tumoral diseases. In this context, physiological processes of gene regulation are widely altered. Tumor samples can present a high level of genomic heterogeneity, with cancer specific changes in miRNA expression and dysfunction of miRNA located in regions of chromosomal instability EWS and ETS family genes EWS gene is fused to FLI1EWS-FLI1 chimeric protein described as an aberrant transcription factor that dysregulates specific target genes involved in tumor development EWS-FLI1 gene alone can change cell phenotype from normal to tumorigenic in fully reversible and controlled manner, this is an excellent system for understanding the complex picture of deregulations happening in cancer cells. The availability of high-throughput data for Ewing patients, including transcriptome and miRNAs expression data, allows us to infer new regulatory interactions involved in the EWS-FLI1 network and to clarify the impact of miRNA activity.Later, we applied our method to elucidate the regulatory role of miRNAs in Ewing’s sarcoma. This malignant pediatric tumor is characterized by specific fusions between A series of experiments was performed on simulated datasets to test the antagonism pattern detection procedure. We generated simulated expression-like datasets exhibiting a triangular-shaped pattern. The aim is to simulate a set of expression profiles The effect of two main variables was examined to determine how they influence the assigned antagonism pattern p-value (defined in Methods), namely the number of available samples Appropriate evaluation of our proposed measure requires a set of real positive controls . However, a limited number of bona fide miRNA targets have been experimentally validated so far and they have been verified in various organisms and physiological conditions. We collected a catalogue of experimentally validated miRNA-target interactions from two main manually curated resources, namely miRecords and miRTarBase To compare results of the two methods, a ranking based evaluation was applied. We plotted the cumulative frequence of true predicted targets (hits) as a function of the prediction rank for the two methods . ResultsThis study includes miRNA and mRNA expression data from 40 samples of Ewing’s sarcoma patients. We collected expression levels of 267 miRNAs and 15651 mRNAs in 40 tumor samples using Illumina human-6 V2 BeadChip and Affymetrix GeneChip HG-U133A/HG-U133Plus2 oligonucleotide microarrays respectively. Normalization and pre-filtering procedures have been applied as described in Methods.We performed pair-wise antagonism pattern detection to evaluate potential regulatory interaction between each miRNA and each transcript. To determine whether the observed antagonism pattern is significant, the p-value was obtained by using permutation method described in Methods section. In simulated experiments with 40 samples, p-values range between Significant antagonism pattern has been detected for 7994 miRNA-mRNA pairs, creating a network linking 264 miRNA with 3747 different genes. Almost every miRNA in the initial dataset presents at least one link in the network. Hence, the combinatorial interactions among miRNAs and their targets are probably necessary to specify more precisely the co-regulating nodes and the set of affected targets of each miRNA. We characterized the reconstructed network by the connectivity distribution of miRNAs and genes, defining a list of miRNA hubs and a list of target hubs (details in Methods). According to the outlined distributions, the list of miRNA hubs and target hubs have been compiled and reported in The observation of significant antagonism pattern in expression data can rise from secondary effects rather than from direct regulatory relationships. As most expression-based inference methods, the antagonism pattern detection cannot distinguish between miRNAs that actually regulate a gene  and miRNAs that show significant antagonism pattern with a gene due to indirect effects. To address this issue and verify that the antagonism pattern captures information about miRNA-gene reliable interactions, two validation analysis have been carried out. In the first one, we verified that miRNA targets identified by antagonism pattern are enriched for binding sites with extensive 5′ miRNA seed pairing in their 3′UTR. A second independent strategy relates to evaluate significantly enriched GO categories for the target sets associated to each miRNA.The principle of 5′ seed pairing in miRNA-target binding is well supported by experimental data. Large scale transcriptomics and proteomics studies have recovered gene sets that are enriched in seed matches To assess the biological relevance of the identified interactions, we tested for enrichment of miRNA targets involved in a specific process or pathway. The assumption that some miRNAs function as master regulators by downregulating a dense network of genes in the same pathway is supported by several experimental works In a subsequent step, we reconstructed the target sets associated to each miRNA taking into account both antagonism expression pattern and miRNA seed occurrence. Combining these two independent criteria, based respectively on expression profile analysis and DNA sequence information, we expect to increase the reliability of predicted direct targets and to reduce false positive predictions. We scored GO enrichment within the newly defined target sets and report statistically significant results in Multiple studies report systematic correlation between expression level of miRNAs and their target genes. Correlation of expression profiles is helpfully used to assist identification of miRNA-mRNA regulatory relationships Pair-wise correlation coefficients have been computed for the same dataset of 267 miRNAs and Since antagonism pattern is expected to particularly highlight miRNA degradation effect on the target mRNA, we compared the reconstructed network based on antagonism pattern to correlation-based network, only considering the negatively correlated component. In order to obtain two networks of comparable size, we increased correlation p-value threshold up to GO enrichment analysis has been repeated for correlation-based target sets, to evaluate the hypothesis that the two compared methods reveal complementary target sets consistently associated to common biological processes. The results of GO analysis for correlation-based targets are reported in We completed the comparison between antagonism-pattern and correlation-based miRNA-mRNA association, analyzing the seed enrichment for miRNA targets identified by correlation. The histogram in In this study we propose a novel statistical measure to integrate large-scale expression data of miRNAs and protein-coding mRNAs, in order to infer miRNA-target interactions. The basic assumption is that miRNA-target expression profile can create a recognizable non-linear pattern corresponding to the statistically under-represented event of simultaneous miRNA and target mRNA elevated expression. This triangular-shaped pattern, that we defined as antagonism pattern, points out an alternative and complementary relationship between miRNA and target mRNA expression with respect to linear regression. The measure of antagonism can be considered a particularly suitable method to elucidate the role of miRNA regulation in the context of tumor diseases, where samples can present a high level of genomic heterogeneity and cancer specific changes in miRNA expression. Here we demonstrated the effectiveness of this statistical measure as a good indicator of real miRNA-target biological interaction. A dedicated computational procedure for antagonism pattern detection has been developed and tested using realistic synthetic datasets. The performance of the provided procedure has been assessed, taking into account the variable sample size as well as noise level in expression measures. Once the conditions to successfully evaluate the antagonism pattern have been determined, we tested the performance of the proposed measure on a catalogue of experimentally validated miRNA-target interactions and we compared it to the linear anti-correlation measure. The percentage of real interactions detected by the antagonism pattern is slightly higher than linear anti-correlation, supporting the effectiveness of this novel measure. Furthermore, we observed that the two patterns are likely to identify quite distinct sets of targets. MiRNA-target networks can be viewed as composed of interactions reflecting two alternative patterns in their expression profiles, depending on the influence of additional factors. An interesting work direction can be to give a mathematical description of these two observed patterns showing that the linear relationship between miRNA-target expression can change towards a triangle-shaped function depending on the strength of additional influence factors. Another direction to explore is how to combine the antagonism pattern detection with the linear correlation. An option would be to extend the antagonism pattern detection procedure including the measure of the opposite “agonist” pattern with a new parameter. The distance between the two parameters (antagonist versus “agonist” pattern) could provide a measure of the linear dependence.We used the antagonism pattern detection to analyze miRNA and mRNA expression profiles from Ewing’s sarcoma patient samples. Through the antagonism pattern detection, the global miRNA-gene regulatory network has been inferred. Connectivity properties of this reconstructed network allow us to specify the co-regulating nodes and the set of affected targets of each miRNA. To verify that the antagonism pattern captures information about miRNA-gene reliable interactions, two validation analysis have been carried out. In the first one, we verified that miRNA targets identified by antagonism pattern are enriched for binding sites with extensive 5′ miRNA seed pairing in their 3′UTR. Results show highly significant seed enrichment compared to the randomly permuted miRNA-target dataset. This implies that the antagonism-based predicted pairs are consistently enriched for potential post-transcriptional regulatory motifs. A second independent validation has been performed by analyzing significantly enriched GO categories for the target sets associated to each miRNA. Results allow us to determine important biological functions potentially mediated by miRNA regulation in Ewing’s sarcoma.hsa-miR-1 and hsa-miR-206. GO enriched terms for these target sets are related to sarcomere, myofibril and contractil fiber part. Consistently, hsa-miR-1 and hsa-miR-206, together with hsa-miR-133  are known to be muscle specific expressed, to regulate sarcomere organization and to contribute to rhabdomyosarcoma development hsa-miR-1 preferentially downregulate nonmuscle genes in HeLa cells. The observation of muscle-related gene enrichment in hsa-miR-1 and hsa-miR-133 targets can be explained by a second mode of target regulation in which some muscle genes primarily regulated at the transcriptional level may be tuned by functional miRNA target sites hsa-miR-1 and hsa-miR-133 actively shape gene expression patterns in muscle tissue regulating sarcomeric actin organization hsa-miR-1 and hsa-miR-133 targets.We observed over-representation of GO terms describing several fundamental processes involving miRNA regulation. The highest enrichment was obtained for the target sets associated to hsa-miR-29b, hsa-miR-127 and hsa-miR-369-3p) were linked to targets involved in cell cycle and mitotic spindle organization. The involvement of miRNA-29b in different human cancers is reported in multiple studies NF-kappaB-YY1-miR-29, whose disruption contributes to rhabdomyosarcoma, suggests that miR-29 acts as a tumor suppressor. The miR-127 also exhibits tumor suppressor activity by targeting BCL6 proto-oncogene and it is silenced in various cancer cell lines hsa-miR-369 involvement in cancer is not yet documented, while it is known to play a peculiar role in translational efficiency on cell cycle exit under growth arrest condition hsa-miR-127 and hsa-miR-369 are expressed as part of the same miRNA cluster in the human Dlk1/Gtl2 domain at chromosome 14q32. This domain is expressed in a large non-coding transcriptional unit which is altered in different tumor diseases and in aggressive ovarian cancer hsa-miR-379 and hsa-miR-410). Their targets are associated to extracellular-matrix (ECM) and collagen. Matrix remodeling and collagen protein accumulation are important mechanisms during bone tissue formation from mesenchymal cells. Numerous gene products involved in the cytoskeleton and the ECM contribute to describe mesenchymal stem cell features of Ewing tumors hsa-miR-148b, hsa-miR-30d, hsa-miR-324-3p, hsa-miR-379, hsa-miR-410. The role of miR-30 in matrix remodeling is already confirmed by experimental evidences and one predicted target gene (CTGF) has been validated as direct target of this miRNA Besides muscle specific terms, GO annotations strictly related to basal mechanisms of cancer were identified. Three miRNAs . This effect can be partially explained by miRNA-mRNA genomic co-localization as well as by intronic miRNAs showing correlated expression pattern with their host genes The antagonism based network has been compared only with the Pearson correlation network. There are methods that use non-parametric correlation or mutual information for assessing non-linear dependences in expression data. The major problem in mutual information analysis of biological data is the reliable estimation of entropy-like quantities from small datasets. Mutual information is difficult to estimate accurately with limited amount of noisy samples. The impact of the entropy estimation on the quality of the inferred transcriptional networks has been recently studied taking a minimum of 50 required samples In summary, the antagonism pattern detection can be successfully used to integrate large-scale expression data of miRNA and protein-coding mRNAs, in order to infer crucial miRNA regulators and miRNA-target interactions. The antagonism pattern can identify alternative relationship between miRNA and target mRNA expression not properly detectable by correlation analysis. Indeed, it can be considered a valid statistical approach to discover new insights in the miRNA regulatory mechanisms.Ethics approval was provided by the locally appointed ethics committee from the Institut Curie, Paris, France. All participants involved in the present study provided written informed consent.Total RNAs issued of 40 Ewing tumors were used for mRNA and miRNA microarray analyses. The mRNA data were collected using the Affymetrix GeneChip HG-U133A and Affymetrix GeneChip HG-U133Plus2. We normalized and combined information across different platforms using the standard functions provided by Bioconductor packages. We used the combineAffyBatch function in the matchprobes library to merge data from HG-U133A and HG-U133Plus2 GeneChips and we applied RMA normalization to the merged dataset. The miRNA expression profiling panel was performed by Integragen  using Illumina human-6 V1 BeadChip (based on miRbase release 9.1) and average normalization method have been applied according to the manufacturer’s suggested procedure Before running antagonism pattern detection and correlation analysis, we filtered low abundant miRNAs and transcripts in order to gain more confidence in the results. We also filtered those data with little variation across the 40 samples because the absence of high variation will result in correlations mainly due to noise. Variation across samples has been evaluated by IQR, using the median IQR as cutoff. These criteria left 267 miRNAs and 15651 probe sets for the analysis.The proposed approach requires as input the genome-wide expression profile of miRNAs and mRNAs from the same set of samples. As final result of our analysis we obtain miRNA-mRNA pairs showing statistically significant antagonism pattern, which we consider as potential miRNA-target interactions.The antagonism detection procedure can be described as follows. Once converted miRNA and mRNA expression values to We define the antagonism coefficient The statistical significance of the antagonism coefficient Once The JAVA code implementing the algorithm is available on request from the authors.To generate expression-like datasets exhibiting a triangular-shaped pattern, we created bidimensional vectors To generate randomized miRNA-target pairs, the assignment of miRNAs to their antagonism-based targets has been randomly permuted. This procedure allows us to obtain randomized miRNA-target pairs with the same 3′UTR nucleotide composition and length as the original pairs. MiRNA-target assignments has been permuted by a shuffling ubiased algorithm implemented in PERL, so that every permutation is equally likely. Randomized miRNA-target pairs have been generated 1000 times and 8-mer, 7-mer and 6-mer seed counts have been computed at each time. We tested the significance of the 8-mer, 7-mer, and 6-mer counts obtained for real data using as null model a normal distribution with mean and variance obtained in randomized count data.The expression profiles of 267 miRNAs have been correlated with changes in mRNA expression for all 15651 transcripts according to Pearson coefficient. When dealing with a limited group of 40 patient derived samples, the presence of outliers could be a crucial problem affecting the correlation analysis. We applied a leave-one-out strategy to identify frequent non-consistent measures in transcripts and miRNA expression data. Outliers are recognized as measures that dramatically change the correlation estimation. The correlation coefficient between the miRNA The reconstructed antagonism-based network has been characterized by the connectivity distribution of genes and miRNAs, in order to identify miRNA hubs (miRNAs linked to a high number of genes in the networks) and target hubs (genes linked to a high number of miRNAs). Using a thresholding and unweighted procedure to define the connectivity, we sum the number of statistically significant links assigned to each node Figure S1False discovery rate (FDR) as a function of the Antagonism p-value obtained in Ewing’s sarcoma study.(PDF)Click here for additional data file.Figure S2GO enrichment p-value distribution for randomized target sets obtained by shuffling real miRNA-target relationships. The dotted vertical line is drawn at the p-value threshold used for GO analysis in real data.(PDF)Click here for additional data file.Table S1Public datasets used for the test on experimentally validated miRNA targets.(PDF)Click here for additional data file.Table S2Detailed results obtained for the test on experimentally validated targets.(PDF)Click here for additional data file.Table S3(A) List of miRNA hubs in the antagonism based network of Ewing’s sarcoma. (B) List of target hubs in the antagonism based network of Ewing’s sarcoma.(PDF)Click here for additional data file.Table S4P-values obtained for the seed enrichment analysis of the antagonism based network.(PDF)Click here for additional data file.Table S5(A) GO analysis results for antagonism based target sets. (B) GO analysis results for target sets based on both antagonism and miRNA seed occurrence. (C) GO analysis results for correlation based target sets. (D) GO analysis results for target sets based on both correlation and miRNA seed occurrence.(PDF)Click here for additional data file.Table S6Comprehensive table with results of antagonism detection and seed enrichment for all genes and miRNAs included in the Ewing’s sarcoma study.(TXT.GZ)Click here for additional data file.Table S7List of miRNA hubs in common between the antagonism-based network and the correlation-based network.(PDF)Click here for additional data file.


Heat shock proteins (HSPs) have been known for decades for their ability to protect cells under stressful conditions. In the 1980s a new role was ascribed for several HSPs given their ability to elicit specific immune responses in the setting of cancer and infectious disease. These immune responses have primarily been harnessed for the immunotherapy of cancer in the clinical setting. However, because of the ability of HSPs to prime diverse immune responses, they have also been used for modulation of immune responses during autoimmunity. The apparent dichotomy of immune responses elicited by HSPs is discussed here on a molecular and cellular level. The potential clinical application of HSP-mediated immune responses for therapy of autoimmune diseases is reviewed. Expression of HSPs is generally upregulated in cells in response to a variety of stressful conditions including nonphysiological temperature, nutrient deprivation, and hypoxia . It is tThe classical and current view of antigen processing and presentation by MHC I can be summed up as follows: production of peptides occurs within the cytosol by the large multi-subunit catalytic body called the proteasome. The proteasome ingests polypeptides and trims them down to small peptides usually with the correct C-termini, but extended N-termini, for MHC I binding. The transporter associated with antigen processing (TAP) pumps peptides into the ER in an ATP-dependent manner. Additional N-terminal trimming proteases such as the ER-associated aminopeptidase (ERAP) are present in the ER for final processing . For a pThe initial event in priming T-cell responses to cancer or infectious disease involves the transfer of antigens from the cells that harbor them to antigen presenting cells. This pathway is called cross-presentation and allows for the presentation of antigens in the form of peptides to T cells in the context of MHC molecules. Cross-presentation of antigens by APCs also directs that the antigen is presented in the context of costimulation, which is a combination of cytokines and a series of APC-T cell interactions through receptors and their corresponding ligands. The specific costimulation received by the naïve T cell dictates the type of T cell response that is primed. HSPs have been shown to play a critical role both in cross-presentation of antigens and in provision of a dynamic set of signals for costimulation. in vivo, it has been modeled in vitro in several antigenic systems in mice and humans over a period of many years  initiated by CD91 upon HSP stimulation involves the activation of NF-κB and p38 MAPK although other molecules are yet to be identified. Downstream of intracellular signaling, a number of cytokines are released by HSP-stimulated APCs including TNF-α, IL-1β, IL-6, IL-12, and GM-CSF [HSP-chaperoned peptides can be cross-presented by professional APCs; however, presentation and recognition of antigen alone by T cells are not sufficient to prime adaptive immunity. In order to prime T and/or B cells, help is needed from an expanding family of costimulatory molecules on APCs. The cytokine milieu provides additional signals for activation and expansion of these effector cells. The immunogenic HSPs were the first endogenous molecules proven to be particularly adept in stimulating APCs to provide costimulation . Studiesd GM-CSF . Other sd GM-CSF . The comCD91 thus has a role in signal 1 (cross-presentation) and 2 (costimulation) that is provided by the APC to T cells in response to extracellular HSP. Similar to HSP-mediated cross-presentation of peptides, other receptors besides CD91 have been suggested to be signaling HSP receptors. However there is abundant published literature that the suggested TLR2/4 receptors were implicated because of the use of endotoxin-contaminated HSP preparations, especially from recombinant sources. A discussion of HSP receptors has been published elsewhere . The fleβ, IL-6, and TNF-α among others  to detect the presence of abundant intracellular molecules (HSPs). We draw many similarities between the innate immune responses elicited by PAMPs through PRRs and those by HSPs through CD91 in terms of costimulation for T-cell priming. Indeed the HSP-CD91 network has been well documented not only in mammals but also in amphibians. While CD91 is a well-studied receptor for HSPs , there m


Development of the vasculature is a complex, dynamic process orchestrated by a balance of pro and anti-angiogenic signaling pathways. The same signaling pathways are mis-regulated and exploited during pathological angiogenesis in cancer, inflammation and cardiovascular diseases and contribute to disease progression. In the last decade, small non-coding RNA molecules termed microRNAs (miRs) have emerged as key regulators of several cellular processes including angiogenesis. It is becoming clear that miRs function in complex networks and regulate gene expression both at the mRNA and protein levels thereby altering cellular signaling responses to specific stimuli. In the vasculature, miRs can function either in a pro-angiogenic manner and potentiate angiogenesis or act as anti-angiogenic miRs by enhancing cell death and decreasing endothelial proliferation. This review aims to provide an update on how microRNAs regulate gene expression and illustrate miR function in the vasculature with a discussion of potential applications of miRs as anti-angiogenic therapeutics. Vascular endothelium is a highly complex organ with a growing list of sophisticated functions. Endothelial cells have been implicated in diverse functions ranging from release of cytokines during influenza infections , maintenMicroRNAs (miRs) are small 20-24 nucleotide long RNAs that regulate gene expression by binding to mRNAs. miRs represent about 1-2% of the genes in several species including mammals . CurrentWhen miRs were initially described, it was proposed that miRs bind to target mRNAs using a seed sequence consisting of 6-8 continuous bases and a few more complementary bases along the miR sequence. In plants, miRs have almost perfect complementarity with the target mRNA and these targets undergo direct RNAse mediated cleavage . HoweverIt has been postulated that miRs maintain robustness of gene expression by insulating biological systems against noise . For exaThe importance of miRs in vascular development was apparent based on loss-of-function studies of Dicer. Dicer hypomorphic mice had severe vascular deformation in both the embryo and the yolk sac and died at E12.5-E-14.5 . Similarin vivo was miR-126. miR-126 is the most abundant microRNA in endothelial cells. Loss-of-function studies in mice and Zebrafish highlighted that miR-126 is important for developmental and pathological angiogenesis and implicated Sprouty Related EVH Domain containing protein 1(SPRED1), a negative regulator of Vascular Endothelial Growth Factor (VEGF) signaling, as a critical target of miR-126 [in vitro and in vivo[The first study to report a putative role for specific microRNAs in endothelial cells was performed by Poliseno et al. The authors used microarray based profiling and identified the most abundant miRs in HUVECs . They re miR-126 . Interes miR-126 . Png andd in vivo. They shWhile the majority of miRs that have been reported function as pro-angiogenic miRs, miR-17 ~ 92 cluster, a well-characterized group of miRs plays a critical role as a negative regulator of angiogenesis ,31. BonaThe miRs highlighted here are examples to illustrate the diverse ability of miRs in manipulating endothelial function. We and others have reviewed the many different miRs that play a role in vascular function ,33. Someet al reported that miR-132 is upregulated with similar kinetics and plays a key role in lymphatic endothelial cells during viral infections [in vivo silencing of miR-132 by the provision of anti-miR-132 nanoparticles to HSV-infected mice led to reduced corneal neovascularization and diminished lesions. Taken together, these observations indicate that miR-132 may play an important role in pathological neovascularization downstream of multiple triggers including tumor-derived growth factors, viral infections and inflammation.We identified miR-132 as an angiogenic miR that functions by regulating p120RasGAP and Ras signaling pathways using a human embryonic stem cell vasculogenesis model and primary endothelial cells . We demofections . More refections . The autSubsequent to our work, other groups have reported the function of miR-132 in perivascular cells. Natarajan and colleagues showed time- and dose-dependent up-regulation of miR-132/212 by Ang II through the Ang II Type 1 receptor . They idAnother recent study  showed tThe vasculature is exposed to several stimuli and stressors during developmental and pathological angiogenesis that trigger distinct miR expression profiles. For instance, miR-210 and miR-424 have been shown to be upregulated in response to hypoxia ,41. We hWhile initial reports of miR function focused on one or two specific targets of miRs, it is becoming clear that the biology of miR-target interaction is much more complicated than simple RNA-RNA binding that is the basis of target prediction algorithms. Recent evidence suggests that evolution of conserved miR/target networks can exert powerful influence on biological functions . Given tFor example, it is well established that VEGF and Notch signaling are key regulators of developmental angiogenesis. Mapping of a few receptors, ligands and transcription factors on this pathway using STRING database  shows hoAlthough miRs were discovered only about a decade ago, they have rapidly progressed to the verge of clinical utility. In fact, there are currently 124 clinical trials listed on microRNAs with 72 of them focused on cancer patients . Many ofAlthough these are exciting times for discovery of miRs and their applications, there are certain limitations to therapeutic targeting of miRs in the context of complex pathologies such as cancer.1) Targeting anti-miR therapeutics to specific tissues. Naked injection of anti-miRs or cholesterol conjugated antagomiRs typically results in excellent delivery to the liver. However, targeting specific anti-miRs to tissues such as tumor vasculature or sites of ischemic injury is still challenging. Nanoparticles and other delivery vehicles with targeting peptides may be useful in delivering anti-miRs for specific applications .in vivo[2) The delivery of double stranded miR/miR mimics to tumor cells is still challenging and faces similar roadblocks as siRNA therapies. Recent work that shows that a single guide strand of RNA is sufficient to mediate RNA interference is exciting. This could lead to more efficient delivery of miRs to tumor cells in vivo,58.3) The mechanism of action of miRs/anti-miRs is complicated by the number of potential targets that could be affected by a miR. There are discrepancies between prediction algorithms and experimental observations, not to mention cell type specific effects and differences in targets between human and mouse. Therefore, global analysis of mRNA and protein levels in cells/tissues that are targeted by the miR therapeutics may be warranted to understand the mechanism(s) of action.Science-Breakthrough of the year 2006’ and the 2010 Lasker award [Vegfa in adult mouse retinal pigmented epithelial (RPE) cells leads to vision loss and ablation of the choriocapillaris, the major blood supply for the outer retina and photoreceptor cells [miR inhibitors can be effective as simple oligonucleotides in ocular disease without the above mentioned requirements for specific delivery systems. Anti-angiogenic therapy, most notably, anti-VEGF antibody has shown significant therapeutic benefit in patients with Age-related Macular Degeneration (AMD) and consequently garnered several accolades including ‘er award . There ier award . While tor cells . It appeInterestingly, our work has identified several promising miRs that are downstream of VEGF signaling pathways that can be pursued as alternatives to VEGF inhibitors in the eye. We demonstrated that anti-miR-132 is able to decrease developmental angiogenesis in neonatal retinas without affecting established vasculature . Since tin vitro and in vivo. The therapeutic potential of these molecules as anti-angiogenic agents has been shown in multiple animal models and we may be just a few years away from using them to target aberrant angiogenesis in humans.The emergence of miRs as regulators of gene expression has undoubtedly altered our understanding of how diverse stimuli affect function in the vasculature. miRs have been shown to alter specific signaling pathways that affect proliferation, differentiation, migration and cell survival in endothelial cells and smooth muscle cells. The challenges moving forward would be to integrate the different miRs and their target networks in the stimulus–response cycles of the vasculature and to understand how manipulating one or more miRs can alter cell function The author declares that he has no competing interests.