Most modelling efforts of transcriptional networks involve estimations of in vivo concentrations of components, binding affinities and reaction rates, derived from in vitro biochemical assays. These assays are difficult and in vitro measurements may not approximate actual in vivo conditions. Alternatively, changes in transcription factor activity can be estimated by using partially specified models which estimate the "hidden functions" of transcription factor concentration changes; however, non-unique solutions are a potential problem. We have applied a synthetic biology approach to develop reporters that are capable of measuring transcription factor activity in vivo in real time. These synthetic reporters are comprised of a constitutive promoter with an operator site for the specific transcription factor immediately downstream. Thus, increasing transcription factor activity is measured as repression of expression of the transcription factor reporter. Measuring repression instead of activation avoids the complications of non-linear interactions between the transcription factor and RNA polymerase which differs at each promoter.
Using these reporters, we show that a simple model is capable of determining the rules of integration for multiple transcriptional inputs at the four promoters of the arabinose catabolic pathway. Furthermore, we show that despite the complex and non-linear changes in cAMP-CRP activity in vivo during diauxic shift, the synthetic transcription factor reporters are capable of measuring real-time changes in transcription factor activity, and the simple model is capable of predicting the dynamic behaviour of the catabolic promoters.
Using a synthetic biology approach we show that the in vivo activity of transcription factors can be quantified without the need for measuring intracellular concentrations, binding affinities and reaction rates. Using measured transcription factor activity we show how different promoters can integrate common transcriptional inputs, resulting in distinct expression patterns. The data collected show that cAMP levels in vivo are dynamic and agree with observations showing that cAMP levels show a transient pulse during diauxic shift.
Early experiments in the utilization of different sugars demonstrated that bacteria will preferentially use glucose over many other carbon sources, a phenomenon termed the glucose effect . Jacques Monod measured the growth curves of Escherichia coli, Bacillus subtilis, and Salmonella enterica in combinations of different sugars, and found that some combinations resulted in a simple growth curve, while others resulted in a biphasic, or diauxic, curve which is the result of preferential catabolism of glucose in the first growth phase, followed by a lag phase during which the catabolic proteins required for using the second sugar are made . Two mechanisms are responsible for the glucose effect; inducer exclusion and catabolite repression, which are mediated by the phosphoenolpyruvate - dependent transport system (PTS). The first mechanism involves the inhibition of numerous permeases by direct protein-protein interaction with the dephosphosphorylated form of enzyme GluIIA , a phenomenon termed inducer exclusion. Also, in the absence of a glucose, phosphorylated enzyme GluIIA will activate adenylate cyclase to increase formation of the second messenger, cyclic AMP (cAMP) [3–5]. cAMP binds to the transcriptional regulator CRP which is involved in regulation of numerous catabolic operons; and it is the maintenance of cAMP at low concentrations during growth on glucose that is the basis of catabolite repression.
The role of the PTS system in the modulation of cAMP levels, and the relative contributions of inducer exclusion and catabolite repression to the glucose effect are controversial. Experiments have shown that while cAMP levels increase during the lag phase, there is no appreciable difference between intracellular cAMP levels during growth on glucose or lactose [6, 7], while conflicting studies show a large difference [8, 9]. Furthermore there is evidence that there is little relationship between glucose flux and intracellular levels of cAMP, and that E. coli increases its intracellular concentration of cAMP before glucose flux decreases , which suggests that the PTS system cannot be solely responsible for regulating intracellular concentration of cAMP. However, more recent work has contested this result, and shown that cAMP levels increase as glucose levels reach 10 μM, in concert with increasing phosphorylation of enzyme EIIAglu Despite its role as a global regulator of nutrient status  involved in modulating the expression of numerous catabolic operons [4, 7, 11–15], the contribution of CRP-cAMP to the glucose effect is not completely understood.
The arabinose catabolic pathway is among those positively regulated by CRP-cAMP and is comprised of four operons, all regulated by the same two transcriptional regulators: AraC and CRP. These operons code for genes involved in regulation, transport, and catabolism of arabinose, and each of their respective promoters have binding sites for the transcriptional regulators AraC and CRP that differ in sequence and location relative to the -35 site. The AraC dimer represses transcription of ParaBAD in the absence of arabinose via loop formation in the regulatory region of the araC/araBAD operon [11, 16–20]. Binding of arabinose to the C-terminal dimerization domain of the AraC dimer induces flexibility in the dimer, allowing the release of the loop and binding of the dimer to the operator that overlaps the -35 region of ParaBAD, leading to transcription initiation at this promoter [17, 21]. ParaC is transcribed divergently from ParaBAD and in this case, AraC acts as a repressor at high concentrations [11, 22]. While transcription of ParaC has a low basal level, the binding of CRP in the araBAD/araC regulatory region increases transcription . The promoters of the arabinose uptake operons, ParaE and ParaFGH, also have binding sites for both transcription factors. The order and position relative to the -35 region of CRP and AraC operators in ParaE are the same as ParaBAD; however, the order of binding sites in ParaFGH is the reverse, with the CRP site overlapping the -35 region . The differences in sequence and position of transcription factor binding sites determine the strength of interaction between transcription factors and the operator, and transcription factors and the RNA polymerase, which in turn govern the overall transcription rate and kinetics of induction. Thus differing gene expression among the four arabinose regulated promoters is the result of varying integration of the common signals from the two transcription factors and RNA polymerase.
Typically, detailed models of promoter activity [13, 26–30] require determining or estimating numerous binding constants, transcription coefficients, degradation coefficients, and intracellular concentrations of each network component, which can be problematic. Alternatively, models can be fit to experimental data in order to estimate parameter sets that explain observed behaviour [14, 31], and functions describing transcription factor activity can be estimated from expression data [31, 32]. However, non-uniqueness of parameter estimates is a common problem, especially where there are multiple parameters to be estimated. Our knowledge of the integration of transcriptional signals, particularly cAMP-CRP, at sugar catabolic promoters is commonly derived from measurements of steady state promoter activity in gradients of its exogenous inducer, cAMP [14, 33]. However, cAMP can be toxic in high concentrations, and evidence has shown that during diauxic shift cAMP levels in vivo vary in a non-linear manner, which has led to discussion about the role of cAMP-CRP in diauxic shift [6, 7, 34–36]. Furthermore, while promoters under positive control by CRP have been used to measure CRP activity within the cell [8, 13], the interpretation of these data is difficult because the interaction between RNA polymerase, cAMP-CRP and the promoter are strongly context dependent, result in non-linear transcriptional activation, and will vary from promoter to promoter. Furthermore, cAMP concentrations within the cell have been shown to increase and decrease quite rapidly  and a positively controlled promoter may not reflect this. Therefore, it is unclear how we can understand transcriptional dynamics in diauxic shift based on knowledge gained from steady state analyses and without a more direct method of measuring cAMP-CRP dynamics in vivo.
Synthetic biology offers an alternative method of measuring the activity of transcription factors in vivo. We report here on the development of synthetic reporters for independently measuring in vivo activities of CRP-cAMP, AraC-arabinose, and RNA polymerase while investigating diauxic shift in the arabinose regulon. The synthetic transcription factor reporters measure transcription factor activity via repression of a constitutive reporter. We use these synthetic reporters to measure transcription factor activity during diauxic shift to determine whether the rules of integration determined from steady state behaviour can be extended to explain dynamic behaviour of sugar catabolic promoters in changing environments.
We analyzed sequences of the I1 and I2 AraC operators and CRP operators of the four promoters in order to design primers for the transcription factor reporters. Sequence alignments of the AraC I1 and I2 sites show strongly conserved residues in the I1 portion of the binding site, but more variability in the I2 site. Interestingly, the consensus of Seabold and Schleif  has a shorter I2 site by one base pair than the sequenced sites from the arabinose regulon, or than the consensus published by Gallegos and coworkers  (Figure 1a), and it was this consensus that provided the strongest binding (Additional file S1, Figure S1). Sequence analysis of the CRP binding site showed that there are five strongly conserved bases (Figure 1b). Based on these calculated consensuses we designed the primers for the transcription factor reporters which included degenerate sites (Figure 1c, Table 1) and cloned them immediately downstream of a synthetic σ70 derivative promoter called synRNAP-σ70 (AATAATTCTTGAAATTTATGCTTCCGGCTCGTATTTTACGTGCAATT). This design resulted in a constitutive promoter whose expression was repressed by the binding of the transcription factor and the inclusion of degenerate sites allowed us to tune the strength of transcription factor binding. A library of reporters was picked and tested for repression in the presence of the inducers (cAMP, arabinose) (Additional file S1 Figure S1, Additional file S1 Figure S2, Additional file S1 Figure S3), and the reporters that showed the strongest repression were named synARA and synCRP and used in all further experiments.
Sequence and Structure of Synthetic Transcription Factor Reporters. A. Predicted consensus of I1I2 operators. The predicted consensus from the four AraC regulated promoters is compared to the consensus from Gallegos et al. 1997. Micro. Rev. 61(4): 393 and from Seabold and Schlief 1998. JMB. 278: 529. B. Predicted CRP operator consensus from four promoters. C. Construct of the synthetic transcription factor reporters.
To test how concentrations of the two inducers affected gene expression we measured the steady state expression of the four arabinose regulon promoters (ParaBAD, ParaC, ParaE, ParaFGH) in a combinatorial gradient of both inducers (Figure 2a). Steady state was considered to have been reached by 44 minutes. ParaE exhibited the strongest expression, and ParaC exhibited the lowest. All promoters required arabinose in order to detect any reporter expression and responded in a largely step-like manner. cAMP served to modulate expression in the presence of arabinose in a graded manner. ParaC exhibited slight inhibition in higher concentrations of arabinose and cAMP (Figure 2a). The three synthetic reporters were also assayed under these conditions. synARA and synCRP exhibited graded repression in the presence of their respective small molecule inhibitors (Figure 3). However, there was also an effect of arabinose addition on the repression of synCRP. At all time points synRNAP-σ70 remained largely unaffected by the concentration of either small molecule effector (Figure 3).
Gradient of expression in four arabinose regulon promoters. Graphs show expression data (CPS/OD) 44 minutes post induction. A. Measured expression of the four arabinose catabolic promoters. B. Predicted expression based on the mathematical model.
Activity profiles of the three synthetic transcription factor reporters. Expression (CPS/OD) was measured in a gradient of cAMP (0-1mM) and arabinose (0-0.2%) at 44 minutes post induction. Activities for synARA and synCRP were calculated using equation #2, and raw expression for synRNAP-σ70 is shown.
The kinetic behaviour of the four arabinose regulon promoters was investigated in conditions of diauxic shift which measures promoter and transcription factor activity in conditions of rapidly changing endogenous cAMP concentrations, from low (high glucose) to high (all glucose consumed) cAMP. Briefly, the optical density and luxCDABE expression of both the catabolic promoters and transcription factor reporters was measured every 4 minutes during exponential phase, in a medium with subsaturating glucose and saturating arabinose. From these data, promoter expression (CPS/OD) and transcription factor activity for AraC-arabinose and CRP-cAMP was calculated according to equation 2.
In the diauxic shift experiments, the exhaustion of glucose varied between experiments. Therefore, we adjusted the start of the experiment to be twenty minutes before the increase in synCRP activity. CRP-cAMP exhibited a strong peak of activity in all assays, which then rapidly returned to baseline activity. Conversely, AraC-arabinose activity increased in an exponential manner after the reduction in cAMP-CRP activity (Figure 4).
Calculated transcription factor activity during diauxic shift. The period of glucose starvation is indicated by the peak in CRP-cAMP activity. Activity is calculated using equation 2 from measurements of synRNAP-σ70, synCRP and synARA expression. These profiles represent mean expression from n = 7 experiments with time 0 normalized to the start of glucose exhaustion.
The model of promoter expression as a function of measured transcription factor activity (Figure 5) was fit to data of steady state expression using non-linear least squares fitting (procedure nls, R ). The results of the fit are presented in Table 2. All models had statistically significant estimates for α and γ; however, the estimates for β for ParaBAD, ParaC, ParaE and ParaFGH are all insignificant at p = 0.05. In these cases the model was refit by removing this term and the residual squared error (RSE) and Akaike information criterion (AIC) were calculated. In every case, both RSE and AIC showed improvement in model fit by removing this term. Model fit was tested by predicting gradient expression given measured synthetic transcription factor activity (Figure 2b). These simulations showed that the fit predicted steady-state gradient expression in all four transcription factor reporters quite well.
Simple mathematical model of promoter expression in arabinose regulon. The promoter, P, can be bound to AraC-arabinose (A*), CRP-cAMP (C*), or both. Transcription is theoretically possible in any of these states and is described by transcription activation rates α, β and γ respectively. Note that these activation rates include binding equilibria for transcription factor binding, and there is no cooperativity.
Fitted coefficients from fit of model to steady state expression.
The models derived from this fit were then tested for their ability to predict induction in diauxic shift (Figure 6). The predicted expression was calculated by using the estimates for α and γ derived from each fits to steady state data (Table 2), and the calculated transcription factor activity measured by the transcription factor reporters (Figure 4). The models predicted expression that mimicked measured expression in both magnitude and timing, with the exception of ParaFGH. In this promoter, the measured magnitude of expression was approximately two-fold higher than predicted. Also, the dynamics of expression in ParaC were slightly underestimated by our model.
Observed and predicted kinetics of expression (CPS/OD) of four AraC regulon promoters, P araBAD , P araC , P araE , P araFGH , during diauxic shift. Predictions are calculated using TF activity profiles (Figure 4) and transcription activation rates estimated from model fits to steady state expression data (Table 2).
Three synthetic transcription factor reporters, synARA, synCRP, and synRNAPσ70, were used to measure the in vivo steady state activity of AraC, CRP, and RNA polymerase in gradients of arabinose and cAMP. This information was used in a mathematical model to determine the rules of integration of common transcriptional inputs at the promoter level. The test of the model was to determine whether these rules could predict promoter behaviour during diauxic shift when AraC-arabinose and cAMP-CRP activity were measured in vivo. At steady state, the transcription factor reporters showed a graded response to the concentration of their respective small molecule effector, demonstrating that repression is an effective method for measuring transcription factor activity that obviates the need for estimating various binding and degradation constants. There was a slight amount of cross talk between small molecule effectors; for example, the synCRP reporter showed slight inhibition as the concentration of arabinose increased (Figure 3). This may be due to the internal regulation of cAMP as the arabinose is catabolized. The synRNAPσ70 reporter remained largely unaffected by arabinose or cAMP concentration (Figure 3). A simple mathematical model based on measured transcription factor activities was sufficient to reproduce steady state behaviour (Figure 2), and furthermore, could predict dynamic behaviour during diauxic shift using measured transcription factor activities (Figure 6). These results suggest that the repression of a constitutive promoter is an efficient and accurate method to measure transcription factor activity in vivo, without the need to measure binding coefficients and intracellular concentrations biochemically.
Induction during diauxic shift is the most biologically realistic environment to test the response of the four promoters to transcription factor activity. Furthermore, the role of cAMP in transcriptional activation during diauxic shift is in question due to the complex behaviour of cAMP as glucose is consumed [6, 7, 34–36]. The model fit to steady state expression data is capable of predicting the major aspects of induction in diauxic shift, and suggests that both CRP and AraC are capable of binding their operators and interacting with RNA polymerase as long as repression by AraC is relieved. The significance of the γ term (Table 2) suggests that both arabinose and cAMP are required for full transcription (AND logic), similar to earlier results [39, 40]. However, the additional significant α term (Table 2) indicates that AraC alone can modulate transcription independently of cAMP. This suggests that the logic function performed by all the arabinose catabolic promoters is more complex, similar to that measured for PlacZ.
The measurement of cAMP-CRP activity during diauxic shift in this study (Figure 4) confirms other observations of cAMP concentration in these conditions . Before the decline of growth rate, decrease in glucose uptake, or reduction in glucose flux, cAMP concentration within the cell changes rapidly, rising quickly, then soon returning to basal activity once the secondary sugar has been detected . This burst of activity has been demonstrated by other investigators . Because cAMP changes so rapidly, it was unclear to us whether models derived from steady state activity could be used to understand dynamical behaviour. Surprisingly, the model derived from steady state behaviour does predict the salient aspects of diauxic shift, though model fits for ParaFGH and ParaC are not as accurate as those for ParaE and ParaBAD (Figure 6). This may reflect specific aspects of transcription in these promoters that is not properly modeled. For example, the relative positions of the CRP and AraC operators in ParaFGH are opposite that of ParaE, with the CRP operator overlapping the -35 site . This suggests that the effect of CRP-cAMP at this promoter may be stronger than AraC, and may include some cooperativity that is not included in the model. Also, at ParaC, AraC can act as a repressor at the O1 site [11, 22]. Repression was not included in this analysis and indeed it is clear that the drop in expression at high arabinose concentrations at this promoter is not adequately modeled. The role of AraC as a simple activator may be oversimplified, as it remains unclear what role AraC plays in the kinetics of induction, given that it is most active at time points after the promoters have been activated (Figure 4). This may indicate that the primary role of arabinose binding to AraC is to relieve repression. That the predicted expression is higher at early time points than measured suggests that there are elements of repression via AraC that the model does not reproduce. AraC has been demonstrated to be required to initiate transcription by helping to recruit RNA polymerase to the -35 site [17, 18, 20, 41, 42]; however, the protein is always present at I2 which is immediately adjacent to the -35 site. Therefore, changes in concentration of AraC, which would be measured by our reporter, may happen long after the resident AraC has fulfilled its role. Conversely, the low early activity of synARA may simply be an artefact of this reporter being relatively low strength.
Mathematical models of transcriptional regulation have either relied on estimates of in vivo binding affinities, concentrations and reaction rates , or have estimated these "hidden functions" [31, 32] from transcriptional data. The first technique has the complication of having to measure numerous quantities in vitro, which is both difficult and hard to relate to in vivo conditions. The second technique has the possibility of estimating non-unique parameter sets and remains a substitute for being able to measure in vivo transcription factor activity in real time. While cAMP concentrations in vivo have been estimated from non-native CRP-dependent promoters [8, 13], the interaction between RNA polymerase and the transcription factor at the promoter results in a non-linear activation of transcription. The non-linearity in transcription activation complicates the interpretation of the rate at which in vivo cAMP concentrations are increasing or decreasing. For these reasons, determining in vivo concentrations of small molecule activators, or the activity of their cognate transcription factors, has been exceedingly difficult. Therefore, we used synthetic biology techniques to develop transcription factor reporters that can give an indication of transcription factor activity in real time. The synthetic transcription factor reporters consist of a constitutive promoter followed by the operator binding site for the transcription factor of interest. It is relatively simple to develop synthetic reporters for intercellular concentrations of effectors by cloning a library of reporters with degenerate operator binding sites  (Figure 1c, Additional file S1, Figure S1, Additional file S1 Figure S2, Additional file S1 Figure S3). Experiments showed that not only were the reporters capable of giving sensitive, linear and real time indications of in vivo transcription factor activity (Figures 3, 4), but that simple models were capable of relating these measurements to promoter activity (Figure 2) even in a dynamic experiment such as during diauxic shift (Figure 6). In other words, using these promoters we measured real time in vivo transcription factor activity and determined their rules of integration at the promoter level. This is a marked improvement on methods of measuring small molecule concentrations in real time.
Preferential catabolism of sugars and signalling of the nutritional state is regulated by complex interactions of sugar intake systems, adenylate cyclase, and transcriptional regulation. This work demonstrates the development of novel synthetic reporters that measure the transcription factor activity in vivo and obviate the need for the estimation of numerous parameters. Because cAMP activity during diauxic shift shows a rapid pulse of activity, its role in regulation of catabolic promoters during diauxie has been contentious [7, 34–36]. However, the integration of information from common transcription factors is mediated by the relative strength of α, β and γ, and this analysis shows that understanding these rules is sufficient to predict the salient features of gene regulation during diauxic shift, without in vitro estimation of numerous biochemical coefficients.
The Escherichia coli strain used in this study was MG1655. All strains were cultured aerobically at 37 °C in Luria Bertani (LB) broth (Invitrogen Canada, Burlington Ontario) or M9 minimal medium (Becton Dickinson Canada Inc., Mississauga, Ontario, Canada) supplemented with 0.1% casamino acids (Becton Dickinson Canada Inc.) and either 0.5% glycerol or 0.1% glucose. Kanamycin was included in liquid and solid media at a concentration of 50 μg/mL as required.
The complete intergenic region for the promoters ParaC, ParaBAD, ParaE and a truncated fragment of the araFGH intergenic region including all known regulatory elements were PCR amplified using primers listed in Table 1. The PCR products were purified, digested with Xho 1 and Bam H1 (New England Biolabs, Ipswitch MA.) and ligated into the low copy luxCDABE reporter plasmid pCS26-Pac . Positive recombinant plasmids were identified by luciferase expression and confirmed by DNA sequencing.
Previous work describes the development of a library of synthetic σ70 promoters of varying strengths (Pabbaraju and Surette, unpublished results). The transcription factor reporters were built from a constitutive σ70 promoter from this library that exhibited a medial level of expression called synRNAP-σ70 (AATAATTCTTGAAATTTATGCT TCCGGCTCGTATTTTACGTGCAATT). Alignments of the AraC binding site and CRP binding site in the AraC regulon were done in the program AlignX (Invitrogen, Canada) and these consensus binding sites were the basis of the transcription factor reporter libraries. The consensus binding site, with four degenerate bases, was added to the 3' primer for the synRNAP-σ70 promoter. This design placed the operator region immediately downstream of the -10 position of the constitutive synRNAP-σ70 promoter, and the addition of degenerate bases allowed us to screen a large number of clones for different levels of activity. Primers designed for synARA clones included binding regions that were based on a single I1 site, or combinations of I1I1 and I1I2 sites with and without degenerate bases (Table 1). We also tested the slightly different I1I2 consensus sequence of Seabold and Schlief . The PCR products were purified and cloned into the pCS26-Pac plasmid. The ligation reactions were transformed into Electromax electrocompetent DH10b cells (Invitrogen Canada), the plasmid library recovered from the liquid cultures, and retransformed into chemically competent MG1655. The library was picked into 96 well plates and screened for responsiveness to the addition of the relative effector. The transcription factor reporters showing the strongest repression were selected and named synARA and synCRP.
Overnight cultures of each reporter were diluted 1/600 into fresh LB medium and grown to half exponential phase (three hours, OD600 0.1-0.2). 50 μl of each reporter was then added to each well in a 96 well plate containing 50 μl LB and gradients of arabinose (0-0.2%) and cAMP (0-1 mM). The plates were read in a multiwell plate reader (Wallac Victor 1420 multilabel counter) at 4 minute intervals, with agitation (30 seconds, 2.0 mm orbital shake prior to measurement) for an hour and a half. This allowed for fine-resolution temporal mapping of promoter activity over 96 different combinations of arabinose and cAMP concentrations (after ). Data was normalized to optical density prior to analysis.
To test for specificity of the transcription factor reporters, luciferase expression was measured in a gradient of both arabinose and cAMP. Overnight cultures of synARA, synCRP and synRNAP-σ70was diluted 1/600 into fresh LB medium and grown to half exponential phase (three hours, OD600 0.1-0.2). 50 μl of each reporter was then added to each well in a 96 well plate containing 50 μl LB and gradients of arabinose (0-0.2%) and cAMP (0-1 mM). OD and luminescence were measured using a multiwell plate reader (Wallac Victor 1420 multilabel counter) at 4 minute intervals, with agitation (30 seconds, 2.0 mm orbital shake prior to measurement) for an hour and a half. This allowed for fine-resolution temporal mapping of transcription factor activity over 96 different combinations of arabinose and cAMP concentrations .
For diauxic shift assays, the cultures were grown overnight in M9 medium plus glucose, diluted 1/600 in the same medium and grown to half exponential phase (three hours, OD600 0.1-0.2). The cultures were then rinsed twice in M9 medium without a carbon source, then added to the 96 well plate with saturating arabinose (0.1%) and subsaturating glucose (0.0001%, 0.0002%, 0.001%, 0.005%, 0.01%). The plate was measured in the same manner as above. Luciferase expression in four minute intervals was measured as before for three hours. All gene expression assays were performed in triplicate, and data was normalized to optical density prior to analysis.
Because transcription of this reporter (SA) and the basal transcription rate (T) are measured quantities, and the activation rate is by definition the difference between fully repressed and fully unrepressed expression  and thus can be estimated from measured data (SAt - TsynRNAPσ70 t), we can easily calculate the quantity A* for every combination of inducers, or for every time step in a diauxic shift experiment. In instances where equation 2 resulted in a negative value, the results were normalized to 0. Identical arguments apply to calculating the activity of cAMP-CRP from the measured expression of synCRP.
then data evaluated from equation 2 can be substituted in equations 3 and 4. The two terms in equation 6 are not equivalent, thus the values estimated for α, β and γ do not exactly correspond to activation coefficients but rather include the ratio of the binding of the transcription factors to the synthetic promoters (K1syn) to the binding of the transcription factors to the promoters in their natural context (K1). However, the transcriptional activity measured by the synthetic promoters is proportional to actual activity in vivo, so that coefficients estimated by model fitting illustrate relative strengths of inputs at each promoter. The coefficients α, β and γ in equation 3 are determined by model parameterization. If any of these coefficients resolves to zero or is insignificant, then the corresponding term can be dropped from the equation and a simpler model refit to the data. Models were fit with nonlinear least square fitting using the function nls in the statistical package R . Model fit was evaluated by comparing residual sum of squares and Akaike's Information Criterion.
The functional form and scaling coefficients predicted from fitting expression data to measured steady state transcription activity should be sufficient to predict kinetics of induction in a dynamical model. Thus the fitted coefficients were then used to predict induction both in steady state and during diauxic shift using measured transcription factor activities and equation 4. Data from multiple diauxic shift experiments and gradient expression assays (n = 7 induction from diauxic shift, n = 3 gradient expression) were averaged.
CJD Performed all cloning, experiments and data analysis, developed and fit the model, and wrote the manuscript. AN Assisted with development of the model, edited and approved the manuscript. MGS Developed original concept, guided experiments, edited and approved the manuscript.


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New traditionalists
Staff Writer February 22, 2021, 10:24 am February 22, 2021 0
Australia's world-leading Performance-Based Standards Scheme continues to gather momentum as more operators realise the benefits. The National Heavy Vehicle Regulator Chief Engineer, Les Bruzsa, provides an update outlining just how much the high productivity system has improved and grown in recent times.
PBS four-axle quad trailer, FTE split quad axle and Volvo FH16, PBS A-Double and Prime Mover.
In spite of a decidedly lacklustre year on a number of fronts, the uptake of Performance-Based Standards (PBS) by a broad cross section of the trucking industry has continued unabated as operators seek out ways to improve productivity and efficiency. PBS is a world-leading program that enables Australia's heavy vehicle industry to use the most productive vehicles for specific freight tasks. It fosters innovation in vehicle design to improve productivity while achieving safer operation and making minimal impact on road infrastructure.
A PBS retrospective
The National Transport Commission (NTC) implemented the current PBS scheme in 2008. Now 12 years down the track, it is more than delivering on the opportunities that were foreshadowed at the outset to unlock productivity gains and improve safety outcomes. Indeed, it would appear the exponentially increasing success of the PBS scheme over the past decade is a clear testament to the freight industry's desire to innovate and be smarter.
As the freight task continues to grow, PBS will be an essential tool in meeting this growth in the safest, most productive and efficient manner possible.
The Australian Road Transport Suppliers Association Institute (ARTSA-i) and the National heavy Vehicle Regulator (NHVR) want to see further barriers removed that potentially limit this innovation to ensure future generations of PBS vehicles are safer, cleaner and even more productive.
To achieve that goal the current scheme framework needs to be continuously improved and hurdles to the use of PBS equipment need to be tackled to ensure Australia continues to lead the world in innovative regulation of heavy vehicles. When it comes to talking about PBS and the benefits it can deliver to the industry, none are more passionate than Les Bruzsa, Chief Engineer at the NHVR.
Enter the Quadfather
As an Automotive Mechanical Engineer, his background includes over three decades of experience in various fields of the road transport industry both locally and overseas.
Between 1995 and 2013 he was employed by the Queensland Transport Department and Transport and Main Roads as Principal Engineer, responsible for assessing new vehicle combinations, developing technical standards, policies and guidelines for vehicle use, access and management.
National Heavy Vehicle Regulator Chief Engineer, Les Bruzsa.
In his current position, Bruzsa provides engineering and technical leadership to the strategic development and improvement of the PBS Scheme and leading the development of NHVR engineering and technical standards related to heavy vehicle safety and productivity.
Speaking about the current state of play with PBS, Bruzsa said a significant milestone was reached early this year with approval granted for the 10,000th PBS combination.
"That number has continued to grow significantly this year, reaching 11,350 at the end of October, and it's very important to celebrate that achievement because it far surpasses original predictions when the Scheme was first implemented," he said.
Bruzsa explained that when the fledgling PBS Scheme had its first review in 2009 the prediction was that there would be 12,000 PBS combinations registered by 2030. Instead, that number is set to be reached some ten years earlier.
Has Covid-19 impacted PBS?
In addition to the higher than expected uptake over the last decade or so, Bruzsa also noted that the effects of Covid-19 and a weaker economy have not significantly lowered the PBS uptake this year. In fact, he states that year-on-year results comparing this year's forecasted total with that of 2019 are surprisingly similar.
"I estimate that this year we will end up with a total of 1,600 to 1,650 combinations compared with around 1,700 last year," he said. "This is no mean feat considering the whole country is in an economic downturn with around a 20 per cent drop in new truck registrations.
"The fact that the PBS uptake rate is still increasing, rather than reducing in line with the drop in new vehicle registrations, shows that the industry is supporting PBS and selecting these PBS vehicle options when available."
Expanding further, Bruzsa said over the past three years around 20 per cent of the total new heavy vehicle market has been PBS approved which he describes as very good market penetration.
Innovative combinations have their perks
"What's also interesting is that the median age of PBS vehicles is 4.3 years compared with 11 years for prime movers – and around 13 years, for rigid trucks and trailers.
"What this means," he elaborated, "is that the PBS fleet is fitted with superior safety technologies and designed to higher level technical standards."
This observation aligns with data garnered by the NTC which suggests a 46 per cent reduced crash rate can be attributed to the use of PBS-approved heavy vehicles.
The split between the four PBS levels (L1, L2, L3 and L4) of different PBS combinations is also a point of interest.
"L1 accounts for 16 per cent while 42 per cent is L2 and 35 per cent has dual L1 and L2 approval, the latter being vehicles approved for two different levels," Bruzsa explained. "Adding these figures together means that around 93 per cent of the PBS fleet are the shorter combinations in the general access vehicle and B-double classes, with 56 per cent coming in under 20m overall length and largely comprising truck and four-axle dog combinations."
He added that this should allay any fears that some road managers may have had that PBS would be responsible for putting 'monster' trucks on the road.
Of the common criticisms of the PBS Scheme, excessive time taken to grant approvals has been arguably the most prevalent. However, Les is adamant that big strides have been made in this area, particularly over the last three years, and will continue to be made moving forward.
"We have made a lot of internal process changes and improvements to help make the approval process more efficient. In terms of the design and vehicle approvals, we are monitoring our response times to ensure expediency," Bruzsa said. "We have also introduced the PBS pre-advised design approval process.
"For instance, more than 70 per cent of the PBS design applications are now handled under the pre-advised process which means that the NHVR is able to approve the combinations for these specific designs within two to three business days."
Bruzsa said this is a huge improvement compared with just a few years ago when this same approval process took 20 to 25 business days.
"I think the industry really appreciates this process improvement and is generally trusting the NHVR to be responsive and to deliver reliable and quick services to the industry," he said.
In sum, from the comprehensive rundown Bruzsa provides, it appears that trucking operators and the NHVR are working more co-operatively than ever before to achieve the best PBS outcomes.
It seems that the industry has recognised that selecting pre-advised designs is a sure-fire way to ensure a speedy approvals process which, in turn, enables operators to get the most productive combinations quickly on the road and earning them better profits.
A PBS B-quad combination.
AustraliaPBSperformance based standardsroad train

redpajama_set_name: RedPajamaCommonCrawl


Great curb appeal! This well maintained 3 bed, 2 bath, 1,953 sq ft ocean view rancher backs on to Lynwood Park and is located on a quiet, no thru road. Step into the foyer of this south facing home and immediately notice the bright rooms filled with natural light. Open concept living includes a cozy gas fireplace, plenty of windows and a kitchen island perfect for entertaining friends and family. This home has lots of extra storage both in the large pantry, crawlspace, and triple car garage including space for a workshop. You will appreciate the convenience of the built in vacuum, home security system and programmable in-ground irrigation. Enjoy the private patio surrounded by a well manicured and tiered yard. Plenty of room for RV and/or boat parking.


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Progress on the path to reinventing the toilet
The Nano Membrane Toilet on trial in South Africa (© Pedro Talaila)
The UK's Cranfield University took up the Gates Foundation's Reinvent The Toilet challenge in 2012. From that has come a self-contained, waterless solution, which combines a number of technological innovations. Keith Hayward got a progress update from Dr Alison Parker.
There is still important progress to be made before the UK's Cranfield University can say it has met the goal set by the Bill & Melinda Gates Foundation to reinvent the toilet. But seven years after it received a grant under the foundation's Reinvent The Toilet challenge, the university is closing in on a solution – progress reflected by the fact that the technology won first prize in last year's IWA Project Innovation Awards, claiming the Kiran and Pallavi Patel Grand Innovation Award.
Cranfield refers to the technology as the Nano Membrane Toilet (NMT). "It is called a toilet, but really it is a miniature sewage treatment works. It provides complete treatment for human waste onsite, in the home," says Dr Alison Parker, one of a project team that has involved around 50 researchers. "We are not there yet – we are still working on it – but that is our vision," she adds.
The NMT is being developed in one of a number of projects to have been selected for support in 2011-2013. The project website acknowledges that inspiration was provided by the 2011 round of recipients, including EAWAG, Loughborough University, the University of Toronto, and TU Delft.
"What excites me about this is the potential to fill a big technological gap in the way we think about managing human waste," says Parker. On one side of the gap is the conventional approach, involving centralised sewage treatment. "This works well for a good number of people, but there is a large proportion of the world for whom that doesn't work," she says. On the other side is onsite sanitation, typically pit latrines. "Emptying pit latrines is very challenging. Even with new technology, it is still incredibly messy and incredibly unhygienic," Parker continues, adding: "Other types of onsite sanitation, such as different kinds of composting toilets, have definite positive points, but lots of people don't want to use compost or don't have a use for compost locally to them."
Combining innovations across technology areas
The NMT combines technical breakthroughs in a number of areas. In particular, it does not require a water connection but, at the same time, does provide a flush. "That gives the user a great experience, so they can't see or smell the waste," says Parker.
Then there is the ability to separate liquids from solids, for which membranes are one of a number of technologies used. "When you start looking into any treatment method, separation of solids and liquids is quite critical," says Parker.
The NMT will also be powered by the energy contained within the waste itself. "The combustion of solids and transformation into ash in an energy efficient way – and in an incredibly miniaturised, small-scale way – is probably the other main innovation," she adds.
All of this clearly means the NMT can be viewed as a 'high tech' option – something that can lead to questions about whether it is a valid response to the needs of people in the poorest of communities.
"It is a criticism that is often levelled at us," says Parker. "I think the idea that poor people only deserve low-tech solutions is actually an incredibly alarming and patronising idea."
"The next best option is the pit latrine, which has incredible disadvantages in terms of how the waste is managed subsequently. There has to be a better solution," she continues.
"There are plenty of examples of high technology – smartphones and apps, solar panels, innovative water heaters, and all sorts of things – that are being deployed successfully and are being taken up really widely."
Application is the real test
Parker's team has carried out studies in Durban, South Africa, to look at the way different technologies have been taken up. "We do need to challenge this idea that high technology can't be deployed in these contexts," she says. "Nevertheless, we are trying to make our system as robust as possible, so you won't need a degree in engineering to be able to fix it."
This points to the wider factors, beyond technology, that will determine whether or not the NMT is ultimately a success. These include how people perceive the NMT – whether it is something they want to use – how units will be maintained, and how to pay for the installation and the ongoing maintenance.
Parker explains that the project to date has included initial field trials in Durban to assess the response of users to the flush function of the NMT. "We have had good feedback from the families we have been working with," she says. Work has included extensive trials of toilet paper being used with the NMT.
The project has also been looking at the business model for deployment of the NMT. "We have been thinking about the business model right from the start," says Parker. "We have been collaborating internally with our own social scientists and our school of management, but also externally with experts such as Practical Action and other organisations to get their ideas about the best way the NMT could work in a real situation."
These are the same challenges faced by any new technology looking to serve informal settlements in urban areas. The project is looking to bridge the gap between, on the one hand, informal settlements as challenging environments for utilities to serve, and, on the other, them being locations where it can be very difficult for entrepreneurs to build viable businesses emptying pit latrines. "Informal settlements… need completely new technological and service delivery models," says Parker.
Work continues on the NMT. "There is still some way to go to make it a commercial product," says Parker, which probably means more than one year but less than five. The aim is to work with a commercial partner, either now or once the NMT is progressed further, technically. "We would love to work with a commercial partner at this stage who could help us think about manufacturing and exact applications in different markets," she adds.
For Parker, though, success encompasses how the NMT will be taken up. "The servicing is nowhere near as onerous as a pit latrine – that is kind of the whole point," she says. "Ultimately, [success] is going to be when it is actually being used, in people's homes, and people are supported by good business models." This could be, for example, people aspiring to own one or rent a unit for themselves, or a municipality installing units.
"We haven't quite determined the business model yet," Parker says, but it is likely to require a number of NMTs within a compact geographical area to allow economically efficient servicing.
"I think that is what we would consider a success – clusters of NMTs supported by a technician," she concludes.
How the Nano Membrane Toilet works
The aim of the Nano Membrane Toilet (NMT) is to treat human waste on site, without external energy or water. It is designed for single-household use, or the equivalent of 10 people. It will accept urine and faeces as a mixture – an important distinction from urine separation toilets. The flush uses a unique rotating mechanism without using any water. This simultaneously blocks odour and the user's view of the waste.
Separation of solids, i.e. faeces, is achieved through sedimentation. Loosely bound water (mostly from urine) is separated using an innovative hollow fibre membrane with a low glass-transition temperature. The unique nanostructured membrane wall facilitates transport of water as vapour rather than as a liquid. This ensures a high rejection of pathogens and some odorous volatile compounds. The water will be collected for reuse for household or irrigation applications.
After release of unbound water, the residual solids are transported by mechanical screw into a combustor that will convert them into ash and energy. The energy will power the membrane processes, and there may be extra energy available for charging mobile phones or other low-voltage items.
See: nanomembranetoilet.org
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Hernández-Pérez, M., K. Corstorphine, and D. Stephens. "Cartoons Vs. Manga Movies: A Brief History of Anime in the UK". Mutual Images Journal, Vol. 2, Mar. 2017, pp. 5-43, doi:10.32926/2017.2.HER.carto.
This paper has as main objective to explore, adopting a historical and critical perspective, the release of film and anime TV in UK. This would be a first step towards the studio of the peculiar implementation of manganime Culture in Britain.
Compared with other European countries, UK has shown to be slower and even reluctant in importing Japanese television products. Thus, while major markets of anime such as France, Italy or Spain expanded considerably during 1975-1995 period, in a recurrent synergy of television markets, and technological publishing, the implantation of the principal channel (televised anime) in UK has been irregular and unstable. Even today, the catalogue of broadcasting anime is limited to some high success movies, late night television on thematic channels and quite recently, video-on-demand services (Netflix). The offer cannot be compare in importance and diversity to other European countries. This fact, far from being anecdotal, has had an impact on the subsequent implantation of media Japanese cultures such as manga, anime, video games and cosplay.
What can be the reason of this irregular development of Japanese visual culture in United Kingdom? Characteristic having the television market and / or the UK audience? Main hypothesis in relation to these issues can be considered to be of sociological character, but are reflected in the idiosyncrasies of British television culture and production system.
Thus, compared to other Western markets (including the US) which saw the opportunity to purchase economic products for children's television audiences in the late 70s and early 80s, the British 'telly' already offered a broad catalogue (Roobarb, Super Ted, Danger Mouse, etc. ...). The only exception to this children's 'made in Britain' programming was the co-production model. This caused a leak of few products that were not even considered as "Japanese" (Seven Cities of Gold, Godzilla) but mere 'cartoons'. This competition with the British children production as well as the wide catalogue of other forms of British Popular Culture would explain why the film, domestic video and later adults programming would be the marginal routes of entry for manganime.


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Negative Equity Becoming a Problem as New-Vehicle Sales Close in on 2015 Record
The good news, according to Edmunds.com, is the industry is on pace to top last year's record new-vehicle sales total. The bad news, the firm noted in a separate release, is car buyers are rolling over negative equity from their trade-ins in record numbers.
Millennial Car Buyers Busting Gender Stereotypes
Millennials are bucking the stereotype that women are damsels in distress and men are confident decisionmakers, according to new research from Edmunds.com.
Edmunds Predicts April Record for Industry Sales
The vehicle information site said this week that April sales are on pace to eclipse the April 2005 sales record of more than 1.5 million units. It also believes the industry is on pace to shatter last year's 17.5 million-unit sales record.
Edmunds: EV, Hybrid Owners Ditching Vehicles for SUVs
An Edmunds analysis found that only 27.5% of all hybrid and EV trade-ins in 2016 have been applied to the purchase of another hybrid or EV, an 11.3% drop from last year's rate of 38.5%. A larger percentage, 33.8%, of trade-ins are going toward the purchase of SUVs.
2015 a Record Year for Used-Vehicle Market, Edmunds Reports
Automotive dealers sold 38.3 million used vehicles in 2015, the highest single-year total since 2007. And they did so despite the price of an average used car rising 4.6% to $18,600.
Edmunds, CUneXus Partner to Develop New Car-Buying Solutions
Edmunds is teaming with CUneXus to build new shopping and purchasing solutions. The latter offers a "click-to-accept" consumer loan platform that allows finance sources to deliver loan offers to in-market consumers through their mobile devices.
Certified Pre-Owned Sales Post Best Q3 Performance, Edmunds Reports
The number of consumers willing to pay a premium for the benefits of a certified pre-owned vehicle is growing, Edmunds reported this week. Between July and September, certified pre-owned vehicles accounted for 22% of used-car purchases at franchise dealerships -- the largest share of any third quarter.
Millennials Prefer Leasing, Edmunds Finds
Millennial car buyers are leasing vehicles at a higher rate than the overall car-buying population, according to analysis from Edmunds.com.
Edmunds Opens Registration for Used+ Program
Edmunds.com has opened registration for its Used+ used-car sales program, which offers shoppers a vehicle-protection plan and a $200 gas card. Asked if the program might lead Edmunds into F&I product sales, a spokesperson said '... It is an option we would be open to exploring.'
September Sales to Maintain Healthy Growth in 2014, Edmunds Predicts
Projected September sales would be an 11% increase from the previous year, with Edmunds predicting that about 1.26 million new cars and trucks will be sold during the month.
Auto Sales to Reach Highest July Mark in Eight Years, Edmunds Predicts
Edmunds.com forecasts that about 1.46 million new cars and trucks will be sold in the U.S. market in July, the highest July total since 2006.
June New-Car Sales Strong Despite Forecasted Decline, Says Edmunds
Edmunds.com forecasts that about 1.36 million new vehicle will be sold in June, a 15.4% decline from May and a 3.1% decline from June 2013. Analysts, however, say the declines don't mean June will be a bad month for car sales.
Honda, Acura Earn Edmunds' Best Retained Value Awards
Honda won in the nonluxury division with a projected residual value of 50.1% after five years, while Acura took home the luxury crown with a projected five-year residual value of 45.4%.
Ford, Chevy and Toyota Lead Edmunds' Inaugural Popular Vehicle Awards
Edmunds.com presented its inaugural 2014 Most Popular on Edmunds.com Awards at this week's New York International Auto Show, with Ford, Chevrolet and Toyota taking home top honors.
Seven Business Leaders Selected to Judge Edmunds.com's Hackomotive Event
The vehicle information site names the seven judges who will oversee its second annual Hackomotive event, a three-day contest in which participants develop products and ideas that promote a better car-shopping experience.


"There's always a first time!" That's a saying each of us has come across at some point in our lives. Be it tackling a real life problem or a technical issue, there will always be something that we've never done before. And today our discussion revolves around one such technical issue – reformatting a Mac. While purchasing your brand new Mac did you consider the thought, that for some reason you might have to reinstall Mac OS X? Most probably, your answer to that would be 'No'. And thus you wouldn't have purchased a DVD from which to re-install OS X. Even if that thought did cross your mind, your next thought would have been that if such a situation arises, you will take the Mac to an Apple Service Center and get it fixed.
Practically speaking, taking the machine to a service center is both inconvenient and costly. Also, sooner or later, you will want to reformat the Mac, be it because of an OS crash or because your machine has become too sluggish to work on. Though there are ways to boost up its pace but finding out the causes one by one and eliminating them is more time consuming than reformatting. Now, when you reach a situation where you want to reformat the machine at home, the question of having a DVD arises. And since you don't have it, you land in a scoop!
Worry not! Thankfully, Apple thought of this scenario before-hand and created a utility through which your Mac can be good as new again. Here's a step-wise process of how you can reformat your Mac OS X without a recovery DVD for manual installation of OS X Yosemite 10.10, OS X Mavericks 10.9, OS X Mountain Lion 10.8 and so on.
This is the most important thing to do before you start the reformat process. This also acts as a perfect opportunity to clean up your disk. Delete all old, unwanted and unused files that are eating up disk space unnecessarily and save the remaining ones. You can save all important files through Time Machine or some OS X clone utility. Also, make a list of all installed applications so that you can download their setups and re-install them after the formatting is complete. Save any setup files that you already have so that you don't have to re-download them.
Once you are satisfied that you have backed up all necessary items, recheck them. Pay the most attention here because if you miss something out at this step, consider it gone forever! Then take a deep breath and restart the machine.
Note: If you are not able to see the OS X Utilities window displaying the above four options and your Mac boots to your desktop instead, restart and hold the key combination specified above for a little longer than you did the last time. Do not leave the keys until you see the Apple logo.
When you reach the OS X Utilities window, click on the Disk Utility option displayed on it. Another window will pop up listing the drives on your machine on the left and a volley of things you can do on the right. Select the hard drive where you want to install the OS X and then click the Erase tab on the right. Then follow the instructions to erase your hard drive. Once erased, exit the disk utility. Moreover, to better clean your Mac hard drive, you can install third party software tools as well, such as CleanMyMac3, which is very useful for cleaning up Mac hard disk.
Re-installing OS X without a recovery DVD requires downloading it from the Apple server. As such, you need to make sure you are connected to the Internet before you proceed further. For this, you can either plug in an Ethernet cable to the machine or connect to a Wi-Fi network by clicking on the Wi-Fi menu in the upper-right corner of the screen. Select and connect to a preferred network.
After you exit the Disk Utility window you will see the OS X Utilities window again. Now select the Reinstall OS X option. Follow the directions shown next and soon enough you will have a freshly installed copy of OS X.
1. The OS X Utilities window is a part of the OS X Recovery system pre-installed on each Mac machine. In case your Mac does not have this installed or if this has been corrupted or erased somehow, you will have to use the OS X Internet Recovery option. This option enables you to operate the Recovery directly from the Apple server over the Internet.
You're probably wondering how you will come to know if the pre-installed Recovery system is not present. Well, you don't need to know. Your Mac will automatically connect to the Internet and start using the Internet Recovery if it is not able to detect the pre-installed version on your machine.
2. The Internet Recovery option will also need you to connect to a Network. Make sure an Ethernet cable is plugged into your machine or you are connected to a Wi-Fi network.
3. The time required to download OS X varies depending upon the version of OS X you are downloading and your Internet connection's speed.
4. Make sure your Internet connection does not block the download of OS X. If you have some setting which is not supported by OS X Internet Recovery, disable that setting for the duration of the download and re-enable it later.
5. The OS X Recovery installed on your machine installs the most recent version of your operating system available. The Internet Recovery installs the version of OS X that your machine originally came with.
Other than these steps and pointers, if by any chance you are not able to reformat your Mac, either purchase an OS X Recovery DVD or consult an Apple Service Center executive. But do give the process a shot from your end. It is pretty simple and can save your money.


Wish we could all be with you to toast your birthday!
All the Best James, and A great vacation in Italy!
Happy Birthday Jim!!!! Don't celebrate too much.
hmmmm......Happy Birthday, James. I know you won't be around tomorrow so I'm sending big hugs your way a day early!
HAPPY BIRTHDAY JIM!!! You are one of the most amazing people I know. You deserve a great birthday!
My friend says happy birthday too! Have a great birthday weekend with your family!
I hope the monkey does not see these guys!
Happy Birthday Jim!!!!!! Hope you have a great day!!
Awww thanks guys! Headed south to Rhode Island with my kids today for the weekend. This was really a great thread to wake up to this morning!!
HAPPY BIRTHDAY!!!!! Enjoy your day!
Belated birthday wishes Jim, hope you had a good weekend with the kids.
I am back from Rhode Island, and just am checking on Gente posts. Thank you all sooo much. I had the most amazing weekend, this thread is the icing on the cake.


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            log4net SDK Documentation - Microsoft .NET Framework 4.0

SecurityContextProviderAttribute Properties

The properties of the SecurityContextProviderAttribute class are listed below. For a complete list of SecurityContextProviderAttribute class members, see the SecurityContextProviderAttribute Members topic.

Public Instance Properties

ProviderType	 Gets or sets the type of the provider to use.

TypeId (inherited from Attribute)	 When implemented in a derived class, gets a unique identifier for this Attribute.

See Also

        SecurityContextProviderAttribute Class | log4net.Config Namespace

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          Copyright 2004-2011 The Apache Software Foundation.

redpajama_set_name: RedPajamaGithub


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Eric M Bader
Eric M. Bader, MD
Cardiovascular Disease, Internal Medicine
Affiliated With: Yale Medicine
Biography - Eric M. Bader, MD
Eric Bader, MD, is a cardiologist who specializes in electrophysiology, which he describes as the management of complex medical arrhythmias, including atrial and ventricular arrhythmias, by means of both medication and catheter ablation.
Cardiac conditions can be difficult to live with, and Dr. Bader says many of his patients feel justifiable anxiety and substantial discomfort as a result. "That said, no field in medicine is better understood or more rigorously studied than the cardiovascular system," he adds.
"The challenge of understanding complex arrhythmia processes and using this understanding to achieve arrhythmia cures—in the form of catheter ablation or the implantation of cardiac rhythm management devices such as pacemakers—can result in dramatic lifelong symptom improvement," Dr. Bader says. His practice includes the implantation and management of cardiac devices such as pacemakers and implantable cardioverter defibrillators (ICDs) for the prevention of sudden cardiac death, and cardiac resynchronization therapy (CRT) to treat heart failure. He also is a member of Yale's internationally recognized lead management team, which involves performing extractions of infected and malfunctioning pacemakers and defibrillators.
Dr. Bader's research interests include leadless pacing and cardiac resynchronization. Recently, he has been investigating the heart rhythm impact of COVID-19 infections.
But Dr. Bader says his favorite part of his work is talking to patients who feel dramatically better after a medical intervention. "I appreciate the opportunity to see how patients adjust to their new lives and to share in their milestones," he says.
New York University School Of Medicine
Jacobi Medical Center
St Luke's-Roosevelt Hospital Center;Montefiore Medical Center
ANTHEM BC/BS
BERKELEY CARE NETWORK
COMMUNITY CARE NETWORK
FIRST HEALTH NETWORK
FOCUS HEALTHCARE
GREAT WEST
TRICARE/CHAMPUS
Yale Cardiology
800 Howard Ave
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325 Boston Post Rd.
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Are you Eric M. Bader, MD?


In the process of introducing her 1997 anthology, Black British Feminism: A Reader, Heidi Safia Mirza offered what may appear to be an irrefutable proposition: "Gender is not experienced in the same way when you are positioned as working class or black, or both." (1) It would seem equally uncontroversial to assert that virtually nothing is experienced in quite the same way, depending upon one's race and class. That Mirza felt compelled to say this, however, suggests that the truth of such a statement about the importance of acknowledging difference was by no means self-evident to everyone in Britain, even as late as the mid-1990s. Indeed, as Mirza added, "To be black and British is to be unnamed in official discourse," (2) and with namelessness came continued invisibility. Three years later, Yasmin AlibhaiBrown confirmed this assessment: "we Black Britons remain barely noticed and on the periphery." (3) Black British women--that is, British women of color whose ancestry was African and/or Asian--who were also working-class often felt themselves doubly overlooked, if not deliberately excluded, as the new millennium neared.
But it was not merely from the "official discourse" of governmental pronouncements about Britain and Britishness that recognition of differences in race and class tended to disappear, for the same was true in the unofficial discourse of popular culture. This was so especially when it came to the many filmed recreations of British history that proliferated in the 1990s--representations of those who had played significant roles in the story of British life. Two decades of white British feminist activism had succeeded in making gender visible and, perhaps more important, marketable. Cinematic adaptations of literary texts by and about Englishwomen of the past--particularly about so-called "strong" and "independent" female characters--proved commercially viable in Britain and saleable abroad, so long as those figures were white and of the educated classes. One result of this phenomenon was the mid-1990s boom in both feature films and made-for-television costume dramas based on novels by Jane Austen and set in the Regency. Media critics now identify the year 1995 as a turning point in Austen's cinematic fortunes. Melissa Sue Kort, for instance, writing recently in Ms. magazine, has spoken of "a revival of interest in her work that began in the mid-1990s," (4) while Nancy Franklin has looked back with some amusement at Austen's selection as "one of People's '25 Most Intriguing People of 1995'." This "1995 honor" by an American magazine devoted to celebrating the existence of celebrities came "in recognition of ... that year's productions," imported from Britain, of "a TV miniseries of 'Pride and Prejudice' ... and a film adaptation by Emma Thompson of 'Sense and Sensibility.'" (5) For Kort, the appeal of such representations--and of Austen's novels themselves--was their invitation to audiences to "lose" themselves "in a well-ordered, meticulously described, seemingly simple and secure past," but also their ability to provide revelations about the "complexity" of women's lives and to hold up a mirror to "the complications of our own times." (6) Yet, as Heidi Safia Mirza's remark about gender suggests, the question of which women's lives--either in the Regency or in the present--were or weren't being addressed remained largely unexamined as the Austen adaptations of the mid-1990s proliferated.
Yet even Rozema's "radical" Mansfield Park (10) did not reflect the extent of the black presence in Britain itself during the Regency. In the Introduction to her edition of The History of Maw Prince, a West Indian Slave (1831), Sara Salih cites historians who estimate "the Black population in Britain at this time" to have been between 10,000 and 20,000. (11) Although some were sailors and merchants from Africa, many were former slaves (or the descendants of former slaves) who had been brought by their owners from the British colonies. After the 1772 ruling by a British court that, under English Common Law, slavery in England itself had no legal standing and could not be enforced, they lived in England as free persons. During the period of the "long" Regency, the Slave Trade Act of 1807 outlawed slave trafficking throughout the British Empire, but this legislation did not affect the ownership of slaves in British colonies. Slavery, especially in the Caribbean, continued unabated until the passage of the Slavery Abolition Act of 1833.
Thus, the Regency--the period glamorized in mid-1990s cinematic representations through visions of pastoral landscapes and drawing-rooms furnished in Empire furniture, all populated by decorative young white women in laundered muslin--was roiled by debate over the legal and moral legitimacy of slaveholding, an issue made visible through the presence, especially in London and in port cities and towns, of free black workers. At the same time, both the countryside and the urban spaces alike were the sites of large numbers of poor white Britons (along with smaller gypsy communities), living in destitution. Vagrancy laws that permitted magistrates to confine the homeless forcibly to workhouses also forbade begging and allowed those found guilty of it to be sentenced to prison or even subjected to corporal punishment.
If the Austen adaptations of the mid-1990s failed to dramatize these other sorts of historical realities, and if Rozema's Mansfield Park gestured toward them but still foregrounded upper-class white responses to slavery, rather than black experiences of it, that does not mean issues of race and class, as well as of gender, were absent from British television and cinema or from what is known as the "heritage industry." The year 1995 may have been an annus mirabilis for one kind of Regency representation, but so was 1994 for another.


The Biodesign Institute is master-planned as four interconnected buildings that will comprise 800,000 sq. ft. Currently, three buildings (554,000 sq. ft.) are complete and house over 1,300 faculty, staff and students. Biodesign was the first facility in Arizona to earn platinum-level LEED certification from the US Green Building Council for environmentally friendly design.
The environmentally friendly aspects of the institute's facility have earned it numerous awards, listed below. Special honors include 2006 Lab of the Year by R&D Magazine and earning Arizona's first platinum-level certification from the U.S. Building Council for Leadership in Environmental Engineering and Design (LEED). Learn more in the building design section.


April 10, 2019 By heather Leave a Comment This post may contain affiliate links. Read my disclosure policy.
Old fashioned egg custard is such a simple yet elegant dessert. This baked custard is silky smooth, creamy perfection.
This custard recipe is one that brings back memories for me. It's something that my Nana would make when we were growing up. Even though this dessert doesn't require many ingredients, there are a few tips to ensure a smooth consistency every time.
Custard is something typically made with eggs, milk and sugar. It usually has a thicker consistency than pudding and uses egg as the thickening agent.
This first step, scalding the milk, might not necessarily be "necessary" but it is the traditional way.
Pour milk into a saucepan. Turn heat to medium and stir the milk until the temperature reaches 180 degrees F. If you don't have a thermometer, watch for tiny bubbles to form around the edges. (Make sure to constantly stir to prevent film forming on top of milk.) Remove from heat.
Whisk together eggs first. I like to beat them for at least one minute. Then whisk in sugar, vanilla, cinnamon and salt.
Slowly whisk in warmed milk to the egg mixture while continuously whisking. Keep slowing adding milk until all incorporated.
Pour the mixture through a strainer to get out any unwanted lumps.
Divide the mixture into 4 ramekins and place them in a 9×13 pan.
Pour 3 cups of warm water into the pan, around the ramekins.
Remove from oven and carefully remove the ramekins from the water bath. Let them cool on the counter. Optional to dust with powdered sugar, nutmeg or cinnamon. Serve warm or cool in the refrigerator.
Be sure not to overheat the milk.
Slowly add warmed milk into egg mixture. If you add to fast it could cause eggs to curdle.
Pouring through the strainer helps remove any possible cooked egg or lumps.
These can be served warmed but I prefer mine chilled. Let them completely cool and then place in the refrigerator.
Why is my egg custard watery?
If the eggs become too hot from a temperature that is too high weeping can occur. Cooking this custard in a water bath helps to bake them gently and evenly.
When I make this dish I use four of these ramekins.
Little pots of deliciousness bests describes this dessert. Garnish with a few pieces of slices strawberries and mint for an added touch of color.
I also love this custardy Magic Cake from JoCooks.
Easy, creamy old fashioned egg custard.
Beat eggs until creamy with a whisk for about a minute.
Whisk in flour, sugar, salt, vanilla.
Pour milk into a saucepan and turn heat to medium. Stir until milk reaches 180 degrees F or bubbles starting forming around the edge of pan.
Slowly pour a splash of the milk into egg mixture and whisk. Continue slowly whisking in milk until all is combined.
Pour milk and egg mixture through a strainer into another bowl. Divide the mixture into 4 ramekins and place them in a 9x13 baking dish.
Pour 3 cups of warm water into the baking dish around the ramekins.


We are family owned and operated since 1957, the year we came into existence and since then we have been serving the Northern Ohio area and have increased our service to include replacement windows and garages. For over 53 years our customers have been using our service. We are service oriented and provide the best possible pricing that we can. We feature only the best products in windows, patio doors, garages and doors.


The Actiontec M1000 ATT U-Verse approved modem is top tier when it comes to reliable DSL modems supported by Centurylink (and other) internet service providers. With the attachable W1000 wifi adapter (sold separately) you can turn your M1000 into a high speed modem/router combo device! If you're looking for an ADSL 2 or ADSL 2+ modem, get this highly rated Actiontec M1000. Get online and STAY online! Great option as a ATT U-Verse approved modem.
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On the heels of a great customer event in LA in February (thanks again to Children Hunger Fund's Nobeleen Alkhato for presenting), we're bringing the fun to NYC on May 24. Don't miss this opportunity to pick the brains of our staff and learn from the success stories of other customers in an intimate, informal setting—with free appetizers and drinks!
What: Hands-on training on the latest iMeet Central features, an exclusive review of our upcoming product roadmap, and a presentation by Suellen Anderson, the Director of Agency Operations at RAPP, on how her team uses iMeet Central and why they decided to migrate away from Workfront to iMeet Central as their core solution for project management.
Where: A Voce, an Italian restaurant helmed by a "young pasta wizard" and offering a "grand new setting" for enjoying Central Park (quotes by New York magazine, which made it a Critics' Pick).
When: 3:00-6:00pm on Tuesday, May 24.
Who: RAPP's Suellen Anderson brings more than 20 years of direct marketing and project management experience to her role as Director of Agency Operations. During her tenure, she has managed multi-channel direct-response initiatives focused on customer acquisition, retention and loyalty across several categories, including retail, travel and hospitality, banking and personal insurance.
RAPP is the only agency that specializes in high-precision CORE DIALOG™: those critical, direct and high-value relationships that link people and brands across the fast-changing digital landscape. The RAPP network spans the globe, housing a diversified and growing family of 2,000 innovators, integrators and implementers. RAPP is a deeply integrated part of Diversified Agency Services, a division of Omnicom Group.
And don't forget to mark your calendar for Collabosphere, the ultimate collaboration conference and the crown jewel on our customer event calendar. After a great year in Texas, Collabosphere will be returning home to Los Angeles this September. Join us for two action-packed days of inspiration and information… and parties. Expect much more information to come soon!


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News and features about Seward County Community College in Liberal, Kansas
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Surgical Technology lab replicates reality
Posted by Rachel Coleman on January 22, 2015 February 25, 2016
When Bridgette Horner backed her vehicle up to the east entrance of the Surgical Technology Lab at Seward County Community College/Area Technical School, she'd arrived to dispose of usable expired supplies from Morton County Health System in Elkhart.
That's not how Surgical Technology Instructor Carmen Sumner saw it, though.
As Horner unloaded packages of disposable surgical supplies, Sumner summed it up in one word: "Jackpot!"
Horner serves as Surgical Technologist, Central Supplies Manager and Sterile Processing Department Director at MCHS, but she's also an alumna of the College. A member of the first-ever surgical technology class to graduate in 1998, she knows exactly what will help surgical technology students learn.
"I'm excited to put these items to use," Horner said. "They're expired, so there's no way the hospital can use them. This is one way they won't be wasted, and they'll help support health services in the region."
The donation is not the first of its kind. Allied Health Services routinely receives gifts from health care providers throughout the region, from defibrillators to disposable materials. It's a win-win situation in a geographically remote area that often struggles to attract healthcare professionals, noted Ed Anderson, who instructs Respiratory Therapy students.
"I'd say 95 percent of the Respiratory Therapists in the region came through our program," he said.
For her part, Horner enjoyed the opportunity to see the changes in the SCCC/ATS surgical technology program. The white, metal-sided building located adjacent to the Epworth Allied Health center at the corner of Washington Ave. and Fifth Street presents a modest exterior. Inside, however, students practice what they've learned in simulated surgical rooms, with interactive manikins and ever-changing scenarios.
"This is awesome," said Horner. "It's so impressive! When I was a student, we had to go to the hospital and use their C-Section room for practice labs." Occasionally, she recalled, labs were rescheduled because the room was needed for real-life operations.
Sumner routinely witnesses the benefits of hands-on practice that can take place without scheduling conflicts — the more the better, she said.
"I tell the students, 'You can make mistakes here and there's no harm done,'" she said. "They'll be simulating a surgery and if we make a mistake that would normally harm a human, we can start over again and practice the correct technique. Our manikins are very forgiving and let us keep working on our skills until we get them perfected."
While many people view the role of a surgical technologist as a passive one — on television programs, they're routinely told, "hand me the scalpel" — Sumner said the reality is all about taking an active role.
"You have to anticipate," she said. "It's not just knowing the instruments —"
" — it's knowing the surgery," Horner agreed. "Every procedure. You use what you've learned, what you know."
Horner, who was logged nearly two decades as a Surgical Technologist, viewed the student lab with approval.
"I would have been much less intimidated when I started my clinical if I had been immersed in this," she said. During her first surgery, she recalled, the surgeon's hand brushed lightly against her cap as the team prepared to go to work. Horner had to speak up in spite of her nervousness. The surgeon, irritated, had to rescrub and put on fresh, sterile gloves.
"That's the surgical conscience," Sumner said. "That's what students have to learn. We all keep an eye out for those situations. The problem isn't contamination. The problem is what to do when we recognize it is happening."
Unlike a few decades ago, when hospitals washed and sterilized gowns, drapes and other required materials used in surgery, today's healthcare providers rely on disposable materials.
"The instruments are reusable, but that's about it," Sumner said. Suppliers now package individual gowns, drapes, gloves, sutures, staples — everything that a surgical team will need for a specific procedure — into bundles that can cost thousands of dollars. To further complicate matters, multiple manufacturers vie for the market, and surgeons often develop preferences for a particular brand of supplies.
What that means for Sumner's students is a larger body of "unknowns" when they step into a real-life surgery.
"The gowns are different, the packages are different, and I've had students experience a moment of panic, where they're scrubbing in and they are unfamiliar with the particular gown that facility uses," Sumner said. "We can't afford to purchase all the different kinds, so we truly rely on donations from area healthcare providers. It's a wonderful supplement to our program."
SCCC/ATS Surgical Technology is now accepting applications for the program, which begins in August. Packets for that program, along with Medical Laboratory Technician, Nursing, Respiratory Therapy and Medical Assistant programs, are available at the Epworth Allied Health Education Center, 520 N. Washington. For more information, call Sumner at 620-417-1411.
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Campus to open late Jan. 17 due to weather January 17, 2020
Sharp Family Champions Center to cut ribbon Jan. 29 January 17, 2020
Public invited to ribbon-cutting December 16, 2019
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College hosts Thanksgiving feast for public November 25 November 21, 2019
• Seward County Community College notifica que esta institución no discrimina a personas en forma de raza, color, origen nacional, discapacidad de acceso o tratamiento, edad, o sexo, en la admisión o acceso a sus programas y actividades. Cualquier persona que tenga preguntas sobre el cumplimiento de los requisitos de las regulaciones que implementan el Título VI, Título IX o Sección 504 están dirigidas a comunicarse con el Vicepresidente de finanzas y operaciones, (620) 624-1951.
• SCCC does not discriminate on the basis of race, religion, color, national origin, gender, age, or disability in admission, access to, treatment or employment in its programs and activities. For more information about Title VI, Title IX, or Section 504, call the VP of finance & operations, (620) 624 -1951.
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A leading businessman has died following a "serious" fire at a large mansion house near Falkirk.
Christophe Ferrand, 44, a senior manager at Aegon Asset Management, was rescued from the blaze, but died later on in hospital.
A 45-year-old woman and two children - a 14-year-old boy and eight-year-old girl - were also taken to hospital, where the boy remains.
The fire was discovered at the house in Dunmore Park, Airth, at about 0500 BST.
Mr Ferrand was chief administrative officer of the UK division of Aegon's asset management business.
The company said in a statement that he had been an "inspirational" figure in the company and would be "sorely missed" by colleagues.
The statement read: "It is with regret and sadness, Aegon Asset Management can confirm that a great friend and colleague, Christophe Ferrand, died in a fire at his home in Scotland last night.
"Christophe, who was the chief administrative officer of the UK division of the asset management business, had recently taken on an expanded role with responsibility for legal and finance in the UK as well as his existing responsibilities."
It added: "Our thoughts are with his family, friends and colleagues. We would please ask that you could please respect their privacy at this very sad time."
The woman and two children were all treated for the effects of smoke inhalation.
Det Insp Ronnie Isles said: "Our inquiries are at a very early stage and the cause of the fire is still to be determined.
"However, due to the remote locality of the house we appeal for anyone who may have been in this area around the time of the fire to come forward."


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Q: Swift how to distance two rightbar button items I'm going to get straight to the point. The below code adds two bar button items without a problem, however I was wondering how I can put a distance between these two bar button items? They are too close to each other, that's why I'm looking for a way to manually set their distance to each other. I'd appreciate any help! Thank you very much in advance!
let btnPlus = UIBarButtonItem()
        btnPlus.title = "+"

    btnPlus.setTitleTextAttributes([NSAttributedStringKey.font : UIFont(name: "Arial", size: 24)!], for: .normal)
    btnPlus.setTitleTextAttributes([NSAttributedStringKey.font : UIFont(name: "Arial", size: 24)!], for: .selected)
    btnPlus.target = self
    btnPlus.action = #selector(OnbtnPlusTouched)
    //btnPlus.setTitleTextAttributes([NSAttributedStringKey: UIFont(name: "Arial", size: 14)!], for: UIControlState.normal)

    let btnRating = UIBarButtonItem()
    btnRating.title = "Oy Ver"

    btnRating.setTitleTextAttributes([NSAttributedStringKey.font : UIFont(name: "Arial", size: 24)!], for: .normal)
    btnRating.setTitleTextAttributes([NSAttributedStringKey.font : UIFont(name: "Arial", size: 24)!], for: .selected)
    btnRating.target = self
    btnRating.action = #selector(OnbtnPlusTouched)
    //btnPlus.setTitleTextAttributes([NSAttributedStringKey: UIFont(name: "Arial", size: 14)!], for: UIControlState.normal)

    navigationItem.rightBarButtonItems = [btnPlus, btnRating]

A: You can add a UIBarButtonItem with a fixed space or a flexible space:
let btnPlus = UIBarButtonItem()
let btnRating = UIBarButtonItem()
let fixedSpace = UIBarButtonItem(barButtonSystemItem: .fixedSpace, target: nil, action: nil)
fixedSpace.width = 15

navigationItem.rightBarButtonItems = [btnPlus, fixedSpace, btnRating]

There is also the possibility to use .flexibleSpace.

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