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Genome-scale metabolic models are available for an increasing number of organisms and can be used to define the region of feasible metabolic flux distributions. In this work we use as constraints a small set of experimental metabolic fluxes, which reduces the region of feasible metabolic states. Once the region of feasible flux distributions has been defined, a set of possible flux distributions is obtained by random sampling and the averages and standard deviations for each of the metabolic fluxes in the genome-scale model are calculated. These values allow estimation of the significance of change for each reaction rate between different conditions and comparison of it with the significance of change in gene transcription for the corresponding enzymes. The comparison of flux change and gene expression allows identification of enzymes showing a significant correlation between flux change and expression change (transcriptional regulation) as well as reactions whose flux change is likely to be driven only by changes in the metabolite concentrations (metabolic regulation). The changes due to growth on four different carbon sources and as a consequence of five gene deletions were analyzed for Saccharomyces cerevisiae. The enzymes with transcriptional regulation showed enrichment in certain transcription factors. This has not been previously reported. The information provided by the presented method could guide the discovery of new metabolic engineering strategies or the identification of drug targets for treatment of metabolic diseases.
Author Summary
The sequencing of full genomes and the development of high-throughput analysis technologies have made available both genome-scale metabolic networks and simultaneous transcription data for all the genes of an organism. Genome-scale metabolic models, with the assumption of steady state for the internal metabolites, allow the definition of a region of feasible metabolic flux distributions. This space of solutions can be further constrained using experimental flux measurements (normally production or uptake rates of external compounds). Here a random sampling method was used to obtain average values and standard deviations for all the reaction rates in a genome-scale model. These values were used to quantify the significance of changes in metabolic fluxes between different conditions. The significance in flux changes can be compared to the changes in gene transcription of the corresponding enzymes. Our method allowed for identification of specific reactions that are transcriptionally regulated, and we further identified that these reactions can be ascribed to a few key transcription factors. This suggests that the regulation of metabolism has evolved to contain a few flux-regulating transcription factors that could be the target for genetic manipulations in order to redirect fluxes.
Citation: Bordel S, Agren R, Nielsen J (2010) Sampling the Solution Space in Genome-Scale Metabolic Networks Reveals Transcriptional Regulation in Key Enzymes. PLoS Comput Biol 6(7): e1000859. https://doi.org/10.1371/journal.pcbi.1000859
Editor: Jennifer L. Reed, University of Wisconsin-Madison, United States of America
Received: March 18, 2010; Accepted: June 14, 2010; Published: July 15, 2010
Copyright: © 2010 Bordel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by the European Research Council. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Systems Biology aims to use mathematical models to integrate different kinds of data in order to achieve a global understanding of cellular functions. The data to be integrated differ both in their nature and measurability. The availability of DNA microarrays allows for the comparative analysis or mRNA levels between different strains and conditions. These data provide genome-wide information, and changes in expression at different conditions are expressed in statistical terms such as p-values or Z-scores that quantify the level of significance in transcriptional changes. The availability of annotated genome-scale metabolic networks allowed mapping of the transcriptional changes in metabolic genes on to their corresponding metabolic pathways and defining significantly up or down regulated sub-networks [1]. Even though this allows for identification of transcriptional hot-spots in metabolism, this does still not provide information about whether there are any changes in metabolic fluxes in these pathways, as it has been shown that in general there is no clear correlation between gene expression and protein concentration [2] or metabolic flux [3], [4].
Metabolic fluxes are the result of a complex interplay between enzyme kinetics, metabolite concentrations, gene expression and translational regulation. Metabolic fluxes can be directly measured using 13C labeling experiments [5]. However, flux data obtained using this approach differ from gene expression data in two main features: 1) their determination is only possible for a relatively small subset of all the reactions in a genome-scale metabolic network and 2) they are indirect data in the sense that the fluxes are quantified obtained by fitting measured labeling patterns using a simple metabolic model. The complexity of the mRNA-flux dependence and the disparity in the nature of both kinds of data make their integration an important challenge.
In this paper we propose a method to integrate gene expression data with flux data by transforming a limited amount of quantitative flux data into a genome-scale set of statistical scores similar to the one obtained from DNA microarrays. In order to do that, a set of experimental exchange fluxes are fixed for each of the studied conditions or for each of the strains investigated, and a sampling algorithm is then used to obtain a set of flux distributions satisfying the experimental values. This approach allows for obtaining means and standard deviations for each flux in the genome-scale network. From the mean and standard deviation it is possible to derive statistical scores for the significance of flux change between conditions [6], [7]. Random sampling in the region of feasible flux distributions has been previously used to study the statistical distribution of flux values and determine a flux backbone of reactions carrying high fluxes [8] as well as to define modules of reactions whose fluxes are positively correlated [9], [10]. Also mitochondria related diseases have been analyzed using random sampling [11]. All the works published so far used the Hit and Run algorithm to perform the sampling [7].
By dividing the average difference among two conditions (e.g. carbon sources or mutant strains) by its standard deviation, it is possible to obtain Z scores for each metabolic flux. These scores can be transformed into p-values that measure the significance of change of each flux (see methods). By comparing these p-values with the p-values derived from gene-expression arrays, the enzymes in the network can be classified as: 1) enzymes that have a significantly correlated change both in flux and expression level (reactions showing transcriptional regulation), 2) enzymes that show a significant change in expression but not in flux (we will refer to them as showing post-transcriptional regulation) and 3) enzymes that show significant changes in flux but not a change in expression (metabolic regulation). Hereby we provide a framework that allows for global classification of reaction fluxes into those that are transcriptionally regulated, post-transcriptionally regulated and metabolically regulated (see Fig. 1). This will have substantial impact on the field of metabolic engineering where changes in gene-expression are often used as the key means to alter metabolic fluxes. In the paper we show the use of the presented framework for the analysis of the yeast Saccharomyces cerevisiae grown at different growth conditions and for the analysis of different deletion mutants.
The fluxes can be regulated at the level of mRNA transcription, by the concentrations of the metabolites or by intermediate steps such as translation or activation of the enzymes.
The combined use of random sampling of genome-scale metabolic networks and expression data allows for global mapping of reactions that are either transcriptionally or metabolically regulated. This information can be used to guide the engineering of microbial strains or as a diagnosis tool for studying metabolic diseases in humans. In particular we should highlight that reactions in which there is no relation between gene transcription level and metabolic flux are not suitable targets for flux increase via gene over-expression. Through analysis of different data sets the method revealed that many changes in gene expression are not correlated with a corresponding change in metabolic fluxes. The use of gene-expression data alone can therefore be misleading. However, our method allowed for identification of many specific reactions that are indeed transcriptionally regulated, and we further identified that the expression of these enzymes is regulated a few key transcription factors. This fact suggests that the regulation of metabolism has evolved to contain a few flux-regulating transcription factors that could be the target for genetic manipulations in order to redirect fluxes.
Comparisons between different sampling methods
Here we propose a sampling method that finds extreme solutions among the feasible flux distributions of the metabolic network. These solutions correspond to the corners in the region of allowed flux distributions, and in mathematical terms they are elements of the convex basis of the region of feasible solutions (which is a convex set). The COBRA Toolbox [12] includes a random sampling option that uses the Hit and Run algorithm [13] to obtain points uniformly distributed in the region of allowed solutions. The difference between the two sampling methods is illustrated in Fig. 2.
The blue points illustrate the uniform random sampling inside the space of allowed solutions.
In order to assess the accuracy of our sampling method to estimate the average fluxes and their standard deviations, we compared a set of internal fluxes measured with 13C labeling [14] with predictions using 500 sampling points obtained using the sampling method in the convex basis and 500 sampling points obtained using the sampling algorithm implemented in the COBRA Toolbox. The results are summarized in Table 1 where our method is labeled Convex Basis (CB), because it samples elements of the convex basis of the region of allowed solutions (see above), and the method from the COBRA Toolbox is labeled Hit and Run (HR). The Z values in the table are the number of standard deviations that the real value is deviating from the calculated mean.
The means obtained by the two sampling methods are very similar for most of the reactions; however the standard deviations found using the HR algorithm are significantly smaller. With the HR method the real values for the fluxes in many cases deviate several standard deviations from the mean, A high value of Z indicates that the real value has a very low chance of being obtained using the considered sampling method (or in other words: the real value does not belong to the family of solutions that is generated by the sampling method). The number of samples with the HR algorithm was increased up to 5000 to check possible effects of the sample size on the standard deviation. Only small increases were observed for the standard deviations of the studied fluxes.
Using the CB algorithm we obtain higher standard deviations and the real flux is for most reactions less than one standard deviation away from the mean flux. We can therefore conclude that the CB sampling method gives more realistic standard deviations for the fluxes. This is important if we want to compare the significance of flux changes between conditions. An underestimated standard deviation would make some flux changes appear as being significant even though they may not be in reality, and our method therefore gives a more conservative list of significantly changed reaction fluxes than the HR algorithm.
Comparisons between different carbon sources and mutant strains
To evaluate our method we used data for the yeast S. cerevisiae. Data from growth on four different carbon sources (glucose, maltose, ethanol and acetate) in chemostat cultures and five deletion mutants (grr1Δ, hxk2Δ, mig1Δ, mig1Δmig2Δ and gdh1Δ) grown in batch cultures were used. The exchange fluxes and gene expression data for the mentioned conditions have been published earlier [15]–[17].
Our method obtains probability scores for each enzyme in the metabolic network (see methods) and this allowed us to classify the enzymes as transcriptionally regulated (correlation between flux and gene expression), post-transcriptionally regulated (changes in gene expression don't cause changes in flux) and metabolically regulated (changes in flux are not caused by changes in gene-expression). The cut-off chosen for this classification was a probability score above 0.95. Tables 2 and 3 show the 10 top scoring enzymes in each group (or fewer when less than 10 enzymes had a score exceeding 0.95). The method is illustrated in Fig. 3.
Two kinds of data are extracted from fermentations, gene expression data and production and consumption rates of different metabolites. The gene expression data are transformed into significance scores and p-values for the expression change of the metabolic genes. The measured fluxes are used to constrain the solution spaces corresponding to different conditions. A sampling among the allowed solutions gives averages and standard deviations for each reaction rate in the metabolic network. These values can be obtained to obtain significance scores and p-values for the changes in reaction rates. The p-values for changes in expression and in reaction rates can be combined to obtain the probabilities for a correlated change between both values (transcriptional regulation), changes in rate not correlated to transcriptional changes (metabolic regulation) and changes in transcription that are not correlated to changes in rate (which we refer to as posttranscriptional regulation).
The method to identify the significance of flux changes relies on a set of measured external fluxes, and in some cases strains that don't show significant changes in external fluxes have changes in internal fluxes [18]. These changes cannot be identified with our method, and our estimations of the significance of flux changes can therefore be seen as conservative estimates. The lists of transcriptionally and metabolically regulated reactions are therefore more reliable than the list of post-transcriptionally regulated reactions (in which some fluxes may be changed in reality but their change pass undetected).
The reactions showing transcriptional regulation form a set of putative targets where enzyme over-expression or down regulation will influence the flux through these reactions. The reactions showing metabolic regulation points to parts of the metabolism where the pools of metabolites are possibly increasing or decreasing in connection with transcriptional changes and hereby counteracting possible changes in enzyme concentration as a result of transcriptional changes. This knowledge can be used to identify whether one should target changes in enzyme concentration (vmax changes), e.g. through over-expression, or changes in enzyme affinity (Km changes), e.g. through expression of heterologous enzymes, in order to alter the fluxes.
Effects of different carbon sources.
In the glucose to maltose transition, only two enzymes showed transcriptional change correlated with their flux. The α-glucosidase MAL32, responsible for the breakdown of maltose into glucose was up-regulated and the glucose transporter HXT4 was down-regulated. The metabolic adjustment in terms of fluxes was minimal and only enzymes directly related with substrate uptake and utilization were detected. The changes in gene expression were also low and only 11 metabolic enzymes were significantly perturbed (without significant flux changes).
The glucose to ethanol and the glucose to acetate transitions showed widespread flux and expression changes, and they are therefore more interesting study cases. In the glucose-ethanol transition 19 enzymes showed transcriptional regulation and 22 other enzymes changed in expression but not in flux. For the glucose-acetate transition the same numbers were 33 and 23 respectively. We can see that about one half of the genes that changed in transcription level also showed significant flux changes, this proportion is higher than in the case of deletion mutants (as it will be discussed later). Among the enzymes showing transcriptional regulation, 14 were shared between the glucose-ethanol and glucose-acetate transitions. Interestingly, no overlap was found between the sets of enzymes that don't change in flux. Metabolic regulation was observed in 21 reactions for each case, among which 8 overlap.
The enzymes showing transcriptional regulation clearly show a down regulation of enzymes involved in glucose uptake and utilization (e.g. Glucose transporter HXT4 or Hexokinase 2) and the up-regulation of enzymes involved in the gluconeogenesis (e.g. Fructose-1,6-biphosphatase) and the TCA cycle (e.g. Succinate dehydrogenase or Citrate synthase). The AcCoA synthetase 2, responsible for supplying AcCoA to the TCA cycle is also transcriptionally up-regulated as well as the ATP synthetase (involved in the respiratory chain) and the external NADH-ubiquinone oxidoreductase 2, which supplies the necessary NAD+ to oxidize ethanol or acetate in the cytoplasm and maintain the redox balance in the cell. Isocitrate lyase, a key component of the glyoxylate cycle, is also transcriptionally up regulated and this allows for net formation of malic acid that can further be converted to phopshoenolpyruvate (via oxaloacetate) that fuels the gluconeogenesis. All these changes in fluxes are consistent with general knowledge about the changes in metabolism from glucose to C2 carbon sources like ethanol and acetate, but what is interesting to see is that not all the reactions associated with these flux changes are transcriptionally regulated, but the cell have selected a few key reactions to regulate at the transcriptional level and these are identified using our method.
In order to make a deeper analysis, we performed an enrichment test to compare the transcription factors involved in the expression of the enzymes showing transcriptional regulation and the enzymes showing changes in expression but not in flux. We found three transcription factors that were strongly overrepresented in the metabolic genes showing transcriptional regulation. In the glucose-ethanol transition, the transcription factors Gcr1 and Gcr2 both appeared in 11 transcriptionally regulated genes and in none of the other genes, whereas the transcription factor Hap4 appeared in 11 transcriptionally regulated genes and 5 of the other regulated genes. For the glucose-acetate transition these numbers were 15-0, 11-0 and 15-0 for the same transcription factors. This means that certain transcription factors are especially involved in the transcriptional regulation of metabolic fluxes (the same kind of enrichment was observed in the deletion mutants, as will be discussed later), and to our knowledge this has not been previously reported. It basically implies that there is global regulation of major flux alterations, which is similar to what has experimentally been shown to be the case for galactose metabolism [19].
The top scoring metabolically regulated reactions, both for glucose-ethanol and glucose-acetate, are the Fructose biphosphate aldolase and the Triosephosphate isomerase. These reactions are known to operate close to the equilibrium and are therefore very sensitive to changes in the metabolic pools, which is consistent with metabolic regulation of the fluxes. In the considered cases the direction of these reactions is inverted. This can only be explained by a decrease in the fructose-1,6-diphosphate pool and an increase in the glyceraldehyde-3-phosphate and dihydroxyacetone pools. This hypothesis is supported by the fact that in chemostat cultures, there was not found any correlation between the glycolytic flux and the expression of the genes encoding these two enzymes [20].
The results for the glucose-ethanol change are summarized in Fig. 4.
The underlined metabolite pools are those that are expected to increase (red) or decrease (green) according to the observed metabolic regulation.
Effects of gene deletions.
As mentioned above several different deletion strains were evaluated and the number of enzymatic reactions showing transcriptional regulation was 26 for the grr1Δ strain, 25 for the hxk2Δ strain, 11 for the mig1Δ strain, 8 for the mig1Δmig2Δ strain and 0 for the gdh1Δ strain. The reactions showing post-transcriptional regulation were 73, 70, 46, 36 and 89 for the same strains, respectively. These numbers clearly show, that in contrast to growth on different carbon sources, most of the transcriptional changes do not result in correlated changes in metabolic fluxes. This indicates that many transcriptional changes are indeed happening in order to minimize the metabolic adjustment resulting from a gene deletion, and the most extreme case is the gdh1Δ strain, where no transcriptional changes seem to be correlated to flux changes. This behaviour is consistent with the MOMA (Minimization of Metabolic Adjustment) hypothesis [21].
There is a substantial overlap between the strains grr1Δ and hxk2Δ, 15 transcriptionally regulated reactions were shared between both strains and 28 post-transcriptionally regulated reactions were also shared. An enrichment test was performed in order to find transcription factors regulating the enzymes showing transcriptional regulation. For the grr1Δ, the most significantly enriched transcription factors were Pho2 (which regulates the expression of 10 of the 26 enzymes with transcriptional regulation and only 6 of the 73 enzymes showing post-transcriptional regulation) and Bas1 (which regulates 10 out of 26 and 7 out of 73 enzymes in the mentioned groups). For the hxk2Δ strain the most significant transcription factor enrichment was found for the factors Pho2 (with 7 and 5 enzymes in each of the groups) and Bas1 (with 7 and 6 enzymes in each of the groups).
Pho2 and Bas1 are partner proteins that regulate the transcription of genes involved in purine and histidine biosynthesis [22]. It is possible that the slower growth rate observed in grr1Δ and hxk2Δ with respect to the wild type is due to a down-regulation of purine and histidine biosynthesis resulting from lower activities of Pho2 or Bas1.
Strains grr1Δ and hxk2Δ show specific growth rates of 0.23 and 0.22 h−1 respectively [16], however the biomass yields were 0.09 g-DW g−1 and 0.2 g-DW g−1. The specific glucose uptake rate for the hxk2Δ strain is significantly lower than for the grr1Δ strain as well as for the reference strain. This is associated with the observation that the glycolytic flux in the hxk2Δ strain shows transcriptional down-regulation of five enzymes. In the upper glycolysis, the Hexokinase 2 has been deleted and the Phosphofructokinase 1 is down-regulated. The Phosphofructokinase 1 was also strongly down-regulated in the grr1Δ strain but the decrease in flux was not as large as in the hxk2Δ strain. In the lower glycolysis of the hxk2Δ, all the three iso-enzymes of Phosphoglycerate mutase were down-regulated as well as the phosphoglycerate kinase. No down-regulation for these enzymes was seen in the grr1Δ strain. The glycerol-3-phosphate dehydrogenase has two iso-enzymes. The first of those isoenzymes was up-regulated in the grr1Δ and the hxk2Δ strains; however its expected flux decreased in both cases (more significantly in hxk2Δ). The second iso-enzyme did not show important changes in grr1Δ but was down-regulated in hxk2Δ. This is not the only case in which different iso-enzymes show different regulatory patterns, and in these cases our method for having flux estimations independent from transcriptome analysis is particularly useful.
The strain hxk2Δ showed a strong decrease in ethanol production compared to grr1Δ. All the alcohol dehydrogenase iso-enzymes were down-regulated in a similar way in both strains. The explanation of the differences in flux towards ethanol should be found in the pyruvate decarboxylase. Pyruvate decarboxylase 3 was strongly down-regulated in both strains, however, pyruvate decarboxylase 2 was up-regulated. This up-regulation was much more significant in grr1Δ, which could explain a higher flux from pyruvate to AcCoA in this strain. The results for the hxk2Δ mutant are summarized in Fig. 5.
The underlined metabolite pools are expected to increase (red) or decrease (green) according to the observed metabolic regulation. The transcription factors controlling the down-regulated pathways are also underlined in green.
The mig1Δ mutant shows a higher specific growth rate than the wild type. In general it is transcriptionally very similar to the wild type [16]. An enrichment test in transcription factors between transcriptional regulated and post-transcriptional regulated reactions was performed and the factor Sfp1 was found. This factor is known to regulate ribosome production and is nutrient sensitive [23]. This could mean that the deletion of MIG1 activates a response against starvation that results in an increased specific growth rate. Among the transcriptionally regulated reactions, a slight down-regulation of the PP pathway is observed together with an up-regulation of several amino-acid production pathways.
In the mig1Δmig2Δ mutant there is a slight decrease in the specific growth rate. All the 8 transcriptionally regulated reactions were down-regulated and belonged to amino-acid biosynthesis pathways. The enrichment test found the factors Cbf1 and Gcn4 (represented in 4 and 1 out of 8 reactions and 5 and 6 out of 36 reactions). Gcn4 is known to regulate amino-acid biosynthetic genes [24] and it seems that the up-regulation of amino-acid biosynthesis due to the deletion of MIG1 is cancelled by an opposite effect due to MIG2.
In all the mutants discussed above, the AcCoA carboxylase and the fatty acid synthesis showed metabolic regulation. This could indicate that in the studied cases the AcCoA pool was the main parameter responsible for adjusting the rate of lipid biosynthesis to match the changes in specific growth rates.
The experiments for the gdh1Δ mutant were performed in chemostat cultures [17] with the same dilution rate. The only observed change in exchange fluxes was a small decrease in glycerol production and only a few significant changes were identified in the metabolic fluxes. However, there were significant transcriptional changes in many metabolic pathways. This again points to the hypothesis that changes in transcription mainly results in altered metabolite levels such that metabolic homeostasis can be maintained. This is supported by metabolome analysis of this mutant, which showed that there were many changes in the metabolite levels [25] and in fact many of these changes were associated with changes also in the transcription of associated enzymes [26]. It is possible that the chemostat conditions, by imposing the same specific growth rate, forced the mutant strain to important transcriptional changes in order to keep the fluxes unchanged.
In Fig. 6 we aim to provide a global visualization of the changes for all the studied metabolic conditions.
The metabolic processes are defined in the same way as in the iFF708 model. The brightness of the color is proportional to the probability of a reaction in the corresponding process to show transcriptional, post-transcriptional and metabolic regulation respectively. The black correspond to 0 and the white to 1.
Sampling in the region of feasible solutions
The steady state condition and the irreversibility of some reactions impose limitations on the flux distributions attainable by the cell [18]. The set of feasible solutions can be further constrained by fixing some fluxes to their experimental values. In general, the fluxes most accessible to experimental determination are those corresponding to uptake or secretion rates. After fixing a subset of fluxes, genome scale models still have a large number of degrees of freedom. In this study we used the genome scale model iFF708 for S. cerevisiae [27]. Random sampling has previously been performed [7] by enclosing the region of allowed solutions in a parallelepiped with the same dimensions as solution space (the null space of the stoichiometric matrix) and generating random points inside this parallelepiped. The points that lie inside the region of possible solutions are then selected. The COBRA Toolbox [12] uses a Hit and Run algorithm to generate random points in this way. In this work instead of sampling inside the region of allowed solutions we sampled at its corners.
In order to obtain corners in the space of allowed solutions we used the simplex method with a random set of objective functions to be maximized. The maximization of each of these objective functions will give a corner in the space of solutions. The constraints imposed upon each optimization are:(1)(2)(3)The values of the measured fluxes (vexp) are different between conditions. This fact changes the shape of the region of feasible solutions between different conditions. S is the stoichiometric matrix of the network.
In order to reduce the effects of internal loops we first identified all the reactions that can get involved in loops using the FVA (Flux Variability Analysis) option in the COBRA Toolbox. The reactions that can be involved in loops are unbounded and show the default maximal or minimal value set in the COBRA Toolbox (1000 or −1000). If these bounds were kept, the means and standard deviations for these reactions would be unrealistic [6] and cannot be used for further analysis. In order to reduce the effect of loops, the default maximal and minimal fluxes for the reactions involved in loops, were set to a smaller value in order to reduce the loop effect. In order to select an appropriate value the bounds were increased from 0 in steps of 0.1 until the minimal value that allows obtaining flux distributions consistent with the experimental fluxes is found. These values went from 1 to 15 mmol h−1g-DW−1 depending on each condition. Also no weights (eq. 4) were assigned to the reactions involved in loops in order to avoid objective functions that maximize the activity of loops.
Random objective functions were generated by selecting random pairs of reactions and assigning them random weights (the reactions involved in loops were excluded from these choices). The weights (wi) assigned to each reaction were generated by dividing a random number between 0 and 1 by the maximal flux for this reaction obtained using FVA. This normalization was made to account for the different size orders of the different reactions. The objective functions take the form:(4)One solution is obtained for each of the objective functions generated.
Our objective is to obtain means and standard deviations for each flux in each of the compared conditions and use them to get a Z-score quantifying the significance of change in each flux between the considered conditions. This score is equal to the difference between the means in each of the conditions divided by the standard deviation of this difference (note that the variance of the difference is the sum of the two variances and the standard deviation its square root).(5)The difference between averages in the numerator follows a normal distribution (according to the central limit theorem) with a standard deviation equal to the standard deviation of the flux (the denominator in eq. (5)) divided by the square root of the number of samples. Therefore, Z itself follows a normal distribution with a standard deviation equal to the inverse of the square root of the number of samples.
The Z score measures the significance of change in terms of standard deviations. If the error in the Z score is lower than 0.15, no information would be lost in terms of classifying a reaction as significantly changed or not. The order of size of a genome-scale model is about 1000 reactions. A reasonable accuracy for the Z-scores would be to expect errors higher than 0.15 on the Z score only for 1 reaction in the whole model. This means a p-value of 0.001. If we want to keep the error on the Z score under 0.15 with a probability of 0.999 we need 500 samples, and this was therefore selected as the sampling number.
Classification of enzymes according to their changes in flux and expression level
The Z-scores can be transformed into probabilities of change by using the cumulative Gaussian distribution. Once we have Z-scores for the significance of flux changes and Z-scores for the significance of gene-expression changes we can obtain probabilities of having correlated expression and flux changes for each enzyme.
An increase in enzyme expression can result in an increase of flux (transcriptional regulation). In order to evaluate the probability for a reaction of being transcriptionally regulated we multiply the probability of its enzyme level changing by the probability of its flux changing in the same direction (obtained using the cumulative normal distribution).(6)(7)If there is a decrease in expression and a decrease in flux, both Z-scores are negative and we will use the absolute values of the Zs in eq. (6). If there is an increase in expression and a negative flux becomes more negative, we will use the absolute value of the Z-score for the flux change. If the direction of the flux changes between conditions, this change must be driven by the metabolic concentrations and no by transcriptional regulation, therefore a Ptri of zero is assigned by default.
In the same way as in eq. (6) we can define probabilities for the expression level changing and for the flux not changing (post transcriptional regulation).(8)(9)Now we use the error function because we want to evaluate the probability of change in any direction. The absolute value of Z is used in all the cases.
The probability of a change in flux but not in transcription (metabolic regulation) can be obtained for each reaction as follows:(10)Each of these three probabilities can be associated to each enzyme in the metabolic network.
Table 4 summarizes the criteria to assign each type of regulation.
Author Contributions
Conceived and designed the experiments: SB JN. Analyzed the data: SB RA. Wrote the paper: SB.
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- 25. Villas-Bôas SG, Moxley JF, Åkesson M, Stephanopoulos G, Nielsen J (2005) High throughput metabolic state analysis: the missing link in integrated functional genomics of yeasts. Biochem J 388: 669–677.
- 26. Cakir T, Patil KR, Önsan ZI, Ülgen KÖ, Kirdar B, et al. (2006) Integration of metabolome data with metabolic networks reveals reporter reactions. Mol Syst Biol 2: 50.
- 27. Förster J, Famili I, Fu P, Palsson BØ, Nielsen J (2003) Genome-scale reconstruction of the Saccharomyces cerevisiae metabolic network. Genome Res 13: 244–253.


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Doing all that reviewing of online image editing software really got me thinking about what else can we do with all those neat images once we've done massive editing and reworking of them? So, I decided to look into some of the many online photo storage sites, several of which I already belong to, and write a quick review of some of my personal favorites! … [Read more...]
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October 27, 2016.
Tags: Pattaya, Guide, Beach, Attractions
Pattaya is a well-known district in Chonburi. Pattaya is a vibrant city with various types of activity.
Around the city is full with entertainments, attractions and shopping. Those who love to stay in energetic environment would love to stay in Pattaya. Pattaya is not only perfect for tourists, but also people who work at outlying district of Bangkok and Laem Chabang area.
There are several famous beach in Pattaya such as Pattaya beach, Jomtien beach and Wong Amat beach.
Pattaya beach
Pattaya beach has the length of 4 kilometers. It is a combination of 3 beaches which are North Pattaya beach, Central Pattaya beach and South Pattaya beach. Along these beach there are walking street that always crowned from Thai and foreign tourists along with entertainments that are live all night long.
Jomtien beach
Jomtien beach is not far from South Pattaya beach with the length of 5 kilometers. It is a perfect place for sunbathe or just enjoy quieter atmosphere than Pattaya beach. At Jomtien beach is also more suitable for water activities such as jet ski, parasail and banana boat. Even though it is quite, still there are lots of shops and convenience stores along the beach.
Wong Amat beach
Wong Amat beach is located 2 kilometers to the north of North Pattaya beach. In Na Klua area, Wong Amat beach is considered as the most peaceful beach. The beach is stretching out like a crescent shape. The white sand beach of Wong Amat beach is even more scenic with a view of Koh-Lan and Sanctuary of truth. If you seek for ultimate relaxation, Wong Amat beach is ideal with private and serene atmosphere. Especially if you stay at Baan Plai Haad, a view of clam and clear water from Wong Amat beach will be first view for every morning of yours.
Art in Paradise
Art in Paradise is the most visited attraction by both tourists and locals. It is an illusion exhibition created by Korean artist. There are several themes of 3 dimensional painting for you and your family to interact and take photo. Art in paradise is perfect for those who travel with couple, family and friends.
Location: On Pattaya Sai 2, goes through Soi 1 around 20 meters and Art in Paradise is on the left-hand side.
Sanctuary of Truth
Sanctuary of Truth is a gigantic construction located on seashore at Phra Chan Bay with the height of 105 meters. It is a famous building representing Thai architecture. The whole construction is made of wood without any metal or screw. The building itself is a modern architecture with a mixture of architecture from late Ayutthaya to early Rattanakosin. Inside of the building is displayed with wooden sculptures telling a story of eastern philosophy, mostly about religion.
Sanctuary of Truth is a place for visitors who seek privacy and quiet side of Pattaya. This place will give a break for you to absorb the sophisticated story that telling through the architecture. Moreover you can take a ride to go around and see the whole building or if you stay at Baan Plai Haad, a beach view room can also give you a perfect picture of whole building of Sanctuary of Truth.
Location: Almost the end of Soi Na Klua 12, Pattaya Street
Pattaya walking street
Pattaya walking street is located on the south of Pattaya. Walking street of Pattaya is a street that opens for transportation during the day and closes on the evening for the market, starting from 6:00 PM. Along the street is full with shops, restaurants and pubs for you to chill out. It is a common meeting point for everyone.
It is also convenience for transportation as the market is easily access via many routes especially if you stay at THE BASE Central Pattaya that located in central Pattaya, still only 300 meters from the beach.
Location: From Sukumvit Street, turn right to the south of Pattaya to Laem Bali Hai pier. If you stay in the city of Pattaya, 2-taew is also available to take to Pattya walking street.
Pattaya is surely a perfect place to stay both for short term and long term rentals. Especially for those who seek for energetic and lively environment. With tourist friendly type of district, it will ease your life while stay at Pattaya.
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Researcher at Department of Historical, Philosophical and Religious Studies Units: History
A, Humanisthuset, HF112 Umeå universitet, 901 87 Umeå
Affiliated as researcher at Centre for Demographic and Aging Research at Umeå University (CEDAR) Units: CEDAR Research
Norra Beteendevetarhuset, 4 tr, Umeå universitet Umeå universitet, 901 87 Umeå
Glenn Sandström is a Docent/Associate Professor researching family dynamics in historical and contemporary perspective.
Glenn Sandström is an Associate Professor in historical demography at the Department of Historical, Philosophical and Religious Studies and Centre for Demographic and Aging Research (CEDAR) at Umeå University, Sweden and he is also a Research Affiliate, at the Stockholm University Demography Unit (SUDA), Department of Sociology, Stockholm University.
Sandström research covers a wide range of issues regarding changes in family dynamics and gender relations such as the long-term development of family stability and fertility in Sweden and other European countries from the late nineteenth century up until today.
Currently, Sandström is working on a project focusing on the social changes underlying the increase in divorce in Sweden since the early 20th century up until today. Apart from historical topics Sandström also coordinates the work package Single Living, Co-Residence and Family Complexity among the Elderly of Today and Tomorrow. This research focuses on the living conditions of individuals living alone in contemporary Sweden and is part of the FORTE-program Ageing well - individuals, families and households under changing demographic regimes in Sweden (Forskningsrådet för hälsa, arbetsliv och välfärd, FORTE, DNR: 2016-07115) lead by Professor Gunnar Andersson Stockholm University Demography Unit (SUDA).
Published open access software is found on my GitHub page:Close


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Capstone Projects
Planning and Future Management of the Adirondack Land Trust Glenview Preserve
Sat, 05/11/2019 - 08:51
Abstract: The Adirondack Land Trust purchased a 238 acre parcel of land, Glenview Preserve, from the Trevor family in 2016. The Glenview Preserve is located along New York State Route 86 in Harrietstown. The Glenview Preserve already consists of a heavily visited scenic vista of Whiteface Mountain, the McKenzie Range, and other Adirondack High Peaks. The property borders the Bloomingdale Bog, the third largest boreal peatland in New York State. The Glenview Preserve can be a perfect spot to incorporate a trail system to allow visitors the opportunity for non-consumptive recreational activities. When creating a trail system for recreation it is important to examine the impacts on local flora and fauna. A clearly designated trail system is the number one way to mitigate impacts on soil, vegetation, wildlife, and water created by visitors. Within the preserve interpretive signs will be implemented to educate and inform visitors on proper usage of the trail system. The unique thing with the area is the gradual grade of only 50 feet so the breathtaking views are virtually accessible to everyone who visits. Recreational activities allow people to get outside and enjoy nature and hopefully build personal environmental stewardship. The trail system will not only allow recreational opportunities for visitors but establish a sense of an outdoor community.
Access: Yes
Literary Rights: Off
Major: Natural Resources Conservation and Management
Year: 2019
File Attachments: Capstone_Murray 2019.docx
Managing a Successful Bakery
Mon, 12/10/2018 - 16:15
Abstract: For this capstone we were given the responsibility of running the A.P. Smith's Bakery for eight weeks. These responsibilities included the management of student employees, and the creation of menus, production lists and ordering sheets.
Access: Yes
Literary Rights: Off
Major: Baking and Pastry Arts
Year: 2018
File Attachments: Final Capstone Paper.docx
Lower St. Regis Lake Survey: A Comparative Study of Fish Population Structure and Function over Time
Thu, 12/13/2018 - 14:24
Abstract: Lake surveys are performed on bodies of water to provide a health analysis of fish populations over time. Lake surveys can be conducted in a variety of ways to attain specific data. Lower St. Regis Lake was surveyed to determine the fish community composition and to understand fish population traits. Using fyke nets placed at six predetermined locations for 24 hours, as well as fishing, we collected data for age, length (mm), weight (g), and parasites present. Data was analyzed in the lab using Excel to form graphs and tables to demonstrate our findings. Catch rates were lower compared to years before and comparing our data to New York State Department of Conservation data found that our length-at-age data was lower for the six-species sampled. Pumpkinseed and yellow perch were the only two species to have over twenty fish sampled. Decreased air temperatures brought in by a cold front during the week of our sampling may have been a reason for our lower number of fish caught. Mesh size is also a bias while using these nets as smaller fish can escape, and predatory fish can prey on smaller fish while in the net. Some species of fish such as black crappie may be more susceptible to capture due to its habit of associating with structure.
Access: Yes
Literary Rights: Off
Major: Biology, Environmental Sciences, Environmental Studies, Fisheries and Wildlife Science, Natural Resources Conservation and Management
Year: 2018
File Attachments: Capstone_Final.docx
A Comparison of Terrestrial Invertebrate Communities among Impacted, Minimally Impacted, and Reference Sites: Implications for Shoreline Vegetation Restoration
Fri, 12/14/2018 - 12:19
Abstract: The problem that this study attempts to address is the possibility of terrestrial invertebrate communities being negatively impacted by man-made disturbances along shorelines. This is a relevant issue because terrestrial invertebrates play multiple key roles in an ecosystem such as pollinating and serve as a food source for both aquatic and terrestrial organisms. The objective of this study is to provide a preliminary observation of terrestrial invertebrate communities between impacted, minimally impacted, and reference conditions sites so that further research can be conducted and add another aspect to shoreline management and restoration decisions. The impacted and minimally impacted sites that were sampled were located along the shore of Lower Saint Regis Lake on Paul Smith's College property. The reference condition site sampled in this study was located along the shore of Black Pond on Paul Smith's College VIC property. Invertebrates were sampled using pitfall traps and sweep nets. The invertebrates were then identified by taxonomic family and preserved in vials of alcohol. The findings of this study may imply that there may be inverse relationship between insect populations and arachnid populations based on impact level. Due to seasonal conditions of the Adirondacks, the results of this study could have been influenced by weather conditions, so a similar study conducted during a different season may produce vastly different results. The information collected in this study can be used to determine shoreline restoration and management decisions in the future.
Access: Yes
Literary Rights: Off
Major: Natural Resources Conservation and Management
Year: 2018
File Attachments: Final Capstone Report.docx
Affordable Sustainable Housing in the Urban Environment
Sat, 05/06/2017 - 21:26
Abstract: This paper addresses the lack of sustainable affordable housing in the urban environment. The focus of this study is the urban environment due to an increased rate of migration from rural areas to urban areas. This increase has led to a growing need for cities to become more sustainable in order to support the increasing population density. Focusing on the residential sector, cities have made great strides towards providing green-supportive and sustainable housing for their residents. However, the accessibility to sustainable housing in the urban environment for low-income residents is limited. The primary research questions this study addresses are what are the barriers to sustainable affordable housing in the urban environment, and how can we overcome these barriers? Through using secondary sources, the findings for this study were that the greatest barriers to developing sustainable low-income housing is the need for costly retrofits, and a lack of government subsidies. Addressing the second research question, the solutions to overcoming these barriers include: technological innovation, public-private partnerships, encouraging grass-roots groups, mixed-use development, and micro-apartments.
Access: Yes
Literary Rights: Off
Major: Natural Resources Conservation and Management
Year: 2017
File Attachments: Affordable Sustainable Housing in the Urban Environment.docx


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McCann Time Capsule: Rebounding in the 1980s
August 8, 1979, was one of the darkest days in McCann's, and especially McCann New York's, history. A number of top executives had recently left the agency at that time to start their own shop, and, on that summer day, Miller Brewing, one of the agency's largest accounts, announced that it was moving all three of its brands from McCann to that new agency.
How bad was it? As "Business Week" wrote: "Madison Avenue was taking bets that the agency would never recover."
With billings in the $80-$90 million range, Miller represented 15 percent of the U.S. agency. Not only was it arguably the single largest account loss ever suffered by any agency, but the circumstances were stunning.
The talent that had left in June to start up the Backer & Spielvogel agency were top management, including: Bill Backer, the McCann vice chairman and longtime creative leader known for commercials like Coca-Cola's famous "Hilltop" spot; and Interpublic vice chairman Carl Spielvogel, who had first joined McCann in 1960 and rose successively to top positions. Then they were soon joined at their agency by other top creative, management, account and media leaders from the McCann New York agency.
Moreover, Miller was a showcase account for McCann with high-visibility creative and strategic work. This included Miller Lite, which created the light beer category in the 1970s with the "It's Everything You Always Wanted in a Beer. And Less" work; Miller High Life's "If you've got the time, we've got the beer" campaign; and Lowenbrau's "Here's to good friends" commercials.
The company's reaction to the Miller loss was major and immediate. McCann was restructured and split again, as it had been in earlier years, into two separate units —a U.S. and an International company—each reporting separately into Interpublic, accompanied by a series of top management changes.
In January 1981, Bill Mackey, who led the McCann International side, was named the new CEO of the once-again globally integrated McCann-Erickson Worldwide. Mackey, one of whose past jobs had been as CEO of IPG's Marschalk agency, brought Bob James, his CEO successor at Marschalk, over to McCann to head U.S. operations and ultimately to succeed him as worldwide CEO. And Bob James in turn brought over from Marschalk John Dooner, who would become McCann's worldwide CEO after Bob James.
The agency also started to add new accounts. Kicking off the momentum in early 1981 was the $20 million global Texas Instruments account. TI, a high tech innovator whose product line also included educational technology (such as that shown in the accompanying 1985 ad), appointed McCann in a worldwide consolidation that replaced 26 foreign and domestic agencies. Additionally, McCann began to see marketers like Nestlé and Nabisco returning as U.S. clients and new assignments from other multinationals, such as Gillette, Johnson & Johnson, and Unilever.
So what began as a somber decade ultimately turned into one of enormous revitalization. Between 1980 and 1990, McCann would almost triple in size from billings of $1.8 billion to $5.0 billion, with the U.S. rising from $430 million in billings to $1.4 billion.


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Our SportLadder is now better than ever with a NEW and IMPROVED material –guaranteed to never slip! It is made of our patent pending non-slip material blend of plastics and rubber. Perfect for all of your on and off court training needs.
Challenge: I need a ladder I can use on or off the court that won't slip!
Solution: The proprietary OnCourt OffCourt SportLadder is arguably the best footwork ladder in the world. The SportLadder has flat-bottom rungs to minimize slipping if accidentally kicked, but the rungs are also rounded on the top to guide athletes to pick up their feet for ideal movement biomechanics. Note that while flat rung ladders are also popular, the rounded top of our SportLadder automatically guides athletes towards better running and movement habits. If you care about your athletes, this is the investment to make!
Rungs are 18" apart and 16" long. This 30-foot ladder comes in 2 parts and is brilliant yellow for increased focus and intensity. Also includes a highly durable zipper case.


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Peris is a sexy young blonde who loves to play. Dressed in pink panties and a pink top, she is hot. She strips to show her petite tits and shaved pussy, but her pink dildo is her best friend. She to lay somebodys her bearded clam deeply with her dildo and orgasms madly.» Click Here to Download the full video «
Category: Uncategorized


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The number and variety of things you can do in Puerto Rico often amazes me.
Visit Monkey Island
If we told you there was an island full of monkeys in Puerto Rico, you would think we'd had one too many piña coladas -- but it does exist! The technical name for the place is Cayo Santiago, and it's home to a colony of around 800 free-ranging rhesus macaques. The monkeys are cared for by the Caribbean Primate Research Center (CPRC) and the University of Puerto Rico. The Cayo, or Key, is located just off the eastern coast of the island. Barefoot Travelers Rooms can get you there by kayak (although not physically on the island... the monkeys need their privacy!).
Drive a Thing
Need a car while you're traveling? You can rent a minivan almost anywhere. Except on Culebra Island. Here, you can rent a Thing. What's a Thing, you ask? It's a noisy, rickety, long-past-its-prime Volkswagen convertible that Dick & Cathie rent to help you get around the island. While some people complain about the racket they make, we found them to be tons of fun.
Do the Superman
Seriously, it's as close as you'll get to wearing red underwear and putting a cape on (although you can still do that if you want). Flying like Superman comes courtesy of Toro Verde in Orocovis. This eco-park features an aerial network of zip-lines, culminating in The Beast, one of the longest zip-lines in the world. You'll be strapped in, face-down, and soaring through the sky like ... you know.
Go Treasure Hunting
Along with much of the Caribbean, Puerto Rico has had its run-ins with pirates. The island's most famous buccaneer was Roberto Cofresí, who, according to legend, was a Robin Hood-type character who shared his spoils with the needy. The pirate Cofresí is rumored to have left his buried treasure in caves near his hometown of Cabo Rojo, or somewhere in Toa Baja. Another treasure chest may be found in Mona Island, which briefly hosted Captain Kidd in 1699. Of course, no one has ever found any buried treasured... or if they have, they're keeping it quiet.
Visit the U.S. Presidents
Did you know you could visit President Barack Obama in Puerto Rico? Or Lyndon B. Johnson? Even Teddy Roosevelt? They are three out of eight life-like statues located behind the Capitolio just outside Old San Juan, and they pay homage to the eight sitting presidents who visited Puerto Rico.
Watch a Cockfighting Bout
This one's a little controversial, but among the various spectator sports in Puerto Rico, cockfighting is a longstanding tradition. While it has its opponents on the island, it's still popular, especially in the interior. And if you want to check it out while you're in San Juan, call the Club Gallístico de Puerto Rico, which is located on 6600 Isla Verde Avenue.
Visit Indigenous Ruins
The ancient civilizations of the Caribbean (the same ones "discovered" by Columbus) date back millennia, but many islands hold scant traces of their lost legacies. In Puerto Rico, however, you can visit a the Tibes Ceremonial Center near Ponce. The archaeological complex includes nine ball courts and three ceremonial plazas, a large indigenous cemetery, all from the Igneri and other Pre-Taino Cultures. It's just one great reason to visit Ponce.
Go Whale Watching
From January to March, those of you visiting Puerto Rico's west coast, or Porta del Sol are in for an annual treat. Migrating humpback whales can be seen from the shore, and up-close encounters can be arranged with local boatmen.
Snuba® Dive
This may be a reflection of my ignorance, but until I came to Puerto Rico I had never heard of Snuba®. This hybrid between snorkeling and scuba diving (which is supposedly easier than both) allows people to explore below the surface at their leisure while breathing through a regulator, with their air supply floating on a raft. And you can try it out with Aqua Adventure and Scuba Puerto Rico are among the outfits that offer Snuba excursions.
Visit the Grave of Juan Ponce de Leon
Most of us know Juan Ponce de Leon; the man synonymous with the Fountain of Youth was also a key figure in Puerto Rico's early history; in fact, his family lived on the island for many generations after Juan himself died while hunting for eternal youth. You can visit the ancestral home of the Ponce de Leon family in Old San Juan, and you can also visit the grave of the legendary man at the Catedral de San Juan.


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We will be staying in the Youth Hostel Bodenwerder. Bodenwerden is located about 60km south of Hannover in the Weser Hills (Weserbergland) and is the birthplace of Baron Munchhausen - the Baron of Lies. The fairy tale charakkter exploits focus as a sportsman, soldier, and traveller on impossible achievements, e.g. riding a cannonball, fighting a forty-foot crocodile, and travelling to the moon.
We will be sleeping in four and six bed rooms where each room is equipped with an own washing facility as well as showers and toilets on each floor. An upgrade to a single or double room is not possible. The accomodation is included in the conference fee so that you do not have to find a place to stay yourself which is pretty complicated while Hannover Fair is taking place.
For more information about the Youth Hostel have a look at their webpage.
Please mind that an individual arrival at the Youth Hostel by public transport is not possible! Staying in a hotel somewhere in Hannover is not possible since we cannot warrant transport from the socail events back to Hannover in the evenings.
Youth Hostel Bodenwerder
37619 Bodenwerder


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By Robert Musantry,
Sports Business Writer
Adidas has officially passed the Jordan brand for the second spot on the top selling sneakers list for the United States, now only behind Nike. This sudden surge surprised many, as Adidas has struggled compared to other top companies in recent years. Now, experts say that Adidas sales have seen quite some growth during 2017. According to the NPD Group, a market-research firm, Adidas has held a 11.3% share of the US Market through August of this year. Comparatively, Adidas had only a 6.6% share over the same period in 2016, just one year ago.
Nike, who still own the top spot, have actually fallen in terms of market share, from 39% at this point last year to just 37% this year. Jordan, although they were surpassed by Adidas, have actually seen some growth as well, with a slight increase from 9.4% to 9.5%. Matt Powell, an NPD sports industry analyst, said that he has "never seen a brand in the sneaker industry grow this fast."
This period of rapid growth can be attributed to many things, among them Adidas' new line of running shoes, the UltraBoost. These shoes were introduced as the "ultimate running shoe" and have also become popular for those concerned with the fashion side of sneakers. The sportswear industry has seen a trend towards more casual wear, and Adidas has adapted to this shift better than competitors like Nike.
Another trend has been the drop in sales of basketball sneakers, which, according to Yahoo Finance, have dropped 22% as a category in 2017. Once again, Adidas has been able to adapt to change, having many other products that are separate from their basketball sneakers, such as their Originals, Boost, and NMD shoe lines, their large selection of clothing, and even some accessories like hats, sunglasses, and phone cases.
Finally, many people have attributed the return of Adidas to their high-profile partnership with Kanye West. After leaving his partnership with Nike in 2013, West released his first Adidas shoe in 2015. The Yeezy Boost has since become a top selling line for Adidas, and has brought the brand major international recognition.
However, Powell has warned against holding West accountable for the growth, tweeting that Adidas actually had negative sales for two years after signing a deal with West.
Regardless of how much West has impacted the brand's sales, the final result has been Adidas regaining their hold on the second spot and company shares growing an impressive 31.3% over the year. This spot may be temporary however, as Adidas' quick rise could be replicated by a competitor, and their market share may shrink once again. But as of now, they sit only behind Nike in the United States.
A version of this article appeared in the Tuesday, September 26th print edition.
Contact Robert at


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When I ask people what are they currently struggling with, almost always the answer is balance.
See life balance is one of those tricky little suckers…..
We are all convinced that everyone else knows the answer. We see these other people that seem to have it all together, and then wonder why can't we figure out our own life.
But in reality having that "Instagram worthy" life balance is a complete LIE!
Truth is, no one has it all together. We all struggle with balancing everything and we all long for more balance in life.
It's funny because I'm often asked "how do you have time for everything?" "How are you doing so much" …. And my reaction is always "…..well you haven't seen the amount of dirty clothes that have yet to be washed."
In all seriousness though I am the first to admit that I do not have it all together…. But there are a few things that have helped me find just a little bit more balance in life.
3 tips for more balance in your life
Stay true to your values
Staying true to your values is a huge way to be more balanced, simply because staying true to your values help you make your decisions that are truly important to you.
We try to do everything and still end up taking more and more, leaving us exhausted and overwhelmed….convincing ourselves that it is a never ending cycle. Y'all…. It's okay to say no to things, especially things that can bring on extra stress in your life. Know your values and let them guide you. Only do the things that are truly important and align with your values.
Live with intention
When you decide that something is really important to you, be sure that you do those things with intention. If you choose to take a break from work to take your kids out for a date at the local bakery….allow yourself to be fully present and enjoy those precious moments with your little ones. If you are a work at home mom and decide to have set work hours while your kids play in the room next door, allow yourself to work and not feel guilty about it. When you start to live with intention, you become more focused and then you don't feel bad about working, or taking those breaks when you need to.
Show yourself lots of grace
Be sure to show yourself lots and lots of grace. We can't possibly do everything without letting go of something. Just do your best and show yourself lots of grace. There will be times where you fall short and that is okay, you are only human, but give yourself the grace to know that you tried and that you will learn and move forward.
We all want to have more balance in our lives, but it takes work. We have to admit to ourselves that we can't do everything and that is okay.
We must let go of this idea of full life balance and learn to take little steps to help us along the way.
Stay true to your values
Live with Intention
And be sure to show yourself lots of grace.
Life is too lovely to stress it away, just do the best you can and enjoy life.


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Discover The World of Classical Music with Grammofy
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Verdi's Otello – Aria Code Episode 3
We All Have Demons, But Sometimes The Demons Have Us
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The legendary Plácido Domingo says he's not a jealous guy, but he must know something about it because he's played the part of Othello more than 200 times.
Shakespeare called jealousy the "green-eyed monster" and no opera captures what happens when that monster takes over better than Verdi's adaptation of the tragedy.
In this episode, host Rhiannon Giddens and her guests consider how jealousy always seems to know where to find us: at work, in our friendships, and of course, in love. Plácido Domingo takes you through his career-defining role, sharing the tragedy of a man undone by his demons, plus you'll hear him sing the complete aria "Dio mi potevi Scagliar" from the Metropolitan Opera stage.
The Guests
Plácido Domingo is not only Rhiannon's favorite singer, but he is also one of the most influential artists in the history of opera. He just celebrated fifty years of performing at the Met and has played the role of Otello (by his count) 225 times.
Robert L. Leahy is teaches at Weill Cornell Medical School and is the director of the American Institute for Cognitive Therapy in New York. He's written 27 books, including The Jealousy Cure.
Ruben Santiago-Hudson is an actor, playwright and director. He may best known for his work on TV and on Broadway, but this year he directed Othello for Shakespeare in the Park at the Delacorte Theater.
Virginia Mason Vaughan is a member of the faculty at Clark University in Worcester, Massachusetts. For more than three decades, she's studied the construct of race in Shakespeare's time and is the author of Othello: A Contextual History.
Thanks, too, to Justin Cunningham who read the part of Othello. He's a Julliard-trained actor and you'll see him next in the new Netflix series about the Central Park Five.
Aria Code is a new podcast that pulls back the curtain on some of the most famous arias in opera history, with insight from the biggest voices of our time, including Plácido Domingo, Diana Damrau, Sondra Radvanovsky, and many others.
Each episode dives into one aria — a feature for a single singer — and explores how and why these brief musical moments have imprinted themselves in our collective consciousness and what it takes to stand on the Met stage and sing them.
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Verdi's Otello – Aria Code Episode 3


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Financial experts warn the same type of up and down cycles happen with too many people's credit cards, resulting in financial chaos.
Charlese Antoinette is careful how she uses a credit card these days, but that wasn't always the case.
"I would just, like, buy whatever I want and charge it up," said Antoinette.
She'd push her plastic to the limit.
But the biggest problem? Once she'd pay the cards off, she'd just rack up charges again.
Experts have a term for this: yo-yo debtor.
"A yo-yo debtor is someone who treats their debt like a yo-yo; they pay it off, and they reel it back in then they release it and they get back into debt," said Tonya Rapley, Millennial Debt Expert.
And while yo-yo debt impacts all ages, a recent survey shows nearly a third of millennials have maxed out a credit card.
"That doesn't mean that they're not intelligent or not financially literate. They just might not know that it's not a healthy practice to be a part of," said Rapley.
Research also reveals that 43-percent of millennials have sub-prime credit -- more than other age groups. Experts say the problem is often spending without planning.
"It's, typically, there is no budget in place. Therefore, they're running a budget deficit over the course of time, which, in turn, balloons the credit card debt up," said Mark Higgins, Credit Counselor.
Tips to stop the yo-yo'ing:
- Budget monthly
- Know your credit limit
- Pay more than the minimum
- Increase savings, even while you are in debt.
Charlese says she reached her limit when she didn't even have enough credit to rent an apartment. She got a credit counselor, paid off all but one card and boosted her credit score more than 200 points -- all in less than a year.
"I'm really glad that I figured out how to be more financially responsible. I'm like, 'Okay. I got it.'"
If you or someone you know is having credit problems there are a number of low and no-cost credit counselors available.
Find out more at www.usa.gov/debt


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December in Winnipeg. The shortness of the days. The crisp mornings and long twilight of the afternoons. It's a season of extremes. The holidays are a time I look forward to. For me this is a time of year where the focus is more about just getting together with people, renewing connections and catching up. Professionally it's my month of greatest extremes. December is usually quiet through the early part of the month and then a scurry of people wanting to get in while they are off work. It's also very nice to see people who visit from out of town. Some people make coming for a reading part of their holiday tradition!
In my own family the kids are grown, there's not a lot of presents to buy (other than for my Mother) and, thankfully, most of us have what we need. A few years ago we started putting the emphasis more on just getting together. I don't much relish the craziness of shopping. I also volunteer a bit around different community areas. I usually help decorate a community centre and other stuff.
For a lot of people this season is challenging. Maybe because there's so much thrown at us about how it OUGHT to be and it's easy to feel we have fallen short if we aren't living some sort of Hallmark card version of the season. Also we often see a lot of old patterns arise as we go "home for the holidays". I see a lot of struggle 'round this time of year, but I also see a lot of progress and hope.
One of the symbols of the season that I have come to have a different appreciation of is the little nativity scene my mother puts out every year. She often threatens to give it away and I always get antsy when she does. It's one of our little struggles but it means something to me. She bought it when she worked at Eaton's back when I was very little. Eaton's was an amazing big old department store in it's day. The toy land at Christmas was (in my memory) a huge otherworldly adventure. It didn't have the same feel as the big box stores of today. Amid all the over the top stuff that our family went through at Christmas time, the little nativity scene or "creche" as my mom calls it, is one of the few things still around. The papier mache figures that have survived many moves and jostling. I think we lost a barnyard animal or two and St. Joseph has a chip on him. The year our basement flooded we found the box waterlogged. Mom very carefully unwrapped each figure and let them dry, they could have crumbled at the touch.
My mom always did her best to make Christmas special for us ( I have three older brothers). In the years when she was a single parent, working secretarial jobs and, for the last while as an inspector on an assembly line in a computer plant, she did her best to keep things together. By the time I was about 13, my older brothers had moved out and it was just her and I for a few years. I remember one afternoon about this time in December, I was home alone after school and there was a knock at the door. A guy from the Christmas Cheer board was there with a hamper. I thought he must have the wrong address and told him so. He had our names and suggested I call my mom and check before going away. I called my mom at work and when she heard why there was a long pause and she said "it's not a mistake". I had not known at the time how hard some things were. Someone had submitted our name for that turkey.
What that little Nativity scene represents to me more than anything now, is endurance, survival and hope in times of challenge. I'm looking forward to spending time with my mother this holiday season, like the little creche, we've come through a lot.
I'm including a link, for those who may be interested, for the Christmas Cheer Board's website, it gives information on how you can donate and the good things they do;
Winnipeg's Christmas Cheer Board
In a few weeks the days get longer again. Whatever our individual spiritual beliefs and traditions, it is a time for renewal and appreciation of what we have, what we can share and what we are here to do. To all my friends and clients, best wishes of peace, comfort and joy and to a good new year, cheers!


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This new scheme – officially launched in November 2017 – enables euro credit transfers with the funds made available on the account in less than ten seconds at any time and in an area that will progressively span over 34 European countries. All payment service providers ( ) are invited to adhere to the scheme as soon as possible. The technical information for implementing the scheme is available on this page.
The Instant Credit Transfer ( Inst) rulebook in effect until 17 November 2019 is the 2017 Credit Transfer Inst rulebook version 1.2.
Read more about the 2017 SCT Inst rulebook version 1.2
Read more about the maximum amount per SCT Inst instruction under the 2017 SCT Inst rulebook version 1.2
The Implementation Guidelines for the version 1.2 are based on ISO 20022.
Read more about the SCT Inst scheme Customer-to-Bank IGs 2017 version 1.0
Read more about the SCT Inst scheme Interbank IGs 2017 version 1.2 (applicable in November 2018)
Do you want to use the 's Intellectual Property Rights related to payment schemes' Rulebooks and Implementation Guidelines for non-euro transactions? Click here for more information.
Annex IV of the 2017 Inst rulebook version 1.2 lists all changes compared to versions 1.0 and 1.1. No changes impacting the business and operational rules or the related implementation guidelines were made in this version.
The first change is the updated definition of the rulebook term 'Major Incidents'. This definition change constitutes a regulatory change. The updated definition of this term now refers to the Eurosystem major incident reporting framework for payment schemes and retail payment systems entering into force on 01 January 2019. The new definition also points to the document 190-18 'Details about major incident reporting for scheme participants'.
This document outlines the detailed assessment criteria for each scheme participant to determine whether an operational or information security incident fits the criteria of a major incident and needs to be reported to the or not. The document 190-18 is not publicly available. The shares this document directly with all existing and future scheme participants.
Furthermore, this rulebook version also includes the updated version 4.2 of the Scheme Management Internal Rules (SMIRs) which are an integral part of each scheme rulebook. The updated SMIRs include a new section 184.108.40.206 on voting by written procedure by the Scheme Management Board (SMB) and several rewordings in the section 18.104.22.168 on the National Adherence Support Organisation.
The new SMIRs also contain a new section 184.108.40.206 covering the dialogue between the and its Overseer, and a new section 4.5 describing the process for making changes to the Risk Management Annexes. These SMIRs no longer make any reference to the Certification Authority Supervisory Board (CASB) set up for the e-Mandate option under the Direct Debit scheme rulebooks. The SMB decided to disband the CASB as of 1 January 2019.
The 2019 Inst rulebook version 1.0 enters into force on 17 November 2019 at 08:00 CET and is applicable until November 2021.
Read more about the 2019 SCT Inst rulebook version 1.0
Read more about the maximum amount per SCT Inst instruction under the 2019 SCT Inst rulebook version 1.0
The Implementation Guidelines are based on ISO 20022.
Read more about the SCT Inst scheme Customer-to-Bank IGs 2019 version 1.0
Read more about the SCT Inst scheme Interbank IGs 2019 version 1.0
The 2019 rulebook contains a clarification about which scheme participants must comply with the mandatory Customer-to-Bank (C2B)
of the rulebook.
The C2B also include usage rules on how a Beneficiary can make a 'Repayment' to transfer back funds to the Originator when the Beneficiary has not received the IBAN of the payment account of the Originator in the original Inst transaction.
A Inst transaction containing an IBAN but not the related BIC, can be transmitted by the Originator to the Originator Bank, even if one of the two Inst scheme participants covered by that transaction is based in a non- country.
The 2019 rulebook foresees a third rulebook option for scheme participants if they wish to agree on a shorter hard time-out deadline among themselves. Several changes have also been made to the procedures for Recall and for Request for Recall by the Originator.
Clarifications in Inst rulebook on the concrete point in time where an Inst instruction turns into an Inst transaction, and that the current maximum amount applies to both Inst instructions and Inst transactions have been added.
Annex IV of the 2019 Inst rulebook contains a table listing all changes made compared to the 2017 Inst rulebook version 1.2.
Clarification paper on SEPA Credit Transfer and SEPA Instant Credit Transfer rulebooks
Guidance on reason codes for SEPA Instant Credit Transfer R-transactions
Advisory Group on Market Infrastructures for Payments (AMI-Pay): best practice on value dating instant payments
SEPA requirements for an extended character set (UNICODE subset) - best practices


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Great food, great entertainment, and a great night's sleep await you on one of our fabulous Willersley Castle Themed Nights. Sunday night starts with a delicious themed dinner, followed by an evening of entertainment in keeping with the theme. A good night's rest is followed by a hearty breakfast in the morning – all for just £55 per person!
20th January: Best of British
10th March: 1960's Night
7th April: Spanish Night
2nd June: Best of British
14th July: Tropical Beach Party Night
1st September: Wild West Night
15th september: Best of British
22nd sept: Matlock Bath Illuminations
3rd november: 1960's Night
Expires:3rd Nov 2019
Hotel: Willersley Castle Hotel
Read our 2019 Holidays & Hotels brochure here on our website or download your own copy.


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Thanx SE, for givin' us the this terrific phone. I've borrowed my cellphone and I don't remember if it has plugin for normal earphones, does it have? As i remember, it hasn't, but could you tell me, pleez?
Or you have to use the SE earphones included with the phone? Thanx 4 your helppin'!! =)
I own a k750i with the latest FW i.e R1L002. Have had it for a week.No problems so far no hanging no freezing.Its perfect. For all those who dont know yes it plays video in full-screen but destroys the quality of the video. people say that the ear volume is low this can be solved. Enter the code >*
It sounds a great phone but i just wish sony erikkson would make somethin a bit more stylish as it seems to be lacking in that department. iam thinking of getting the samsug e720 which looks better but is the functions on the phone not a touch on the erikkson? pls reply with thoughts. kels
This phone is awesome better then all the rest it has everything u need I even using it a mp3 player after adding 1GB external memory
Hey everyone, I know this wonderful Sony Ericsson can't be beat. Nice, wonderful features and easy to use! I love this phone! Friendly features, bright- colorful screen, big expansion slot and a huge internal memory! Theres no way you can take all the space of the 1 gigabyte Memory Stick Duo! This is like a compact computer... its great, wonderful... But I'm having questions..... How do you put MP3's to this phone..... I know its possible but how? please respond... thanks KLG2992..... :-)
hey pplz!
Can sum1 tel me if this fone is beta than samsung D600?
My last fone waz a sony ericsson n it wa crap, the gear stik broke it didnt accept my calls and my text messages came 24 hours later than the were sent!
I took it to be repared and thy said it wa a common fault in all sony ericssons!
So i am usin an old fone now n i am gona but a new 1!
But i dont which 1 2 get!
answer me plz sum1 xxxxxxxxxxxxx
The 2mpx camera is awesome! All pictures in my gallery were taken with my sony ericsson k750i (contrast & color balance have been photoshoped)
the gallery: http://www.guilty.be/dumb/
Most of you go on like only smartphones can be good, your all wrong, just because you like smart phones and dislike others dosen;t make them crap, so stop your lame comments that keep making peoples choices harder, and give friendly, advice where you jsut don't diss a phone simply because YOU didn't like it!
Unknown, the K750i you got must have bin fake because my K750i comes out with extrodianray pictures, great quality video clips, great performance etc, so K750i in my eyes is still the best phone around!
If you want there are some applications you may find for this phone, not so many though as you already said that it's not a smartphone but if want to buy a smartphone that supports many applications you may choose p910 or even nokia 9500 communicator as long as the phone cam is not an issue for you. As for this mobile cam it's the best still camera equipped in any mobile that is available out there you may take it or you may leave it but that's it. As for the video quality it's average between most of the mobiles that has video recording capability but not the worst cause i've seen a lot worse. As for your next step my advice to you dude buy the best blain mobile - as you descriped it - and the best camcorder out there and they will cost you more less than this daylight robbery 900$.
Geez man is it singing and will it communicate or interactive with me for being "smartphone" to bay 900$ in a phone. Put more 400$ and buy a laptop, at least it will truly interactive with you.
A question to the wind ... What does being smartphone has to offer more than some supported applications only some business men & quick moving people will need to work with !! and by the way such people have their own laptops and pda's already.
I was tempted by the apinions about the camera quality of the phone so I went to buy it. Thanks god I had the option to return it after a week.
So much discussed "camera" turned out to give nothing more than the average pictures, and terrible video clips (so i am signifficantly better off having my pentax optio S all the time with me (and is approx the same size as the phone) - yas it is another thing to carry but at least the quality is satisying.
but it is not thr point I write here as the cammera I could stand the problem is operating system (and in fact lack of it) - I am used to have smartphones and having this phone I felt as if I am having a calculator - no possibility to ad software is really a disaster - I did not realise that "plain" phones are soooo dumb
so the next step in my "phones story" is a N90 - a excellent smartphone with a good came and very good clip taking posibilities
I've just got this awesome yesterday n it totally rocks...cool features, awesomely clear camera n it brings wonderful music 2 ur ears.....love the design, way better than k700 which i had half a year ago.love the 'front' back design...
thanx @DoUL buddy.......if i have any query i will e-mail you for more guidance, if u dont mind..thanx once again...........Dominic
Low quality because of low resolution used to capture the clips with, not low quality for the camera, so don't get me wrong.
As I told you man your clips can go as long as you want till the last bit in your memory. Because of the low quality & the format that this device captures the clips with - 3GP -, any clip you'll take won't take much memory. You already have 38 MB internal in the phone + 64 memory stick comes with the bundle = 102 MB. For me I captured a long clip 7:09 minutes and it only took 3.95 MB !!.
As for the low quality and format I see the clips aren't that bad. Yes some distortion arise with the very quick moves but other than that it still can capture the moment very well ;)
thanx @doul for your answer..than i think Nokia 3230 has a 1 hour video recording by default amd it only has a 32 MB card.....is ther any way we can extend the video shooting part to atleast 1 hour.....without expanding the memory card.i am really worried before i buy this gadget.....thanx one again..
Regards Dominic
Yes I believe this is the latest final released firmware. There is another one R1N005 that is still a beta testing firmware so not recommended at all upgrading to it yet.
Others maybe will have the firmware R1J002 - including me - which is basically the same as your one but the only difference between them is a bug was encountered at R1J002 in the GPRS with the O2 service provider but other than that they are the same, only some performance differences here and there, this one is faster in some benchmarks while the other is faster in another some of benchmarks. - The Common Differences - but they both have the same camera version & LCD version.
My advice to all the guys, Do Not upgrade your firmware as long as your device is acting well !
Thanks @ Burim
to @Doul, what is latest firmware? mine is R1L002...is this the newest firmware?
Located here in the Phil..
The most useful feature of the phone is that it has fast synchronization on Outlook 2003...calendar, task, notes ,and contact..perfect partner!
Can't imagine that my k750i has the same super MEGABAsS sound when playing MP3 with my CAR...nice one!!!! i love it..
to those ppl who encounter short battery life, try to lessen the Brightness of the screen.
Ofcoarse, since the phone is new and you keep on playing with it, no question why it lowers the battery life...funny isn't it? Mine, I make all the necessary adjustments...power mode, lower brightness, turn off blutooth, infrared...
If you use it for phone calls only, then the longest time you will be talking to your recepient is 6-7hours....not too bad right? lol!!! Masakit sa tenga!