In the global condition, the practitioner focused on the connection and support from the foot to the rest of the body. Before (baseline) and after each intervention , we measured brain activity during intermittent pushing/releasing with the left leg and during resting state. Independent localizer tasks were used to identify regions of interest (ROI). Brain activity during left-foot pushing did not significantly differ between conditions in sensorimotor areas. Resting state activity  increased from baseline to post-local in medial right motor cortex, and from baseline to post-global in the left supplementary/cingulate motor area. Contrasting post-global to post-local showed higher ReHo in right lateral motor cortex. ROI analyses showed significant increases in ReHo in pushing-related areas from baseline to both post-local and post-global, and this increase tended to be more pronounced post-local. The results of this exploratory study show that a short, non-intrusive sensorimotor intervention can have short-term effects on spontaneous cortical activity in functionally related brain regions. Increased resting state activity in higher-order motor areas supports the hypothesis that the global intervention engages action-related neural processes.We use functional magnetic resonance imaging to investigate short-term neural effects of a brief sensorimotor intervention adapted from the Feldenkrais method, a movement-based learning method. Twenty-one participants  took part in the study. Participants were in a supine position in the scanner with extended legs while an experienced Feldenkrais practitioner used a planar board to touch and apply minimal force to different parts of the sole and toes of their left foot under two experimental conditions. In the  In the global condition, the manipulation explores the connections from the foot to the rest of the body, focusing on the function of the foot for body support. We use functional magnetic resonance imaging (fMRI) to investigate potential short-term effects of these two forms of the artificial floor on neural activity during a functionally related motor task  as well as during resting state. We hypothesized that the local manipulation would lead to increased activity in primary sensorimotor areas representing the stimulated foot, while the global manipulation would engage more widespread and higher-level motor areas.The Feldenkrais method is a movement-based learning method aimed at improving organization of the body in action  while maintaining other systemic functions . One assumption of the Feldenkrais method is that general improvement in movement organization can be achieved by clarifying the functional relation between components that need to be integrated in action. This can be approached, for instance, by exploring the coordination between different body parts, by systematically varying postural and balance constraints, by guiding somatosensory attention to different aspects of a movement , or by mental imagery. Feldenkrais lessons often begin with small-amplitude and slow movements in order to enhance processing of sensory information and facilitate exploration of alternatives to habitual movement and perception patterns. This is in line with Nikolai Bernstein's theoretical work on motor learning: “Certainly, the most sensible and correct training would be organized in a way that combined a minimization of effort with a large variety of well-designed sensations and that created optimal conditions for meaningfully absorbing and memorizing all these sensations” , but the manipulation can also have a more global influence, reaching other body parts via legs and spine, such as the pelvis, trunk, head and shoulders. Whether and how movement is transferred to different body parts, depends on the stimulation  and the neuromuscular state of the body . Importantly, the goal of the practitioner is not to induce substantial movement in the foot or along the body, but to provide the participant's nervous system with sensory information about the connection from the foot to the rest of the body and, ultimately, with a sensation of the foot's function for body support under various orientations and contact points of the support surface. To achieve this, the practitioner adjusts the direction, timing and applied force to the configuration of the participant's foot and other body parts, as well as to the participant's responses to the stimulation.During the artificial floor manipulation  guiding the interactive manipulation. It should be noted that both forms of the artificial floor are typically used in Feldenkrais lessons, not as separate interventions but in combination. The distinction between the local and global manipulation is made in the present study in order to elucidate the role of the practitioner's attention to functional relationships between body parts.Application of the artificial floor in a complete Feldenkrais lesson usually proceeds from a more The artificial floor manipulation reverses the usual relation between body and support surface: it is not the feet that “look for support” from the floor, but a “support surface”—a planar board moved by the practitioner's hands—that approaches and makes physical contact with the feet. Feldenkrais assumed that this allows the participant to experience the use of the foot for body support under various conditions of contact area and orientation, thereby eliciting a learning process that may influence subsequent use of the foot in standing and walking. Two major, non-trivial assumptions are that this is possible even though the participant is lying supine, without need for active postural stabilization, and despite the fact that the forces applied to the foot during the manipulation are much smaller than forces during bipedal standing or walking.Tactile perception and proprioception from foot and ankle play a crucial role in stabilization of upright posture  shares an essential feature with the use of the foot for body support, as it aims to generate sensory information emphasizing the connection from the foot to other body parts. As a consequence of bi-directional action-perception links, we expected the global manipulation to elicit a corresponding neural action pattern (of using the foot to support the body) in the participant's nervous system.Close links between action and perception have been postulated early in the history of experimental psychology Lotze,  and are Resting state fMRI offers the possibility to study spontaneous brain activity in the absence of an instructed task. It may therefore be especially appropriate to investigate potential changes in neural dynamics induced by behavioral or neural interventions , such effects would have to be due to changes in sensorimotor organization at the level of the central nervous system. The aim of the present exploratory study is to investigate these hypothesized neural changes in terms of brain activity in a functionally related foot-pushing-task as well as during resting state. In addition, we hypothesized that a subtle variation in the application of the artificial floor affects the way in which the manipulation engages the participant's nervous system and thereby influence subsequent neural activity during related motor tasks or during resting state. More specifically, we predicted that the local, global) described above, we use fMRI to measure brain activity during a motor task mimicking the use of the foot for body support in a supine position  as well as resting state activity before and after each intervention (with the feet standing on the same support surface). The intervention was carried out by an expert Feldenkrais practitioner with more than 30 years of professional experience . In order to minimize perturbation due to repositioning, the intervention and all tasks were carried out while participants were lying in the MR scanner. An independent localizer task before the experiment was used to determine regions of interest (ROIs) activated during the foot-pushing task. Based on the above reasoning, we predicted that application of the local artificial floor would lead to an increased activity in regions (both during pushing and resting state) in sensorimotor areas representing the stimulated foot. For the global artificial floor intervention, we predicted more widespread activity including higher-level motor areas, reflecting a more function/action-related neural processing.To assess potential changes in neural organization following the two forms of the artificial floor manipulation  took part in the study after written informed consent and approval of the Ethics committee of the German Psychological Society (DGPs). According to self-report, all participants were right-handed and did not have any history of neurological disorder, chronic pain, or medical conditions impairing movement or balance. Participants were also selected to have previously participated in MR studies, in order to minimize the likelihood of physical or emotional discomfort during the experiment. Prior to the study, participants were informed that the goal of the study was to investigate the link between perception and movement in tasks involving being touched at the foot sole and gently pushing the foot against the floor, respectively. No reference was made to the Feldenkrais method.functional localizer task (6 min), performed at the beginning of the experiment, participants had both knees bent, the feet standing on a solid, horizontal support surface, positioned at the same vertical level as their body. Participants were (previously) instructed to repeatedly push the foot into the support surface and release again, performing this movement gently and with as little effort as possible while sensing any resulting somatosensory sensations along their body. The pushing movement was performed in 20-s blocks, randomized for the left and right foot, with 20-s resting periods between active blocks. Eyes were open during this task. The tasks were cued by visual stimuli, with a black screen denoting rest period and a green screen with centrally presented letter L (R) denoting pushing with the left (right) foot.A highly experienced and internationally recognized practitioner and teacher of the Feldenkrais method  instructed participants for the active movement tasks, repositioned participants' legs between conditions, and carried out the intervention  in the scanner. Table baseline resting-state period (3 min), during which participants had both knees bent, with the feet standing on the support surface, and with the instruction to close their eyes and rest.Subsequently, participants performed a push/release task  with only their left foot standing on the support surface . Participants were instructed to keep their eyes closed during this task in order to enhance somatosensory attention to the body  was followed by the pushing task with the left foot (4 min) and the resting state task (3 min) described above. The order of intervention conditions  was counterbalanced across participants. The Feldenkrais practitioner was only informed about the order of the conditions  before the first intervention with each participant, such that her initial interaction with the participant was not influenced by this information.During the artificial floor intervention , any discomfort during the experiment, and any potential previous experience with the Feldenkrais method.www.adni-info.org) . Functional images, both for the functional localizers and resting state analysis, were collected using a T2*-weighted echo planar imaging (EPI) sequence sensitive to blood oxygen level dependent (BOLD) contrast .Images were collected on a 3T Magnetom Trio MRI scanner system  using a 32-channel radiofrequency head coil. The structural images were obtained using a three-dimensional T1-weighted magnetisation prepared gradient-echo sequence (MPRAGE) based on the ADNI protocol . For the functional analysis (pushing task), the first four volumes of all EPI series were excluded from the analysis to allow the magnetisation to approach a dynamic equilibrium. Data processing started with slice time correction and realignment of the EPI datasets. A mean image for all EPI volumes was created, to which individual volumes were spatially realigned by means of rigid body transformations. The structural image was co-registered with the mean image of the EPI series. Then the structural image was normalized to the Montreal Neurological Institute (MNI) template (resampling voxel size of 3 × 3 × 3 mm), and the normalization parameters were applied to the EPI images to ensure an anatomically informed normalization. Participants showing head motion above 3.5 mm of maximal translation  and 2.0° of maximal rotation during scanning would have been excluded (this was not the case for any participant). A spatial filter of 8 mm full-width at half maximum (FWHM) was used. Low-frequency drifts in the time domain were removed by modeling the time series for each voxel by a set of discrete cosine functions to which a cut-off of 128 s was applied.For the resting state analysis, the first five volumes were discarded to allow the magnetisation to approach a dynamic equilibrium, and for the subjects to get used to the scanner noise. Part of the data pre-processing, including slice timing, head motion correction  and spatial normalization to the MNI template, were conducted using the Data Processing Assistant for Resting State fMRI toolbox  was convolved with a hemodynamic response function and head movement parameters were included in the design matrix. We were interested in the contrast comparing left and right pushing  as well as left pushing compared to rest . Resting state activity was analyzed in terms of regional homogeneity. Based on the fact that fMRI activity is typically spatially clustered .Brain activity during the pushing tasks was analyzed at the first (within-subject) level using regressors for T-tests at the second (between-subject) level. For the functional localizer task, the resulting SPMs  were thresholded at p < 0.05 with familiy-wise error correction (FWE). For the pairwise comparisons of resting state activity (ReHo) and functional activations in the pushing task before and after the intervention, the resulting statistical maps  were thresholded at p < 0.005 (uncorrected) with an additional cluster size threshold of k = 42. The required cluster size was determined by Monte Carlo simulation . Mean ReHo values in the three conditions  in these ROIs were extracted using MarsBaR toolbox  were defined based on brain activation during the functional localizer task at the beginning  and differences in ReHo between conditions are shown in Figures left-push to rest  as well as more lateral clusters . Comparing left-push to right-push .Push-related activity in the functional localizer prior to the experiment was analyzed in two ways. Comparing t Figure , we founh Figure  revealedleft-push > rest; Figure  shows similar activation to the functional localizer at the beginning of the experiment, peaking in right medial motor cortex . Comparison of push activation (left-push > rest) for each pair of time points showed a significant cluster in the occipital lobe for post-local > post-global  and in the left temporal lobe for baseline > post-local . A ROI-based analysis  did not reveal any significant differences of the activation during pushing between the three time points .Aggregating pushing-related activations  as well as additional clusters in temporal cortices . The ROI-based analysis showed that ReHo increased in the ROI defined by the functional localizer prior to the experiment  as well as the activation in the push-trials during the experiment  and marginally significant in the ROI defined by the pushing-related activation across the three time points during the experiment . Decreases in ReHo from baseline to post-global were found in lateral sensorimotor areas .ReHo increased from l Figure  in left post-global and post-local  and one additional cluster in right temporal cortex . The difference was marginally significant in the ROIs defined by the functional localizer  and significant in the ROI defined by the push-activation across the three time points during the experiment . The opposite contrast  did not show any significant differences in the whole-brain or ROI-based analyses.Comparing ReHo between l Figure  showed aN = 21 above) were excluded from all analyses.None of the participants reported prior experience with the Feldenkrais method. One participant experienced discomfort while lying in the scanner. Data from this participant . Moreover, several participants reported differences between the left and the right leg during bipedal upright standing (outside the scanner) after the experiment. For instance, participants described the left foot and leg as feeling “lighter but in clearer contact with the floor,” having a “wider contact area,” feeling “more relaxed” or “more stable, better able to keep balance,” compared to the right side.About half of the participants reported changes in subjective experience during performance of the pushing movement after the artificial floor intervention. Among other things, participants described that their “foot felt more relaxed, resulting in more pushing with the ball of the foot rather than just the heel,” that “the pushing felt easier,” that “it was easier to control the movement,” that “the contact area of the foot on the support surface was larger,” or that they “generally felt more relaxed” after the intervention.  than the local intervention (mobility within the foot). We did not observe reliable changes in pushing-related activity in sensorimotor brain regions from pre- to post-intervention. However, resting state activity , changed in primary and higher-order motor regions in distinct ways for the two forms of the intervention.We set out to study potential changes in neural activity during a gentle foot-pushing task (related to body support) and resting state, induced by two forms of the artificial floor intervention: the Brain activity while gently pushing the foot onto a horizontal support surface (and releasing the push) was measured prior to and during the experiment, as an active motor task hypothesized to be functionally related to the artificial floor intervention. Brain activity was comparable for left-foot pushing across these conditions, including broad areas of primary sensorimotor cortices and higher-level motor areas. Contrasting left- to right-push showed significant differences in right medial primary motor cortex, consistent with somatotopic representation of the foot  cannot be portrayed as mere after-effects of different ways of active pushing, but instead as consequences of the artificial floor intervention.baseline (pre-intervention) to post-local and post-global differed in spatial location. The local intervention induced an increased ReHo in right medial primary motor cortex, consistent with somatotopic foot representations found in previous studies  but in a more anterior part of motor cortex  that has been related to higher-level aspects of motor control  differential effect of two ways of performing the artificial floor intervention: local and global intervention. We can therefore not rule out the possibility that observed differences in ReHo may be explained by systematic differences in low-level parameters of the intervention. Also, the push-forces exerted by the foot on the floor in the active conditions, could not be recorded in the scanner, and we did not assess potential changes in movement patterns after the intervention outside the scanner. Thus, further research is needed to characterize the artificial floor intervention in terms of the physical interaction between board and participant's foot as well as to assess potential effects of the intervention at the motor-behavioral level.Unfortunately, our experimental setup did not allow for measurements of the movement of the board, the contact forces and area between board and foot, or the resulting  movement in the participant's body. In particular, we are unable to report potential differences in these parameters between the p < 0.005, uncorrected), probability of type-I error was controlled for by requiring an appropriate minimal cluster size determined by Monte Carlo simulation  and are consistent with anecdotal reports of experienced practitioners about frequently observed effects of the artificial floor intervention. Typical Feldenkrais sessions last longer  than the intervention used here (2 × 3 min) and involve more body parts and functional relationships between them. For instance, a Feldenkrais session involving the artificial floor manipulation would generally connect the support/push function of the foot more explicitly to the rest of the body, for instance by asking participants to perform related active movements by themselves  or by providing additional manual touch and guidance at other parts of the body. Further, studies are needed to investigate potential effects of more complete Feldenkrais interventions on the nervous system as well as subsequent motor behavior.local) or functional use of the foot for body support , differentially affected subsequent resting state activity. Increased resting state activity in higher-order motor areas supports the hypothesis that the global intervention engages action-related neural processes.The results of this exploratory study show that a short, non-intrusive sensorimotor intervention based on the Feldenkrais method can have effects on spontaneous cortical activity in functionally related regions. Moreover, two variants of performing the artificial floor manipulation, focusing on either foot mobility (One of the authors (EA) is a Feldenkrais practitioner and teacher, directing professional training programmes in the Feldenkrais method. One of the authors (JV) took part in one such training but is not working as a practitioner. The other authors are not associated in any way with the Feldenkrais method. The current study does not address the effectiveness as such but investigated the short-term influence of a brief sensorimotor manipulation motivated by the Feldenkrais method. We therefore do not see any actual conflict of interest.


Transposable elements (TEs) comprise approximately half of the human genome, and several independent lines of investigation have demonstrated their role in rewiring gene expression during development, evolution, and oncogenesis. The identification of their regulatory effects has largely been idiosyncratic, by linking activity with isolated genes. Their distribution throughout the genome raises critical questions—do these elements contribute to broad tissue- and lineage-specific regulation? If so, in what manner, as enhancers, promoters, RNAs? Here, we devise a novel approach to systematically dissect the genome-wide consequences of TE insertion on gene expression, and test the hypothesis that classes of endogenous retrovirus long terminal repeats (LTRs) exert tissue-specific regulation of adjacent genes. Using correlation of expression patterns across 18 tissue types, we reveal the tissue-specific uncoupling of gene expression due to 62 different LTR classes. These patterns are specific to the retroviral insertion, as the same genes in species without the LTRs do not exhibit the same effect. Although the LTRs can be transcribed themselves, the most highly transcribed TEs do not have the largest effects on adjacent regulation of coding genes, suggesting they function predominantly as enhancers. Moreover, the tissue-specific patterns of gene expression that are detected by our method arise from a limited number of genes, rather than as a general consequence of LTR integration. These findings identify basic principles of co-opting LTRs for genome evolution, and support the utility of our method for the analysis of TE, or other specific gene sets, in relation to the rest of the genome. The acquisition of cell- and tissue-specific patterns of gene expression is central to morphological and physiological differentiation during development and evolution. The gene regulatory basis of phenotypic differences is often addressed by comparing the levels of gene expression between tissues within, or in the case of evolutionary divergence, between, species. Such comparisons appear relatively straightforward when single genes are of primary interest. However, tissue divergence into characteristic phenotypes involves suits of genes, coregulated in their spatiotemporal expression, often through shared flanking regulatory mechanisms. Transposable elements (TEs) have been proposed to play such coordinating role in development and evolution; yet detecting their tissue-specific regulatory signatures has been difficult. In contrast, obtaining profiles of tissue- or taxon-specific gene expression is relatively undemanding given modern sequencing technologies. Here, we present a straightforward approach to screen for tissue-specific signatures of TEs using transcriptomic data.TEs have entered the genome in past viral invasions and comprise 50% or more of the human genome . AlthougTEs often manifest lineage- and tissue specificity of effects. The potential for tissue-specific or developmental stage-specific effects is prominently revealed in cancers, where particular TEs are often found to be highly expressed . Yet conAlthough transcriptomes are readily mined for the transcripts initiating in, or including TE sequences , the tisThe approach used in this article was developed to identify tissue-specific signatures of gene subsets at the transcriptome level. We focused on gene subsets with a particular LTR within 10 kb upstream of the transcriptional start site, in the same orientation of the linked gene transcripts, and considered whether the expression of these LTR-associated genes is potentially affected by the presence of the particular LTR element. In the event of genome-wide effects of the element on the transcriptional regulation in a particular tissue, we would expect that the expression pattern of LTR-associated genes in that tissue differs from expression pattern of other genes in the genome (detail below).The transcriptome data used in this study were mapped by In order to arrive at meaningful conclusions from comparing gene expression across samples, it is desirable that the variance in gene expression is uniform across all levels of expression, rather than being a function of the mean expression level. To this end, data are usually transformed, and the appropriate transformation depends on the type of measurement  or lowly (negative correlation) expressed in tissue B. Lack of coexpression of a set of genes between tissue A and all other tissues implies tissue specificity of gene regulation.A). The tissue expression levels of each gene  define the tissue vector. The length of each vector can be calculated as iG is the standardized measurement, like FPKM or transcripts per million [TPM] score), the length of each transcriptome vector reduces to A).The gene expression profile of a cell type or tissue can be conceptualized as a vector in a high dimensional space spanned by the gene axes A. The ti tissues . It foll tissues A.Fig. 1B).Within this space, capturing a subset of genes, which differ between the transcriptomes, entails identifying a subspace of genes, in which tissue vectors are particularly dissimilar. In this study, the specific subspace was chosen a priori as the set of genes potentially regulated by an LTR. We set out to test whether this subset of genes manifests lowered similarity of expression among tissues, relative to other genes B.supplementary files S1–S4, Supplementary Material online, show the heat plots in which each square represents the odds ratio to observe the value for LTR+/LTR− as low or lower as the one observed. The odds ratio was calculated based on the null distribution of the statistics for each pair of tissues compared. Null distribution was generated by randomly resampling 5,000 times the set of genes corresponding to the number of LTR-associated genes and calculating the statistics . The observed value was compared with this null distribution to determine the significance of the observation. We have chosen to present the heat map with odds ratios rather than the values of the statistics itself, because the distribution of the statistics is specific to each square of the matrix, and hence the same value of the statistic may imply a significant observation in one, and a very common observation in another pair of tissue. The visual effect can in this case be misleading. The code for the described calculation is written in R (ver. 3.0.1) and is available from the authors.To capture this potential effect of LTRs, we compared the correlation between tissues calculated from the LTR-associated genes (LTR+), and the correlation between tissues calculated from LTR-absent genes (LTR−). The result can be expressed as a ratio between the two similarity measures, LTR+/LTR−, for every pair-wise tissue comparison. This summary statistic allows us to assign the effect of an LTR in each tissue pair a single value, namely the correlation of the subset of genes relative to correlation across all other genes. We use absolute correlation values, because the magnitude, rather than the sign, is of interest. The resulting value ranges from 0  to a potentially undefined number . The latter is not expected to be problematic, as while the similarity between tissues can be small, it is unlikely to be 0. The rationale for this summary statistics is that if there is no detectable effect of an LTR on gene expression pattern in either of the two tissues, the coregulation of LTR-associated genes will not differ significantly from the coregulation of remaining genes in the genome. To determine whether a small number of genes are driving the expression pattern, we examined the effect of single genes on our measurement using jackknife, thereby removing single LTR-associated genes from the set of genes and recalculating the ratio of similarity between the subset and whole genome. The difference between the value based on a reduced set and the value based on a full gene subset is an estimate of the particular gene’s contribution. We recorded genes as contributing candidates if their removal increased the coregulation statistics by more than 5% of the value observed with full subset.The interval of 10 kb upstream of the gene location was chosen arbitrarily in this work. Gene expression is known to be affected by the LTRs located further upstream of the genes. To test to what extent the detected signal is robust to changes in the length of genomic interval considered, we reanalyzed the data using intervals of 20 and 50 kb upstream of the coding genes and included TEs residing in these intervals. Longer interval inevitably results in a larger number of LTR-associated coding genes.In a closer examination of detected signals, we focused on the placental, lung fibroblast, and skeletal muscle myoblast transcriptomes. To examine whether the tissue-specific function of an LTR is also reflected in LTR transcription, we counted the number of reads mapping one or multiple times to the full genomic sequence of a particular LTR, and compared these between the tissue with which the LTR was associated in previous analyses, and tissues with no such association. To this end, we downloaded all genomic sequences for every LTR studied using UCSC table browser, and used Bowtie2  to map rTo determine tissue-specific effects of LTRs, we asked whether a set of genes with transcription start sites within 10 kb downstream of an LTR (LTR-associated genes), manifest a distinct gene expression pattern in a tissue—a pattern that differs from the expression pattern of these genes in other tissues. As a metric, we used the degree of coexpression of LTR-associated genes between tissues, relative to coexpression of LTR-unassociated genes. We refer to this metric as the relative coexpression . The rationale of this approach is that the coexpression of genes represents similarity in their regulation between tissues, and therefore a group of genes with significantly lowered correlation is likely differentially regulated between tissues. We consider an LTR to have a tissue-specific regulatory effect when LTR-associated genes manifest significantly lower coexpression in comparison with multiple other tissues.P). Significance values were generated in nonparametric tests separately for each pair of tissues. Each element of the matrix thus reports the odds of observing the value of the relative coexpression between tissue pairs as low or lower, when a gene subset is randomly selected from the genome. supplementary file S1, Supplementary Material online. We report the significant LTR-tissue associations in We tested 62 LTR elements  in 18 tiEven when LTRs show a tissue-specific signature, it is unlikely that all LTR-associated genes are involved in this signal. We determined the effect of single genes on the relative coexpression using jackknife. To this end, we measured the change in the score upon removing LTR-associated genes one at a time . This test was performed for all strong associations between LTR and tissue where significant effect on coexpression was shown in >50% of pairwise tissue comparisons (minimum 9 of 17). The main drivers of tissue-specific effects are listed in There is no apparent pattern to the distances between the LTR and the genes, within the 10-kb interval. We measured the distance between the 3′-end of the LTR and 5′-end of the adjacent downstream gene, for the elements in which the association was found. The median distances for the different TEs range from 3 to 7 kb. Within the genes significantly associated with the particular element, the distances of single elements mostly manifest the full range, from 0.1 to 10 kb.A very common LTR may be associated with genes driving the expression pattern in a certain tissue by coincidence, especially if only a few genes are responsible. To test whether the effect of LTR-associated genes may be due to the genes themselves irrespective of the presence of the LTR, we repeated the analysis with the orthologous gene sets in mouse. Where possible, we specifically searched for the presence of the same signature in the expression of orthologous mouse genes, when the associated LTR is absent in mice. This test is complicated by the availability of matched transcriptomes. We found in two cases that the genes themselves maintained the same effect, meaning that the effect is likely not driven, even if potentially enhanced, by the presence of LTR in the human genome. This is the case for the effects of MER11-associated genes in ovary and breast, as well as the LTR2-associated genes in liver.We observed moreover that even when LTRs are shared between mouse and human, the coexpression pattern is often not shared. One reason is certainly that between mouse and human, LTRs are rarely colocalized with the same set of genes (not shown). It has been noted previously that recruited LTRs often have a taxon-specific regulation even if they are present in multiple species , respectively, 4.5-fold (for 50 kb) greater number of LTR-associated coding genes. The results are qualitatively congruent with the results from the 10-kb interval. However, the signal frequently weakens with increased length of the interval, implying that the genes contributing to the expression pattern are likely within a short interval of the LTR. In some cases, new associations can be revealed using longer intervals, such as the association of MER11A with placenta, or MLT1C with heart. These are likely due to effects of the additional organ-specific genes that have been included into the analysis when longer intervals are included. As LTR have been associated with long-distance effects, this is not surprising. Overall, the result supports the robustness of the approach. The heat maps corresponding to 20- and 50-kb intervals can be found in We examined in greater detail a subset of LTRs predicted to function in the placenta. A signal for placenta-specific effect on gene expression was detected for five LTRs: MLT1F2, MLT1A, MER39B, MER21A, and MLT1B. Notably, the effects of MER21A and MER39B in placenta have been reported previously (see Discussion).x axis in the histograms). To examine whether putatively functional LTRs—those within 10 kb upstream of the genes—share common sequences, we characterized the sequence conservation by comparing them to the consensus sequence (retrieved from Repbase ). We useWe also examined to what extent the LTR sequences themselves are expressed as an indicator of promoter , and whether the LTR-containing transcript is specific to the tissue with which they are associated. We examined the levels of transcription of the placenta-associated LTRs , as well as several LTRs that were not associated with placenta. We compared these levels among placenta, lung fibroblast, and skeletal muscle myoblast transcriptomes (ENCODE data set). Repeats pose a difficulty for transcriptome alignment, as many mapped reads cannot be uniquely assigned to a single genomic position. Thus it is not clear from the above result whether the reads stem from the overall baseline transcription, or from high transcription at only a small number of elements. We approached this question by focusing on the portion of reads that could be mapped unambiguously to single genomic loci. We found that of these loci, most manifest very low number of mapped reads, however few single repeats are moderately to highly transcribed (>10 FPKM). Although this pattern only considers unique loci, the few highly transcribed uniquely mapped loci account for a high proportion of the total reads that map to the genome: up to 85% of mapped reads in placenta, 51% of mapped reads in lung fibroblast, and 30% of mapped reads in skeletal myoblast, in the case of the elements examined here. It is also these highly expressed loci that account for differences in expression between tissues. The expressed LTR loci are located in various genomic compartments : most arThe approach used in this article was developed to identify tissue-specific signatures of gene subsets at the transcriptome level. We used our computational tool on gene subsets with a particular LTR within 10 kb upstream of the transcriptional start site, in the same orientation of the linked gene transcripts. We considered that the expression of these LTR-associated genes is potentially affected by the presence of the particular LTR element. In the event of genome-wide effects of the element on the transcriptional regulation in a particular tissue, we would expect that the pattern of LTR-associated genes in that tissue differ from the coexpression pattern of other genes in the genome.The method is a straightforward explorative tool, conceived to detect reliable signatures for subsequent experimental studies. Note that the interval of 10 kb was chosen arbitrarily in this study. We know that regulatory regions can be located at a greater distance from the gene. Two important factors influence the reliability of the results, one biological and another technical. First, transcriptomes represent snapshots in the development of cells, tissues, and organs. The effects of LTRs may occur at various times in development and may or may not leave a lasting signature on the expression of associated genes detectable at other developmental stages or physiological conditions. Not finding an effect therefore cannot exclude the role of an LTR in a particular tissue, as it can take place at different developmental times and under different conditions. The second factor affecting the results is technical. The method depends on the combinability of transcriptome data used. Transcriptional data have various sources of technical noise such as sample collection and preparation as well as the sequencing itself. FPKM measurement used here attempts to correct for the sequencing depth by normalizing to the total number of mapped reads. We relied in this study on previously reported FPKM values. However, Two further aspects are important for signature interpretation. First, the correlation may be strongly driven by single genes with high expression. In such cases, the effect may or may not be associated with a functional LTR, as colocalization with a single gene may be coincidental. Such effects will be detected as single gene effects by the jackknife approach discussed above. Second, the degree of specificity revealed depends on the number and choice of tissues included in a study. In general, a greater number of tissues provides more fine-grained information about the uniqueness of a particular transcriptome and hence the specificity of the effects of particular LTRs.This study suggested several cases of tissue-specific roles of LTRs. Using these signatures, we furthermore identified the genes with major contributions to this expression pattern. The relatively low numbers of genes colocalized with any one of the LTR elements preclude meaningful large-scale functional analyses such as gene ontology. To independently validate the results, we therefore examined previously documented functional associations between LTR-associated genes and the tissue for which they appear to be specifically regulated.RCAN in muscle (HSD17B (MER21A), BAAT (MER11A), DHRS2 (LTR12D), APOC1 (LTR2), ST6GALI, and CYP19A1 (MER21A), whose expression has been empirically found to be regulated by the associated LTRs, were also identified in our approach as the main contributors to the detected tissue-specific expression pattern. It is noteworthy that not all genes regulated by an LTR necessarily act in a tissue-specific manner. Because this approach focuses on tissue specificity only partial replication therefore may be expected. We found many novel associations that to our knowledge have no empirical evidence so far and will be of interest in future studies.Indeed, we were able to replicate several of the LTR- gene-tissue associations that have been empirically determined previously  can be contrasted to the remaining genome in a similar way to detect tissue-specific or taxon-specific effect of an upstream regulation among tissues within an organism, or between homologous tissues in a phylogenetic context. Similarly, sets of candidate genes can be tested for changed coregulation across developmental stages, treatments, or pathological conditions.http://genestation.org/Ohio_Collaborative.HumanBodyMap transcriptomes were retrieved from public data bases . The reproductive tissue transcriptomes  are available from GeneStation database at Vanderbilt University: Supplementary files S1–S4 are available at Genome Biology and Evolution online .


This paper puts forward a framework for probabilistic and holistic cost-effectiveness analysis to provide support in selecting the least-cost set of measures to reach a multidimensional environmental objective. Following the principles of ecosystem-based management, the framework includes a flexible methodology for deriving and populating criteria for effectiveness and costs and analyzing complex ecological-economic trade-offs under uncertainty. The framework is applied in the development of the Finnish Programme of Measures (PoM) for reaching the targets of the EU Marine Strategy Framework Directive (MSFD). The numerical results demonstrate that substantial cost savings can be realized from careful consideration of the costs and multiple effects of management measures. If adopted, the proposed PoM would yield improvements in the state of the Baltic Sea, but the overall objective of the MSFD would not be reached by the target year of 2020; for various environmental and administrative reasons, it would take longer for most measures to take full effect. Deterioration of marine environments due to various anthropogenic pressures –3 has beThe implementation of the MSFD calls for adoption of a form of ecosystem-based management (EBM) in which marine protection and delivery of the ecosystem goods and services are taken care of concurrently , 6. ThusEarlier experiences of economic analyses applied in implementing the Water Framework Directive (WFD) show thatA fundamental feature of a holistic cost-effectiveness analysis is the substantial uncertainty on both costs and effect of different measures on closing the gap. A successful CEA would require access to relevant numerical models. Ideally, the analysis would be carried out using an ecosystem-economic model accounting for the 11 descriptors and their interlinkages, but such models do not exist. As the second-best option, cost-effectiveness could be analyzed by individual management measure and by the GES descriptor it relates to. This was the principal approach applied in corresponding WFD analyses . UnfortuA study comparing qualitative and quantitative approaches to CEA for the WFD argues that CEA should meet three core requirements: 1) transparency, 2) pragmatism and 3) usefulness . By tranThe contribution of the present study is to develop a probabilistic approach for cost-effectiveness analysis of marine protection projects that is in line with the EBM. The framework will be demonstrated through its capacity to rank candidate measures and identify cost-effective combinations of measures that can be incorporated into the final Finnish PoM. In light of the iterative nature of the planning of measures and of the Directive, which follows an adaptive management cycle of six years, the framework has been designed to allow for numerical computations with limited data and limited human resources, and to be flexible enough to be amended during subsequent cycles. The framework is general in that it shows the steps needed to execute a theoretically sound CEA and provides a pragmatic application of such an analysis. Thus the framework is applicable in other contexts, such as water quality or fisheries management, biodiversity conservation or marine spatial planning, where decision support tools are needed to analyze complex ecological-economic trade-offs under uncertainty , 22–25.The rest of the paper is organized as follows. The second section describes the step-by-step CEA framework for implementation of the MSFD as well as an application of the framework–including data collection—for Finland. The third section presents the results, which include a ranking of the measures as well as cost-effective combinations of measures. The last section concludes the study and paves the way for further development of CEA methodology.The eighth step is to compute a cost-effective combination of measures that will meet the minimum requirement for GES under an exogenously given budget constraint. The resulting candidate PoM is then analyzed in terms of whether it contributes to closure of the gap in a balanced manner across different GES descriptors and within the budget. Where new measures are identified or the budget constraint changes, steps 4–8 are repeated until a satisfactory PoM is found.In Finland the MSFD has been transposed by updating the Act on Water Resources Management , the lawThe sub-working group whose mandate was to conduct the CEA was established at a rather late stage, when steps 1–5  had alreFrom the very beginning of its work, the socio-economic working group realized that the potentially applicable ecological-economic models available covered only few of the GES descriptors. Moreover, the models available for non-indigenous species (D2), commercial fisheries (D3) and eutrophication (D5) would require substantial modifications if they were to be applied in the MSFD context –32. ThisIn our application, effectiveness is defined in terms of a probability distribution describing the likelihood that a particular measure will bridge given proportions of the gap between the present status and the minimum threshold for reaching GES. Note that GES is not a synonym for undisturbed virgin state of marine ecosystem, but rather includes states that tolerate some pollution and consumptive use. The advantages of this approach are that 1) the probability distribution can be flexibly parameterized using data sets, models, expert judgments or a combination of these; 2) probabilistic analysis allows us to study uncertainties related to the success of the candidate PoMs, an important feature called for in the earlier literature , 33, 34;The particular challenge that MSFD poses for CEA is the large number of GES descriptors. A multidimensional environmental objective such as GES makes data collection a laborious and demanding task. First, it is very likely that several management measures affect more than one descriptor, and any analysis should account for and estimate all these multiple impacts. A second and even more challenging consideration is that measures may have antagonist, additive or synergetic effects on each other . Given tThe proposed definitions of the costs and effects of the measures and the assessment method were tested and further developed after a pilot email questionnaire conducted spring 2014. Based on the survey findings, it was decided that the cost and effect data would be acquired using group interviews following the procedure set out in The effectiveness of a candidate measure was defined using a discrete conditional probability distribution . The exphttp://www.tenk.fi/en/ethical-review-human-sciences/ethical-review#_ftnref1. The study is based on material collected in working groups appointed by the Finnish Ministry of Environment; therefore consent was not explicitly recorded. Workshops did not include any personal questions and the material underlying this study is the outcome of group discussions. The material  was publicly available during the public hearing for the Finnish PoM.According to the guidelines of the Finnish Advisory Board on Research Integrity the nature of this research did not require ethical review. URL: There are several alternative ways to formulate the problem of developing economically justified suggestions for candidate PoMs. For example, the choice of measures can be based on maximizing improvement in the environmental state under some exogenously given budget constraint, or minimizing the aggregate cost under some minimum requirement. Multi-objective optimization maybe used in the case of conflicting objectives. The selected formulation may reflect the aims or aspirations of society, the scarcity of resources, the nature and profusion of the objectives involved or the coverage and quality of data available for analysis.E[U], under a particular budget constraint. Societal utility reflects the improved provision of marine ecosystem services to society and the marginal value of these services. Utility is described here as the ability of the focal PoM to close the gap between the current and the desired state of the marine environment:In a general form, the management problem can be defined as one of selecting the combination of candidate measures that maximizes the expected aggregate welfare or utility, n = 31 measures to closing the gaps in the case of m = 11 GES descriptors are given byfij. The decision variables, xi are binary variables, and they denote whether the ith measure becomes part of the PoM or not. The implicit assumption is that the GES descriptors are separable and that the societal utility derived from improvements in the 11 GES descriptors are additive, meaning that the goals are interchangeable and complementary.The probability distributions describing the contribution of δj, denotes the relative severity of the gap in the case of each GES descriptor, and it may be given as a proportion expressing the value of the current state vis-à-vis the value envisaged by the descriptor. Another scaling factor, γj, denotes the relative weight society gives to meeting the GES descriptors and reflects the marginal value of the ecosystem services provided if the gap is closed.Initially, the gap between the present environmental status and GES might differ from descriptor to descriptor. A scaling factor, cidenotes the probability distribution for the cost of the ith measure and ć denotes the overall budget constraint. In addition, a decision maker may set some minimum requirement The joint effect of measures is maximized subject to a budget constraint:The expected costs and effects of measures imply a risk-neutral decision maker. However, as the costs and effects of measures are expressed as probability distributions, risk aversion and rankings based on stochastic dominance may also be taken into account.31) to be comprehensively evaluated and thus a heuristic was employed to search for the optimal solutions and utility possibility frontiers for various budget constraints. The solutions were derived by first ranking alternative measures based on the cost-to-effect ratios of meeting individual GES descriptors and then including the most promising measures in the focal PoM first. For comparison, the outcomes were computed for a large number of randomly selected combinations of measures, as well as for combinations of randomly selected measures and the best candidate measures.The choice of the optimal combination of measures is a binary optimization problem. Such problems are computationally challenging. There is no way of guaranteeing that the global optimum is reached unless the outcomes of alternative combinations of measures can be exhaustively explored. In this study, the number of alternative candidate PoMs was too high , society’s preferences regarding different GES descriptors  or any other component of the modelling framework. The framework also allows for considering the problem of a risk-averse decision maker who, instead of looking at expected outcomes, may require that GES be pursued in the case of certain core descriptors for which the probability of success is higher. Such an objective may be attributed to the quest for a “safe minimum standard”. In addition, the model allows the societal utility function to be concave, rather than linear, and to include discontinuities.All the computations can be repeated as soon as new candidate measures are identified or new data on the effectiveness or costs of measures become available. Moreover, the analysis is easily updated for new knowledge on the relative severity of the gaps Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Conventional polyHEMA-based and silicone hydrogel soft contact lenses were soaked in drug solution  membrane without corneal epithelial cells, a PET membrane with a monolayer of human corneal epithelial cells (HCEC), and a PET membrane with stratified HCEC. In the cell-based in vitro corneal epithelium models, a zero order release was obtained with the silicone hydrogel materials (linear for the duration of the experiment) whereby, after 48 hours, between 4 to 6 in vitro corneal model that is more representative of the physiological conditions in the eye to more adequately characterize ophthalmic drug delivery materials. Our results demonstrate how in vitro models with corneal epithelial cells may allow better prediction of in vivo release. It also highlights the potential of drug-soaked silicone hydrogel contact lens materials for drug delivery purposes.In this study, we compared, for the first time, the release of a 432 kDa prostaglandin  Ocular drug delivery is either intended to target the ocular surface to manage superficial conditions such as dry eye, microbial keratitis and conjunctivitis, or to treat intraocular disorders such as glaucoma, and age-related macular degeneration. Eye-drops are still the most common drug delivery method, comprising 90% of ophthalmic medications, followed by ointments and gels in situ forming gels Studies show that only about 1 to 5% of the applied dose penetrates the cornea It is accepted that simple “soaking” of a contact lens in a topical drug solution may be insufficient for adequate elution on the ocular surface; therefore, it is considered to have a low potential for success in vitro fixed volume model compared to in vivo experiments For the most part, drug release has been studied in a fixed volume of deionized water (DI), Phosphate Buffered Saline (PBS) or artificial tear solutions in vitro corneal epithelial model will allow replication of the relevant factors of the in vivo environment. Human corneal in vitro models offer a cost effective and more standardizable substitutes Considering that the primary drug permeation route to the front of the eye is through the transcorneal pathways, it is also important to consider the role of the cornea in drug release studies in vivoin vitro. The objective of this study was to investigate the release of Latanoprost by commercially available contact lenses using in vitro models containing corneal epithelial cells.Pharmacokinetics of most prostaglandin The lens doping solution was prepared by dissolving latanoprost and latanoprost free-acid  in PBS . The concentration of the stock drug solution was Four commercially available contact lens materials, galyfilcon A, senofilcon A, omafilcon A, and balafilcon A were used. The properties of the four lens types are presented in HPV-immortalized human corneal epithelial cells, a generous gift from Dr. May Griffith (Integrative Regenerative Medicine (IGEN) Centre, Linköping University, Sweden) in vitro models were used to assess drug release from commercially available contact lenses in-cluding diffusion through a) a Polyethylene Terephthalate (PET) membrane  for latanoprost . Following the EIA kit instructions, each collected sample was analyzed in duplicate and at two different dilutions. To determine the uptake amount by the contact lenses, samples were also aliquoted from the original drug stock solution as well as the remaining drug solutions after soaking the lenses.The release results represent the concentration of the drug on the other side of the PET membrane, meaning that the drug has been released from the contact lens material on top of the membrane, then diffused through the cells, if present, and the culture membrane. Note that the EIA kit does not distinguish between the free-acid form and ester form of the drug.As mentioned above, to measure the amount of released drug, samples were taken from the bottom of the wells and replaced by fresh solution at each time point. Refreshing a fraction of the medium in the bottom at each time point affects measurements. Therefore, it is necessary to account for the dilution effect and adjust the measured concentrations to provide an accurate measure of the concentration without the dilution effect.Assuming the fraction of total volume of medium in the bottom which is being aliquoted is “i-th time point (In Eq.(1), i-th step can be found as below:The adjusted concentration, The proposed method to estimate adjusted concentrations neglects the effect that dilution might have on the diffusion rate. However, for small difference between calculated and measured concentrations, the change in diffusion rate will be insignificant.in vitro corneal models and time points, an analysis of variance (ANOVA) was performed, followed by multiple pair-wise comparisons using the Holm-Sidak test .Results are presented as the mean of six experiments for latanoprost and three experiments for latanoprost free-acid Preliminary studies showed that there was no decay of latanoprost and latanoprost free-acid in the culture medium or buffered solution used in the current research (results not presented), thus enabling the use of the enzyme immuno-assay method to measure drug concentrations in both solutions for up to 48 hours. All the results presented have also been adjusted according to Eq. (4), to take into account the small dilution that may occur as samples are taken out and fresh medium is added.The uptake analysis showed that 95% of the dissolved latanoprost was taken up by the galyfilcon A and senofilcon A silicone hydrogels and 98% by the balafilcon A  was substituted with PBS. When compared to KSFM, the latanoprost release decreased significantly in the no-cell model in PBS in vitro models; a significantly higher amount of latanoprost was released in the monolayer and multilayer models in vitro corneal models, an extended release of drug was observed over time  in the amounts of the drug that has been released from galyfilcon A and senofilcon A silicone hydrogels  form of latanoprost contributed to only 4% of the total drug diffused through an Different latanoprost release profiles were observed among the hydrogel contact lens materials tested. Compared to the silicone hydrogel materials, the high release of latanoprost from omafilcon A in spite of the lower drug uptake may be explained by the low affinity of the latanoprost (an hydrophobic compound) toward the omafilcon A contact lens material, which is a high water content hydrogel. The large partition ratio results in a low uptake by this material when soaking in aqueous solution of hydrophobic drugs, as well as relatively fast release rates in the release solution.Using latanoprost esterified form, i.e. the active drug compound, latanoprost free-acid, affected results in all models. Higher water solubility of the latanoprost free-acid led to higher amounts of drug being released from the silicone hydrogel lens materials in the no-cell model. As a more polar molecule, latanoprost free-acid has a lower partition ratio between the hydrophobic silicone hydrogel contact lens materials and the aqueous solution when compared to latanoprost. While higher amounts of latanoprost free-acid were released in the no-cell model, lower releases were observed in the presence of cells. With latanoprost free-acid, epithelial cells now act as a barrier against the diffusion of the latanoprost free-acid, and hence limit the diffusion of the hydrophilic drug.in vitro models with cells when studying release of a prodrug that requires to be metabolized before diffusion through the tissue to the site of treatment.As one compares the latanoprost and latanoprost free-acid release results, it becomes evident that similar drug release profiles cannot be obtained by replacing the prodrug with the drug, even in the no-cell model. Not only are the amounts released significantly different by an order of magnitude, but while all silicone hydrogel materials released similar amounts of latanoprost, balafilcon A released significantly more latanoprost free-acid compared to the other two silicone hydrogels. The balafilcon A/latanoprost free-acid results are likely due to the fact that balafilcon A material has an overall net negative charge due to the incorporation of some acidic material components in vitro studies on prostaglandin analogues, our results can only be compared to the release of drugs from the contact lens materials with similar size and hydrophobicity. Previous in vivo studies have shown a prolonged release of relatively hydrophobic drugs such as ketotifen in vitro using a fixed volume release model in vitro models correlates well with the extended release profiles of the hydrophobic drugs observed in vivoin vitro models of drug release likely contributes to the contradiction between these in vitro and in vivo studies Due to the lack of previous in vitro corneal model is in the order of in vitro models have a significant impact on the outcomes of testing ophthalmic drug delivery materials. Our in vitro study suggests that silicone hydrogels have the potential to deliver latanoprost effectively over an extended period of time.Poor release results from commercially available contact lens materials soaked in hydrophobic compounds such as latanoprost have been obtained with fixed volume release models similar to the no-cells model used here. However, we have demonstrated, using drug-soaked silicone hydrogel materials, that the amount of drug diffusing through an


The eruptive phase of a submarine volcano located 2 km away from the southern coast of El Hierro Island started on October 2011. This extraordinary event provoked a dramatic perturbation of the water column. In order to understand and quantify the environmental impacts caused, a regular multidisciplinary monitoring was carried out using remote sensing sensors. In this context, we performed the systematic processing of every MODIS and MERIS and selected high resolution Worldview-2 imagery to provide information on the concentration of a number of biological, physical and chemical parameters. On the other hand, the eruption provided an exceptional source of tracer that allowed the study a variety of oceanographic structures. Specifically, the Canary Islands belong to a very active zone of long-lived eddies. Such structures are usually monitored using sea level anomaly fields. However these products have coarse spatial resolution and they are not suitable to perform submesoscale studies. Thanks to the volcanic tracer, detailed studies were undertaken with ocean colour imagery allowing, using the diffuse attenuation coefficient, to monitor the process of filamentation and axisymmetrization predicted by theoretical studies and numerical modelling. In our work, a novel 2-step segmentation methodology has been developed. The approach incorporates different segmentation algorithms and region growing techniques. In particular, the first step obtains an initial eddy segmentation using thresholding or clustering methods and, next, the fine detail is achieved by the iterative identification of the points to grow and the subsequent application of watershed or thresholding strategies. The methodology has demonstrated an excellent performance and robustness and it has proven to properly capture the eddy and its filaments. Mesoscalar oceanic eddies are ubiquitous structures that can be found anywhere in the world’s oceans, with a predominance of anticyclonic over cyclonic vortexes . SeveralIn the North East Atlantic Ocean, the presence of the Canary Archipelago acts as an obstacle to the oceanic and atmospheric flows, originating a rich mesoscale variability mainly represented by anticyclonic and cyclonic eddies spun off from the islands’ flanks ,6. The mIn this context, active and passive satellite sensors have been operating to investigate the variability of mesoscale features in the ocean. In particular, sea level anomaly fields from altimeters  have sysetc. These oceanographic remote sensing data also played a fundamental role during field campaigns guiding the Spanish Government oceanographic vessel to the appropriate sampling areas [The eruption of a submarine volcano at El Hierro Island provided a unique and outstanding source of tracer that enabled us to study a variety of structures. El Hierro, an island off the Atlantic Ocean coast of North Africa was rocked by thousands of tremors and earthquakes since July 2011. On October 10 of the same year an underwater volcanic eruption started 300 meters below sea level. ng areas .Traditional eddy detection methods using altimeter data can be categorized into three classes : (i) thoetc. Several image segmentation techniques have been developed for a variety of applications in various fields of image analysis [In our work, as we deal with higher resolution optical ocean colour data, the detection approaches are different, mainly addressing segmentation techniques. Segmentation is performed based on different attributes of an image such as size, colour, texture analysis ,25,26,27This paper illustrates the capabilities of multispectral satellite remote sensing systems to improve the understanding and monitoring of submarine volcanic processes and, mainly, it presents a novel image processing methodology for the segmentation of oceanographic structures. To that respect, automated detection methods are essential for understanding the complex movements and the dynamic characteristic of mesoscale eddies.The organization of contents is structured as follows: Satellite data from AQUA/TERRA-MODIS and ENVISAT-MERIS sensors have been the main source of remote sensing image information for monitoring El Hierro submarine volcanic processes. The NASA Moderate-resolution Imaging Spectroradiometer (MODIS) has a viewing swath width of 2330 km, it images the Earth in 36 spectral bands, ranging from 0.405 µm to 14.385 µm, and it collects data at three spatial resolutions: 250, 500, and 1000 meters. The ESA Medium Resolution Imaging Spectrometer (MERIS) has a swath width of 1,150 km, measuring the solar radiation reflected by the Earth in 15 spectral bands from 0.4125 µm to 0.9 µm and with a pixel field of view of 0.019°, generating full resolution data at 300 m resolution.Chl-a) concentration and diffuse attenuation coefficient (dK).We have implemented a methodology for providing multitemporal and multisensor imagery information, at 1000 m (MODIS) and 300 m (MERIS) resolution, on the concentration of specific oceanographic parameters, mainly chlorophyll-a , while for the rest, the volcano activity had temporarily stopped or was very low in the sensed area.(i)dK images. Next, the initial segmentation stage is applied with the possibility to select different clustering and thresholding algorithms. Finally, a post-processing is required to remove isolated noisy pixels or to fill holes in the eddy structure due to clouds. After the initial detection, the structure is well identified; however the fine detail of the emerging filaments cannot be preserved without incorporating unwanted areas.Initial eddy detection: First, a pre-processing is applied to the MODIS/MERIS (ii)Eddy growth: The precise delimitation is of fundamental importance when analysing the structure and its evolution, so controlled growing techniques are applied only where filaments appear. Points to grow and their directions are found using the skeleton of the initial structure detected. At each point, a window with the corresponding orientation and size masks the image and new pixels are added to the structure after the application of thresholding or watershed region growing algorithms. This process is iteratively applied to achieve the desired fine segmentation.Methods for detecting eddies are very important and can help to better understand the processes involved in eddy generation, evolution and decaying, as well as their impacts on marine environment and climate change. In addition, such eddy segmentation allows for the evaluation of the numerical modelling performance in terms of eddy centre, positions, sizes and motion. The precise detection approach we have developed is presented in The different modules that integrate the methodology are next explained in detail.etc. So, a cloud mask (CM), as well as the land mask (LM) are applied to the geometrically corrected (GC) Kd image. Despite the good performance of the cloud detection algorithm [median) can be applied to the corrected and masked image. This filtering method has also been selected because of its smoothing effect without edge deterioration. Thus, the pre-processed image  were incorporated to the eddy segmentation tool. In our study, satisfactory results to segment marine structures are achieved by techniques like Riddler, Otsu, Gaussian Mixture Modelling, Pun or Li. In particular, Otsu algorithm provides an intermediate, yet accurate, solution and it can be selected as a compromise technique, providing robustness and excellent results for the majority of images. After the application of the segmentation algorithm, we get a binary image corresponding to the Initial Segmented Eddy (ISE):Several segmentation algorithms have been developed over the years, however oceanographic structures are difficult to be properly detected with conventional algorithms. After testing a variety of techniques and analysing their results with expert oceanographers, the following methods for the preliminary eddy segmentation have been selected: ding. In ,31 a detSome examples of possible automatic segmentations are included in dK threshold to different values (0.04 to 0.06) it is not possible to achieve at the same time the fine detail of the complete filaments without adding undesired lower levels of d.KTrying a segmentation using manual thresholding techniques provides the results included in RH) operation usually to fill cloudy pixels belonging to the eddy. The resulting initial segmented eddy  of the segmented eddy is used to identify the points to grow and it provides, as well, the growing direction  are obtained by selecting the end-points of the branches of the skeleton [The growing points The growing directions are determined by the angle of the first order polynomial that better fits, in a least-squares sense, the medial axis in the neighbourhood of each growing point.For each point to grow, a window with the appropriate orientation and size  is generated and used to mask the previous segmented image, as displayed in Thresholding techniques use a threshold for each window that is applied to get the grown segmented eddy (GSE), as:kGP are the growing points obtained from the grown structure at the k-iteration, w is the window generated at each growing point and Thr represents the thresholded result after applying the corresponding approach. Specifically, two different approaches have been included to decide the appropriate threshold: (i) its value is automatically obtained, for each window, using one of the automatic thresholding methods as previously indicated; or (ii) the threshold is the minimum dK value allowed to be incorporated to the eddy during the growing process.The watershed segmentation [entation , has theRG indicates the region growing technique, SP is the spatial proximity function applied and WS represents the watershed transform.We apply the watershed segmentation to every windowed portion of the image, once the previously detected structure within the window has been excluded. Note that the growing process is guided towards the direction defined in the previous step. The complete structure growing process previously described at Equations (5) or (6) is iteratively applied until no new pixels are added to the main structure or the required number of iterations has been completed d. etc.) and its filaments , whose results are included in next section.Finally, it is worth to mention that there are two specific modules, at the output of each step of the methodology, to analyse and extract the maximum information about the eddy . Only a few iterations are presented to illustrate the growing process on a specific filament. In the last figure, the skeleton (yellow) and the growing direction (red) are included. dK images processed from the MODIS and MERIS sensors. The first row includes the contour of the eddy after the initial detection module while the final contour after the growing stage is shown in the second row. We can appreciate that filaments have been properly captured, even those having low diffuse attenuation coefficient values (greenish tones), without the inclusion of noise associated to other areas having similar low dK levels . Notice that the spiraling filaments detach form each vertex of the elliptical shaped core. Image sequences also show how the spiraling like eddy rotates around its centre of mass. Therefore, superposed to water parcel orbits around the eddy centre, there is a low frequency anticyclonic rotation of the ensemble constituted by the elliptical core and attached filaments. This low frequency rotation may be appreciated by the deformation of the filaments through sequences of Detailed information about the morphology and location of the core of the eddy has also been extracted for the images under analysis. In October 2011, an underwater eruption gave rise to a novel submarine volcano south of the island of El Hierro, in the Canary Islands, Spain. During the eruption, large quantities of mantle-derived gases, solutes and heat were released into the surrounding waters. Through the months the eruption lasted, satellite remote sensing imagery has been of fundamental importance to guide the governmental oceanographic vessel during field campaigns and also to monitor the impact of the eruption on the marine ecosystem.Specifically, the periodic processing of MODIS and MERIS data was performed to provide information on the concentration of a number of biological, physical and chemical parameters. Apart from low-resolution data, the volcano activity and effects were also examined using very high resolution from the Worldview-2 satellite.On the other hand, the eruption of the underwater volcano has provided an exceptional source of tracer that allowed the study a variety of structures. In particular, eddies are of great interest as they can exert an important role on the global climate and local oceanic dynamic due its influence in the transport of ocean heat and momentum, as well as, in the enhancement of vertical shear mixing. It is important to emphasize that the Canary Islands’ area is a very active zone of long-lived eddies.Thanks to the volcanic tracer, detailed studies could be undertaken using ocean colour imagery. In our work we have considered MODIS and MERIS images and, in particular, diffuse attenuation coefficient products have been processed to allow the analysis of such structures at much higher resolution. The material released by the volcano has provided a unique high resolution picture of an anticyclonic mesoscale eddy. This has allowed observing for the first time in the nature the process of filamentation and axisymmetrization predicted by theoretical studies and numerical modelling.To that respect, a novel 2-step methodology has been developed. It is composed by an initial structure detection module followed by a fine detail growing process. The methodology obtains the initial eddy segmentation and achieves the maximum detail of the structure, especially for the emerging filaments, after the iterative identification of the points to grow and the subsequent application of growing strategies. The method incorporates different segmentation algorithms and region growing techniques providing enough flexibility to allow the oceanographers to get the best result and to obtain detailed information about the morphology and location of the core of the eddy, as well as the length and width of each filament.dK values, without the inclusion of noise associated to other areas having similar low dK levels.This detection approach has been validated over a database of MERIS and MODIS oceanographic products and it has demonstrated an excellent performance and robustness to noise and weak gradients. Specifically, it has proven to properly capture the filaments, even those having low In summary, the underwater volcano has allowed the precise analysis of an anticyclonic eddy, providing valuable information at a resolution that cannot be reached using sea level anomaly fields from altimeters.


While community health workers are being recognized as an integral work force with growing responsibilities, increased demands can potentially affect motivation and performance. The ubiquity of mobile phones, even in hard-to-reach communities, has facilitated the pursuit of novel approaches to support community health workers beyond traditional modes of supervision, job aids, in-service training, and material compensation. We tested whether supportive short message services (SMS) could improve reporting of pregnancies and pregnancy outcomes among community health workers  in Malawi.We designed a set of one-way SMS that were sent to HSAs on a regular basis during a 12-month period. We tested the effectiveness of the cluster-randomized intervention in improving the complete documentation of a pregnancy. We defined complete documentation as a pregnancy for which a specific outcome was recorded. HSAs in the treatment group received motivational and data quality SMS. HSAs in the control group received only motivational SMS. During baseline and intervention periods, we matched reported pregnancies to reported outcomes to determine if reporting of matched pregnancies differed between groups and by period. The trial is registered as ISCTRN24785657.Study results show that the mHealth intervention improved the documentation of matched pregnancies in both the treatment  and control  groups relative to the baseline period, despite differences in SMS content between groups. The results should be interpreted with caution given that the study was underpowered. We did not find a statistically significant difference in matched pregnancy documentation between groups during the intervention period . mHealth applications have the potential to improve the tracking and data quality of pregnancies and pregnancy outcomes, particularly in low-resource settings. The field of global health has set ambitious goals, including the Millennium Development Goals and the recently defined Sustainable Development Goals . These iResearchers have demonstrated the importance of supervision and incentives to improve motivation and performance among CHWs –13. MobiThe Institute for International Programs (IIP) at Johns Hopkins University and the Malawi National Statistical Office (NSO) collaborated to implement a community-based vital event documentation system  using Health Surveillance Assistants (HSAs), government trained and paid CHWs with a scope of work set by the Malawi Ministry of Health . We implAn estimated 45% of under-five deaths occur in the neonatal period and many of these deaths are not reported due to cultural and social norms –31. To pWe conducted the data quality mHealth intervention with the RMM project implemented in the Balaka and Salima districts of Malawi. The intervention period for the mHeath trial was from November 2012 to November 2013, and the intervention period for the RMM project was from January 2010 to December 2013. The districts in the RMM project were selected for their high under-five mortality, high fertility, ease of access for the study team, average population size relative to other districts in the country, and full coverage by HSAs deployed in the district. Each HSA in Malawi is assigned to a catchment area of approximately 1,000 inhabitants and its associated health facility, covering a radius of eight kilometers except in district-defined hard-to-reach catchment areas . We randThrough the RMM project, HSAs received training on the documentation of pregnancies, births, and deaths, a task within the HSA scope of work set by the Ministry of Health . At the We designed the intervention as a mobile support for HSAs using one-way short message services (SMS) sent by the mHealth coordinator at the NSO to HSAs in the RMM project. The mHealth intervention had a three-week pilot phase and a twelve-month implementation phase divided into two phases. The study team modified the intervention after eight months of implementation to incorporate feedback from the HSAs suggesting that the variety of SMS and their frequency should be increased . The intFor both periods, we pretested the SMS among purposively selected HSAs . To meet the 160-character limit of the SMS, established abbreviations were used and punctuation dropped in ways that pilot testing confirmed did not affect the HSAs’ comprehension of the message. For both treatment and control groups, the SMS were randomized and scheduled as per the schedule. The repetition of SMS presented in a randomized sequence was done to reinforce important data quality messages in the treatment group yet avoid alert fatigue.We selected Frontline SMS as the messaging platform because it is open source and fit the technical and management capacities of the IIP and NSO data management team . FrontliPrior to the start of the intervention, the lead mHealth researcher trained two RMM data management team members at the NSO to serve as field coordinators for the study. During phase one, the two field coordinators alternated responsibility each week. During phase two, one coordinator retained lead responsibility and was supported by the other coordinator when conducting field visits. The lead mHealth researcher supervised the initial implementation of the intervention, maintained daily communication with the mHealth coordinators throughout the intervention, and assisted in resolving technical and logistical challenges.At the September 2012 data review meetings of the RMM project, the NSO data management team introduced the mHealth intervention as a mobile support to HSAs and supervisors. We did not train HSAs in sending or reading SMS, because SMS were an established method of communication in the RMM project. At the start of the SMS pilot period and the second phase of the mHealth intervention, the field coordinator sent SMS with a brief description of the intervention expectations and schedule.Prior to each scheduled transmission of SMS to HSAs, the field coordinator sent a test SMS to three NSO RMM team members to ensure proper functioning of the Frontline SMS platform. Once receipt of the test SMS was confirmed by at least one of the recipients of the test SMS, the field coordinator sent the scheduled SMS. For HSAs with two phones, the field coordinator sent the same scheduled SMS to both numbers to increase the likelihood that the SMS was received. After the field coordinator sent the SMS, the Frontline SMS platform was kept open and the USB GSM modem was kept connected in the event that an HSA responded with a question or clarification.Although the intervention was unidirectional, HSAs occasionally sent response SMS to the field coordinators. The field coordinator documented SMS communications and airtime costs each day SMS were sent and tallied totals at the end of the week . Each weAs per the scope of work set by the Malawi Ministry of Health, HSAs track pregnancies, births, and deaths using their Village Health Registers (VHRs). For the RMM project, we asked HSAs to extract these events monthly onto a RMM extraction form that was submitted to the NSO. Details of the data collection process are available elsewhere ,27. The The primary outcome measure was the improvement in matched pregnancy documentation between groups during the intervention period. Possible pregnancy outcomes included adverse events , live birth, and out-migration of the pregnant mother. The secondary outcome measures were the improvements in matched pregnancy documentation by group between baseline and intervention periods. We also evaluated changes in matched pregnancy documentation by HSA residency in their assigned catchment area.Pregnancies and outcomes were matched using the six-digit HSA code and the woman’s unique 11-digit ID. Matching results included three outcomes: (i) pregnancies matched to an outcome (ii) live births and adverse pregnancy outcomes without a pregnancy match (iii) pregnancies without a matched outcome. Only matched (i) and unmatched pregnancies (iii) were used to analyze the change in documentation of matched pregnancies, our primary and secondary outcome measures. We exclude unmatched outcomes (ii) from the analysis since they can be used to address a different research objective, the characteristics of women missed by HSAs during pregnancy.We were restricted to the sample size of the RMM project, i.e. the number of pregnancies and outcomes that occurred within 160 randomly selected catchment areas of Balaka (n = 80) and Salima (n = 80) districts affiliated with thirty health facilities and 160 HSAs. We included only 156 of the 160 catchment areas in the randomization since four catchment areas did not have HSAs at the time of randomization . We randWe calculated the study power using published data from comparable studies conducted in Africa. We estimated that HSAs documented pregnancy outcomes for 70% of pregnancies at baseline, and HSAs in the control group would maintain this percentage during the intervention period . We estiTo improve balance in treatment and control arms, we stratified by district and used a +/- 10% relative restriction to constrain on three variables: average catchment area population per HSA; total cluster population; and baseline timeliness of extraction form submission. The validity of the acceptable randomizations was checked by evaluating the number of times any two randomization units appeared in the same arm together . From th10.7281/T1F769G3). HSA characteristics included in the mHealth intervention analysis were collected in August 2013 through the Village Health Register Verification assessment conducted for the RMM project. In catchment areas with HSA turnover, characteristics of the HSA interviewed for the assessment were used.The data clerks performed double data entry in CSPro on edited forms . AbortioWe did not conduct intention-to-treat analysis among HSAs who left RMM or transferred between RMM catchment areas, in order to minimize contamination in groups. HSAs who moved from a health facility cluster in one group to a health facility cluster in other treatment group received messages consistent with their new group. HSAs who transferred out of their RMM catchment area were instructed to stop submitting RMM extraction forms and were dropped from the mHealth intervention. Their truncated data was included in the analysis. HSAs were masked to their treatment group allocation. It was not possible to mask the field coordinators to treatment group allocation since they were responsible for maintaining an updated contact list on Frontline SMS.We used a population-averaged panel data model with generalized estimating equations to analyze the effect of the mHealth intervention on matched pregnancy documentation using Stata 12 . The modWe obtained ethical approval in the U.S. from the Institutional Review Board (IRB) at the Johns Hopkins University Bloomberg School of Public Health, and in Malawi from the National Health Sciences Research Committee. We obtained a waiver of written consent from the IRB. Approval letters are available upon request.In the initial design, 76 HSAs affiliated with 15 health facilities were allocated to the treatment group and 80 HSAs affiliated with 15 health facilities were allocated to the control group . Two HSADuring the intervention, one HSA in the control group left the RMM project. Eight catchment areas experienced HSA turnover. One HSA replacement included the transfer of an HSA from a catchment area in the treatment group to a catchment area in the control group. The analysis was conducted with and without catchment areas with HSA changes. There were no substantive differences between the two sets of results, and the final analysis includes catchment areas with HSA changes during the intervention period.During the baseline and intervention periods, HSAs reported 7,407 matched pregnancies, of which 4,140 were reported in the intervention period (55.9%). This mHealth intervention, designed and analyzed at the cluster level, led to an improvement in HSA documentation of matched pregnancies in each group between baseline and intervention periods. However, we did not find a statistically significant difference in matched pregnancy documentation between groups during the intervention period. Overall documentation of pregnancies and outcomes improved during the intervention period, with notable increases in the documentation of out-migration and adverse pregnancy outcomes.A range of factors can influence CHW performance, but translating these factors to mobile applications is novel and results vary ,43. ReseThe improvements in the control group were not expected based on research showing no effect with simple, repetitive SMS . We tailThe increase in the documentation of abortions, miscarriages, and stillbirths is noteworthy, because these events are difficult to capture in low-income countries ,51. HawsIn a study conducted among HSAs in the Mwanza district of Malawi, Kok & Muula found that HSAs identified transportation challenges as one of the main work dissatisfiers . TranspoA major limitation to the study is limited power of the study due to a restricted study population. However, this design allowed for a pre-post intervention design with complete baseline data. This also allowed calculation of three ICC coefficients that can be used by researchers designing clustered studies. Another limitation is the change in design of the intervention midway through the trial to increase the dose of SMS in the treatment group from three SMS a week to five. We made this decision with great consideration. We agreed that the modest increase in SMS frequency in the intervention group responded to the needs of the HSAs. The change in intervention may have introduced bias for assessing the intervention effect. We expect the bias to be positive relative to phase I results and negative for phase II results in the treatment group assuming the increased dose improved matched pregnancy documentation. We did not measure the effect of the change due to the underpowered study design and absence of notable differences between phases in both treatment groups. This further highlights the importance of qualitative assessments in complementing quantitative results to elucidate HSA perceptions on the effect of any form of SMS modifications ,45.The Frontline SMS system did provide confirmation that SMS were sent though the cellular network, but we did not have confirmation from HSAs that the message was received, read, and understood, or about the delay between the arrival of the SMS and its reading. This required the assumption that a SMS confirmed by Frontline was received and read by the HSA in a timely manner. A bidirectional SMS intervention would have allowed us to confirm receipt and quantify delays, but the intervention would no longer be a reminder system and require a higher level of engagement from the field coordinator at the NSO and from the HSAs. If this intervention is reconsidered for HSAs in Malawi, researchers should consider pilot testing a bidirectional system to determine its feasibility and acceptability given the more interactive design.pilotitis” that is plaguing mHealth interventions, but also generated important information that can be used to develop an improved design prior to scale-up [This intervention was not designed to be scaled up as implemented through the RMM project. New investments would be required to maintain the intervention and to scale it up. This pilot study is an example of the “scale-up .The ubiquity of mobile phones and growing access to mobile networks in hard-to-reach areas makes mHealth a very attractive tool, especially in low-income countries. Our results reveal that regularly scheduled SMS with basic motivational messages, both with and without data quality content, can improve performance of CHWs in the documentation of pregnancy outcomes. The study was underpowered so results should be interpreted with caution. These results are still important given the growing interest in mHealth applications and respond to calls for their rigorous evaluation . ResearcWe observed measurable improvements in the documentation of matched pregnancy outcomes associated with regular, unidirectional SMS of two intensity levels sent to CHWs in rural Malawi. Improvements were noted in each group regardless of whether or not the motivational SMS additionally included content explicitly related to data quality. The study was underpowered so results should be interpreted with caution. We did not find a difference in the documentation of matched pregnancies between groups during the intervention period. The effect of continuous guidance and support to CHWs via SMS on the data quality of pregnancy outcomes should be further investigated using randomized field studies. In particular, further research is needed to exploit the motivational effect of SMS communications, their content, and frequency. We believe that mHealth tools have the potential to improve the tracking of pregnancies and the documentation of pregnancy outcomes, particularly in low-resource settings.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.