Individual perceptions are essential when evaluating the well-being benefits from urban green spaces. This study predicted the influence of perceived green space characteristics in the city of Szeged, Hungary, on two well-being variables: the green space visitors’ level of satisfaction and the self-reported quality of life. The applied logistic regression analysis used nine predictors: seven perceived green space characteristics from a questionnaire survey among visitors of five urban green spaces of Szeged; and the frequency of green space visitors’ crowd-sourced recreational running paths and photographs picturing green space aesthetics. Results revealed that perceived green space characteristics with direct well-being benefits were strong predictors of both dependent variables. Perceived green space characteristics with indirect, yet fundamental, well-being benefits, namely, regulating ecosystem services had minor influence on the dependent variables. The crowd-sourced geo-tagged data predicted only the perceived quality of life contributions; but revealed spatial patterns of recreational green space use and aesthetics. This study recommends that regulating ecosystem services should be planned with a focus on residents’ aesthetic and recreational needs. Further research on the combination of green space visitors´ perceptions and crowd-sourced geo-tagged data is suggested to promote planning for well-being and health benefits of urban green spaces. Societal benefits supplied by urban green spaces (UGS) to city dwellers are vital to maintain and increase urban citizens’ quality of life (QoL) ,2. UGS aConsequently, sensory dimensions and experiential contacts with the environment are crucial building blocks of area-based perceptions ,21, and The second pillar of our study centres on the possible suitability of frequency of crowd-sourced geo-tagged data, recorded within UGS, to gauge visitors’ perception of UGS. Scholars who have researched the sensory dimensions of UGS have tended to use conventional data sources to identify associations and causalities in the human attitude towards UGS . TherefoDespite the successful application of questionnaire-based analytical methods, it is timely to test the capabilities of data crowd-sourcing to understand visitors’ attitudes towards UGS. The authors’ decision to use crowd-sourced data was three-fold. Firstly, UGS visitors of recent years can use location-aware technologies to spatially log their activities; for example, UGS visitors can geo-tag their photographs that they take during their UGS visits, or log trajectories of their physical activities. Second, geo-tagged user-generated content (UGC), a form of Big Data, is able to expose patterns and relationships which would be hidden from questionnaire-based methods ,35. ThirResearch Question 1: To what extent did perceived green space characteristics influence visitors’ levels of satisfaction with the visited green spaces and the self-reported QoL contributions of the study areas?Research Question 2: Was the frequency of geo-tagged photographs and running trajectories statistically related to visitors’ perception of UGS?The present study aimed to support urban planners and green space managers from the city of Szeged, Hungary, with information on the role of the city UGS in citizens’ QoL. Therefore, expert interviews were conducted with urban planners and environment managers of the Szeged City Council to assess their needs. Their interest was to understand how green space characteristics influence the use and perception of the city green spaces. The study was designed and conducted accordingly and aimed to answer the following two research questions:To reveal the role of green space characteristics in visitors’ self-reported levels of satisfaction and the perceived QoL contribution of the studied green spaces.To statistically explore the potential of frequency of geo-tagged photographs and running trajectories in revealing human perceptions of UGS.The aims of the study were:The study areas were five urban green spaces (UGS) within the city of Szeged , selecteA survey was conducted with visitors of the five UGS to examine attitudes towards the study areas. Thirty questions evaluated the perceived quality of life (QoL) contributions, and the answers were measured on a Likert scale . The details of the survey were reported by Kothencz and Blaschke . At the Running trajectories are the ground paths of runners as they move through space. All running trajectories available on the crowd-sourced running path sharing website, Futótérkép , for SzeTrajectories running through those parts of the UGS where they were not supposed to occur  were not deleted, as they also counted as a logged path even though the GPS receiver of the runner was not very accurate. The number of running trajectories (NoOfTrks) per UGS were added together and used in the subsequent analysis  as indepPhotographs geo-located on the grounds of the UGS or within their one-block-wide perimeter were downloaded from the photo sharing website Panoramio  and inpuThe number of photographs in the five categories imaging green space aesthetics were summed up for each UGS. The proportion of images picturing green space aesthetics (AesthPct) to the total number of photographs per UGS were calculated for each study area. The percentages per UGS were used as independent variables representing aesthetic appreciation of the study areas .Each row of the table inputted in the analysis represented individual-level data; the questionnaire record of a single respondent. The unique identification code of each park, of which the particular person responded, was added to the records. The total number of running trajectories per the relevant UGS and the proportion of images picturing green space aesthetics per the relevant UGS were also added to every row of the input table.Ordinal logistic regressions were applied using two dependent variables representing individual-level perceptions of the UGS: visitors’ level of satisfaction with the UGS and the perceived QoL contribution of the UGS. For each respondent, individual regressions were performed.Y represents the categorical ordered variable, and x and b represent the independent variables and their coefficients, respectively.Ordinal logistic regressions are logistic generalized linear models (GLMs) where the dependent variable is a categorical ordered variable. GLMs can be expressed as follows (Equation (1)):For each dependent variable two models were performed. One model included the seven perceived green space characteristics as subjective area-based independent variables from the questionnaire: the perception of nature, noise abatement, capacity for recreation, microclimate regulation, habitat, air purification, and the visual appearance.In the second model two variables extracted from the crowd-sourced geo-tagged data were added to the subjective independent variables. These were the proportion of images picturing green space aesthetics and the number of running trajectories, representing objective green space popularity through quantitative data. Therefore, their values for each UGS were assigned to the records of the seven subjective area-based independent variables.The results of the logistic analyses are shown in The relative odds of experiencing a very positive satisfaction with the green spaces are 1.66 times greater for respondents who perceived high degree of nature in the study areas than for those who did not.For survey participants perceiving high recreational capacity of the green spaces, the relative odds of experiencing very positive green space satisfaction are 1.47 times greater than for those that reported poor recreational capacity.The relative odds of experiencing positive satisfaction with the green spaces are 1.89 times greater for respondents with a high aesthetic satisfaction of the green spaces than for those who perceived low aesthetics.The odds ratios obtained from Model 1 can be interpreted as follows: Interpretation of the Akaike information criterion (AIC): Considering the four models, Model 1 had the best performance indicated by the lowest AIC value. If models only with questionnaire-based variables are concerned, still Model 1 performed better. If the extended models are compared Model 2 performed better.The present study fulfilled two goals. Firstly, it uncovered the role of perceived green space characteristics in visitors’ level of satisfaction and the perceived quality of life (QoL) contribution. Secondly, the research demonstrated the potential of frequency of geo-tagged photographs and running trajectories in revealing human perceptions of urban green spaces (UGS).The results demonstrated the extent to which the seven perceived green space characteristics influenced visitors’ levels of satisfaction and the self-reported QoL. Every model revealed that visual appearance of the UGS was the most crucial predictor of visitors’ levels of satisfaction and the self-reported QoL . The perception of nature was the second most influential green space characteristic that highly affected visitors’ satisfaction in Models 1 and 2 already at 1% level of significance. The third most crucial green space characteristic was the perceived recreational capacity. This positively influenced visitors’ level of satisfaction with the UGS in Models 1 and 2 at 5% level of significance, and the perceived QoL contribution of the five UGS in Model 4 at 5% level of significance. Of lesser importance, habitat contributed to visitors’ levels of satisfaction in Models 1 and 2 at 10% level of significance. Another notable perceived green space characteristic, the microclimate regulation, affected visitors’ self-reported QoL in Model 3 at 10% level of significance.Our work identified that regulating ecosystem services were largely underrated by survey participants. Noise abatement, microclimate regulation and air purification services supplied by the study areas seemed to have only minor significance on visitors’ perceptions. Likewise, habitat was underappreciated. There are two possible arguments to explain these results. The first is the mainly ornamental purpose of Szeged’s city centre green spaces means they are likely to contribute less to regulating ecosystem services than those which are located away from the city centre. The second possible reason stems from a lack of societal awareness of the fundamental importance of regulating ecosystem services, in that the benefits derived from regulating services are not directly “tangible” or perceivable. Even though cultural ecosystem services such as the scenery and the recreation are also intangible, green space visitors can directly benefit from them through aesthetic appreciation, relaxation and physical activity. Therefore, visitors feel more attached to cultural services than regulating or habitat services. These findings are confirmed by results of earlier studies. Tyrväinen, et al.  found reOur findings suggest that visitors' perception of the UGS were effectively conveyed through the seven questionnaire items, while frequencies of geo-tagged photographs and running trajectories were only partly related to individual-level perceptions of the study areas. The number of running trajectories and the proportion of images picturing green space aesthetics were found significant at 1% and 5% level of significance, respectively, in relation to the perceived QoL contribution of the UGS in Model 4. However, the two crowd-sourced area-based variables were not strong enough to support survey participants’ satisfaction with the study areas. Consequently, the potentials of frequency of geo-tagged photographs and running trajectories were limited in revealing human perceptions towards the studied UGS. On the other hand, geo-tagged photographs and running trajectories exposed spatial patterns in their distribution. As it can be seen in The findings of our work are in line with those from previous studies by showing that Big Data, in the present case geo-tagged UGC, is suitable to identify general relations ,43, whilAesthetics are a contextual feature affecting personal perspectives and have implications for landscape and ecological planning and intervention . TherefoHence, it is vital that urban planning and management implement development of regulating ecosystem services with a consideration of cultural services in their strategic and operational goals. In addition, education is needed to raise environmental awareness of intangible ecosystem services as this would enable a better appreciation of them as crucial sources of our well-being benefits .There are interventions in green space planning and management that require qualitative knowledge, for example, to understand visitors’ perception of the scenery of the UGS. To achieve this goal, questionnaire-based methods are still required. However, green space managers need to be aware of the measured green space usage and appreciation, and this is the specific area where geo-tagged UGC can contribute valuable information to help management decisions. Frequency and spatial concentration of running trajectories may function as measured recreational green space usage , whilst Even though geo-tagged UGC has potential planning implications ,69,70, cPrior to the work the authors assessed the needs of urban planners and environment managers to understand residents’ perception of well-being benefits they derive from UGS. Future studies will follow in which urban planners and green space managers will be involved in all phases of the work, from the study design to the data acquisition and analysis to the result implementation. This would guarantee the full compatibility of the methodology and the results in decision support practices.Individual-level objective variables, such as respondents’ socio-economic status, affect individual perceptions often leading to very different self-reported satisfaction ,72. TherThe authors conducted the study as a pilot in Szeged. As the methodology is easily transferable to other cities, the authors would like to pilot it in other urban areas to gain more experience and possibly detect differences in preferences in perceived well-being benefits derived from urban green spaces.The present study can be considered as a basis to understand the city as a complex system. The methods applied here for UGS can be transferred to the city scale. Future research may apply methodologies that go beyond green spaces and use questionnaires and crowd-sourced geo-tagged data for a better comprehension of other urban resources and services.This study used answers from a questionnaire survey together with the frequency and spatial distribution of crowd-sourced geo-tagged images and running trajectories of five UGS in the city of Szeged, Hungary. The aim was to predict the influence of perceived green space characteristics, reported by surveyed green space visitors, on two well-being variables: visitors’ levels of satisfaction with the green spaces and the perceived quality of life contribution of the respective area. The ultimate goal was to inform urban planners and green space managers of Szeged about the effects of green space characteristics on well-being benefits that the citizens obtain from UGS. Four models of ordinal logistic regression analysis were applied. They disclosed that perceived green space characteristics with direct well-being benefits strongly influenced visitors’ green space attitude. The two well-being variables were particularly dependent on the perceived scenery of the study areas. Recreational capacity, which has direct well-being benefits, was also able to predict the two dependent variables. Perception of nature, an indirect well-being benefactor, was a strong predictor of visitors’ levels of satisfaction with the green spaces. Perception of green space characteristics referring to vital regulating ecosystem services with provision of indirect well-being benefits had a low influence on green space visitors’ levels of satisfaction and self-reported quality of life contribution of the study areas. The two crowd-sourced area-based variables, the geo-tagged photographs and running trajectories, were able to explain the perceived quality of life contribution of the green spaces but failed to explain visitors’ green space satisfaction. In turn, the geo-tagged UGC revealed fine-scaled spatial patterns within the study areas which highlighted the site popularity and real green space usage.The following conclusions can be drawn for urban planning and management: Consideration of site aesthetics and recreational services is crucial for UGS development, and regulating ecosystem services should be planned accordingly. Awareness of the importance of regulating ecosystem services should be raised by environmental education. The joint use of questionnaire surveys, delivering perceptual information on UGS, and geo-tagged UGC, revealing real recreational green space usage and aesthetic appreciation, is recommended for further investigation. When technically ready, the approach could promote informed decisions in urban planning and management. This study has been carried out in close collaboration with the administration of the city of Szeged and the results will be presented to the relevant administrative units in order to assure citizens’ needs-centred planning.


Metabolic abnormalities are identified in over 90% of stone formers and the institution of preventative dietary and medical measures has resulted in substantial reduction in stone recurrence rates. We review the contemporary approach to metabolic evaluation of urolithiasis.A careful medical and dietary history, stone analysis, serologic tests, and urinalysis constitute the initial screening regimen in patients who have been diagnosed with stones. Risk stratification of patients, based on the outcome of the initial screening tests, determines the need for and extent of urinary evaluation in individual stone formers. Conservative dietary measures or a simple metabolic evaluation and treatment has been described for first-time or low-risk stone formers, although the number of 24-h urine collections needed is debatable. A more extensive metabolic evaluation is recommended for recurrent or high-risk stone formers or for those in whom empiric treatment or medical therapy based on simplified evaluation is unsuccessful.Regardless of etiology, all stone formers should be counseled on dietary measures for stone prevention. The need for medication is determined by the results of 24-h urine analysis and the risk level of the patient. Cost effectiveness of the metabolic evaluation and treatment is strongly influenced by recurrence rate and efficacy of therapy.Metabolic evaluation and treatment has clearly been shown in randomized trials to reduce stone recurrence rates. Further study will determine the extent of evaluation necessary and the need for selective vs. empiric medical therapy for first-time and recurrent stone formers.


For the newly introduced class of integrase inhibitors, a huge discrepancy between these two measures of efficacy was observed. Hence, a thorough understanding of the relation between these two measures of drug efficacy is imperative for guiding future drug discovery and development activities in HIV. In this article, we developed a novel viral dynamics model, which allows for a mechanistic integration of the mode of action of all approved drugs and drugs in late clinical trials. Subsequently, we established a link between in vivo and in vitro measures of drug efficacy, and extract important determinants of drug efficacy in vivo. The analysis is based on a new quantity—the reproductive capacity—that represents in mathematical terms the in vivo analog of the read-out of a phenotypic assay. Our results suggest a drug-class specific impact of antivirals on the total amount of viral replication. Moreover, we showed that the (drug-)target half life, dominated by immune-system related clearance processes, is a key characteristic that affects both the emergence of resistance as well as the in vitro–in vivo correlation of efficacy measures in HIV treatment. We found that protease- and maturation inhibitors, due to their target half-life, decrease the total amount of viral replication and the emergence of resistance most efficiently.Predictive markers linking drug efficacy to clinical outcome are a key component in the drug discovery and development process. In HIV infection, two different measures, viral load decay and phenotypic assays, are used to assess drug efficacy  in vivo measure of drug efficacy, whereas phenotypic assays are used to assess drug efficacy in vitro. The recent development of novel HIV drugs resulted in a huge discrepancy between viral load decay and in vitro predictions of drug efficacy. We used a mathematical modelling approach to resolve this discrepancy by introducing a new quantity, the reproductive capacity, that allows a transfer of the in vitro drug efficacy measure into the in vivo context, enabling a direct comparison. We developed a novel model of viral dynamics that incorporates the mechanism of action of all established and novel antivirals. Based on the model, we analyzed the ability of the viral infection to replicate under different drug treatments, and estimated class-specific times until virological failure. We conclude that the half life of the targeted viral stage is an important class-specific attribute that impacts on the overall success of a drug in vivo. Our findings have direct implication for the drug discovery and development process.To guide drug discovery and development, measures of drug efficacy that are linked to clinical outcome are of key importance. In HIV treatment, decay of plasma viral load is typically used as an  Since 1996, human immunodeficiency virus (HIV) infection is treated with a combination therapy, known as highly active anti-retroviral therapy (HAART) in vivo potency of novel antivirals is usually assessed by viral load decline in small clinical trials of monotherapy, e.g., in vitro potency of antivirals is typically assessed by using phenotypic/single-round infectivity assays During therapy, plasma viral load (HIV RNA per mL blood plasma) is recommended by the National Institute of Health as a marker of therapy success in vitro, using phenotypic/single-round infectivity assays, and in vivo, using viral load decline, was observed in vitro efficacy is amongst the lowest Investigation of novel drug targets for the treatment of HIV infection resulted in the development of new drug classes. In 2003 and 2007, the fusion inhibitor (FI) enfuvirtide in vivo dynamics of HIV. Sedaghat et al. in vivo potency.Mathematical modelling of viral dynamics has lead to many insights into the pathogenesis and treatment of HIV. It is a valuable tool to interpret the time course of virological markers  during HIV treatment Consequently, viral load decay may be misleading for assessing the potency of integrase inhibitors (and other novel inhibitors) in comparison to existing drug classes. However, an alternative, more appropriate measure of drug efficacy, which allows to directly compare drugs from different drug classes is still missing.mechanistic way; and (iii) to analyze determinants of drug efficacy critical for drug discovery and development. The proposed measure of drug efficacy, termed reproductive capacity, extends the established in vivo marker, plasma viral load, by incorporating additional infectious viral stages, and the in vitro phenotypic/single-round infectivity assays by taking into account host specific defense mechanisms. This enables us to understand the observed discrepancies between in vitro and in vivo efficacy for integrase inhibitors, and to elucidate and quantify the role of immune-system related clearance mechanisms in drug action. The results presented herein are of particular value to categorize different molecular targets in the HIV life cycle and are expected to be of significance for guiding future HIV drug discovery and development.The objectives of this article are (i) to develop a novel, generic measure of drug potency that facilitates comparison across different drug classes; (ii) to develop a novel mathematical model of the viral replication cycle that incorporates the action of established and novel drugs in a We derived a detailed virus-target cell interaction model as depicted in gag-pol-polyprotein contained) enzymes. During the final step, the viral protease, which is packed into the correctly assembled, immature virions Target cells are produced by the immune system with some constant rate Depending on the stage of the life cycle, the host organism has different abilities to clear the virus. It was assumed that infectious, immature and defective virions The system of ordinary differential equations (ODEs) describing the rate of change of the different viral species and target cells in the detailed model (depicted in Gag-polyprotein) The actions of the different drug classes within the viral life cycle are shown in ed model , PIs theWe used the detailed virus-target cell interaction model to predict the effect of the different drug classes on the distinct stages of the viral life cycle. In order to enable a direct comparison between the different drug classes, we artificially eliminated the feedback by keeping the uninfected target cell hown in  , while dwn in   and finally incorporated the viral mutation process (resulting from erroneous reverse transcription) into the overall model. Whether the longer lived cell population consists solely of macrophages The model enabled us to mechanistically incorporate the action of all drugs that are approved or in late clinical trial. The impact of a compound on a corresponding (lumped) parameter in the model is specified by process ,  denotegiven in . Since sThe production of infectious offspring is crucial for the survival of a viral population. The phenotypic single-round infectivity assay measures the amount of infectious offspring after one round of replication. For a given drug, the assay quantifies the drug's efficacy by measuring the reduction in viral offspring relative to the drug-free situation. We defined a new quantity—termed the reproductive capacity The basic reproductive number Based on the two-stage virus dynamics model, the basic reproductive number in vitro reproductive capacity, corresponding to the read-out of the phenotypic assay in vivo measure, the in vitro measure would not take into account: (i) the clearance of any infective stage by the immune system  is therefore delayed by the viral life cycle. MIs and PIs do not interfere with the total amount of virus that is being released, but rather shift the ratio of infective to total virus,  see , inset, load see , when asload see , in agreload see  yield siIn contrast to other inhibitor classes, InIs decrease the amount of late infected cells  see , which hnds  see . Sedaghands  see , which din vivo efficacy of an antiviral drug depends on many different factors, particularly the ability of the virus to adapt to the pharmacological challenge by developing resistance mutations. The ability to develop drug resistance is strongly dependent on the induced pattern of resistance mutations against a particular drug, but might also be influenced by the velocity at which replication competent compartments are removed from the body. However, viral load decay focusses on only one single variable, namely the total output of virus, whereas other infectious stages  and (S31), Supplementary The mechanistic mode of action of a compound at its target site can be elucidated by cell free assays that use purified viral protein, e.g. reverse transcriptase for RTIs. The influence of viral mutation, the immune system and pharmacokinetics are absent in this type of assay. However, it is possible to deduce the pharmacodynamic mode (e.g. Eq. (1), see also in vivo pharmacokinetic data is available . We did not consider them in this study, since they are expected to contribute little to the dynamics analyzed herein (the first and the second decay phase).in silico. In its current form, the reproductive capacity requires detailed knowledge about (i) the composition of the viral population, and (ii) the fitness of the different viral strains under a given treatment  and (16)–(19)).The reproductive capacity is a useful concept to analyze and monitor drug efficacy in vitro, e.g., by phenotypic assays. We model strain specific fitness The fitness of certain viral strains can be assessed ce e.g., , usuallyAcquisition of detailed knowledge about the composition of the viral population might, due to recent advances in sequencing technology t = 0), the effects of the drug treatment were simulated until the viral population size reached 90% of its pre-treatment value, i.e., virological rebound occurred. During a simulation, the stochastic partitioning of the reaction system was dynamically updated and stochastic reaction events were realized accordingly. Every numerical calculation was computed with a relative error tolerance of The overall virus dynamics in our model comprise different viral strains with copy numbers that can vary over several orders of magnitude. For this reason we have chosen a hybrid (stochastic deterministic) setting for numerical simulation. This approach (i) takes stochastic fluctuations in the slow reaction processes into account; and (ii) reduces the computational costs for the simulation of the fast (deterministic) system dynamics. We used the direct hybrid method proposed in Text S1This file contains the derivation of the simplified model  from the(0.30 MB PDF)Click here for additional data file.Figure S1Delay in the onset of viral load decay, exemplified for PI treatment. Simulation results (red line) using the novel two stage virus dynamics model and simulating 100% effective PI treatment are shown together with median clinical data (black diamonds) from PI (RTV) monotherapy.(0.91 MB EPS)Click here for additional data file.


The scientific community is showing increasing interest in characterizing the function, evolution and language of these sequences. Despite this, there remains little in the way of user-friendly access to a large dataset of such elements in conjunction with the analysis and the visualization tools needed to study them.Comparative genomics is currently one of the most popular approaches to study the regulatory architecture of vertebrate genomes. Fish-mammal genomic comparisons have proved powerful in identifying conserved non-coding elements likely to be distal . In an interactive and intuitive way the website displays data on > 6800 non-coding elements associated with over 120 early developmental genes and conserved across vertebrates. The database regularly incorporates results of ongoing in vivo zebrafish enhancer assays of the CNEs carried out in-house, which currently number ~100. Included and highlighted within this set are elements derived from duplication events both at the origin of vertebrates and more recently in the teleost lineage, thus providing valuable data for studying the divergence of regulatory roles between paralogs. CONDOR therefore provides a number of tools and facilities to allow scientists to progress in their own studies on the function and evolution of developmental cis-regulation.Here we present CONDOR (COnserved Non-coDing Orthologous Regions) available at: By providing access to data with an approachable graphics interface, the CONDOR database presents a rich resource for further studies into the regulation and evolution of genes involved in early development. This is predominantly because we know so little about their language, mode of action and origin, and traditional ways of identifying them are difficult and laborious. The burgeoning field of evolutionary developmental biology (termed evo-devo) is increasingly shifting its emphasis towards the importance of gene regulatory networks in the evolution of developmental pathways [In recent years, although great advances have been made in our ability to annotate gene sequences, the annotation of sequence elements that control complex gene expression is lagging far behind. With exons accounting for only a tiny proportion of the genome < 1.5%) there remains a vast amount of unannotated sequence, containing an unknown number of functional elements. In particular, the annotation of the complex network of .5% therepathways -5). ThesThe confidence with which putative functional non-coding elements can be identified depends on the evolutionary distance between the species selected, and a balance must be found between eliminating background noise and retaining sufficient sequence similarity for detection . The incFugu rubripes, with its highly compact non-coding regions, has been commonly used as a model genome to increase the efficacy of detecting putative functional sequences. The similarities between mammals and Fugu are recognisable despite their large evolutionary divergence. Both vertebrates, they share not only the defining characteristic of a backbone, but also many basic anatomical and physiological similarities and patterns of development. The strong parallels in early developmental patterning are likely to be under control of a common core set of genes and regulatory elements. Significantly, a strong spatial association between highly conserved non-coding elements (CNEs) and genes involved in transcriptional regulation and/or development (termed trans-dev genes) has been described in many studies [in-vivo function reproducibly to up-regulate tissue-specific expression of reporter genes during development.The large evolutionary distance between fish and mammals means that even the slowest evolving DNA has diverged sufficiently to significantly improve the signal to noise ratio in genomic alignments. In particular the genome of the pufferfish,  studies ,4,7-9. F studies ) or usin studies  are cons studies  and zebrcis-regulation in early developmental genes. Here, such elements are rigorously defined by optimal alignment strategies for sequences as divergent as fish and tetrapods, ensuring that sequences are derived from orthologous regions. In addition, we cater for the growing number of researchers in evo-devo interested in sets of experimentally verified developmental enhancers. Thus we couple both bioinformatics and functional data in a visual and searchable form.With an increasing supply of functional data, we have created CONDOR (Conserved Non-coDing Orthologous Regions), a database resource for the study of evolutionary conserved Fugu-mammal multiple alignments, in particular the CNEs identified from them. The second stores functional (lab-based) data from in vivo enhancer assays carried out in-house. While these two datasets are integrated in the database their derivation will be outlined separately.The relational database structure can be divided conceptually into two main parts, representing the two main sources of data stored within it. The first relates to the computationally derived dataset from Fugu genome and four mammalian genomes including human, mouse, rat and dog. The full methodology used to identify CNEs can be seen in Figure Fugu scaffolds using additional assembly data (such as BAC ends and sequence from older assemblies) from the Fugu Information Network [Fugu and human genomes [trans-dev genes. The CONDOR database is therefore designed around these distinct regions. Multiple alignments are more sensitive and better suited for identifying conservation between highly diverged sequences such as fish and mammals than more commonly used pairwise alignments [Fugu) encompassing 129 trans-dev genes conserved in synteny with these clusters in both fish and mammals. The chromosomal locations of developmental gene regions currently in the database can be seen in Figure The core of the CONDOR database is made up of a set of more than 6800 CNEs identified through a combination of sensitive multiple and multi-pairwise alignments of orthologous regions between a baseline teleost  Network . The ali genomes . These cignments  and wereignments . Currentcis-regulatory elements can exert their regulatory influence large distances from their target gene [trans-dev gene(s) but rather by the initial whole-genome CNE synteny map resulting in the identification of CNEs that may be located large distances from the trans-dev gene or within the introns of neighbouring genes  they arvergence . CNEs thvergence . The empvergence . The firvergence . The higvergence  means in targets .PAX8 region to 342 in the IRX3/5/6-SALL1 region, possibly reflecting differences in regulatory complexity and the extent to which regulation has been conserved between fish and mammals as well as the possible presence of overlapping regulatory domains. CNEs in CONDOR range in length from 21–864 bp (mean = 117 bp) and cover some 750 Kb of human sequence, or around 0.05% of the non-repetitive genome. Therefore, although this set represents a tiny fraction of the ~5% of the human genome thought to be under selective constraint [Numbers of CNEs in each region in CONDOR vary considerably, ranging from two in the nstraint  it is prnstraint ,11.Fugu). Specific identifiers are only assigned to sequences in these species (as opposed to any of the other vertebrate genome in which a CNE is present), as they are only sequences that can be strictly guaranteed to derive from orthologous regions.It is important for future analyses that published sets of tested CRMs identified through species comparisons can in some way be traced back to a sequence repository and thus properly defined, similar to the definition of sequences in protein or transcript databases. Commonly in currently published work, functionally characterized conserved non-coding sequences are referred to by some arbitrary name and by their location in the genome or the distance from the start of a gene. This often changes with the release of updated genome assemblies and makes it difficult for other researchers to track the sequence back from the literature. In response, CONDOR is the first database to use specific identifiers for conserved non-coding elements in each genome, independent of their current location in a genome assembly. Unique identifiers are used both to refer to a CNE orthologous unit (akin to a cluster of orthologous genes), e.g. CRCNE00000054, which is useful in defining a CNE in any vertebrate genome to which it is conserved, as well as individual sequences within the CNE, e.g. CRCNEAC00000172 (in human), from sequences used to define the element in the original multiple alignment .The central search page allows CNE-containing gene regions to be located from a pull-down list or searched more generally through a text search by gene name, protein accession number, GO term etc for any genes contained within these regions. Once located, CNEs within a gene region can be visualized via an interactive graphical browser (termed 'CONDOR View') or in a tabular form (termed 'Text View'). Each gene region analysed extends as far as the shared synteny and gene order between fish and mammals, allowing for long-range 'Text view' displays information on the CNEs in a tabular format providing additional data to that in 'CONDOR View', giving details of conservation in other vertebrate genomes Figure  and highin silico and experimental annotations can be readily viewed. A similar track in the UCSC Genome Browser is also planned in the future.The database is also available as a permanent DAS source within the most current human, mouse and rat assemblies at the Ensembl Genome Browser  so that The vast majority of CNEs exhibit no sequence identity to any other sequence in the genome ,28,29. Hcis-regulatory elements complementing and adding to the growing list of enhancers tested in the mouse model [CONDOR will enable greater accessibility to ongoing functional data of se model  with thetrans-dev paralogs and offer an important resource for studying cis-regulatory element evolution after duplication and how this influences paralogous gene function. It is worth noting that since CNEs can act over a long range, identifying their target gene can become complex when studying a region that contains more than one developmental gene. In the case of dCNEs, the retention of both element and gene after duplication, strongly implies association [Whilst the vast majority of CNEs conserved across vertebrates are unique in the human genome ,29 an inociation . CONDOR Fugu from alignments where both Fugu gene regions are compared independently to the same  region in mammals. Recent comparative analysis of CNEs in these regions reveals assymetrical evolutionary rates and a pattern of retention and loss of CNEs between duplicated genes indicative of regulatory subfunctionalization [In addition, the CONDOR database is the first resource to make available data on CNEs identified from a number of teleost-specific duplicated regions within lization . To faciIn addition to providing data on the locations and sequence information of each CNE, CONDOR also provides a number of other features to help study the function and evolution of these sequences:in vivo or in vitro analysis, a number of automatically generated PCR primer predictions using the EPrimer3 program [• To facilitate the amplification of CNEs from a genome of choice for  program  are prov• Sensitive BLAST alignments of any CNE against a variety of nucleotide sequence databases can be displayed dynamically . Database searches include a variety of vertebrate genomes  allowing lineage or species specific mutational events to be detected. The output produces the multiple alignment in a number of formats  that can be directly viewed in the browser or downloaded for further downstream analysis.• CNEs from each of the main genomes can be downloaded in bulk as a set of sequences in FASTA format or as a tab-delimited file with substantial sequence and positional data. Bulk downloads can also be filtered by genome, gene region or CNE length and conservation.The database is run on a Solaris platform and housed using the MySQL (v1.4.0) database management system at the School of Biological and Chemical Sciences, QMUL, London. The web interface is run on an Apache web server that serves a set of CGI scripts and static web pages. The "CONDOR-View" graphical browser is created using the Bioperl Bio::Graphics module . CNEs arThe identification and characterisation of distal CRMs that control the complex gene regulatory networks in early vertebrate development remain one of the greatest challenges of the post-genomic era. It is of increasing interest to the scientific community for datasets of predicted CRMs that are well defined, easily accessible and manageable in number. Here we present CONDOR, a database resource of both predicted and experimentally verified orthologous non-coding elements conserved across the vertebrate lineage and associated with early developmental regulators. This resource is designed both for the increasing number of experimental groups interested in prioritising a set of elements for experimental verification and computational users interested in training sets with high regulatory potential.. A full tutorial can be found on the website. Please cite this paper when publishing data using computational data from CONDOR. Use of functional data in publications is only permitted with prior agreement from the corresponding author. Positions of CNEs within vertebrate genome assemblies are constantly updated as new versions are released and in vivo zebrafish enhancer annotations are added to the database automatically once results for each experiment are collated.The CONDOR database is publicly available without payment or registration at in vivo data collection. DG, JC, SS, PS, HC and GE developed and carried out the in vivo enhancer assays. DG and GM helped write parts of the manuscript. GE conceived the database, and participated in its design and coordination.AW carried out all multiple alignments, computational data extraction and post-processing, designed and implemented the database schema and web front end and wrote the first draft of the manuscript. JC designed the zebrafish expression sub-domains and colour-coding system involved in the


These manifold functions impose significantconstraints on the C-peptide structure that are conservedin evolution. After cleavage of proinsulin, the intactC-peptide is stored with insulin in the soluble phase of thesecretory granules and is subsequently released in equimolaramounts with insulin, providing a useful independentindicator of insulin secretion. This brief review highlightsmany aspects of its roles in biosynthesis, as a prelude toconsideration of its possible additional role(s) as a physiologicallyactive peptide after its release with insulin into thecirculation in vivo.The C-peptide links the insulin A and B chains in proinsulin,providing thereby a means to promote their efficientfolding and assembly in the endoplasmic reticulum duringinsulin biosynthesis. It then facilitates the intracellulartransport, sorting, and proteolytic processing of proinsulininto biologically active insulin in the maturing secretorygranules of the


Plasmodium berghei through Anopheles stephensi and the survival of the mosquito both depend non-linearly on parasite density. These processes regulating the development of the malaria parasite within the mosquito may influence the success of transmission-blocking interventions (TBIs) currently under development.The combined effects of multiple density-dependent, regulatory processes may have an important impact on the growth and stability of a population. In a malaria model system, it has been shown that the progression of An individual-based stochastic mathematical model is used to investigate the combined impact of these multiple regulatory processes and examine how TBIs, which target different parasite life-stages within the mosquito, may influence overall parasite transmission.The best parasite molecular targets will vary between different epidemiological settings. Interventions that reduce ookinete density beneath a threshold level are likely to have auxiliary benefits, as transmission would be further reduced by density-dependent processes that restrict sporogonic development at low parasite densities. TBIs which reduce parasite density but fail to clear the parasite could cause a modest increase in transmission by increasing the number of infectious bites made by a mosquito during its lifetime whilst failing to sufficiently reduce its infectivity. Interventions with a higher variance in efficacy will therefore tend to cause a greater reduction in overall transmission than a TBI with a more uniform effectiveness. Care should be taken when interpreting these results as parasite intensity values in natural parasite-vector combinations of human malaria are likely to be significantly lower than those in this model system.Plasmodium species infecting humans in malaria endemic regions, it would be important to quantify the variability and duration of TBI efficacy to ensure that community benefits of control measures are not overestimated.A greater understanding of the development of the malaria parasite within the mosquito is required to fully evaluate the impact of TBIs. If parasite-induced vector mortality influenced the population dynamics of  Plasmodium berghei through Anopheles stephensi depends non-linearly on parasite density [Density-dependent processes that regulate population growth are common in host-parasite systems and can influence the resilience of an infection to control interventions . In a mo density . Two typ density . This mePlasmodium may influence the life-expectancy of the mosquito [An. stephensi was dependent on its age and on the presence and density of P. berghei [P. berghei-An. stephensi model system, may operate in other parasite-vector combinations [In addition to this density-dependent sporogonic development, the mosquito's (and thus parasite's) survival could be influenced by parasite density. Indeed, there is growing evidence that the mosquitoes survival depends on its age ,4 and thmosquito  . In the berghei . These dinations ,9, incluinations -14. TherP. berghei sporozoites at the bite site and the probability that the mouse host will go on to develop blood infection [Plasmodium yoelii-An. stephensi system, despite substantial variability in the number of sporozoites injected between different mosquitoes and over time from the same mosquito. Notwithstanding such a correlation, the presence of parasite-induced, density-dependent vector mortality would make parasite density important so both sporozoite presence and density shall be presented in this paper.The intensity of malaria transmission in endemic areas is typically measured by the entomological inoculation rate (EIR); the annual number of infectious bites received by a person living in such areas. This metric makes the assumption that the infectiousness of a mosquito is the same irrespective of how many sporozoites are within the salivary glands. However, recent evidence indicates that there is a correlation between the number of nfection . The impnfection  recentlyPlasmodium life-stages within the vector [Density-dependent parasite growth may impact upon various malaria control strategies. A number of promising control measures are being developed with the goal of reducing the incidence of human malaria by blocking transmission to and from the mosquito vectors. These include transmission-blocking vaccines (TBVs), which target antigens expressed on the malaria parasite within the mosquito , biologie vector . UnderstP. berghei-An. stephensi model system. Since deterministic models can underestimate the cumulative effect of multiple non-linear functions, an individual-based stochastic model is used to capture the changes in parasite density over the different stages of sporogonic development. The highly overdispersed distribution of parasites recorded among mosquitoes [et al [Plasmodium.In this paper, mathematical models are used to investigate the potential effect on malaria transmission of the multiple density-dependent processes that have been identified in the squitoes ,16,21-23squitoes . The ress [et al  to invess [et al  it was aj within mosquito i, be it macrogametocytes ingested  the excess mortality in the mosquito. Ookinetes could increase susceptibility to bacterial infection as they perforate the mosquito midgut [It is unknown which stage(s) of o midgut , whilst o midgut , nutrieno midgut ,27 or coo midgut . Assessio midgut .If vector mortality were determined by parasite load and not just parasite presence or absence, any variability in intervention efficacy would influence its overall effectiveness. Vaccinated populations typically generate a wide range of antibody responses  and the By way of illustration, the variability in efficacy of all the interventions investigated in this paper was modelled on the range of responses expected from a single immunogenic TBV. Though the distribution of antibody titres generated by any TBV is not well known, it is expected to be similar to that in other human vaccines, which tend to show a log-normal distribution of antibody concentrations ,31. The To aid interpretation of the complex concepts investigated in the paper, a measurement of parasite transmission is devised based on the contribution of an individual mosquito to onwards transmission over its lifetime . This method of estimating the lifetime transmission potential of a mosquito has been used in other mathematical approaches, though the exact definitions vary ,33. In tP. berghei through the mosquito makes the relationship between the number of macrogametocytes ingested and the presence or number of salivary gland sporozoites highly non-linear . To illThe results of the above model were used to re-fit the relationship between parasite density and mosquito mortality rate using the same data and methodology presented in  see Add. The modThe complex relationship between the aggregated parasite distribution and the range of efficacies generated by a potential TBV makes the impact of parasite-induced vector mortality far from intuitive. Ultimately, the impact of parasite-induced vector mortality will depend on the time it takes for an infected mosquito to become infectious, the shape of the relationship between parasite density and mosquito mortality and the distribution of parasites within the population (both before and after an intervention). If vector mortality is determined by the number of oocysts, a mosquito that ingests 100,000 macrogametocytes will bite, on average, 21% fewer times from 16 days after blood-feeding onwards  to prioritize as target(s). The complex relationships between different positive and negative density-dependent processes mean that the life-stage that is best to target may also vary between settings. However, some general conclusions can be drawn from this analysis. Interventions that reduce ookinete density beneath a threshold are likely to have auxiliary benefits as they utilize positive density-dependent processes naturally restricting sporogonic development at low densities. TBIs which fail to reduce ookinete density beneath this threshold will have a lower overall impact on transmission than the efficacy achieved at the targeted life-stage. Unless the intervention successfully clears the infection, any reductions in parasite density could potentially increase the life-expectancy of the mosquito, enhancing transmission. Therefore partially effective interventions with a high variance in effectiveness will be more effective than those that have a more uniform efficacy.P. berghei-An. stephensi laboratory model and this work needs to be repeated in naturally found parasite-vector combinations to draw definitive conclusions about human malaria. Similar density-dependent processes influencing sporogonic development within the mosquito have been identified in other parasite-vector combinations [P. falciparum indicate a sigmoidal relationship between gametocyte density and mosquito infectivity [Model systems provide a powerful tool for identifying testable hypotheses that may be applicable to the transmission of human malaria. The processes investigated in this paper have been identified and quantified in the inations ,36. Indeectivity -14, indiPlasmodium falciparum are lower than those in the experimental system investigated here, with a significant proportion of human hosts having densities too low to detect through standard microscopy [This paper has examined the effect of different potential TBIs on mosquitoes fed on blood with known gametocyte concentrations. More accurate estimation of an intervention's impact at the population level will require information on the distribution of gametocytes within the bitten human population, and this is likely to vary temporally , geograpcroscopy . This maThe chance that a TBI actually increases the average number of infectious bites a human host population is exposed to will depend on the severity of parasite-induced vector mortality, the distribution of parasites within the human host and mosquito population before and after the intervention, and the shape of the relationship between gametocyte density and mosquito infectivity. The probability of infecting a feeding mosquito plateaus at moderate gametocyte densities, though the exact shape of this gametocytaemia-infectivity relationship may vary -41. TherPlasmodium within Anopheles) even a small increase in mortality of this age group may have a significant impact on transmission.The model has been parameterized using mosquito mortality data from laboratory experiments where the insects will live much longer than they would do under natural conditions. In the wild, mosquitoes are likely to make relatively few bites more than 10 days after becoming infected, so malaria transmission will be highly sensitive to small changes in mosquito mortality. In addition, older mosquitoes are much more susceptible to changes in parasite density . Since tThe results of this analysis indicate that interventions with an intermediate or low efficacy are more likely to cause an increase in transmission. Although it is unlikely that any TBI with a low efficacy would be deployed in a public health programme, the effectiveness of an intervention may wane over time so it is important to consider how these low efficacies will influence transmission dynamics. For example, TBV efficacy may decline relatively rapidly as the concentration of antibodies within the bloodstream falls. This means that unless vaccine efficacy is regularly boosted, antibody concentration may drop, causing the level of transmission to increase above pre-intervention levels. This is in additions to any enhancement of mosquito infectivity that may occur as the concentration of serum antibodies falls -44.Plasmodium may benefit from eliciting a mild transmission-blocking immune response. If malaria transmission is determined by sporozoite presence (rather than density), then reducing parasite density once the mosquito is infected will tend to increase overall transmission by reducing the mortality of the insect vector. Pre-fertilization antigens are known to be a target of the natural immune response and it may be more plausible to ascribe this type of immunity to an evolutionary response by the parasite and not the human host.It is interesting to note that if parasite-induced vector mortality operates in natural combinations of medical importance, then Further work is required to identify whether parasite-induced vector mortality acts in malaria endemic regions so that any perverse outcome of TBIs can be thoroughly understood and contained. Taking into account the possible non-linear relationships between different sporogonic life-stages will increase the chance of identifying any changes in mosquito mortality caused by the parasite. Re-fitting vector mortality data indicates that later sporogonic stages are the most likely cause of parasite-induced vector mortality. However, it is likely that more than one parasite life-stage will play a role, as oocysts or sporozoites cannot have caused the elevated mortality seen in mosquitoes immediately after blood feeding.Research into possible TBIs should consider how the development of the parasite within the mosquito may influence study results. The standard method of assessing the efficacy of a TBV is to compare the number of oocysts within mosquitoes fed on immunised/unimmunised blood ,46. ThesIn light of the possible impact of parasite-induced vector mortality, lab-based studies measuring intervention efficacy should compare the survival of mosquitoes between treatment groups over their whole lifetime. This is because vectors that ingest a lower number of parasites may have had a higher probability of surviving long enough to be dissected. Failing to control for this may cause the intervention efficacy to be overestimated. It is also important to investigate the impact of an intervention on the survival of older mosquitoes as they make the greatest contribution to overall transmission. Studies should quantify the distribution of efficacies caused by a TBI (both within the human host and the mosquito) to allow the full impact of a control measure to be estimated. It is also important to understand how the efficacy of an intervention may change over time. Phase II clinical trials of possible TBV candidates should quantify the rate and variance of antibody decay in a range of environmental settings . In addition, ideally the duration of both Phase II and Phase III trials should be lengthened until target antibody concentrations have returned to pre-intervention levels, allowing the full impact of starting an intervention but failing to sustain it to be quantified.Plasmodium may influence mosquito biting frequency [There is evidence that other regulatory processes acting upon the malaria parasite within the mosquito may also be density-dependent in natural populations. For example, requency , feedingrequency  and physrequency . Other frequency , and therequency ,52 and lrequency -55.This paper has highlighted the importance of understanding the population dynamics of the malaria parasite within the mosquito as processes regulating the development of the parasite may enhance or impede the effectiveness of a control strategy. Mathematical models should be used to investigate how an intervention influences both parasite development and vector mortality in order to fully evaluate the effectiveness of the new transmission blocking interventions currently under development.TBI: (Transmission-blocking interventions); TBV: (Transmission-blocking vaccine).The authors declare that they have no competing interests.TSC carried out the mathematical modelling and drafted the manuscript. TSC, EJD and MGB conceived the study. EJD, RES, GKC, JCK and MGB participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.Description of the mathematical model and table of parameter values.Click here for fileThe impact of varying which parasite life-stage causes mosquito mortality on the mean number of potentially infectious bites made by a mosquito in its lifetime. Parasite mortality is dependent on the mean number of ingested ookinetes (blue line), oocysts at day 10 (green line) or salivary gland sporozoites on day 21 (red line). All bites made 10 days after the first bloodmeal are defined as being potentially infectious. The relationship between ookinete density and the later life-stages was estimated using the full model and used to re-fit the mosquito mortality data from [ata from .Click here for fileThe relationship between gametocytaemia and transmission for a Poisson-distributed parasite population. The potential for malaria transmission is dependent on either salivary gland sporozoite density  or prevalence . The mean number of salivary gland sporozoites available to be injected per mosquito and the mean number of infectious bites per mosquito are both estimated assuming the mosquito is infected at its first bloodmeal and correspond to the total during the mosquito's lifetime. The model was run with either no intervention (black line) or representing a TBI which reduced the production of a life-stage by 60%: gametocytes (yellow line); ookinetes (blue line); oocysts (green line); salivary gland sporozoites (red line). The thin dotted-dashed grey line (which overlaps with the red line in A) indicates an overall reduction in sporozoite density/prevalence efficacy of 60% as a benchmark for comparison. The number of parasites of each life-stage among the mosquitoes follows a Poisson distribution (i.e. Click here for file


The development and simulation of dynamic models of terpenoid biosynthesis has yielded a systems perspective that provides new insights into how the structure of this biochemical pathway affects compound synthesis. These insights may eventually help identify reactions that could be experimentally manipulated to amplify terpenoid production. In this study, a dynamic model of the terpenoid biosynthesis pathway was constructed based on the Hybrid Functional Petri Net (HFPN) technique. This technique is a fusion of three other extended Petri net techniques, namely Hybrid Petri Net (HPN), Dynamic Petri Net (HDN) and Functional Petri Net (FPN).The biological data needed to construct the terpenoid metabolic model were gathered from the literature and from biological databases. These data were used as building blocks to create an HFPNe model and to generate parameters that govern the global behaviour of the model. The dynamic model was simulated and validated against known experimental data obtained from extensive literature searches. The model successfully simulated metabolite concentration changes over time (pt) and the observations correlated with known data. Interactions between the intermediates that affect the production of terpenes could be observed through the introduction of inhibitors that established feedback loops within and crosstalk between the pathways.Although this metabolic model is only preliminary, it will provide a platform for analysing various high-throughput data, and it should lead to a more holistic understanding of terpenoid biosynthesis. Biological networks are complex and may consist of hundreds of reactions that directly and indirectly affect each other. In order to understand the relationship between these reactions in detail, it is necessary to view the network as a whole . ComputaVarious approaches can be taken to building and simulating a biological pathway model . The mosWe chose to model the terpenoid biosynthetic network using Hybrid Functional Petri net with extension (HFPNe). This method allows easy modelling of complex biological information using a graphical approach and also allows various types of entities to be modelled together. The technique is derived from the traditional Petri net theory that was first described by Carl Adam Petri in his 1962 PhD dissertation . HFPNe iCaenorhabditis elegans.HFPNe-based biological models are built based on three basic entities called places, transitions and arcs; these are shown in Figure Gallus gallus and Rattus norvegicus, whereas the MEP pathway takes place in the plastids of eubacteria such as Escherichia coli and Streptomyces cerevisiae, as well as in the chloroplasts of plants .-48.46-48To simulate this mutually antagonist inhibitory feedback loop, the 'MEV_switch' component is set to 'true' and the other two switches are set to 'false.' The parameter that controls the production of ATP is adjusted to a higher value of 30 unit/pt from 10 unit/pt, resulting in overproduction of ATP by 3-fold and activates the inhibitory arc. The threshold value for the inhibitory arc that suppresses the production of ATP is set to 80 units to allow the inhibitory effect to take place.The second feedback loop identified in the MEV pathway is a positive feedback mechanism. It is initiated when FPP levels become high enough to inhibit the catalytic activity of mevalonate kinase . MK catalyses the fourth reaction in the MEV pathway, phosphorylation of mevalonate to produce mevalonate-5-phosphate (MEVP) . Suppression of DXR activity reduces the rates of the downstream reactions in the MEP pathway, affecting the production of monoterpenes Figure  51]. Th. Th51]. To simulate the crosstalk mechanism, the 'MEV_switch' component is set to 'false.' The 'MEP_switch' component is set to 'true' while the 'crossSwitch' component is given a script value. The script value enables the activation of the 'fosmidomycin' component after 150 pt, thereby establishing the crosstalk mechanism. The simulation result successfully described oscillatory changes in the concentrations of the metabolites involved over time.The simulation and validation processes performed using the model are consistent with known biological information and data. The intuitive approach introduced by the HFPNe technique enables intricate modelling tasks to be viewed and solved in a graphical perspective. The model serves as a tool for better understanding the reactions involved in both pathways and how they affect each other. Perturbations performed on the model provide insight into the effects of manipulation of a single reaction on the whole network and thus should facilitate both industrial and biomedical terpenoid bioengineering.The biological information and data needed to construct this model included information on the substrates, products and enzymes involved in the MEV and MEP pathways, as well as detailed enzymatic reaction information such as reaction stoichiometry and enzyme mechanisms. Consumption and production rates were also taken into consideration in developing the dynamic model. This information was used to design the model layout and parameter assignments for each of the HFPNe elements. The data were obtained through the use of extensive literature and biological databases including KEGG, BRENDA, ENZYME, IUBMB, MetaCyc and PATHWAY DATABASE ,52-56.The HFPN elements  represent the metabolites and processes that comprise the MEV and MEP biosynthetic pathways. Continuous places represent the substrates, products, inhibitors and enzymes in both subnetworks, whereas continuous transitions represent biological processes such as catalysis, phosphorylation and degradation. Generic places represent the on/off switches and parameter modulators. The normal arcs then connect places  to transitions while inhibitory arcs connect inhibitors to their prospective inhibited processes. All these components were arranged according to the reaction stoichiometry and enzymatic mechanism in their order of occurrence in the respective pathways Tables  and 2.Each of the HFPNe elements possesses different attributes that characterise its function in the model. Parameter values are assigned to each of these attributes to control the behaviour of the model during simulation. These values are generated based on the biological information obtained and are translated into virtual concentration values , ordinary differential equations (speed formulas of transitions), numeric values  and scripts (on/off switches) ,18,22-25The test-driven models were simulated and validated against known data obtained through literature searches. Simulations were carried out under five different conditions as explained in the Results and Discussion section. Simulation results were displayed as concentration (unit) versus time (pt) graphs. Petri net time (pt) indicates virtual Petri net time units that do not correspond to real time; concentration also is given in general concentration units (unit) that do not specifically correspond to actual concentration units such as mM and μM. The changes in metabolite concentrations (unit) over time predicted by each simulation were validated against known biological data to identify gaps and inconsistencies. Uncorrelated biological behaviours were re-examined and previous developmental steps  were repeated. The simulation process was then re-executed and revalidated against known biological information. The entire process was carried out iteratively in order to rule out inconsistencies and obtain an optimum system.ADP: (adenosine diphosphate); ATP: (adenosine triphosphate); DMAPP: ; DXR: (deoxyxylulose-5-phosphate reductoisomerase); EC: (enzyme commission); GPP: (geranyl diphosphate); HDN: (Hybrid Dynamic Petri net); HFPNe: ; HPN: (Hybrid Petri net); IPP: (isopentenyl diphosphate); FPN: ; FPP: (farnesyl diphosphate); pt: (Petri net time); MEP: (methylerythritol-5-phosphate); MEV: ; MEVP: ; MEVPP: ; MK: ; ODE: ; PMK: .AHH performed the research and drafted the manuscript. ZAMH formulated the study, gave valuable insights, participated in its design and helped to draft and refine the manuscript. All authors read and approved the final manuscript.Two simulation files to complement Figure 3.Click here for file


LOXL1 is a member of the lysyl oxidase family of enzymes, which are essential for the formation, stabilization, maintenance, and remodelling of elastic fibers and prevent age-related loss of elasticity of tissues. LOXL1 protein is a major component of exfoliation deposits and appears to play a role in its accumulation and in concomitant elastotic processes in intra- and extraocular tissues of XFS patients. This discovery should open the way to new approaches and directions of therapy for this protean disorder.Exfoliation syndrome (XFS) is an age-related, generalized disorder of the extracellular matrix characterized by the production and progressive accumulation of a fibrillar extracellular material in many ocular tissues and is the most common identifiable cause of open-angle glaucoma worldwide. Exfoliation syndrome plays an etiologic role in open-angle glaucoma, angle-closure glaucoma, cataract, and retinal vein occlusion. It is accompanied by an increase in serious complications at the time of cataract extraction, such as zonular dialysis, capsular rupture, and vitreous loss. It is associated systemically with an increasing number of vascular disorders, hearing loss, and Alzheimer's disease. Exfoliation syndrome appears to be a disease of elastic tissue microfibrils. Directed therapy simply means devising specific treatments for specific diseases. There was little incentive to attempt to distinguish between various open-angle glaucomas if the treatments were essentially the same. However, this view also prevented the application of directed therapy in those instances in which such was available and applicable. Pilocarpine has multiple beneficial actions in eyes with XFS. Not only does it lower IOP, but by increasing aqueous outflow, it should enable the trabecular meshwork to clear more rapidly, and by limiting pupillary movement, should slow the progression of the disease. Theoretically, miotics should be the first line of treatment. Pilocarpine 2% q.h.s. can provide sufficient limitation of pupillary mobility without causing these side effects. In 2007, two common single nucleotide polymorphisms in the coding region of the  Exfoliation syndrome (XFS) is an age-related disease of the extracellular matrix characterized by the production and deposition of a fibrillar extracellular material in numerous ocular tissues. It is now recognized as the most common identifiable cause of open-angle glaucoma, affecting an estimated 60 to 70 million people worldwide.1Exfoliation syndrome is diagnosed by the presence of typical exfoliation material (XFM) on the anterior lens surface or pupillary border. All anterior segment structures are involved. The classic pattern of deposition on the lens consists of three distinct zones that become visible when the pupil is fully dilated, a central disc, intermediate clear zone created by the iris rubbing XFM from the lens surface during its physiologic excursions, and a granular peripheral zone. The material on the anterior lens capsule causes rupture of iris pigment epithelial cells leading to pigment dispersion, trabecular meshwork hyperpigmentation, iris transillumination defects at the pupillary margin and loss of the pupillary ruff. Pigment dispersion after dilation can lead to marked IOP rise and may be one of the earliest manifestations of the disease. Exfoliation material is often found at the pupillary border.Patients with XFS are twice as likely to convert from ocular hypertension to detectable glaucoma.The prevalence of XFS increases with age in all affected populations. Approximately two-thirds of patients have unilateral disease on clinical examination, however, XFS is often detectable in the contralateral eye with conjunctival biopsy.The pathogenesis of XFS is defined by the accumulation of an abnormal fibrillar material biochemically related to the composition of the extracellular matrix that results either from excessive production and/or insufficient breakdown. The product has a unique structure as seen by electron microscopy.78Two common single nucleotide polymorphisms (SNPs) in the coding region of the lysyl-oxidase-like 1 (LOXL1) gene on chromosome 15 have been identified and account for virtually all cases of XFS in several populations.LOXL1 is a member of the lysyl oxidase enzyme family responsible for formation, stabilization and remodeling of elastic fibers. They are important for cross-linking of elastin, for providing a scaffold that ensures spatially defined deposition of elastin and for regulating the production of elastin.13XFS is associated with both chronic open-angle glaucoma and angle closure. The zonules and ciliary body are often diffusely coated with XFM and zonules are often frayed and broken. Zonular fragility and poor pupillary dilation contribute to an increased incidence of serious complications during cataract extraction, including zonular dehiscence and vitreous loss. Phacodonesis and even subluxation of the lens or intraocular lens is common.2aIt appears that XFS is etiologically related to cataract formation itself. Decreased aqueous humor ascorbic acid concentrations in XFS,Ocular ischemia is clearly correlated with XFS. An increased frequency of retinal vein occlusion has been noted in XFS26728As the term implies, directed therapy aims to specifically address and interfere with the underlying disease pathogenesis. Ophthalmologists have begun to recognize the inadequacy of therapy limited solely to IOP reduction, particularly with the significant potential morbidities associated with XFS. The widespread systemic and ocular manifestations of XFS underscore the need for a broad and comprehensive treatment paradigm. It is essential that future investigations focus on disease-specific therapeutic modalities.The management of XFS currently hinges on ocular antihypertensive therapy and initially can be achieved with topical therapy in many cases. Prostaglandin analogues such as latanoprost and travoprost reduce IOP at each time point in the 24-hour diurnal curve as compared to untreated IOP, with travoprost conferring a slightly higher level of reduction.41Pilocarpine has multiple beneficial actions in eyes with XFS. Not only does it lower IOP, but by increasing aqueous outflow, it should enable the trabecular meshwork to clear more rapidly, and by limiting pupillary movement, should slow disease progression. Theoretically, miotics should be the first line of treatment. Prostaglandin analogues are complementary in increasing uveoscleral outflow facility. By decreasing outflow, aqueous suppressants may cause longterm worsening of trabecular function that may be particularly detrimental in XFS.42Unfortunately, pilocarpine is underutilized in the treatment of glaucoma due to the possibility of decreased vision or the misconception that it must be used q.i.d. It is our experience that pilocarpine 2% q.h.s. can provide significant limitation of pupillary movement without the inconvenience of q.i.d. dosing and dimming of vision. Pilocarpine can also blunt an early morning IOP spike.43Argon laser trabeculoplasty is particularly effective in XFS.48The investigation of surgical therapy directed at trabecular dysfunction is particularly sensible for XFS. Trabeculotomy has been reported as successful in XFS although additional trials are required.Drugs that alter the structure of the trabecular meshwork may be particularly effective in XFS. Latrunculin B is currently being evaluated in human trials for its ability to bind to free actin in trabecular cells leading, to temporary disintegration of the existing actin cytoskeletin thereby increasing aqueous outflow.5355Future therapy of XFS should be directed at preventing formation of XFM or depolymerizing existing XFM. An intriguing line of research is an investigation of the factors that protect the contralateral eye from developing XFS. If the development of the disease in the second eye is actively suppressed, as by an immune mechanism, then elucidation of this could lead to a novel treatment. The systemic ramifications of XFS and the morbidity associated with these diseases highlight the need for a therapy that prevents aggregation of XFM microfibrils disaggregating the microfibrils.Homocysteine is an amino acid, elevations of which result from a disturbance of methionine metabolism. Hyperhomocysteinemia is a widely recognized cardiovascular risk factor and is reversible with dietary supplementation with folic acid, pyridoxine and cyanocobalamine.60The discovery of the role of LOXL1 polymorphisms in XFS draws attention towards lysyl oxidase and its role in XFM production. Lysyl oxidase is inhibited by homocysteine in vascular endothelia and is also regulated by transforming growth factor-fl 1 (TGF-fl 1).67Patients with XFS have an increased likelihood to have coexisting cerebrovascular and cardiovascular disease, and systemic agents that provide neuroprotection and vasoprotection may prove to be an important therapeutic tactic in the treatment of XFS. Pharmacologic treatment of non-IOP dependent mechanisms in glaucoma has traditionally been limited to calcium channel blockers that are widely used in treatment of hypertension, coronary artery disease, migraines and Raynaud's disease. The effect of calcium channel blockers on visual field progression and ocular blood flow is disputed.71Gingko biloba extract (GBE) has been used in Chinese medicine since 3000 BC for the treatment of aging, dementia, tinnitus, bronchoconstriction and sexual dysfunction with minimal side effects. It improves peripheral, cerebral and ocular blood flow, prevents platelet aggregation and protects against free radical damage.7274Curcuma longa and is a component of the commonly used Indian spice turmeric. Curcumin has antioxidant, anti-inflammatory, anti-angiogenic and anti-neoplastic activity via the inhibition of numerous mediators of inflammation.Curcumin is an anti-oxidant extracted from the plant Resveratrol is a compound found in the skin of red grapes. It is an effective antioxidant and protects against degeneration of neurons in ischemia. It is hypothesized that resveratrol is responsible for the “French paradox”, the observation that the French have a lower incidence of heart disease despite a high fat diet due to the consumption of red wine. It is an effective antioxidant and prevents neuron degeneration.7980XFS is a systemic disorder with components of low-grade inflammation and oxidative damage leading to accumulation of XFM. The ocular morbidity associated with XFS far exceeds that of open-angle glaucoma, and a host of systemic diseases with considerable morbidity and mortality are associated with XFS. Further elucidation of the genetics and pathophysiology of XFS is crucial to our ability to treat this disease effectively. There is a role for non-IOP lowering treatment modalities in XFS as there is significant evidence of generalized ischemia and neurodegeneration. With this directed investigation, there is hope that XFS will be both a preventable and curable disease.


Esophageal perforation is associated with a significant risk of morbidity and mortality. We report herein a case of lye-induced esophageal perforation managed successfully by employing endoscopic T-tube placement with a successful outcome. Esophageal perforation may represent a life-threatening emergency, and delay in diagnosis can result in a significant increase in morbidity and mortality. The diagnosis depends on a high degree of suspicion, recognition of clinical features, confirmation by contrast esophagography or endoscopy and appropriate treatment. The most common cause of esophageal perforation is iatrogenic injury. Other causes include spontaneous perforation (Boerhaave syndrome), caustic chemical ingestion, foreign body penetration, and trauma. The outcome after esophageal perforation is dependent on the cause and location of the injury, the presence of underlying esophageal disease, the interval between injury and initiation of treatment, and the patient's overall health. Mediastinal and intra-thoracic perforations identified within 24 hours are usually managed by surgical repair. Later presentations are managed depending on the condition of the patient, and approaches include chest tube drainage, exclusion and diversion, the insertion of endoscopic endoprosthesis, or thoracotomy with repair and debridement. Because of concern over the failure of primary repair, particularly where surgery is not performed within 24 hours, a controlled esophageal fistula may be established by the insertion of a T-tube at the site of perforation ,2. We adA 51-year old gentleman with a background history of autistic spectrum disorder, depression and obsessive-compulsive disorder was admitted to the intensive care unit (ICU) following transfer from a referring hospital where he initially presented 6 days previously with a 24-hour history of vomiting and abdominal pain following ingestion of a lye detergent. There was no evidence of airway damage, and no initial evidence of esophageal perforation, and he was managed conservatively. On day 5 post admission he became septic, and imaging revealed air and contrast in the mediastinum and right thoracic cavity (Figure He was then transferred to this tertiary centre. On initial admission to ICU he was septic, with fever and hypotension, and signs of progressive respiratory failure. He was intubated and large-bore intercostal drains were inserted bilaterally. Esophagogastroduodenoscopy showed diffuse esophageal mucosal injury and but no obvious sign of perforation, and a repeat CT showed no obvious leak of contrast from the esophagus. A naso-jejunal tube was inserted for nutritional support. A percutaneous tracheostomy was inserted due to an anticipated requirement for prolonged ventilation. On day 30 in the ICU, gastric fluid leakage of 500 ml emerged from the right-sided chest drain. A repeat endoscopy revealed a large esophageal perforation in the lower esophagus opening into the right thorax, and the tip of the chest tube could be seen through this defect in the thoracic cavity.The decision was made to insert a T-tube endoscopically (see Additional file A follow up tubogram showed good flow of contrast into the stomach and esophagus via the T limbs with no leak of contrast from the site of perforation Figure . The T-tEsophageal perforation is associated with significant morbidity and mortality . It is uEsophageal tissue is less amenable to repair after the first 24 hours. In a review, Brinster et al reported that primary repair of early esophageal perforations is associated with a low incidence of leak and a mortality rate of 10%, but that the mortality of surgery beyond 24 hours approaches 40%, and this may be associated with a high incidence of fistula . A numbeIn this case, the delay in diagnosis and the initial assessment of a healed perforation suggested that a non-operative course was appropriate as long as the mediastinal and thoracic sepsis could be adequately drained. When gastric contents presented themselves via the chest drain a full month following the initial insult, we adapted this principle and were able simply to place the T-tube using the combination of access via endoscopy and the intercostal chest tube, thus establishing a controlled fistula. The exclusion criterion for this novel endoscopic treatment is acute esophageal perforation within first 24 hours. It is an option for delayed diagnosis of esophageal perforation. We acknowledge that the subsequent improvement in his clinical condition may have occurred in any case, and the perforation may eventually have healed, but we wish to highlight the ease of this technique and the benefits in terms of advancing his oral intake and a successful outcome.This case report highlights the safety, simplicity and efficacy of T-tube drainage in the management of delayed esophageal perforation, and this may represent a useful adjunct in the non-operative management of selected.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.MAM and FO drafted the manuscript. KTL prepared the figures and movie clip, reviewed, amended and finalized the manuscript. NR provided technical information and reviewed the manuscript. JVR critically reviewed, amended and finalized the manuscript. All authors read and approved the final manuscript.Endoscopic esophageal T-tube insertion for perforated esophagus. The movie clip shows the step by step techniques of endoscopic esophageal T-tube insertion for perforated esophagus.Click here for file


In this study, the various factors determining the out-of-pocket expenditure on child health care by households are discussed to answer the following questions: How much are households currently spending on child health care? Is there any role of socio-economic status of households on expenditure on child health care? What percentage of their income is spent on child health care and is it catastrophic?Four slums with a total a population of 7000 were selected for this study. Households where there is history of illness/ sickness in children under 5 years in last one month were included in the study.There were a total of 218 episodes of child illness in the households. The household's belonging to socio- economic class I and II had higher spending on child's illness per episode as compared to households of socio- economic class III, IV, and V. Socioeconomic status was the key determinant of health care expenditure.In this study, it has been found that almost all the households suffered from catastrophic health expenditure. The relationship between poverty and ill-health is indisputable. There is more recent evidence to show the effect of illness on poverty. Even relIndia has adopted the tax-based model of financing health care. However, decades of under-funding have resulted in poor infrastructure, vacant posts, and poor quality health care. This makes the patients seek private health care for their needs, with its associated out-of-pocket payments. Studies show that about a quarter of Indians who are hospitalized are impoverished because of the medical costs.We tried to study the following factors determining the out-of-pocket spending on child health care by households to answer following questions: How much are households currently spending on child health care? Is there any role of socio-economic status of households on expenditure on child health care? What percentage of their income is spent on child health care and is it catastrophic?Four slums with total population of 7000 adjoining urban health training centre were selected for the study. A total of 658 children under the age of five years reside in the area. All the households having children aged less than 5 years were visited. Only those households where there is history of illness/sickness in children under 5 years in last one month were included in the study to minimize the recall bias. The head of households and the mothers of the sick children were interviewed by using pretested and prestructured questionnaire. Questions were asked on the type of illness the child was suffering, place where treatment was sought, and information on expenditure incurred. The socio- economic classification of household's was done using modified Prasad's scale according to current consumer price index. The data so collected was analyzed using EPI6 software in the department of Community Medicine.Data from 190 households were collected and analyzed, where at least one child aged less than 5 years was ill in the past one month period. There were total 218 episodes of child illness in the households. The mean duration of each episode was 4.6 days. 158 episodes were treated at private hospitals, 53 episodes at public hospitals whereas for 7 episodes no treatment was sought. Out of 190 households, only 13 households were having some form of health insurance. Thirty households (16%) had some plan for health insurance.Our results showed that socio-economic status was the key determinant of health care expenditure. These findings are similar to that reported in multicountry analysis of catastrophic household healthcare expenditure. The poorWe conclude that poorest members of community incurred catastrophic health expenses. Our findings have important policy implications and can be used to ensure better access to health services and a higher degree of financial protection for low-income groups against economic impact of illness.