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Quantitative modeling and analytic assessment of the transcription dynamics of the XlnR regulon in Aspergillus niger
Jimmy Omony1,3,
Astrid R. Mach-Aigner2,
Gerrit van Straten3 &
Anton J.B. van Boxtel3
BMC Systems Biology volume 10, Article number: 13 (2015) Cite this article
Transcription of genes coding for xylanolytic and cellulolytic enzymes in Aspergillus niger is controlled by the transactivator XlnR. In this work we analyse and model the transcription dynamics in the XlnR regulon from time-course data of the messenger RNA levels for some XlnR target genes, obtained by reverse transcription quantitative PCR (RT-qPCR). Induction of transcription was achieved using low (1 mM) and high (50 mM) concentrations of D-xylose (Xyl). We investigated the wild type strain (Wt) and a mutant strain with partial loss-of-function of the carbon catabolite repressor CreA (Mt).
An improved kinetic differential equation model based on two antagonistic Hill functions was proposed, and fitted to the time-course RT-qPCR data from the Wt and the Mt by numerical optimization of the parameters. We show that perturbing the XlnR regulon with Xyl in low and high concentrations results in different expression levels and transcription dynamics of the target genes. At least four distinct transcription profiles were observed, particularly for the usage of 50 mM Xyl. Higher transcript levels were observed for some genes after induction with 1 mM rather than 50 mM Xyl, especially in the Mt. Grouping the expression profiles of the investigated genes has improved our understanding of induction by Xyl and the according regulatory role of CreA.
The model explains for the higher expression levels at 1 mM versus 50 mM in both Wt and Mt. It does not yet fully encapsulate the effect of partial loss-of-function of CreA in the Mt. The model describes the dynamics in most of the data and elucidates the time-dynamics of the two major regulatory mechanisms: i) the activation by XlnR, and ii) the carbon catabolite repression by CreA.
The fungus Aspergillus niger has numerous economic and ecological applications, for example in paper manufacturing, animal feed and human foods. It is used in industrial fermentation processes for the production of organic acids like citric acid [1, 2], gluconic acid [3] and also for the production of enzymes such as amylase, amyloglucosidase, cellulases, pectinases, lactase, invertase and acid proteases in the food industry [4, 5]. Biotechnological products from A. niger are applied in the pharmaceutical, cosmetic and chemical industries [6]. Understanding gene transcription (mRNA synthesis) is crucial for advancing industrial applications and controlling the synthesis of target compounds from A. niger. Transcription of genes coding for xylanolytic and cellulolytic enzymes in A. niger is controlled by the transactivator XlnR [7]. XlnR plays a major role in regulating the expression of plant cell wall degrading enzymes (PCWDE) [8–10]. The target genes of the XlnR regulon encode amongst others the main xylanolytic enzymes xylanases B and C and β-xylosidase, and accessory enzymes like α-glucuronidase A, acetylxylan esterase A, arabinoxylan arabinofuranohydrolase A, and feruloyl esterase A [11]. The XlnR regulon also comprises genes coding for cellulolytic enzymes like endo-glucanases A, B, C and cellobiohydrolases A and B.
Carbon catabolite repression is a regulatory mechanism occurring in microorganisms leading to the adjustment of their carbon metabolism, thereby minimizing energy demands. The carbon catabolite repressor protein CreA was initially described in Aspergillus nidulans [12]. In the presence of high concentrations of D-xylose (Xyl), CreA modulates XlnR-induced expression of the genes involved in xylan degradation [13]. Up-regulation of several PCWDE in A. niger can result from carbon starvation or creA deletion [10, 14]. Mach-Aigner et al. [15] assessed the effect of CreA on transcription of PCWDE-encoding genes in A. niger by comparing the response to Xyl in two strains: the wild type (Wt) and a CreA mutant strain (Mt). They analyzed the transcript levels of genes coding for PCWDE, evaluated their Xyl concentration dependent expression profiles and described the role of CreA and XlnR in regulating transcription in A. niger. They found that genes coding for enzymes with similar function responded in a similar manner to a particular Xyl concentration and noted that utilization of high Xyl concentration is beneficial for the induction of hemicellulase-encoding genes [15].
Regulatory network reconstruction involves the analysis of trends and dynamics in data, and the inference of functional and regulatory mechanisms in biological systems. Mathematical modeling helps us to represent and understand complex interactions in networks. Modeling enables prediction of the resultant effect of nonlinear interactions in a network [16], thereby providing insight into cellular regulatory processes and kinetics [17]. Ordinary differential equations (ODEs) are a popular formalism for modeling time responses in regulatory networks [18–23]. ODEs used in the context of modeling biological networks consist of mechanistic representations of the transcription rates in biological (sub-)systems [19]. Dynamic modeling involves incorporating prior knowledge into models of the regulatory network.
Omony et al. [24] provided a dynamic model based on ODE for the description of the regulation mechanisms and dynamics of the XlnR regulon. Mach-Aigner et al. [15] investigated the role of CreA using time course data (TCD) based on an hourly sampling interval. However, the information in this time interval was not detailed enough to adapt the previous dynamic model [24]. The purpose of the current work is to make amendments to the model based on experimental data from shorter time intervals, that better resolve the transcript dynamics. It also allows for a variety of regulation mechanisms for the XlnR target genes, rather than a single mechanism as proposed in previous models [24]. Additionally, by comparing the Wt with Mt data, an attempt is made to obtain more insight in the mechanism underlying the CreA regulatory influence.
Strains, growth conditions, RT-qPCR
The strains (A. niger N400 (CBS120.49) (Wt) and the CreA mutant strain NW283 that exhibits a de-repressed phenotype (Mt)), growth conditions, RNA-extraction, reverse transcription and the quantitative PCR analyses in this work were described by Mach-Aigner et al. [15]. TCD were obtained for 23 genes for the Wt using a sampling interval of 20 minutes during a period of 5 hours, and for 8 genes for the Mt using an hourly sampling interval (see Additional files 1 and 2). Strains were grown in bioreactors on sorbitol, which was used as a non-inducing/non-repressing carbon source. The experiments involved quantifying expression of: xlnR, genes encoding endoxylanases (xlnB and xlnC), β-xylosidase (xlnD), arabinoxylan arabinofuranohydrolase (axhA), acetylxylan esterase (axeA), α-glucuronidase (aguA), feruloyl esterase (faeA), endoglucanase eglA and eglB, which are XlnR target genes [25]. The other XlnR target genes are: eglC, talB, xdhA, ladA, estA and the Xyl reductase-encoding gene xyrA as well as abfB, bglA and xkiA [26]. XlnR also regulates expression of α- and β-galactosidase-encoding genes (aglB and lacA) [27] and cellobiohydrolase-encoding genes cbhA and cbhB [28].
Previous model formulation for the XlnR regulon dynamics
Based on prior knowledge of the regulatory mechanisms of the XlnR regulon described by de Vries et al. [13], the original model of Omony et al. [29] was formulated as follows:
$$ \left\{\begin{array}{c}\hfill {\overset{.}{x}}_{xlnR}=bu-{k}_d{x}_{xlnR}\hfill \\ {}\hfill {\overset{.}{x}}_i={k}_{is}\frac{{\left({k}_{i1}{x}_{xlnR}\right)}^{h_1}}{1+{\left({k}_{i1}{x}_{xlnR}\right)}^{h_1}}\frac{1}{1+{\left({k}_{i2}u\right)}^{h_2}}-{k}_{id}{x}_i\hfill \\ {}\hfill \mathrm{given}\ {x}_{xlnR}(0),\ {x}_i(0),\ u(0)\hfill \end{array}\right. $$
where x xlnR - activity state for xlnR, u - Xyl concentration, b - input stimulus coefficient, k i2 - inverse of Hill constant for CreA, k d - mRNA degradation parameter for xlnR, x i - activity state for target gene i and \( {\overset{.}{x}}_i \) – transcription rate. The transcription rate is constant when the gene is on, but is reduced by the two switching Hill functions in the first term of the right hand side, one for activation by XlnR and the other for stimulus proportional de-repression.
Omony et al. [29] used Hill coefficients h 1 = h 2 = 1. The h 1 values govern the transcriptional response of target genes to Xyl induction. A small value, h 1 ≈ 1 may lead to a graded response, whereas larger values are more likely to result in a bi-stable switch-like response in gene expression profiles [30]. Ninfa and Mayo [31] established a positive association between high Hill coefficients and transcription dynamics. Many biological systems have large Hill coefficients (h l = 1,2 ≥ 2) [32] which are associated with multi-stability, higher order dynamics, and the number and specificity of transcription factor (TF) binding sites. Our data suggest that steeper switching is more appropriate; therefore, in this work we used higher h 1 and h 2 values. We considered Xyl to cooperatively interact with XlnR at the promoter site of target genes, as opposed to earlier proposed binding mechanisms [29].
New model formulation
To describe the regulatory mechanism of the dependence of the XlnR regulon on Xyl and CreA, we define the xlnR promoter as P xlnR . The normalized transcript levels indicate that the expression of xlnR remains nearly unchanged (values ~1 to 2 relative to the hist gene), irrespective of the used Xyl concentration (Figs. 2H; 3H; 4 W and 5 W). In the model, the resultant protein (XlnR) is considered to ultimately bind cooperatively with Xyl to the promoter of a target gene forming the complex x xlnR u. The concentration of this complex, which is also considered as the active form of XlnR, is denoted by X = [x xlnR u]. This complex dissociates into [x xlnR ] and [u], which hereafter are simply denoted by x xlnR and u, respectively. CreA binds to the promoters of the target genes, thus inhibiting transcription. The number of CreA binding sites varies between the promoters of the target genes, however, we assume that occupation of at least a single binding site suffices to repress transcription.
xlnR is constitutively transcribed, irrespective of the Xyl concentration [15]. It is evident from our data (Figs. 2, 3, 4 and 5) that the pseudo-steady state assumption \( {\tilde{x}}_{xlnR}=b\tilde{u}/{k}_d \) based on earlier proposed models for the expression of xlnR [29] cannot be maintained. Therefore, an improved model based on the mechanism in Fig. 1A is used to explain the network dynamics. The regulation mechanism for the XlnR regulon is modeled using the equations
Regulatory mechanisms in the XlnR regulon and transcription profile classification. a: Model of how XlnR and CreA control transcription of the XlnR target genes. The term TF represents a transcription factor. URS and UAS – upstream repression and activation sequences, respectively. The depicted CreA repression of xlnR is hypothetical. b: The transcription dynamics were classified as: C 1 – a monotonic increasing function, C 2 – a function with a maximum and a lower steady state, C 3 – a function that steadily rises to a maxima, decreases and then again increases (de-repression of transcription), and C 4 – bimodal expression with de-repression. W 1 and W 2 are time window partitions. The term a.u. refers to arbitrary units. Any other parameters are as described in the materials and methods section
$$ \overset{.}{X}={K}_{\mathrm{on}}{x}_{xlnR}u-{K}_{\mathrm{off}}X $$
$$ {\overset{.}{x}}_{\mathrm{CreA}}={k}_1u-{k}_2{x}_{\mathrm{CreA}} $$
$$ {\overset{.}{x}}_i={k}_{is}\left(\frac{X^{h_1}}{K_{i1}^{h_1}+{X}^{h_1}}\right)\left(\frac{K_{i2}^{h_2}}{K_{i2}^{h_2}+{x_{\mathrm{CreA}}}^{h_2}}\right)-{k}_{id}{x}_i $$
given [X(0), x CreA(0), x i (0)]T; k is – mRNA synthesis parameter, k is ∈ {k is,Wt, k is,Mt} – maximum transcription rate in the Wt and Mt, respectively. The state variables depict mRNA concentration, protein abundance and other time-dependent quantities.
Here K on (h-1) and K off (h-1) are the association and dissociation constants, respectively. K on describes the interaction between XlnR and Xyl, and K off the strength of the interaction between the two molecules. x CreA depicts CreA concentration, k 1 and k 2 are constants. K i1 is the expression threshold parameter (half-saturation concentration) that corresponds to activation of target gene i by the complex of XlnR and Xyl. K i2 ∈ {K i2,Wt, K i2,Mt} is an expression threshold parameter (half-saturation concentration). It corresponds to CreA repression in the Wt and Mt, respectively. This allows for a partial or complete CreA loss-of-function Mt to be accounted for in the model. k id is the first-order rate constant for the mRNA degradation for target gene i.
Let \( {\psi}_{\mathrm{CreA}}={K}_{i2}^{h_2}/\left({K}_{i2}^{h_2}+{x_{\mathrm{CreA}}}^{h_2}\right) \) be the Hill function associated with CreA repression. Alternatively, it can be written as \( {\psi}_{\mathrm{CreA}}=1/\left(1+{\left({x}_{\mathrm{CreA}}/{K}_{i2}\right)}^{h_2}\right) \), which illucidates on the switiching level K i2 and steepness parameter h 2. For partial CreA repression, x CreA ≠ 0, with 0 < ψ CreA < 1. The value h 2 ≫ 1 represents increasingly steeper switching around the switching level K i2. We used h 2 = 4 throughout for the modeling. CreA mutation implies that x CreA ≈ 0, therefore, ψ CreA ≈ 1 in equation (4), in which case equation (3) is not required. Alternatively, a partial loss-of-function of CreA can be represented by maintaining equation (3), but adjusting K i2 and/or h 2 (in case of less effective binding sites). We define \( {\psi}_{\mathrm{Xyl}}={X}^{h_1}/\left({K}_{i1}^{h_1}+{X}^{h_1}\right) \) as the Hill function associated to the active form of XlnR, which according to equation (1) is associated to Xyl and xlnR. In the modeling, the basal transcription level for each transcript is considered as negligible compared to the actual transcript levels.
The parameters K i1 and K i2,{Wt|Mt} are related to the chemical affinity between the respective TFs and their binding sites [33]. The model structure is the same for the XlnR targets, except for variations in the levels of CreA repression. The parameters k is,Mt and k is,Wt are used to assess the mRNA levels for gene i in the Mt and Wt. Let λ i := k is,Mt/k is,Wt be the proportional fold-change between the mRNA level in the Mt to the Wt. It is likely that λ i ≫ 1 for most target genes, since higher transcript levels were observed in the Mt than Wt. Transcription in the Mt does not significantly differ from the Wt when λ i ≈ 1. Overall, we tested the hypotheses that: (i) expression of the XlnR targets is not regulated by CreA repression, (ii) not all response patterns C 3 - C 4 (described below) can be generated by the model in equations (2) to (4).
In equations (2) to (4) we assumed: (a) Xyl cooperatively binds with XlnR at P xlnR and CreA binds to the DNA of XlnR targets. The new model formulation allows us to explain higher order dynamics by assuming, (b) negligible time-delay in translation, (c) transcription only occurs if at least a TF binding site is occupied, (d) all transcribed genes are translated into proteins, (e) all TFs bind independently to the target gene promoter, and (f) mRNA and protein degradation is not regulated.
Fitting the model to the data
The model fit to data was conducted separately and sequentially, starting with the Wt followed by the Mt. In each strain, the model was simultaneously fitted to the data corresponding to use of 1 and 50 mM Xyl. We used the sum of squared errors minimization between the measured and estimated data values. The combined goal function is the sum of two functions one belonging to each of the 1 and 50 mM Xyl induction. Equations (2) to (4) were fitted to the data resulting in a single vector of parameter estimates, θ i where
$$ {\theta}_i={\left[{K}_{\mathrm{on}},{K}_{\mathrm{off}},{k}_1,{k}_2,{k}_{is},{K}_{i1},{K}_{i2},{k}_{id}\right]}^T $$
where θ i is a concatenation of vectors ϑ 1 = [K on, K off, k 1, k 2]T and ϑ 2 ∈ {ϑ 2,Wt, ϑ 2,Mt}; ϑ 2,Wt = [k is,Wt, K i1, K i2,Wt, k id ]T and ϑ 2,Mt = [k is,Mt, K i1, K i2,Mt, k id ]T; K i1 and k id are considered as the same in the Wt and Mt. These two parameters were later fixed for the estimation of k is,Mt and K i2,Mt using the Mt datasets. In equation (5), θ i ∈ {θ i,Wt, θ 2,Mt}, where θ i,Wt = [ϑ 1, ϑ 2,Wt]T and θ i,Mt = [ϑ 1, ϑ 2,Mt]T for the Wt and Mt, respectively. The optimization routine consisted of the goal functions: J i,1(θ i ) = min∑ N j = 0 (y ij − ŷ ij )2 and J i,50(θ i ) = min∑ N j = 0 (y ij − ŷ ij )2 for the 1 and 50 mM induction dataset, respectively. The terms y ij and ŷ ij respectively refer to the measured and estimated data value for gene i at time instant j.
The model fit to data was performed one gene at a time. The goal functions were combined according to the expression J comb(θ i ) = J i,1(θ i ) + J i,50(θ i ). The optimization was performed using the lsqnonlin routine in MathWorks Matlab R2013a. The results for \( {\widehat{\theta}}_i \) are given in (Additional file 3: Table S1 and S2). Initially, \( {\widehat{\theta}}_i \) was obtained in the Wt for all genes and the averages of some parameters were fixed at \( {\widehat{\overline{\vartheta}}}_1 \) and the remaining ϑ 2 re-estimated. Here \( {\widehat{\overline{\vartheta}}}_1 \) is the vector of parameter estimates averaged (and fixed) for all target genes. It contains the least sensitive parameters in the models. On the contrary, \( {\widehat{\vartheta}}_2 \) contains estimates for non-fixed parameters, which are more sensitive than those in \( {\widehat{\overline{\vartheta}}}_1 \). The coefficient of variation (CV) is defined as \( {\mathrm{CV}}_{{\widehat{\vartheta}}_2}=\mathrm{S}\mathrm{D}\left({\widehat{\vartheta}}_2\right)/{\widehat{\vartheta}}_2 \) where SD is the standard deviation.
Qualitative model hypothesis based on analysis of the data
Overall, four classes of transcription patterns were observed in our data (Fig. 1b, classes C 1 - C 4). C 1 is a monotonic increasing function; transcription under C 2 reaches a maximum value after which it gradually decreases to a new steady state. In C 3 and C 4 there is strong evidence of de-repression (time window W 2, Fig. 1b). The models proposed in this study describe the transcription dynamics for some target genes, especially those belonging to C 1. In C 2, first, transcription is activated by Xyl, it attains a maximum value and then decreases. This decrease corresponds to Xyl depletion. The transcription dynamics for some genes could not be captured by our models, e.g. abfB, which has no binding sequence for XlnR [25]; hence, we would conclude that it is not part of the XlnR regulon.
Using our observations and the regulatory model for the XlnR targets as shown in the equations and Fig. 1a, the behavior can be qualitatively described as follows. Initially, transcription is triggered by Xyl-mediated induction (insignificant repression of transcription). At this instance, the CreA concentration is low but gradually builds up to eventually repress transcription (Fig. 1a), as long as Xyl is present in the medium. Transcription is reduced again when Xyl gets depleted, as is depicted in class C 2. The conceptual scheme allows differences in gene expression between various target genes by varying the degree of induction or repression for each individual gene.
The XlnR regulon transcription dynamics
An initial experiment was performed using the Mt and analyzing 7 target genes of the XlnR regulon, in order to evaluate manageable sampling intervals and to obtain insight into Xyl utilization. In a second, more extensive experiment, the Wt was used and more detailed TCD for 23 genes could be obtained. We observed that: (i) XlnR is constitutively expressed, (ii) the target gene expression levels reach higher values at a stimulus of 1 mM compared to 50 mM, both in the Wt as in the Mt, and (iii) in the Mt target gene expression levels are generally higher than in the Wt (Figs. 2, 3, 4 and 5). Expression of xlnR in the Mt was marginaly less than in the Wt at most time points (Figs. 2 and 3H; 4 and 5 W).
Model fit to the data obtained for the Mt using 1 mM Xyl. a-g: Black bold line is the model fit to data with all parameters fixed except k is,Mt and K i2,Mt. The green bold line is the fit with all parameters fixed except k is,Mt, K i2,Mt and k id . \( {\widehat{\overline{K}}}_{\mathrm{on}}=14.957 \), \( {\widehat{\overline{K}}}_{\mathrm{off}}=75.541 \), \( {\widehat{\overline{k}}}_1=21.455 \) and \( {\widehat{\overline{k}}}_2=20.065 \) were fixed. The graphs are plotted on different scales to aid visibility and the shaded areas indicate standard deviations. h: transcript levels of xlnR. The red line is the fit to the mean xlnR expression values. The dots represent normalized and averaged RT-qPCR data
Model fit to the data obtained for the Mt using 50 mM Xyl. a-g: The black bold line is the model fit to data with all parameters fixed except k is,Mt and K i2,Mt. The green bold line is the fit with all parameters fixed except k is,Mt, K i2,Mt and k id . \( {\widehat{\overline{K}}}_{\mathrm{on}}=14.957 \), \( {\widehat{\overline{K}}}_{\mathrm{off}}=75.541 \), \( {\widehat{\overline{k}}}_1=21.455 \) and \( {\widehat{\overline{k}}}_2=20.065 \) were fixed. The graphs are plotted on different scales to aid visibility and the shaded areas indicate standard deviations. h: xlnR transcript levels. The red line is the fit to the mean xlnR expression values. The blue dots represent normalized and averaged RT-qPCR data
Model fit to the TCD obtained for the Wt using 1 mM Xyl. a-v: \( {\widehat{\overline{K}}}_{\mathrm{on}}=14.957 \), \( {\widehat{\overline{K}}}_{\mathrm{off}}=75.541 \), \( {\widehat{\overline{k}}}_1=21.455 \) and \( {\widehat{\overline{k}}}_2=20.065 \) were fixed. w: xlnR transcript levels. The red line is the model fit to the mean xlnR expression values. The graphs are plotted on different scales to aid visibility and the shaded areas indicate standard deviations. The blue dots represent normalized and averaged RT-qPCR data. The black lines show the model fit to the data points
Model fit to the TCD obtained for the Wt using 50 mM Xyl. a-v: \( {\widehat{\overline{K}}}_{\mathrm{on}}=14.957 \), \( {\widehat{\overline{K}}}_{\mathrm{off}}=75.541 \), \( {\widehat{\overline{k}}}_1=21.455 \) and \( {\widehat{\overline{k}}}_2=20.065 \) were fixed. w: xlnR transcript levels. The red line is the fit to the mean xlnR expression values. The graphs are plotted on different scales to aid visibility and the shaded areas indicate standard deviations. The blue dots represent normalized and averaged RT-qPCR data. The black lines show the model fit to the data points
A similar up-regulation dynamic of the XlnR target genes occur within the first hour after Xyl induction (Figs. 2 and 3); however, after 1 h, the same genes show varying patterns in transcription dynamic and the intensity of gene expression (Fig. 4). This difference may partly be attributed to a reduction in Xyl concentration. Unlike other target genes, transcription of e.g. xlnD and bglA is bimodal, i.e. they exhibit a repeated up and down pattern of expression (Fig. 4). Transcription values of some target genes (e.g. eglB, cbhB and aglB; Figs. 4 and 5: r, t and v) remained around their steady states, thus showing less interesting dynamics. Some genes exhibited dynamics that cannot be explained using both the previous and newly proposed models, especially using 50 mM Xyl (e.g. xlnD, xyrA, axeA, ladA, abfB, xkiA and lacA, Fig. 5).
Genes with similar or the same regulation mechanisms often have similar expression profiles and response to stimuli. Such genes are expected to cluster together in a dendogram. Dynamic time warping [34] was used to cluster the data, which enables identification of genes with similar in time-evolution profiles (Additional file 4: Figure S1). In the gene expression clusters, the placement for axhA differs in the Mt irrespective of the inducing Xyl concentration (Additional file 4: Figure S1A and B). In the Wt, induction with 1 mM Xyl resulted in 19 genes in a single cluster (Additional file 4: Figure S1C) while induction with 50 mM Xyl resulted in two large clusters each with 10 genes (Additional file 4: Figure S1D). Genes with bimodal transcription pattern like xlnD and bglA also cluster together in the case of both Xyl concentrations (Additional file 4: Figure S1C and D). Since the gene clusters are based on their transcription profiles, they intrinsically provide clues as to which genes might possess similar additional regulation mechanisms.
Model results
After parameters estimation, our model fits quite well to most of the TCD (Figs. 2, 3, 4 and 5, black lines; Additional file 3: Table S1). Hence, it is possible to find parameter values that are consistent with the data, with the exception of some genes. For the 50 mM experiment the model fits very well to some of the gene profiles, e.g. xyrA and axeA and aguA in the case of the Mt (Fig. 3) and xlnB, aguA and talB in the case of the Wt (Fig. 5).
In the parameter estimation during model fitting to data, the covariance matrix had large correlation values between some parameters. Parameters with poorer estimates were fixed before rerunning the model fit; thereby, reducing the parameter degrees of freedom. To assess the influence of k id , the models were fitted with and without fixing the parameters. Though not significantly pronounced, not fixing k id results in a systematic shift of the model fit to the right (compare green line to the black line) for some genes in case of the Mt data obtained with 1 mM Xyl (Fig. 2). In case of the Mt data obtained with 50 mM Xyl not fixing k id does not result in any significant difference in the model fit (compare green line to the black line) for all genes (Fig. 3). Overall, the difference between the model fits in green and black lines is insignificant.
It remains a challenge to accurately quantify CreA abundance. The lack of data on CreA results in some parameters being estimated with less precision (Additional file 3: Table S1). Such lack of data on relevant state variables still remains a huddle in modeling biological systems [35–37]. The number of parameters in equation (5) poses a challenge in the network inference because of the presence of correlated parameters. Another source of bias in parameter estimation is data measurement noise which complicates the surface of the objective function. This may introduce local minima in an already complex search space, especially for nonlinear models [38]. This need not be a major problem if the interest is to describe the transcription dynamics, but only when an attempt is made to attach a biological meaning to the estimated parameters. To unravel the XlnR regulon dynamics, the derived \( {\widehat{\theta}}_i \) were used to analyze the Hill functions. The effects of the role interplay between ψ Xyl and ψ CreA can be seen from the variation in intensity of transcription on XlnR target genes (Additional file 5: Mt: Additional file 4: Figure S2, and S3A to G, Additional file 6: Wt: Additional file 4: Figure S4, and S5A to V), with possible antagonism between ψ Xyl and ψ CreA.
Unlike induction with 50 mM Xyl (Additional file 4: Figure S3 and S5), the evaluation of the Hill function components using the parameter estimates shows that in case of induction with 1 mM Xyl, the effect of CreA is negligible, i.e. ψ CreA ≈ 1 throughout the 5 h time-frame (compare Additional file 4: Figure S4). Otherwise, Xyl plays a major role (0 ≤ ψ Xyl ≤ 1) in activating transcription (Additional file 4: Figure S2 and S4). This effect is generally reversed in the presence of high Xyl levels (Additional file 4: Figure S3 and S5). Unlike high Xyl concentration, for low Xyl induction the activation term ψ Xyl and the mRNA degradation term k id x i also plays a major role in determining the network dynamics; however, at high Xyl concentration the components ψ Xyl and ψ CreA jointly regulate transcription.
The possibility of feedback by CreA
Additional parameter estimation exercises were done in an attempt to improve the fit, including the introduction of a feedback effect in the model (shown in Fig. 1a, but not in the equations). It was found that this did, indeed improve the fit to data for some genes, but also introduces bias in the parameter estimates. The data are not rich enough to estimate the feedback parameters with confidence. Including a feedback loop in the model requires prior knowledge of candidate molecules and their likely direction of impact, as a repressor or activator. Such a feedback effect may be further complicated if the molecules interact with other regulatory proteins that potentially also regulate the same target gene. Another reason for excluding the feedback effect in the models is because it would only further escalate the number of parameters required to describe the dynamics of the XlnR regulon; hence, the reason for using a relatively simple yet sufficiently powerful model to explain the observed transcription dynamics in the data. Therefore, the feedback mechanism was not further pursued in the current modeling.
It was not possible to obtain good fits by merely changing the switch level K i2 in the CreA inhibition function between Wt and Mt. Rather, the Wt and Mt switch levels remain roughly the same between the Wt and Mt. The estimated rate coefficient k is indicates the relative quantity of mRNA molecules synthesized per unit time (Additional file 3: Table S1). Contrary to expectations the uninhibited transcription rate coefficient k is must be set considerably higher in the Mt to obtain good fits. It is unclear which underlying mechanism in the Mt is responsible for the partial loss-of-function of CreA.
The transcription responses to the 50 mM Xyl induction are given in Figs. 3 and 5. Although the Xyl concentration decreases from 50 mM to ~38 mM (Fig. 6b) after around 5 h, the Xyl level is still high. About 80 % of the initial Xyl pulse is left by 5 h, indicating a gradual decrease in Xyl concentration. It was thus expected that the target gene response would follow a monotonic increasing curve, with a possible slow decrease due to decreasing Xyl concentration. The responses in Figs. 3 and 5 deviate significantly from this expectation.
Xyl concentration. Concentration of Xyl during growth in the bioreactor was monitored by HPLC analyses. a: Induction was performed with 1 mM Xyl. b: Induction was performed with 50 mM Xyl. The shaded areas indicate standard deviations and the black lines indicate the mean fit through the data points
Overall, some genes were lowly expressed and others had noisy expression profiles. Some genes were lowly expressed in the Wt, e.g. eglB, cbhA, cbhB and aglB in the case of both Xyl concentrations (Figs. 4 and 5). The gene bglA shows a bimodal transcription pattern with peaks at ~1 and ~3 h (Fig. 4P). The expression of faeA is controlled by CreA and XlnR, and it is also known to respond to other aromatic compounds [39]. To get insight into how the genes grouped in relation to similarity in their expression profiles, we performed a cluster analysis. Clustering of expression profiles (Additional file 4: Figure S1A, B and D) show that faeA falls in a unique group. A similar mechanism might exist for the other target genes, which would explain the complex dynamics in the data, particularly for induction with high Xyl concentration. Using 1 mM Xyl for induction, after 5 h nearly all Xyl is consumed (Fig. 6a).
ODEs can be used to describe regulatory mechanisms like the kinetics of protein-protein or protein-mRNA interactions. The dynamics in the gene expression profiles provide leads for testing candidate regulation mechanisms for the XlnR regulon. The response curves show the four types of behavior depicted in Fig. 1b, which are typical of high order dynamics, and show fast and slow mRNA degradation for some XlnR target genes. Transcription values of some target genes remained around their steady states and thus have less interesting dynamics. Many genes exhibited dynamics that fit our models (Figs. 2, 3, 4 and 5). The transcription profiles for some of the XlnR targets indicate the involvement of further regulatory mechanisms such as post-translational regulation. In such a case, we do not exclude the possibility that such regulatory mechanisms could be either single or multiple post-translational modification steps.
Even if the interaction between XlnR and Xyl was not yet experimentally proven, an interaction between carbohydrates and XlnR was already proposed by Hasper et al. [40]. The same was suggested for the ortholog Xyr1 in Trichoderma reesei and also for the Saccharomyces cerevisiae Gal4 itself [41].
Two regulatory mechanisms for the transcription dynamics were proposed: i) activation by xlnR, and ii) regulation by CreA (main effect ~3 to 4 h). Significant de-repression is observed in the Mt for some target genes, especially using high Xyl concentrations. Overall, the proposed model to a large extent explains the dynamics of the XlnR regulon concerning the opposing effects of XlnR activation and CreA repression. It also provides an explanation for the larger expression rates achieved with the 1 mM stimulus compared to the 50 mM. We have demonstrated that the same model structure can be used to describe the dynamics of most XlnR targets in experiments involving both the Mt and Wt – especially at low Xyl inducing concentration.
Analysis of our model in relation to the data indicates that some regulatory elements or mechanisms controlling the XlnR regulon are missing. For example post-translational modifications of XlnR and CreA have not been considered. Moreover, additional, yet unstudied TFs (both, activators and repressors) are likely involved in regulation of at least part of the XlnR regulon in an indirect or direct manner. The possible auto-regulatory influence of XlnR might play a role in the regulatory network. Finally, the mechanisms of the loss of function of CreA in the mutant may need some further study.
However, the need for re-parameterization between Wt and Mt still leaves room for further structural improvements. Some of the patterns observed in the higher order dynamics using 50 mM Xyl for induction still remain a challenge to be accurately described; however, in the future, uncovering more regulatory components through experiments should enable further refining of the proposed model in our work. Unraveling the complexity in regulatory mechanism of the network is crucial for engineering strains with enhanced ability to produce PCWDE.
In this work we investigate the dynamics of the XlnR regulon using mathematical modeling coupled with experimental data of gene expression profiles. We have shown that the frequent sampling of the high resolution TCD in our work revealed interesting dynamics in the data. These datasets are useful for network inference and mathematical modeling, particularly for the XlnR regulon that has not been extensively modeled before.
The data used for the modeling and supporting the results and conclusions of this article are included within the article and its additional files.
RT-qPCR:
reverse transcription quantitative polymerase chain reaction
TF:
PCWDE:
plant cell wall degrading enzymes
ODE:
ordinary differential equation
TCD:
time course data
HPLC:
high performance liquid chromatography
URS:
upstream repressing sequence
UAS:
upstream activating sequence
Xyl:
D-xylose
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This work was supported by the graduate school VLAG and the IPOP program of Wageningen University. The authors thank Birgit Jovanovic from the Institute of Chemical Engineering at TU Wien for the assistance with the HPLC analyses.
Molecular Genetics Department, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, Groningen, 9747 AG, The Netherlands
Jimmy Omony
TU Wien, Institute of Chemical Engineering, Gumpendorfer Str. 1a, Wien, A-1060, Austria
Astrid R. Mach-Aigner
Biobased Chemistry and Technology, Wageningen University, P.O. Box 17, Wageningen, 6700 AA, The Netherlands
Jimmy Omony, Gerrit van Straten & Anton J.B. van Boxtel
Gerrit van Straten
Anton J.B. van Boxtel
Correspondence to Jimmy Omony.
JO: data analysis, modeling and wrote manuscript. ARMA: performed biological experiments, RT-qPCR analyses and provided biological background for modeling. GvS and AJBvB: contributed in modeling. All authors read and approved the manuscript.
Matlab codes. The Matlab codes used for modeling the Mt. (ZIP 22 kb)
Matlab codes. The Matlab codes used for modeling the Wt. (ZIP 19 kb)
Parameter estimates and standard deviations for the Wt and Mt TCD. Table S2. Model goodness-of-fit R 2 estimates for the Mt and Wt TCD. (DOCX 24 kb)
Additional file 4: Figure S1.
Dynamic time warping clustering of expression profiles. Figure S2. Hill function plots for data obtained from the Mt using 1 mM Xyl. Figure S3. Hill function plots for data obtained from the Mt using 50 mM Xyl. Figure S4. Hill function plots for TCD obtained from the Wt using 1 mM Xyl. Figure S5. Hill function plots for TCD obtained from the Wt using 50 mM Xyl. (ZIP 1939 kb)
Data for Mt. Data used for the modeling. (ZIP 3 kb)
Data for Wt. Data used for the modeling. (ZIP 7 kb)
Omony, J., Mach-Aigner, A.R., van Straten, G. et al. Quantitative modeling and analytic assessment of the transcription dynamics of the XlnR regulon in Aspergillus niger . BMC Syst Biol 10, 13 (2015). https://doi.org/10.1186/s12918-016-0257-4
XlnR regulon
Empirical systems biology


It seemed out of place to put this directly in the Merchants Guild thread, so I am going to try and compile it here.
How much is booth space?
- $50, both for pre-registration and at the gate. There is no difference in price based on how many days you are attending.
- Visit this website: Edhellen. (Pre-registration ended October 3rd).
What information do I need to give for registration?
- Your first and last name, your fighting name, and your booth name.
What does the vendor fee include?
- Entry for one person, vendor booth space, and a vendor certificate that must be displayed in your booth. You will also be allowed to camp behind merchant's row.
What must I provide as a vendor?
- Your own tent, whatever you are selling (obviously), and any additional accouterments such as tables/etc for your booth space.
Are there electrical and water hookups?
Is there a list of vendors that will be there?
The Merchants Row is actually located on the road directly across from the beach and around the corner from the fighting field. The power outlets are 150 feet behind Merchants Row, and water comes from a three way spigot about that same distance behind. There is also camping space readily available behind Merchants Row, but only one or two fire pits. The 'Row comfortably accommodated the four merchants there 4 out of 5 days, but one additional booth set up on Saturday and was sort of squeezed in in front of a burned out tree stump and almost in front of the lone cabin off to the side.
There is no guide to setting up. You don't get a booth number, location, or anything. You just go and set up wherever in that area suits you best/isn't taken.
Last edited by Sparrow on Tue Oct 12, 2010 12:25 am, edited 1 time in total.
I'm going to go ahead and start this early so I have part of it done already. I have a few things to note as a start, though, to help put my final report in perspective.
- The booth I will be at with my boyfriend/partner in crime is called War Paint Artisans. We paint faces and do drawings, so this may affect how my profits are skewed. You folks who make jewelry and other wearable items will very likely get different results (hopefully other vendors at Octfest will share their experiences here so the review isn't skewed).
- We did one Bel event (Numenor Opener) and made zero profits, but a lot of folks were still hanging around our booth. I am going out on a limb here by hoping that more people at this event means more likely we will get paid business. Regardless, we will try to just have fun even if we don't make anything. This is one of our first events where we weren't provided a table, chairs, and canopy, so those expenses are added to help out those looking to get into being a vendor.
- We've only been face painting for about two years, but here is our history as a team: we have done three Relay for Life events (painting for cheap and donating all profits to the event), two Spring events at my workplace (they kept all profits), and two Summer events at my workplace (ditto). Most of those events included plenty of kids, but also several teenagers and adults. We usually make pretty decent profits for whoever we are working for at those events.
- Belegarth in general has a certain amount of folks who get some kind of temporary body marking; a temporary "tattoo" made of Sharpie ink, skin dyeing, etc. We'd like to provide an alternative to some of those that can be difficult to wash away when needed, or can actually be harmful for your body (I don't recommend using acrylics on your skin; it's alright for the 'once in a while' but it really isn't meant for skin).
- Belegarth also has a decent amount of people who have a character of some kind, so they may also be interested in our drawings and sketches.
So far, I can sum up my costs for Octfest minus my prior cost for paint/art supplies (the products, which will be used for other events too), gas/travel, change for the cash box, and food (as I assume that those expenses will be easier for you to gauge on your own). I also did not have a usable canopy, so I had to purchase one which I am including in the expenses.
Cost for extra person: $25 (pre-registered).
Cost for canopy: $80 (at Big Lots, if you are curious).
Cost for tables: $30 + 1 borrowed.
Total cost for booth setup/site/extra person: ~$220 (divided by two people).
Camping expenses will obviously make this total higher, but I am going to count the $220 as the cost to a vendor with no prior set up/anything in addition to attending the event. For someone with more experience, the cost is $50 for a booth, +$25 if you pre-register another person ($30 if you pay at Troll).
Our total profits, between all of our sales and tips: $228.
This means we made back pretty much all of the costs to go, and next event will actually turn a slight profit due to the fact we have purchased items that will be reused (canopy, lights, table). Overall I am feeling pretty positive about our results (I honestly expected the event to pay for itself in general fun rather than cash flow!).
Last edited by Sparrow on Wed Oct 13, 2010 8:15 pm, edited 1 time in total.
So far, from my perspective.
+ Staff was friendly and willing to answer questions.
++ Feasts provided on Friday and Saturday (counted twice because they feed you twice).
+ Cabins and trailers available to those who don't want to sleep in a tent.
+ Location was nice; lots of people passing by on their way to the field or to camp, nice view of the lake (especially pretty at night), right across from the beach.
+ The other merchants were completely amazing people, extremely friendly, and just generally helpful. I felt right at home with them.
- Pre-reg was only posted about September 20, leaving only 1 week and 6 days to pre-register.
- Price for booths was posted the same day; 1 week and 6 days prior to event.
- No clear FAQ was available; all info gathered by Q&A (waste of staff time, some repeated questions; gave Sir Misti a FAQ made of all the answered questions in thread for next year's use). It can take a while for your questions to be answered (assuming due to staff being busy), and it was suggested that it would be easier to get an answer by emailing.
- Power hookups located ~150 feet away from the site, which was not told to us before.
- Power hookups were not marked vendor use only; campers behind us were using the power off those hookups too. The amount of power being used was causing brown outs. Lady Feri, one booth down from us, could only run one griddle at a time for quite a while.
- No readily available information on trailer and cabin rental; you have to seek it out rather than find it easily.
It seems more negative than positive, however the event itself was awesome, the weather was beautiful (despite the heat the last couple days), and the general atmosphere was great. I will most definitely plan to go again and hope to try to help add something special to the event.
Earlier pre-registration, earlier booth prices, info on cabins and booths.
FAQ. For the overall event.
More information on the site itself.
Posted signs indicating that the power outlets are for vendors that need them.
More information about when events are scheduled.
When can volunteers come down? Maybe schedule some volunteer days (don't know if I've missed seeing them somewhere along the way)? I would like to put it into my calendar to come down and assist, but don't know when a crew would be there.
These are just some of the many reasons Dur-Demarion has a Merchant's Guild. The Guild Master is the one person who finds out everything going on and spreads the word to the Guild as a whole so we're not left in the dark, working as a go-between to the Guild and the Administrators so everyone gets the same information.
Excellent point. I think next year I will try and get in touch with whomever is running the event and quiz them on some of the details, then publish my results here or somewhere similar.
In the main thread I suggested maybe making a message board. I think it would be a good idea, anyway.


FHW architects answered the wish of master of the work to enjoy a resolutely contemporary house on the heights of Verviers.
Getting organized in the slope of the ground, the house opens sometimes on the landscape, sometimes on the arrangement(development) of the garden which includes an outside swimming pool and a stretch of water.
FHW knew how to integrate into the architecture the passion of master of the work for exotic fishes and uncluttered design Allier and warm house.


Castle Donington is a a mix of modern shops with dignified Georgian and Regency houses. Several timber framed houses dating from the 17th century and earlier survive along the main road. It is famous nationally for the Donington Park race track, and also the Download music festival. On May Bank Holiday Monday a medieval market is held, comprising of local stalls accompanied by dancing and music.
Castle DoningtonFollow in the footsteps of motor racing legendsWe go for a spin around the historic Donington Park race circuit - something that gets the adrenalin racing.
Download FestivalAccessibility award for DownloadAttitude Is Everything is a disabled led charity dedicated to improving deaf and disabled people's access to live music.
The Co-operativeCo-op cash helps village tackle holiday hungerThe Northern Membership and Community Council handed over £200 to Castle Donington Food Bank to support the launch of a lunch club for children that will provide hot meals during the holidays.
Castle DoningtonRail links hopes are voiced by airport chiefs"It is vital for the UK economy that the there is support for airports like East Midlands that can deliver greater access to world markets."
Castle DoningtonOff the Tracks Festival to celebrate 30th birthdayThe family-friendly festivals are the perfect chance for you to flee to the countryside, let your hair down, chill out and escape the humdrum of everyday life.
Leicestershire County CouncilSpotlight on traffic problemsAs part of its day-to-day work to manage traffic, the council is looking at the effectiveness of current traffic management in the towns, including double yellow line restrictions, residents' permits and limited waiting times.
Download FestivalPatrols in place in Castle Donington during DownloadPolice officers and Police Community Support Officers will be patrolling the village over the weekend and a well managed traffic plan has been put in place by the organisers to keep traffic flowing freely.
Castle DoningtonMan's body found in Castle DoningtonA post-mortem examination is due to take place today (Monday, June 4).
Download FestivalTime to rock with DownloadDownload Festival returns to Donington Park this weekend - and here's what you need to know.
Castle DoningtonAttempted ATM theft in Castle DoningtonThis morning the premises remains cordoned off while officers carry out a full forensic examination of the scene.
North West Leicestershire District CouncilPizza takeaway plan is approvedA number of submissions were made both for and against the application, with the main concerns being traffic and a lack of customer parking.
Download FestivalMystery performances at Download Festival....There will be several Zippo Sessions throughout the weekend giving plenty of opportunities to catch the performances.
Loughborough Town HallCTC show to raise cash for the BHFCTC Presents will be raising money and awareness in aid of British Heart Foundation in relation to their May production of 'The Best Little Whorehouse in Texas'.
Download FestivalDownload Festival reveals major improvements this year"Download Festival is built upon an ethos of inclusivity and it's incredibly important to us that as many people as possible are able to enjoy the festival to the fullest."
ShepshedFire at Shepshed care homeThe call was made at 10.11pm and when firefighters attended the scene at Charnwood Road, Shepshed, the premises was in the process of being evacuated and there was smoke coming from the boiler room.


text:
Design Thinking and Global Startup
Korea Advanced Institute of Science and Technology(KAIST)
4.7 (96 件の評価)
This course offers a systematic engineering design methodology: Stanford Design Thinking for preparing global startup. The course empathizes the whole Stanford design thinking process, "Empathy-Define-Ideation-Prototype-Test," for building a startup with a business model that has a "customer truly need" component. This course focuses on lean "Prototype and Test" stages in Stanford Design Thinking. For this, the course introduces a novel manufacturing methodology by using 4th Industrial Revolution technology such as FaaS (Factory as a service) with AI, Bigdata, and 3D Printing. As a final achievement of this course, students will make their critical tools such as one-liner, lean-canvas, and global IR (investor relations) pitch deck for the first step of a global innovative startup founder. Although the lectures are designed to be self-contained, we recommend (but do not require) that students refer to the book E. Ries, The Lean Startup, Crown: New York, 2011/T. Hopkins, Selling for Dummies, Wiley: Indianapolis, 2011, and The Mechanical Design Process, D.G. Ullman, 4th Ed., McGraw-Hill, 2010, which can be helpful for this course.
I would thank the instructor and it was clear explanation of the Topic and given key feature points so it may be useful for the learners very much.
Super helpful to learn about design thinking and investor relations. Thank you so much Mr. Yoon Yong Jin!
Introduction to Singapore and Silicon-V Startup Ecosystem and Strategy
5-122:26
5-39:30
Yoon Yong Jin
Design Thinking & Global Startup
[MUSIC] Hi, everyone. This is Yoon Yong Jin from KAIST. Now, we are in the first session of the fifth week of the Stanford Design Thinking and ASEAN Startup. Up to now, we go over the whole the process of the Stanford sesign thinking from the empathy to the test. Just please remind that the lectures last week, we study about the later part of the Stanford design thinking about prototyping plus test. We more focused on the prototyping with the introduction of the 3D printing technology and also like introducing about how to combine with the fourth Industrial Revolution. Emerging technology such as artificial intelligence or like some DfAM, Design for Additive Manufacturing or IoT or big data and blockchain, etc. And so now, I assume that you already go over the whole design thinking process together with the ASEAN startup process. Also, we study about combined process hybrid approach to proceed the startup activity. So as our like the class, keywords is the two keywords. There is one in the Stanford design thinking, the other ASEAN startup, global startup. So this week, we are going to like study about how we proceed their startup in Singapore ASEAN and also I want to introduce you to the, share my experience in the startup activity in Silicon Valley. And also like what kind of startup ecosystem is in the ASEAN area and the Silicon Valley area, also comparing with the Korea and then figure out how we really do the global startup with help of the distemper design thinking framework. So this is course is current content is that we start from the introduced about ASEAN global business ecosystem including the the Singapore case and also the other part of the ASEAN areas, such as Vietnam or Indonesia or Malaysia or other place. And second is I will share why the Singapore can be a global business platform for the Korean small and medium enterprise company. Why there is opportunity in Singapore, also in ASEAN area. And after that, I'm going to introduce about the Singapore startup ecosystem and government agency and incubators and what kind of support we can use to expand our business from the Korea to the ASEAN area. And the more that into it, the more detailed information about ASEAN global business platform which is about Korea Innovation LaunchPad Singapore Association and KILSA. And after that, I want to introduce you to the Silicon Valley Startup Ecosystem with my experience and also like my experience at that time was almost ten years ago and also I have activities with Silicon Valley now together with AI application startup. So what's the advantages? What's the disadvantages in the startup if we want to do the startup in the Silicon Valley comparing to the ASEAN and other area? Okay, let me start with the study to talk about why Singapore and ASEAN areas which in Southeast Asia. As you know in recently, President Moon is talking about the new thought and policy which is about like try to expand our Korean like some business opportunity to the ASEAN area from comparing to the 2016. The Korean ASEAN bilateral trade is around $120 billion, but try to increase that to the $200 billion which is more like doubled. Within the four years, try to double the volume now double for the volume of the this bilateral trade between the Korea and ASEAN area. As the President Moon say, I'm determined to the bring the Korea and ASEAN relations to a new height and of scare to the level of the bilateral cooperation. So the ongoing now, 2019 and 2020 is next year. And now, the there's a lot of activity is going on now between the Korea and ASEAN area. So to look at the roster business opportunity in the ASEAn area, this is the number we can look at. The first thing when you consider about startup, we definitely consider about our customer, right? Number of customer, which is about population and demographic are like some opportunity. So this population in ASEAN area is the third largest population in the world followed by the China and India market. And ASEAN area is around to 630 million people live there, including Singapore Malaysia Vietnam, Indonesia, Thailand, Philippines. Those are major countries in the ASEAN area. And other, there's a lot of other country too and I just summarized that those major country in the ASEAN area. So if you look at their like annual growth rate from the 2014 to 2018. And so they still shows the higher level, higher number of this growth rate around like 3% to the 9% something, depending on the area. And for example, Singapore, even their Singapore GDP is very high, two times higher than Korea shows that the high like growth rate. However, this year actually the Singapore shows the very low like growth rate too and the other country like Vietnam af you can see this, the average like or over the 6% growth rate. And in the overall like average of this growth rate in the ASEAN area these days is around the 5.03% percent of to the 2020. So as you see, the ASEAN is very big market as with big population 630 million people live there and with a big economy cider US dollar, 2.4 trillion US dollar market and then with the desires of the market and also growth rate is huge. So I can say there's a lot of opinions opportunity in the ASEAN area. How about technology? This is the technology and business. When you look at this global compact competitiveness of the ASEAN in 2017 to 2018 from the international monetary fund, IMF announced this information. If you look at the political stability and business environment and economic exchange rate, exchange risk and credit rating from the Indonesia to Vietnam. You can see the Singapore score the highest definitely among the ASEAN area. For example for the political stability is very good and business environment is top in the world and also economics is very good. And exchange risk, which is about how about the money comes in and the money out is very stable and credit ranking is also very high. In total in IMF, they ranked world number three in the world, Singapore, as one of the best place for doing the business in the world. And also Malaysia also shows okay, some ranking global competitiveness for the business too. And also in the technology-wise for the IT knowledge and technology and future readiness for the this technology, Singapore shows the top number one in the world in the digital competitiveness. Because I assume that our possible business item for this class, maybe we need to use a big data or the artificial intelligence, kind of some emerging technology about the fourth Industrial Revolution. So this digital technology readiness is very important. So however, Indonesia shows very bad record about IT knowledge and technology and future readiness. Malaysia and Singapore shows very good ranking for this three area so like this, the ASEAN has a lot of opportunity. However, from this previous slide and this slide, we can see the Singapore and maybe Malaysia can be our choice. We can choose those country as a starting point gateway to the ASEAN market. Because other place like Philippines and Thailand and Vietnam and Indonesia still is not ready, a lot of risk to do the business in that area. So my suggestion is that we can start our business from the very safe market in which is Singapore. And then by leveraging this Singapore opportunity, and then you can expand your business to the ASEAN market through the Singapore, this business platform. This Singapore business platform is very well made over the very long time. You can summarize the ASEAN countries' strengths and weakness from this Vietnam, Indonesia, Malaysia and Thailand and also Singapore. For the Singapore, the management of cities and technology readiness and technology regulation and wireless broadband is very well prepared. So these very good strengths in part for doing the some business related to the fourth Industrial Revolution like topics AI or other stuff. However, the immigrant laws and some female researchers and R&D productivity is kind of a weakness of the Singapore part. And also other country is also have their own strengths and weakness. So it's better to know what kind of the strengths and weakness is there among those ASEAN countries. And I summarize why Singapore? When you compare the Singapore and Korea with number, let me compare with, talk about the PPP, purchasing power per capita. You can think if you calculate with the people GDP and then so make money versus and then living cost. So the Singapore shows that this PPP is around 87/88K, one of the highest over the world and Korea shows there's a 35/36K. And then shows there are around 2.5 times difference, which means the Korean people make less money. And also they need more money to survive in Korea. In Singapore, people have more money, make more money, but the living cost is low. So in the end, the purchasing power is like this. So if you want to do some business whether we need to choose between the Singapore, Korea, then we definitely choose. I definitely recommend you to start your business in Singapore because there is much more purchase power in the Singapore. How about startup ecosystem value? Startup ecosystem value is almost four or five times different between the Singapore and Korea. So you can think about this fact that this is about how much the startup ecosystem, how much is there which means whether there is a lot of company can buy if you do the company. If you start your company, you want to sell your company sometimes, right, either IPO or either MNA from the big company. And Singapore, there's a lot of market, a lot of big company is ready to buy your company. Comparing to the Singapore, Korea there is not that kind of active market to the trade to the company so it may be very painful. It would be very painful the procedure if you do the startup in Korea. Now, the Korea also getting better. However, the Singapore is better environment to do the startup. How about global connection index? Each is also like the Singapore shows much higher than the Korea. Definitely, Singapore use the English as the official language. So that's why Singapore shows the better Global Connection index but not only for that, the cultural stuff and also a lot of different country people live in Singapore. So also not only for that, global education, the kind of stuff is Singapore shows much higher number in the Global Connection Index, which means what? If you want to do some global business, it's better to start in Singapore because you can start your startup with English-based. Also, there is a lot of global venture capital or Global MSCC headquarters are in Singapore. So you can have a more chance to expand your business to the global size if you start your business in Singapore. And also early stage funding for the startup also is higher than Korea too. So like this, Singapore shows a very good environment for doing startup. And there is our global startup ecosystem ranking from the global startup ecosystem report in 2017. This research shows that Singapore shows promise over the world and then the Singapore shows very vibrant startup ecosystem with those high ranking. Even you think travel fee is not that high but when you consider this ranking, is ranked with the every cities, not country. So there is a hundred and thousand cities over the world, among that is the Singapore ranking in terms in the world. Especially number one is the talent, talent ranking is number one, is very strange. Because Singapore is very small country with a very small number of people around 60 million people live there. However, they import very talented people all over the world. And so that's why they have a top ranked in the talent for there's a lot of opportunity. You can think in Singapore when you think about the global startup. For the Courier. There is no cities with record in the recent release of to the 30s like cities, but there is no Korean cities still out of the distal ranking. So like this as an Asian Asian cities Singapore is one of top cities in to the Startup Environment. So this those are the summarize of the the region for the why we launched the business in Singapore. The first is the a lot of funding. The Available Venture Capital size around to 24 billion US dollar. And also for the gateway to the access to the market or on 630 million people in the Austin area the world's third largest market in the world. So the Singapore can be a gateway to the go to the dead pig size of the market. And also business infrastructure from the like World Bank ranking number one in the world top four doing the business in the Singapore. So Singapore is a top ranked in the world. And also a lot of government support with a lot of women entrepreneurs. And there is more but those are the major reason why the we need to think about a Singapore as Startup platform for the business. How about Asean area? So potential of the Asean area I can summarize those three, is number one is big problem of the people. There are 630 million people in the 11 different country. This 11 different countries also, very interesting like Singapore is very developed country. However, there's many like a developing country around the Singapore in Asean area. So there's a lot of business opportunity you can think. Not only for the top technology like that kind of top-notch based business. But also you can think about very low-tech or mid-tech. Those kind of for example, like appropriate technology for the poor country that kind of business opportunity is there these days. Many social Ventures getting to the Asean market to expand their like Global Market like market share. Also number two is a tough five South Asean country shows the very big growth rate around 5.06% from the 2017 to the 2021. Which is much higher than the USA or the Europe. And third most important importantly importantly is that60% of the population is very young age population around 15 to 34 years old, which means you can start your business. The high-tech building is much easier than other place because other place many population kind of go to the elderly people. So elderly people is not that used to get news to the those kind of new technology. So those because those are the Asean area many population is young generation. So it's very easier to introduce those that like new like technology for example, smartphone based or other like some ARVR that kind of technologies. And in addition to this the southeast Asia is a fast-growing market and one of the unknown market. So that's why I emphasize that you need to think about Asean Market not only this region. But also if you're Korean because the now the Korean government like push more to the their economic the relationship trying to increase their economic relationship with South East Asia and also even you are not in the Korean like North Korean. You also can think about getting to the Asean Market because there is opportunity in the Asean area. This is the some Global the companies like some startup company. Now they become the big company they start from the start of company become the communication Network company. And then Unicorn company is around there like a valuation over the 1 billion US dollar and those are the Asean based global companies, which is Unicorn company I can say. Maybe grab and VNG and then Garena and Lazada and Indonesia Tokopedia. Those are the some startup company now already they hear the information is quite a little bit not that updated yet the group. He's already go over the much more than 1.5 billion valuation. So the you can do in Korea there is also those kind of startup company. However, due to the market like that kind of I'll population and also like a lot of Young Generation and also like English based. So they try the many Unicorn companies. Unicorn companies can the come out from these Asean area. So I hope you guys also like try to restart your business in the Asean area. Plus you can use a very well-educated like your partner's you can find easily well-educated partner in the Asean area easily too. So that's the also very important point when you talk about global startup like when you talk about doing the Global Startup, okay? This is the first session of the 50 of The Stanford design thinking and Asean Startup. So maybe we'll move on to the next session. Thank you for your attention and see you again.

redpajama_set_name: RedPajamaCommonCrawl


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Thanks for the demo — this is the first I've seen the feature in use. We might have occasion to use it at my mother's WordPress.com site someday. I'm kind of disappointed that there isn't an option to expand to full screen.
You bog-a-holic!! but cool eh?
Nice job on the slideshow! Will it become a regular feature?
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The implementation priorities for an institution are the most important priorities. They are the high-priority actions that are achievable. To determine these, How To Restore Fire Damaged Pictures t is helpful to consider the criteria of impact and feasibility together for each action.
A device that is useful for this is a grid developed by Pamela Darling, which is shown here in a modified form. The impact and feasibility of each action are plotted on the grid shown on the following page. Darling explains that those How To Restore Fire Damaged Pictures actions that are of high impact and can be implemented with little difficulty are placed in Box #1.
Those actions that have high impact but are difficult to implement go into Box #3. Those actions that are not difficult to implement but will have little impact go into Box #2. Those actions that are difficult to implement and have little How To Restore Fire Damaged Pictures impact go into Box #4.
Darling goes on to explain that those How To Restore Fire Damaged Pictures actions in box #1 probably deserve highest priority, since they can be easily accomplished and will have significant impact. Those in Box #4 can often be postponed or even disregarded because they achieve little while requiring great effort. Many of those in Box #2 can also be eliminated because they accomplish little, though some may be worthwhile because they are easy to do.
Box #3 How To Restore Fire Damaged Pictures items need careful consideration: despite their difficulty, they deserve implementation because of their high impact. REMEMBER: One of the most difficult aspects of preservation planning is prioritizing. Planning requires significant people skills and an understanding of the organizational dynamics of the institution. Nowhere is this more How To Restore Fire Damaged Pictures evident than in prioritizing.
You need to bring all your How To Restore Fire Damaged Pictures interpersonal skills to bear on discussions of priorities with your colleagues. You need to listen to what the issues of other departments are and be able to focus on what best serves the needs of the institution as a whole rather than just on the needs of your particular How To Restore Fire Damaged Pictures department or area of expertise.
In the long run, this will best serve your needs as well. At the same time, you need to be a skillful negotiator and a good sales person. As with most other dealings with people, a good sense of humor will ease the process. "Every library collection is established for one or more definite purposes.
A collection development and management program organizes and directs the processes of acquiring materials, integrating them into coherent collections, managing their growth and maintenance, and How To Restore Fire Damaged Pictures deselecting them when appropriate in a cost- and user-beneficial way."
A coherent How To Restore Fire Damaged Pictures collection policy also establishes the parameters within which a systematic preservation program operates. The collection policy is based on the institutional mission statement, which enunciates the goals that the How To Restore Fire Damaged Pictures collections are to achieve.
The collecting priorities that the collection policy establishes help to focus preservation activity on the most important parts of the collections. To put it colloquially, the mission statement tells you where you are going; the collection policy gives you the details of how you will get there; and the preservation policy makes sure that at least the most valuable portions of the baggage do not fall apart en route. Collection policies define the scope of current collections and indicate areas in which future collections may be How To Restore Fire Damaged Pictures developed.
By specifying the How To Restore Fire Damaged Pictures subjects and formats to be included in or excluded from the collections, the policy encourages consistency in selection of new materials and deselection of those that no longer serve institutional goals.
Because it deals with the full range of a library's collections, the policy makes selectors or bibliographers aware of the breadth and variety of materials being collected and thus helps them to see the collections as a whole rather than focus on just those portions for which they are responsible. It thereby How To Restore Fire Damaged Pictures encourages communication and cooperation, identifies areas of weakness, and helps to determine priorities for other library functions such as cataloging and preservation.
A collection policy also looks outward, if only by implication. This means that it takes into consideration the holdings of other repositories, especially in subjects of relatively esoteric How To Restore Fire Damaged Pictures research interest.
Until recently, this How To Restore Fire Damaged Pictures has in most cases meant repositories located fairly nearby which could and would provide convenient access. The advent of the photocopy machine, the growth of automated ILL systems, the steadily increasing number of preservation microfilms of important research How To Restore Fire Damaged Pictures collections, and now the ability to digitize on demand information for electronic transmission have steadily expanded the geographic range from which materials can be obtained with relative ease.
As a result, collection policies and preservation programs tend to place a higher priority on materials of particular importance to the institution's programs that cannot be readily obtained elsewhere. It is not possible to develop a successful preservation program without a clear mission statement and a comprehensive How To Restore Fire Damaged Pictures collection policy.
Ultimately, How To Restore Fire Damaged Pictures preservation is about setting priorities because not even the richest institutions are able or willing to preserve everything they have collected for all time. The collection policy helps to determine preservation priorities because it states the level at which the institution collects in any given subject.
That level is in turn usually determined by the importance of a given collection to the institution's programs and ultimately to its mission. An objective How To Restore Fire Damaged Pictures methodology for determining collection level by measuring the quantity and types of materials in it is often used by collection managers and preservation administrators.
"The RLG Conspectus is a collection assessment method that maps subjects' strengths and weaknesses within an individual library, a consortium of libraries, or a geographical region using How To Restore Fire Damaged Pictures standardized criteria and descriptions."
The Conspectus uses a How To Restore Fire Damaged Pictures numerical scale with standard definitions to describe the types of client activities supported by the collection. These are, in descending order: comprehensive (5), research (4), intermediate (3), basic (2), minimal (1), and out of scope (0). Levels (1) and (2) are subdivided into two levels and (3) into three levels to provide finer distinctions that are useful in describing smaller collections.
The ALA Guide for Written Collection Policy Statements cited in note 1 provides further information on the use of the Conspectus in How To Restore Fire Damaged Pictures collection policies. Collection level is not, however, the only criterion to be noted in determining a collection's importance to an institution as a factor in establishing its priority for preservation.
Another important feature is whether or not it contains materials that are artifactually valuable or have a significant associational value for the institution. In dealing with archival materials, one should also consider How To Restore Fire Damaged Pictures evidential value.
Such value pertains to How To Restore Fire Damaged Pictures materials needed because of their legal, administrative, and/or fiscal significance to an organization. If well thought-out and comprehensive, collection policies provide a vital point of reference for making preservation decisions.
Preservation considerations must also inform a collection policy if it is to serve as a reliable guide to the development and management of collections. "All How To Restore Fire Damaged Pictures collection management decisions, made from the time of acquisition of an item, are embodied in that item's physical deterioration and its ultimate need for preservation intervention."
Thus, at each stage of the process of acquiring, processing, housing, providing access to, maintaining, and eventually deselecting materials, all library staff, and especially those directly involved in collection development and management, should have clearly in mind the preservation implications of their How To Restore Fire Damaged Pictures decisions and actions.


text:
Newton's Hammocks
Email Alerts|
Hammock Rocker Helps Soothe Worries and Ease Tension
Newton's Hammocks Celebrates One Year Anniversary of the Hammock Rocker Launch
Portland, CT -- (SBWIRE) -- 04/27/2016 -- The hammock experience can be mesmerizing. Aficionados can imagine themselves relaxing in a hammock on a warm, breezy afternoon – a quietly exhilarating experience. Lying motionless, at peace with the world and fretting about nothing is the near-perfect escape. The best way to improve this experience is to sway slowly back and forth. According to the U.S. Patent Office, there are numerous "electro-mechanical devices," some with 200+ parts that fit the bill. What these machines accomplish with much sound and fury Newton's Hammock Booster does quietly with just two functional components – a stick and a string. The entire device includes a small clip that connects the string to the stick, and a thin 6-inch piece of wood to wind the string for storage.
"Blue-collar, white-collar or no-collar, it doesn't matter," says Dr. Cherackal Chacko, retired physicist and founder of Newton's Hammocks, "…when workers return home they should be able to relax and recharge, and I have not found a better way to do so than to unwind in a gently swinging hammock."
Newton's Hammocks is celebrating the anniversary of the launch of the Booster by reminding current and prospective customers of two things:
One: improving the functional parts of the Newton's Hammock Booster is not a difficult thing to do. The average person, however, is unlikely to reproduce the Booster 's quality of material and workmanship without spending a fortune on expensive components. The caveat of an improvised rocker is the hygiene. Every time someone use the hammock they are forced to drive the device into the ground – effectively making the cleaning and storage a continual headache.
Two, Newton's Hammock Booster is a thoughtful, green gift, and the company is starting a policy of volume discounts for its customers.
Newton's Hammock is a product distinguishable from competitors in multiple ways:
-No batteries or power source required.
-No switches or buttons to push or turn.
-Minimal effort required.
-Lightweight (less than 6 oz.
-All in perfect silence!
-Green, handcrafted, domestic product
-Self-operated, total privacy, no external intervention needed
Modern life, especially life in America these days, feels like all work and no play. For most of us it is easier to put in eight hours of honest work for our employer than to spend 30 minutes on ourselves in a truly relaxed state. Achieving that release of tension, especially in our nerves and muscles, is a difficult thing to do on a regular basis. However, Newton's Hammock Booster is a special piece of equipment that helps realize the very best in relaxation and soothing sensations – and it is one that is sure to please its clientele for years to come.
About Newton's Hammocks
Newton's Hammocks is a boutique Connecticut-based company that debuted the Hammock Rocker in February 2015. The company has integrated its products into its own offices. At Newton's Hammocks, it is mandatory that every employee spend half an hour — for which they get paid — in a hammock, either at home or at work.
www.newtonshammocks.com
Dr. Cherackal Chacko
Email | Web | Profile
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Source: Newton's Hammocks
Posted Wednesday, April 27, 2016 at 2:00 AM CDT - Permalink
For information regarding the content of this press release please contact the media relations contact listed above directly.
Copyright © 2005 - 2021 - SBWire, a service of ReleaseWire LLC


Dozen Sickened on Flight From Virgin Islands to U.S.
CHARLOTTE – Emergency and fire crews responded to a major North Carolina airport after about a dozen passengers and crew members on a flight from the U.S. Virgin Islands complained of sickness.
WCNC-TV reported that seven US Airways crew members were taken from Charlotte-Douglas International Airport Saturday night after they complained of headaches and nausea.
The crew members had been onboard a plane that had just arrived from St. Thomas in the U.S. Virgin Islands. Five passengers from that flight were treated on the scene.
Shortly after the plane took off from St. Thomas, crew members noticed a slight odor on board and alerted authorities in Charlotte.
Investigators are working to determine what caused the odor, and the plane has been taken out of service.


International Watch Co., Schaffhausen, "Spitfire Double Chronograph"
Case: steel, anti-magnetic protection cap. Dial: two-tone silvered. Movm.: rotorwind movement, Glucydur balance.
Housed in a 44-millimetre stainless-steel case, the 79230-calibre chronograph movement vouches for the watch's mechanical perfection. With its soft-iron inner case for protection against magnetic fields and a sapphire glass secured against sudden falls in pressure, the Double Chronograph has all the features needed by a watch designed for flying.


text:
FN Awarded U.S. Army Contract for the M249 SAW
The company will continue to produce the light machine gun, in service since 1986
By The Editors | Published Sep 24, 2020 6:21 PM
M249 SAW light machine gun. Daniel Scott Booth 2015
The U.S. military has been using the 5.56 NATO M249 Squad Automatic Weapon since 1986. It replaced the M60 machine gun, chambered in 7.62 NATO. Daniel Scott Booth 2015
FN America, LLC has announced it has been awarded a firm-fixed-price contract to supply the U.S. Army with M249 Squad Automatic Weapons, also known as the M249 SAW. The contract is expected to be completed by 2025.
The FN M249 SAW has been a mainstay throughout the U.S. military since 1986 and is currently in service in more than 30 countries globally. FN has been the sole source manufacturer of the 5.56-chambered belt-fed lightweight machine gun.
Designed for front line applications delivering crucial support at the infantry squad/fire team level, the FN M249 SAW provides highly accurate fire from a highly maneuverable light machine gun. The ergonomic polymer buttstock contains a hydraulic buffer that allows SAW gunners to maintain a high rate of fire with accuracy and effect.
"We are honored to continue our dedication as a critical partner to the U.S. military and pleased to announce this latest Army contract award for the FN M249 SAW, a flagship design for FN, in service for more than 30 years. The proven design has served the U.S. military, reliably and without fail," said Mark Cherpes, President and CEO for FN America, LLC. "We look forward to building these machine guns at our production facility in Columbia, South Carolina."
The M249 SAW was originally adapted by the FN Minimi.
Throughout its history, FN has been one of the largest suppliers of small arms to the U.S. military. In addition to the M249 and its variants, the company currently holds contracts for the FN M240 medium machine gun and its variants; the FN MK 46, MK 48, MK 17 and MK 20 SSR for USSOCOM, and various other contracts.
For more information about FN's military product line or current U.S. military contracts, please visit www.fnamerica.com.
FN USA
M249 SAW
Military Contracts


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Bursa Uludağ Üniversitesi (deutsch Bursa Uludağ Universität) ist eine staatliche Universität in der türkischen Stadt Bursa.

Geschichte
Die Bursa Uludağ Üniversitesi wurde erstmal 1970 abhängig von der Universität Istanbul als medizinische Fakultät Bursa gegründet. Nach der Errichtung einer medizinischen Einrichtung in Bursa wurde der Lehrbetrieb 1974 in Bursa fortgesetzt. Nach einer Beschlussnahme des türkischen Parlamentes wurde die Universität 1975 offiziell als Bursa Üniversitesi gegründet und erhielt 1982 ihren heutigen Namen.

Fakultäten
 Medizinische Fakultät
 Fakultät für Wirtschafts- und Verwaltungswissenschaften
 Fakultät für Maschinenbau
 Fakultät für Veterinärmedizin
 Fakultät für Bildungswissenschaften
 Theologische Fakultät
 Fakultät für Künste und Wissenschaften
 Fakultät für Landwirtschaft
 Fakultät für Rechtswissenschaft
 Inegöl Fakultät für Betriebswirtschaftslehre
 Fakultät für Sportwissenschaften
 Fakultät für Architektur
 Fakultät für Gesundheitswissenschaften
 Fakultät für Zahnmedizin

Weblinks

Einzelnachweise

Universität in der Türkei
Bursa
Gegründet 1970
Organisation (Provinz Bursa)

redpajama_set_name: RedPajamaWikipedia


text:
Q: What is the use of indexes in Couchbase server? Can anyone explain what is the use of indexes in Couchbase server in layman terms?

A: Indexes in Couchbase Server are used for the same reason indexes are used in any database: to enable and enhance the performance of queries.

redpajama_set_name: RedPajamaStackExchange


text:
Gambia must release four activists jailed for distributing anti-government T-shirts
Amnesty International today called for the immediate release of four activists arrested over the distribution of T-shirts calling for an end to dictatorship in Gambia.
One activist, Dr Amadou Scattred Janneh, the country's former Minister for Information and Communication, was yesterday sentenced to life imprisonment with hard labour for treason. Three other men - Modou Keita, Ebrima Jallow and Michael Uche Thomas - were each sentenced to three years with hard labour for sedition.
The four were arrested in June last year after distributing T-shirts made by the NGO Coalition for Change - The Gambia (CCG) which featured the slogan "End to Dictatorship Now".
Dr Amadou Scattred Janneh, who has American citizenship, was arrested in his office on 7 June for being in possession of the CCG T-shirts. Michael Uche Thomas, a Nigerian, and Modou Keita and Ebrima Jallow, both Gambians, were arrested on the same day, accused of printing the T-shirts.
The four men were sentenced at the Special Criminal Court in Banjul. Charges were initially brought at the Banjul Magistrate Court on 13 June and the case was later transferred to the High Court.
Amnesty International Gambia researcher Lucy Freeman said:
"The conviction of these men is a violation of their rights to freedom of expression, assembly and association.
"President Jammeh is once again proving that he does not tolerate any form of criticism and is ruthlessly persecuting anyone who speaks out against his regime.
"These men are prisoners of conscience and must be released immediately and unconditionally. Gambia must stop such acts of persecution and allow criticism to be heard in the country."
Journalists and activists in Gambia are routinely subjected to human rights violations such as unlawful arrests and detentions, torture, unfair trials, harassment, assaults and death threats, making it extremely difficult for them to do their work.
According to CCG spokesperson Ndey Tapha Sosseh: "The Coalition for Change - The Gambia is an organisation with the objectives of ending The Gambia's dictatorship and as well as the climate of fear that has so consumed Gambian society."