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The purpose of this article is to analyze the effect of hotel innovations on firm value. Specifically, this study fills a research gap in the previous literature by examining this effect through market value and by distinguishing the potentially different impacts of distinct innovation types: product, process, organization and marketing. This research contributes to consolidating the empirical evidence of hotel innovation and performance by analyzing whether distinct types of innovation lead to different levels of results. The findings show that innovations are perceived to have a positive impact on the future sales of the company: in a four-day period (0,+3), there is an increase in stock exchange returns of 1.53%. In terms of innovation types, process and marketing innovations are found to have a higher positive effect on hotel market value than product and organization innovations; which is explained by potential cost differences among innovations.
This article analyzes the effect of innovation on the firm value of hotels. Innovation is a critical element in today's tourism companies (Sundbo et al., 2007 and Martínez-Ros and Orfila-Sintes, 2009). It certainly helps firms reach and keep higher competitiveness standards (Aldebert et al., 2011, Hjalager, 2002, Kumar et al., 2008, Pulido et al., 2011, Rodgers, 2007 and Zach et al., 2010) as well as growth (Love et al., 2011). In the context of hotel management, Chen (2011) points out that "innovation appears to be the only means for an organization to convert change into opportunities and thus succeed". In a recent article, Hjalager (2010) describes innovation research in tourism as a young phenomenon, suggesting several research gaps that should be tackled in order to get further insights that contribute to consolidating the theoretical underpinnings of tourism innovation. Specifically, this author, among others, points out a research gap in tourism innovation that should attract more analysis: the relationship between innovation actions and their economic performance. This author explicitly raises the question of examining "what types of innovation produce what level of results". In an attempt to add to the extant body of knowledge on the topic, this article analyzes the effect of different types of innovations on the hotel industry's market value. With this objective, the subsequent sections of the article are arranged as follows: Section 2 presents the literature review of innovation in the hotel industry and justifies the hypotheses; Section 3 outlines the methodology and data employed; Section 4 describes the results; and Section 5 presents the conclusions.
This article analyzes the effect of innovation on hotel market value. The results show that innovations are perceived to have a positive impact on the future sales of the company (note that market value is a future-oriented measure of cash-flow). Specifically, in a four-day period (from day 0 to day +3), there is an increase in returns of 1.53%; what is more, 1.53% in four days is equivalent to annual returns of 300% (this annual measure would be compared with other investments' internal rate of return or discounted cash-flow, which are usually presented through annualized figures to use equivalent measures). Also, in an attempt to further explain these returns derived from innovations, the news items are categorized into the four traditional types: product, process, organization and marketing (plus distribution). "Process" and "marketing" (and "distribution") innovations have a higher positive effect on the market value than "product" and "organization" innovations. This shows that each innovation needs to be treated differently and individually, not only between but within categories, on account of cost differences among innovations. 5.1. Implications Beyond the specific results of the empirical application, the study has important implications, both academic and managerial. Regarding academic implications, the finding that a hotel's market value varies when the prospect of innovation activities is announced indicates that this measure can be regarded as appropriate to analyze this type of investment. This fact, along with the outcome that these variations in hotel market value are contingent upon the type of innovation, suggests that, when a comprehensive examination is pursued by the analyst, different types of innovation and distinct kinds of measurements should be employed. While different innovation types are expected to lead to different results, the use of several performance measures might allow the researcher to find hidden effects that, otherwise, would not be easy to uncover. Note that using distinct performance measures implies looking at the company from different perspectives; perspectives that represent facets that might be worth looking into. As for managerial implications, the use of market value facilitates the analysis of the effect of innovation on performance by estimating unbiased market predictions on future profits. This technique employs a forward-looking firm performance measure that overcomes all the difficulties of the traditionally used backward-looking firm profitability, i.e., accounting measures. Specifically, once the innovation is announced, managers can observe the evolution of share prices to determine how valuable the news is perceived to be by the shareholders. If the shareholders' perceptions of it are not as good as the managers would have expected, they may want to see whether this is due to a lack of information (or even to misinformation). If this were the case, a new flow of information should be released, in order to clarify the hotel's innovation. Also, finding that innovation has a positive impact on firm market value means that the market considers that an innovative company is a healthy one. Therefore, any tourism company introducing innovations, no matter whether they are internal or external, should show that it is innovating all the time, by releasing news through a well-executed public relations system. 5.2. Limitation Even though the process followed to collect the innovation news announcements in the hotel industry guarantees that, a priori, all of them must have been detected, the resulting sample size is a clear limitation; not so much because it affects its ability to capture potential reactions in share prices, but because it does not allow the inclusion of a larger number of potential explanatory variables that could shed further light on the innovation-related determinants of hotel market value. 5.3. Future research For further research, several lines can be followed in order to provide a more comprehensive view of the relationship between innovation and market value in the hotel industry: (1) The paper uses the Schumpeterian classification to identify differentiated effects in innovation types; nevertheless, the use of other taxonomies would offer a broader view in terms of academic perspectives (as it would permit the identification of the best explanatory classification) as well as in terms of management perspectives (as it would show decision-makers the best innovation types according to the taxonomy used). (2) A larger sample would permit the use of factors related to a hotel's characteristics; for example, the same innovation might have different effects on hotel performance depending on its category (e.g., number of stars). (3) The analysis of the effect of innovation on distinct tourism-related industries would show whether the same type of innovation has a differentiated influence (hotels, travel agencies, airlines, etc.). (4) The use of specific information on each innovation, especially in terms of costs incurred in the investment could shed more light on the specificities of every initiative; that is, knowing the cost invested in an innovation could determine how costly (per unit) the variations in market value are. (5) Risk analysis. Some innovation decisions are particularly relevant as sometimes they can imply enormous investments; this fact, together with the large amount of fixed costs generally involved in the hotel industry, makes them strongly revenue-dependent, which might lead to profit instability. Consequently, it would be critical to examine the effect of innovation on the risk of a hotel's market value (measured through its volatility, for example). (6) There is a social trend today that favors corporate social responsibility (CSR) actions, so it would be interesting to see whether innovation oriented to enhance a hotel's CSR exerts an incremental effect on performance. That is, as the CSR literature shows that these activities have a positive impact on market value and this article has shown that innovations also have a positive effect on market value, it would be worthwhile to analyze whether a greater effect exists when both activities – CSR and innovation – are linked together (e.g., the announcement of an innovation that allows a more efficient use of solar panels in hotels). (7) Recent literature has proven sequential interdependency between radical and incremental innovations (Martínez-Ros and Orfila-Sintes, 2009). It would be relevant to test the effect of this sequential interdependency on market value, as it would allow managers to determine the best innovation decisions (and their sequence) to optimize the firm's market value. (8) As market value has been shown to reflect innovation announcements, it is possible to analyze the effect of innovation-based competitive reactions on the firm. In other words, as market value captures the hotel's innovation strategy, it could be expected that it will reflect competitors' innovation strategies too. It would facilitate a rivalry analysis between actions and reactions based on innovation strategies. (9) Innovation is supposed to enhance customer satisfaction, and it should be reflected in the "opinion polls" that are shown in most hotels' web pages these days. On the one hand, the value or ranking obtained in these polls can be used as an alternative measure of innovation effectiveness; and on the other, correlation analyses between the value/ranking obtained through these perceptions and a hotel's market value would show whether variations in market value are materialized in real terms too. (10) With the advent of new technologies, other measurements of innovation impact can be used. If "twitter" were treated as a market where information is exchanged, and the number of "tweets" were considered as a measure of repercussion (or hype), it could be interesting to observe the expectations generated by an innovation announcement on a specific day. Paralleling the method employed in this article, it would imply observing whether the amount of "exchanged information" (tweets) derived from a firm's release of news on a given day is abnormally superior to the quantity of "exchanged information" in a normal day, and whether and how many good things are said.
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Quantification of high-specificity cyclic diguanylate signaling
Jonathan P. Massie, Evan L. Reynolds, Benjamin J. Koestler, Jian-Ping Cong, Marco Agostoni, and Christopher M. Waters
PNAS July 31, 2012 109 (31) 12746-12751; https://doi.org/10.1073/pnas.1115663109
Jonathan P. Massie
Evan L. Reynolds
Benjamin J. Koestler
Jian-Ping Cong
Marco Agostoni
Christopher M. Waters
For correspondence: watersc3@msu.edu
Edited by Susan Gottesman, National Cancer Institute, Bethesda, MD, and approved June 25, 2012 (received for review September 23, 2011)
Figures & SI
Cyclic di-GMP (c-di-GMP) is a second messenger molecule that regulates the transition between sessile and motile lifestyles in bacteria. Bacteria often encode multiple diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes that produce and degrade c-di-GMP, respectively. Because of multiple inputs into the c-di-GMP–signaling network, it is unclear whether this system functions via high or low specificity. High-specificity signaling is characterized by individual DGCs or PDEs that are specifically associated with downstream c-di-GMP–mediated responses. In contrast, low-specificity signaling is characterized by DGCs or PDEs that modulate a general signal pool, which, in turn, controls a global c-di-GMP–mediated response. To determine whether c-di-GMP functions via high or low specificity in Vibrio cholerae, we correlated the in vivo c-di-GMP concentration generated by seven DGCs, each expressed at eight different levels, to the c-di-GMP–mediated induction of biofilm formation and transcription. There was no correlation between total intracellular c-di-GMP levels and biofilm formation or gene expression when considering all states. However, individual DGCs showed a significant correlation between c-di-GMP production and c-di-GMP–mediated responses. Moreover, the rate of phenotypic change versus c-di-GMP concentration was significantly different between DGCs, suggesting that bacteria can optimize phenotypic output to c-di-GMP levels via expression or activation of specific DGCs. Our results conclusively demonstrate that c-di-GMP does not function via a simple, low-specificity signaling pathway in V. cholerae.
signaling specificity
The second messenger cyclic di-GMP (c-di-GMP) controls the transition between motile and sessile lifestyles in the majority of bacteria by inducing biofilm formation and repressing motility. c-di-GMP is synthesized from two GTP molecules by diguanylate cyclase (DGC) enzymes encoding a GGDEF domain. Degradation of c-di-GMP is mediated by c-di-GMP–specific phosphodiesterase (PDE) enzymes encoding either an EAL or HD-GYP domain (1). A striking feature of c-di-GMP–signaling systems is the large number of DGCs and PDEs typically encoded in bacterial genomes (2). For example, the human pathogen Vibrio cholerae encodes 40 DGC domains, 20 EAL domains, and 9 HD-GYP domains (2). Therefore, it is unclear whether c-di-GMP signals via a low-specificity central hub or in high-specificity microdomains.
Signaling specificity relates to the degree in which c-di-GMP produced or degraded by individual DGCs or PDEs regulates downstream c-di-GMP–mediated responses. In a high-specificity system, c-di-GMP produced or degraded by individual DGCs or PDEs modulates one or a small subset of c-di-GMP–regulated behaviors. High-specificity signaling would be controlled with molecular mechanisms that segregate signal production with the output response. This could be accomplished with strategies such as signal sequestration in protein complexes, signal localization at distinct cellular sites, or temporal separation of signal production and signal reception (3⇓⇓–6). Furthermore, such mechanisms occur in eukaryotic second messenger–signaling systems (7⇓⇓⇓⇓–12), and mathematical modeling of eukaryotic cells predicts that cAMP-signaling domains as small as 50 nm can be established with precise localization of synthesis and degradation enzymes (13).
Alternatively, in a low-specificity signaling system, c-di-GMP produced or degraded by each DGC or PDE would modulate a common global signal pool. In this situation, the concentration of c-di-GMP is relatively uniform throughout the cell. c-di-GMP–mediated responses would then be regulated at specific concentrations of c-di-GMP determined by the binding parameters of the signal transduction machinery. Production or degradation of c-di-GMP by a DGC or PDE enzyme, respectively, would equally impact each downstream phenotype.
There is currently a lack of consensus on the degree of c-di-GMP–signaling specificity because evidence supports both high- and low-specificity models. A number of studies have concluded that c-di-GMP functions via high specificity based on the observation that the intracellular concentration of c-di-GMP does not directly correlate with phenotypic expression (14⇓⇓–17), an observation inconsistent with a low-specificity system. These studies relied on mutagenesis of respective DGCs. Although informative, these observations are qualitative in nature because deletion of a DGC only produces two states in which the intracellular concentration of c-di-GMP can be analyzed: the native and mutant states. This limitation makes it impossible to quantitatively describe the relationship between the intracellular concentration of c-di-GMP and phenotypic output for different DGCs. Furthermore, in many cases, the concentrations of c-di-GMP and expression of phenotypic outputs were not determined using identical growth conditions.
In contrast, evidence for low-specificity signaling has also been documented. DGC enzymes from divergent species showing no sequence conservation in the variable N-terminal signal recognition domain are able to cross-complement mutations of one another (18). These findings are not consistent with high-specificity signaling models that predict other properties of these enzymes, such as localization signals or protein interaction domains, would be important for activity. In these situations, the complementing DGCs are not expressed at native levels, and it is possible that overproduction of c-di-GMP "floods" the normal cellular pools, although rigorous quantification of the intracellular concentration of c-di-GMP has generally not been performed to examine this possibility. A genetic reductionist approach was used to study signaling specificity in Salmonella Enteritidis (19). In this study, a complete DGC mutant of this species was created, and each individual DGC was individually complemented at natural expression levels. Phenotypic analysis determined that active DGCs functioned redundantly, supporting a low-specificity system, although the intracellular concentration of c-di-GMP in the various DGC strains was not determined.
A rigorous examination of c-di-GMP–signaling specificity requires a determination of the intracellular concentration of c-di-GMP and expression of associated phenotypic outputs generated by different DGCs at multiple states. Importantly, these measurements must be taken from bacteria grown in identical conditions because subtle environmental changes can influence the intracellular concentration of c-di-GMP. Here, we report our studies that incorporate all of these elements to quantify c-di-GMP–signaling specificity in the human pathogen V. cholerae. Our experiments used a systematic analysis to determine the intracellular concentration ranges of c-di-GMP at which multiple DGCs induce biofilm formation and expression of the c-di-GMP–inducible gene aphA. When considering all data points generated by multiple DGCs, no correlation of the total c-di-GMP concentration with biofilm formation and transcription was observed. However, a significant correlation between c-di-GMP production and phenotypic output was observed when examining individual DGCs. These results conclusively show that c-di-GMP functions via high-specificity signaling in V. cholerae. In addition, we observed that the rate of change in c-di-GMP at which different DGCs impact phenotypic output is distinct, suggesting that different DGCs can be used by V. cholerae to generate a fast or slow induction of biofilm formation and transcription in response to changing c-di-GMP.
Rationale.
To differentiate between low- or high-specificity signaling in c-di-GMP–mediated regulation of V. cholerae biofilm formation and transcription, we quantified these phenotypic responses at multiple in vivo c-di-GMP levels generated by seven distinct DGC enzymes. Low-specificity signaling predicts that a significant correlation will exist between total intracellular c-di-GMP levels and downstream effects independent of the DGC examined because c-di-GMP is uniform throughout the cell. However, high-specificity signaling predicts that little correlation will exist between the total in vivo c-di-GMP level and downstream phenotypic regulation by DGCs because individual DGCs contribute to a segregated c-di-GMP microdomain. However, significant correlation will exist between c-di-GMP and phenotypic outputs generated by a single DGC domain. Importantly, our approach allows the quantification of c-di-GMP–signaling specificity without an a priori understanding of the molecular mechanisms involved in maintaining this specificity by directly measuring the input to the system (i.e., c-di-GMP synthesized by different DGCs) and the output (i.e., phenotypic response).
Determination of the Enzymatically Active DGCs in V. cholerae.
Our approach relies on generation of multiple c-di-GMP input states by distinct DGCs. To accomplish this, we constructed expression vectors for all 40 V. cholerae DGCs under the control of the Ptac promoter on the plasmid pEVS143. This promoter is a derivative of the trp and lac promoters and is inducible with the synthetic inducer isopropyl β-D1-thiogalactopyranoside (IPTG). Induction of the Ptac promoter in V. cholerae occurs in a graded response (Fig. S1). Importantly, the expression of each DGC is driven by the same promoter and translation initiation sequence.
DGC enzymes are modular proteins encoding a variety of signal reception domains in the N terminus. Because these signaling domains control the activity of DGC enzymes, typically only a subset of DGCs is active in the cell at any given condition. To determine which DGCs are enzymatically active in the conditions examined here, each of these expression plasmids was conjugated into the wild type (WT) strain of V. cholerae, and biofilm formation was assessed using a minimum biofilm-eliminating concentration (MBEC) biofilm assay. WT V. cholerae grows to the high–cell-density quorum-sensing state, which represses biofilm formation (20, 21). Indeed, the WT strain containing an empty vector control produced minimal biofilms (Fig. 1). Furthermore, biofilm formation of a V. cholerae ΔvpsL mutant, which is unable to synthesize extracellular polysaccharide (22), formed the lowest level of biofilm formation. Overexpression of QrgB, an active DGC enzyme from the bacterium V. harveyi, induced biofilm formation as reported previously (20). Expression of each of the DGCs was induced by addition of 0.1 mM IPTG, and biofilm formation was measured at 8 h (Fig. 1). Biofilm formation was expressed as a fold increase relative to the ΔvpsL mutant. The DGC proteins showed a wide margin of activity ranging from no induction of biofilms to over 26-fold induction of biofilm formation (VC1599). We selected 18 of the DGC enzymes that showed approximately 5-fold or greater induction of biofilms for further examination.
Determining active biofilm forming DGCs. Each DGC from V. cholerae was induced with 0.1 mM IPTG, and biofilm formation was determined using the MBEC biofilm assay. The ΔvpsL mutant is unable to produce VPS and form significant biofilms. A strain containing an empty vector control and expressing a known active DGC (qrgB) are also indicated. Biofilm fold change is expressed relative to the ΔvpsL mutant. Three biological replicates were performed, and the error bars show the SD.
Quantification of Biofilm Formation Using Flow Cytometry.
Because the intracellular levels of c-di-GMP are constantly in flux and rapidly respond to changes in environmental conditions (23), to rigorously examine signaling specificity, c-di-GMP must be quantified from the identical conditions at which the phenotypic outputs are measured. Because it is difficult to reproducibly measure c-di-GMP from a surface attached biofilm, we developed a method that allowed us to quantify biofilm formation and extract c-di-GMP concurrently from cultures of V. cholerae grown planktonically. This assay is based on our observation that hyper–biofilm-forming strains of V. cholerae aggregate in culture, even when grown with vigorous shaking. We reasoned that the number and size of these aggregates could be quantified using the forward-scatter and side-scatter parameters of a bacterial population analyzed by flow cytometry. These parameters measure the relative size and granularity of a particle, respectively. A similar approach has been used to quantify biofilm formation in Pseudomonas aeruginosa (24). To test whether flow cytometry was an appropriate method to quantify biofilms, we induced expression of the DGC VC1599 using eight different concentrations of IPTG consisting of a threefold dilution series from a maximum amount of 1 mM inducer and measured aggregate formation with flow cytometry. Induction of VC1599 showed a dose-dependent increase of the aggregate population present in the P1 gate (Fig. 2). To quantify aggregation, we multiplied the percentage of the total population present in the P1 gate by the average forward scatter of the P1 population to generate a biofilm formation index. We confirmed that aggregate formation as measured by this assay is dependent on expression of the major extracellular polysaccharide matrix in V. cholerae, vibrio polysaccharide (VPS), because induction of DGCs in a vpsL mutant did not produce aggregates (Fig. S2).
Analysis of biofilm formation using flow cytometry. Flow cytometry plots for the induction of VC1599 are shown. The x axis represents side scatter (granularity), and the y axis represents forward scatter (size). The gate P1 brackets the bacterial aggregates in the population. The concentrations (in mM) of IPTG used were: 0.0005 (A); 0.0014 (B); 0.0041 (C); 0.0123 (D); 0.037 (E); 0.11 (F); 0.33 (G); and 1.0 (H).
The 18 DGCs able to induce biofilm formation we identified using the MBEC assay (Fig. 1) were analyzed for dose-dependent biofilm induction at 8 IPTG concentrations by flow cytometry. From these experiments, nine DGCs yielding suitable biofilm-induction curves were selected for further analysis. Two of the 18 active enzymes, VC2285 and VCA0557, were discarded because these DGCs produced robust biofilms when uninduced. This is presumably attributable to high activity of these DGCs such that the low level of transcription driven by the Ptac promoter in the absence of IPTG was sufficient for full induction of biofilm formation. Seven DGCs (VC1370, VC1372, VC1376, VCA0697, VCA0939, VCA0956, and VCA0965) did not show increased biofilm induction in response to IPTG when analyzed by flow cytometry. This finding represents a discrepancy with the MBEC biofilm assay because these DGCs increased biofilm formation in that condition (Fig. 1). We hypothesize that this discrepancy between analysis of biofilm formation by the MBEC assay and analysis of biofilms using flow cytometry arises from differential activity of these DGCs in these two conditions. This differential activity could be attributable to a number of factors, including growth on a surface, density of the culture, oxygen concentration, or degree of agitation. Surface activation of a DGC has been described previously because the DGC WspR from P. aeruginosa forms active clusters when grown on a solid surface (25). This observation further highlights the importance of quantifying c-di-GMP and phenotypic output from identical growth conditions.
The biofilm formation of each of the nine suitable DGCs was then measured by flow cytometry at eight IPTG induction states in triplicate (Fig. S3). Additionally, to test whether the induction of biofilm formation by these DGCs is dependent on c-di-GMP synthesis, we mutated the Gly-Gly residues in the active site of each DGC to Ala-Ala. Indeed, mutation of the active site of each of these DGCs abolished their ability to induce biofilm formation, showing that c-di-GMP synthesis is required for the observed activity of these DGCs (Fig. S2).
Measurement of the Levels of c-di-GMP and Correlation to Biofilm Formation.
In addition to measuring biofilm formation for the nine specified DGCs, we also measured the in vivo c-di-GMP concentration in triplicate from the same cultures at each of the eight IPTG expression states using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) (Fig. S3). We observed that induction of the DGCs VC1067 and VC2224 at the highest concentrations of IPTG produced c-di-GMP levels in excess of 100 μM (Fig. S3). Because this concentration is much greater than the typical concentrations of c-di-GMP in bacteria (26), and high levels of c-di-GMP can inhibit growth of bacteria (27), these two DGCs were excluded from further analysis.
The correlation between biofilm formation and the concentration of c-di-GMP for the remaining seven DGCs at eight expression states was determined. The best-fit line of the dataset that we were able to obtain, giving an r2 value of 0.10, indicated there was little correlation between global c-di-GMP levels and biofilm formation when analyzing the data points in toto (Fig. 3A). However, a reanalysis of the data to determine the correlation between c-di-GMP levels and biofilm formation separately for each individual DGC found significant correlations ranging from r2 values of 0.67 to 0.99 with an average r2 value for all seven DGCs of 0.78 (Fig. 3B). These data suggest that DGCs do not contribute to a global c-di-GMP pool, indicative of low-specificity signaling, but, rather, that each uniquely induces biofilm formation, indicative of high-specificity signaling.
Correlation of c-di-GMP concentrations with biofilm formation and aphA expression. The amount of biofilm formation is plotted versus the intracellular concentration of c-di-GMP for each of the eight induction states of the seven DGCs that were analyzed (A and B). Each data point represents the average of three independent measurements. Error bars are not shown for clarity. The best-fit line for the entire dataset indicates no significant correlation (A), whereas analysis of the individual DGCs shows significant correlation (B). The r2 values for each DGC in B are indicated in the legend at right. Likewise, aphA expression versus c-di-GMP is shown (C and D). The best-fit line for the complete dataset (C) and individual DGCs (D) are shown. Each DGC is indicated by a different color, as displayed in the legend at right.
Interestingly, the slopes of the best fit lines for the analysis of individual DGCs were highly divergent (Fig. 3B and Fig. S4). The DGCs VC1599, VC2454, and VCA0165 had the largest slopes, indicating that these DGCs are capable of producing a large induction of biofilm formation over a relatively small change in in vivo c-di-GMP levels. We name these "fast response" DGCs. Alternatively, the DGCs VC1104, VC1216, and VC1353, exhibited intermediate slope values, termed "intermediate response," whereas VCA0074 had the smallest slope exhibiting a gradual increase in biofilm formation from 2.5 to 25 μM c-di-GMP, termed "slow response." These divergent responses of signal production to phenotypic output exhibited by different DGCs suggest that each enzyme can specifically impact the concentration and rate of change in c-di-GMP at which biofilm formation is induced in V. cholerae.
Correlation of c-di-GMP Concentration with aphA Gene Expression.
To determine whether high-specificity signaling exists in V. cholerae for other c-di-GMP–mediated responses besides biofilm formation, we examined the c-di-GMP–mediated induction of transcription. Two c-di-GMP–dependent transcription factors, VpsR and VpsT, have been identified in V. cholerae. VpsR directly binds to c-di-GMP to increase transcription of vpsT (28). VpsT itself then binds to c-di-GMP (29), and both c-di-GMP–bound VpsR and VpsT are thought to induce expression of the vps genes to generate the extracellular matrix necessary for biofilm formation (30). In addition to vpsT induction, c-di-GMP–bound VpsR also induces expression of the gene aphA (28), which encodes a transcription factor that activates a subset of the low–cell-density quorum sensing regulon and virulence gene cascade (31, 32). To study the impact of c-di-GMP on transcriptional control, we measured induction of a VpsR target gene. However, because multiple positive-feedback loops exist between VpsR and VpsT (30), and both of these transcription factors are c-di-GMP–dependent, we reasoned a more direct assay for VpsR activation by c-di-GMP was quantification of aphA induction.
aphA gene expression was determined upon induction of the seven DGCs described above at eight IPTG concentrations using quantitative real-time PCR (Q-PCR) in triplicate. The correlation between the in vivo c-di-GMP concentration and relative abundance (RQ) of aphA mRNA was determined (Fig. 3C). Similar to our analysis of biofilm formation, no significant correlation (r2 = 0.08) between total c-di-GMP levels and aphA expression was observed when examining all data points. However, analysis of the individual DGCs revealed significant correlation between the c-di-GMP produced by specific DGCs and the subsequent expression of aphA with r2 values ranging from 0.45 to 0.87 and an average r2 of 0.64 (Fig. 3D). Similar to our analysis for biofilm formation, these results indicate that c-di-GMP induction of transcription occurs via high-specificity signaling.
Again, varying slopes of the best-fit correlation lines for individual DGCs were observed, revealing different DGCs generate distinct input/output response rates (Fig. 3D and Fig. S4). In the case of aphA induction, the DGCs VC1104 and VCA0165 displayed the fast response profile with rapid induction of transcription over relatively small changes in c-di-GMP levels. In contrast to these fast response DGCs, VCA0074 again showed a slow response profile with a relatively small change in aphA transcription over a much wider range of c-di-GMP (about 3–25 μM).
One of the major unresolved questions in the field of c-di-GMP signaling is the degree of signaling specificity. Our analysis of seven DGCs each expressed at eight expression levels indicates that no significant correlation exists between the global intracellular c-di-GMP concentration and biofilm formation or induction of aphA expression. However, for both of these phenotypes, significant correlations were observed when DGCs were individually examined. These data suggest that different DGCs contribute to segregated subsets of intracellular c-di-GMP in the cell. Therefore, our findings strongly support that c-di-GMP functions via high-specificity signaling in the control of biofilm formation and VpsR-mediated transcription in V. cholerae.
Our results contrast the conclusion of low-specificity signaling obtained in Salmonella Enteritidis in which the ability of the 12 native DGCs to control c-di-GMP–mediated phenotypes was determined in a complete DGC mutant strain (19). In that study, four DGCs were found to be functionally redundant to induce cellulose synthesis. However, the levels of c-di-GMP produced by the single DGCs reinserted into the genome of the DGC-null strain were not measured, and the relationship of c-di-GMP concentration to phenotypic output was not determined as reported here. A closer examination of this relationship in Salmonella Enteritidis might reveal evidence for high-specificity signaling, or, alternatively, the c-di-GMP–signaling system of this bacterium could be inherently different from that of V. cholerae.
An important question is how the observed ranges of in vivo c-di-GMP levels relate to the normal physiological levels of c-di-GMP in the cell. We typically observe the concentration of c-di-GMP in the WT strain grown in Luria–Bertani (LB) media to a density of OD600 1.0, the optical density of the cultures examined in this study, to be 0.5–2 μM. Mutation of the master high–cell-density quorum-sensing regulator of V. cholerae, hapR, locks the cells into the low–cell-density state, increasing both c-di-GMP and biofilm formation (20, 33). We have found that the concentration of c-di-GMP in a ΔhapR mutant is ∼8–10 μM. However, it is important to note that we do not know whether 10 μM is the maximum physiologically relevant concentration of c-di-GMP experienced by V. cholerae because other natural environments could stimulate higher intracellular concentrations. All of the expression states examined here produced c-di-GMP at concentrations under 15 μM, with the exception of VCA0074, which produced c-di-GMP up to 25 μM. Thus, the intracellular concentration of c-di-GMP examined here are, for the most part, within the expected natural variation of the intracellular concentration of c-di-GMP in V. cholerae.
Interestingly, the rate of input to output response varied greatly between the different DGCs. We hypothesize these different rates provide V. cholerae with flexibility to appropriately modulate and adapt the regulation of behaviors by c-di-GMP in different environments. For example, the biofilm fast-response DGCs VC2454, VCA0165, and VC1599 might function in environments where a rapid increase in biofilm formation versus c-di-GMP concentration is required. Perhaps the environmental cues recognized by these DGCs are indicative of environments where biofilm formation is the most highly adapted lifestyle. In contrast, the slow-response DGC VCA0074 affects biofilm formation over a much larger range of c-di-GMP. Slow-response DGCs could gradually alter biofilm formation when fast responses are not needed or could function as fine-tuning mechanisms to cause smaller phenotypic changes. The subset of active DGCs specific for different environments would be selected either through regulation of protein expression or activation of DGCs through binding to specific cues indicative of the local environment.
The next question then becomes: what are the molecular mechanisms that lead to high-specificity signaling? One possibility is that DGCs and PDEs could be differentially localized to microdomains within the bacterial cell. The DGC PleD localizes to the stalk cell pole in the bacterium Caulobacter crescentus (3, 4). Another means of signaling specificity is association of DGCs with protein complexes. This mechanism was recently observed by the interaction of the DGC DosC and PDE DosP with the RNA modifying enzyme PNPase (6) and the interaction of DGCs with an HD-GYP protein in Xanthomonas campestris (34). Specific localization of PDEs in the cell likely plays an important role in segregating c-di-GMP–signaling pools as has been shown for cAMP signaling in eukaryotic organisms (12). Finally, it is possible that certain c-di-GMP pools are sequestered in the cell. For example, the slow response of VCA0074 for aphA induction might be attributable to the inability of the c-di-GMP that it synthesizes to interact with VpsR. In support of this hypothesis, it was shown that 80% of the cellular c-di-GMP in Gluconacetobacter xylinus is associated with the membrane (35).
The systematic approach we have undertaken is, to our knowledge, the most detailed characterization of the intracellular levels of c-di-GMP generated by multiple DGCs. This approach is widely applicable to study signaling specificity in other bacterial systems. We conclude that c-di-GMP functions via high-specificity signaling pathways in V. cholerae and our results will serve as a framework to probe molecular mechanisms by which high-specificity signaling is mediated.
DNA Manipulation and Growth Conditions.
The protocols for manipulation of DNA were performed as described previously (36). A description of construction of the DGC expression vectors can be found in SI Materials and Methods. Bacteria were grown in LB media with kanamycin (Sigma) at 100 μg/mL at 37 °C with shaking.
Quantification of Biofilm Formation and c-di-GMP.
Static biofilm formation was determined using MBEC plates as described previously (37). MBEC plates were grown for 8 h with gentle rocking before biofilms of V. cholerae were determined by staining with crystal violet. To measure biofilm formation of V. cholerae grown planktonically, 3 mL of LB containing kanamycin in 18 × 150 mm glass tubes was diluted 1/100 with an overnight culture and the inducer isopropyl IPTG was added at a concentration of 1 mM followed by a threefold dilution series seven times to yield eight IPTG-inducer concentrations. This mixture was analyzed by a Becton Dickinson LSR flow cytometer with settings of forward scatter (FSC) and side scatter (SSC) in log at voltages of 548 and 359 V, respectively, with an SSC threshold of 300 V. Biofilm formation at these eight induction states was measured independently in triplicate. Analysis of biofilm formation in Fig. S2 was done similarly, except cultures were grown in 1 mL of LB with kanamycin in 5 mL of polystyrene round-bottom tubes (BD Falcon) and induced with 0.1 mM IPTG. The intracellular concentration of c-di-GMP was determined as described in SI Materials and Methods.
Quantification of aphA Gene Expression.
RNA was harvested from the cultures described above using an RNeasy protocol (Qiagen) according to the instruction of the manufacturer. DNA was removed from extracted RNA using a DNase digestion assay supplied by Ambion according to the instructions of the manufacturer. Subsequent conversion of RNA to cDNA for Q-PCR was performed using reverse transcriptase GoScript supplied by Promega according to the instructions of the manufacturer. Q-PCR was then performed with aphA gene–specific primers and a TaqMan probe on an Applied Biosystems StepOne real-time PCR system. gyrA gene expression was used as an internal reference. Primer sequences are indicated in Table S1. Reagents and protocols for Q-PCR were conducted in accordance with those specified by Applied Biosystems.
Correlating the c-di-GMP Concentration with Phenotypic Output.
Biofilm formation and aphA transcription data were correlated to c-di-GMP levels using a number of different data transformations. When fitting a model to a set of data, we were most concerned whether or not the residuals, defined as the difference of each point from the best-fit model, were normally distributed. To test normal distribution of residuals, we used a Box–Cox transformation (38). For the logarithmic transformation reported in Fig. 3, we found that the residuals are normally distributed using a type one error rate of 0.01. The logarithmic transformation fell within the 95% confidence limit using a Box–Cox analysis; therefore, the transformation reported here was the one deemed best to normalize the residuals and yet still ensure the highest observed r2 values. We attempted a number of other data transformations not reported here. Regardless of the transformation used, the r2 values were similar and the r2 values obtained when fitting individual DGCs were always significantly higher than that of the r2 models obtained when examining all data points. Transformation and model generation were generated using the computer program Prism version 5 (GraphPad).
We thank Kerwyn Casey Huang, Ned Wingreen, Chris Adami, Arend Hintze, and Bonnie Bassler for helpful discussions and early support of this research; Ben Pursley for construction of pBRP157; and the Michigan State University Mass Spectrometry Facility for assistance in measuring c-di-GMP. This work was supported by membership within and support from the Region V Great Lakes Regional Center of Excellence (RCE) [National Institutes of Health (NIH) Award 2-U54-AI-057153], funding from the National Science Foundation (NSF) under Cooperative Agreement no. DBI-0939454, and funding from NIH Grant AI-080937 and Michigan State University (to C.M.W.). J.P.M. and E.L.R. received support from the NSF-sponsored Undergraduates in Biological and Mathematical Sciences Program Grant DMS-0531898.
↵1To whom correspondence should be addressed. E-mail: watersc3{at}msu.edu.
Author contributions: J.P.M., E.L.R., and C.M.W. designed research; J.P.M., E.L.R., B.J.K., J.-P.C., M.A., and C.M.W. performed research; J.-P.C. contributed new reagents/analytic tools; J.P.M., E.L.R., B.J.K., M.A., and C.M.W. analyzed data; and J.P.M. and C.M.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
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text:
Oakland A's could be forced into record payroll for 2022
By David Hill
Aug 31, 2021; Detroit, Michigan, USA; Oakland Athletics left fielder Mark Canha (20) celebrates his two run home run with first baseman Matt Olson (28) during the fifth inning against the Detroit Tigers at Comerica Park. Mandatory Credit: Tim Fuller-USA TODAY Sports /
The Oakland A's are not a team that is known for their high payrolls. In fact, the A's have never had a payroll over $100 million, with a payroll of $92 million as their high water mark. Even their acquisition of Starling Marte had a financial component as they had the Marlins pay the rest of his remaining salary. You know you're pinching every penny possible when the Marlins are picking up the tab for a player.
However, the A's may not have a choice as it comes to their payroll in 2022. While they have just over $14 million committed to salary next year, the A's also have an incredible arbitration class with almost every key piece of their roster due for a raise in the upcoming offseason.
Oakland A's could be forced into record payroll
As it currently stands, the A's have at least 11 players eligible for arbitration. This includes their top two starters in Chris Bassitt and Sean Manaea, first baseman Matt Olson, third baseman Matt Chapman, and outfielder Ramon Laureano. Closer Lou Trivino is also amongst that group, and even if he does not handle the ninth inning next year, is still a key part of the bullpen.
Out of that group, there are some players that could reasonably be expected to non-tendered in Burch Smith and possibly Adam Kolarek if he becomes eligible for arbitration this year. However, that would be a relatively small drop in the bucket compared to the salaries that one could reasonably expect that the rest of the A's arbitration class could receive.
MLBTR projects that class to receive just over $58 million, which already pushes the A's payroll close to their comfort level. This also leaves the A's with just 15 players on the roster, as they would also need to find a way to replace their free agent class headlined by the likes of Starling Marte, Mark Canha, and Josh Harrison.
Even if the A's scour the bargain bin in free agency once again, they will still need to spend some money. They simply do not have the players in their system to be able to step in and handle those roles, at least not just yet. Unless the A's make some difficult trades in the offseason and tear the roster apart, their payroll may move past their previous record.
Next. Moreland will not be back in 2021. dark
The Oakland A's are going to have some tough decisions regarding their players this offseason. The payroll could reach record levels.


Doug is back and retells the story of his harrowing ordeal in dealing with Hurricane Florrence head on (he left the state).
On March 10, 1993, Michael Frederick Griffin murdered Dr. David Gunn in Pensacola, Florida, in the United States. This was the first documented murder of an OB-GYN where the murderer's stated intention was to prevent a doctor from performing abortions.


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Have you ever participated in a recipe exchange? They are a lot like the chain letters we used to try to avoid in the 80's (and probably long before that!). With the advent of e-mail came the "send this to 10 friends and wait to see what you'll receive in a week" e-mails. As much as I'd like to say I always played along, I didn't. Honestly, I don't think many of you did, either. Anyway, I did have a REALLY good experience with a recipe exchange and that's where this recipe came from.
These deliciously moist, tender and juicy pork tacos are called, "Chile Lime Pork with Carrot-Lime Escabeche" and they came to me in the form of an e-mail recipe from my friend, Colleen. Sadly, just like just about every other recipe I "save" in my e-mail, this one got lost in the shuffle until last night. Both the guys I served this to were VERY happy with me, so I'd highly recommend dusting off your slow-cooker and giving this a try. Cinco de Mayo is coming up, and these should definitely be on your menu for next weekend.
Rub the pork with the seasonings, and prep the tomato-garlic-lime juice mixture the night beforehand so that all you have to do in the morning is to dump it all into the slow-cooker.
Mix the chili powder, coriander and salt together on a large plate. Roll pork in mixture, taking up all of the seasoning. Cover and refrigerate overnight.
Chop and store the tomatoes, garlic and lime juice in a container overnight.
Place the pork and tomato mixture in a slow-cooker and cook on high for 8-10 hours.
At least an hour before serving, mix the carrots, onion, 1/2 cup lime juice, oil, cilantro and salt together and allow to marinate to blend flavors.
Serve the pork with the Carrot-Lime Escabeche, sour cream, Cotija cheese, salsa and corn tortillas.
This is an all time family favorite! We love to get it going before we head out skiing for the day and when you walk in the door-yummy smells abound! I've never made this for anyone who did not love it.


The video player above will consecutively play through 8 of our Adult dog videos showcasing the environment in which our dogs live as well as the exceptional quality and excellent temperaments of our adults.
You may press the "PLAY ALL" button in the center of the video to watch through all 11 videos or you may select a particular video to watch from the drop down PLAYLIST menu in the top left corner of this video player.
The video player above will consecutively play through 16 of our puppy videos showcasing the great temperaments and exceptional quality of our puppies as well show the enriching environment in which our pups are raised.
We are so excited to meet you in person and introduce you to your new puppy! We invite each new family to visit our home twice. The first time on Litter Visit Day and again on Litter Pick-up Day. In the meantime you can take the grand tour of our home right here on this page!!!! Below you will find pictures & videos of our home, inside and out.
Please understand we get daily phone calls and Emails with requests to come and meet our dogs and tour our kennel facility but we DO NOT have a kennel facility to tour. All our dogs live in our home as part of our family. We have set up our home and yard to accommodate our dogs and puppies so that they are loved and raised in the family life environment which they deserve.


In this special 2-hour episode of the Australian Roundtable, Ethan and Johnno continue their discussion about the 'Sydney Siege' and look back on the year of 2014. Sub-segments on the 'Siege' include: Channel 7's cover story about the 'sniper'; initial 'eye witness' accounts of the event; anti-gun rights propaganda; Ken Johnson (the grinning father); Martin Place and The Matrix. General discussion topics include: Grant Denyer and news actors; surveillance state and gov/media propaganda; ZeeklyTV and Informed Planet; 2014: Year in review.
This entry was posted in Episodes on December 28, 2014 by admin.
In this special 2-hour episode of the Australian Roundtable, Ethan and Johnno discuss a range of topics related to the 'Sydney Siege': The legislative and recent historical context of events in Australia leading up to the incident; MSM coverage on the day of the 'Siege'; Other significant events which were drowned out of the MSM by the event; Tessa Kum of the 'illridewithyou' hashtag; Inconsistencies of the Official Story, and much more.
This entry was posted in Episodes on December 21, 2014 by admin.
In this episode of the Australian Roundtable, Ethan, Johnno and Lindsay discuss Queensland Police and Santos corporate sponsorship; normalising police militarisation; some of the underlying mechanisms associated with the social destruction of society; CIA torture in Guantanamo Bay and David Hicks; the 2 year anniversary of the Sandy Hook hoax and much more.
This entry was posted in Episodes on December 14, 2014 by admin.
In this special 2.5-hour episode of the Australian Roundtable Podcast, Johnno is joined in the studio by inaugural guest Raja B to discuss Larissa Waters' comments on 'gendered toys' and cultural marxism, higher education 'reforms', the China-Australia Free Trade Agreement, an update on the Hong Kong 'democracy' protests, the release of independent documentary 'We Need To Talk About Sandy Hook', Fabian/socialist subversion of Western society, Russia and the NWO, esoteric inquiry and much more.
This entry was posted in Episodes on December 7, 2014 by admin.


text:
Intex Aqua Xtreme II Unveiled In India, Priced At $151
By Kristijan Lucic June 26, 2015, 3:34am
Like many other India-based smartphone OEMs, Intex releases quite a few low and mid-range smartphones throughout the year. This company has released the Aqua 4G earlier this month, an affordable mid-range handset which offers 4G LTE connectivity. Interesting enough, this is the first Intex-branded handset to offer 4G LTE connectivity. That being said, the company has now released yet another member of their 'Aqua' line of devices, Intex Aqua Xtreme II, let's see what it has to offer.
The Intex Aqua Xtreme II sports a 5-inch 720p (1280 x 720) IPS display along with 2GB of RAM and 16GB of internal storage (expandable up to 32GB via microSD card). MediaTek's MT6592M octa-core SoC clocked at 1.4GHz powers this thing, and a 2,000mAh battery is also a part of this package. The 13-megapixel snapper is located on the back of this smartphone, while there's a 5-megapixel shooter available up front. This is a dual-SIM handset which ships with Android 4.4 KitKat, with some additions by the company on top of it, of course. This handset doesn't offer 4G LTE connectivity, but you do have access to 3G HSPA+ speeds. The Aqua Xtreme II measures 140 x 71.9 x 7.9mm, while it weighs 132 grams.
"At Intex we continuously endeavor to provide the consumers enhanced value by keeping them in sync with ever-changing technology. Aqua Extreme II is yet another example of this effort and steps into the slot vacated by its hugely popular predecessor 'Aqua Extreme'. As the name suggests, Aqua Extreme II will enthrall the consumers with its top of the line specs while giving them excellent value for money," said Sanjay Kumar Kalirona, Mobile Business Head at Intex Technologies.
This handset ships with Intex's Star Launcher on top of Google's OS, and it allows you to access applications straight from your homescreen. A number of apps come pre-loaded on this device, including Intex's Service App, ASK ME, Quikr, Newshunt, Myntra, Start, Clean Master, Teen Patti... and so on, so we can't exactly say this handset's software is close to stock. Anyhow, the Aqua Xtreme II comes in Black, White, Silver and Champagne color options and costs Rs. 9,590 ($151).
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Kristijan Lucic
Kristijan has been writing for Android Headlines since 2014 and is an editor for the site. He has worked as a writer for several outlets before joining Android Headlines, and has a background in writing about Android and technology in general. He is a smartphone enthusiast that specializes in Android applications, and that platform in general. Contact him at Kristijan. [email protected]


All India ITR (www.allindiaitr.com) has launched a mobile app for ITR filing. All India ITR is a free app that will help you to file your income tax return anywhere and anytime without any hassle. This All India ITR mobile app is available for Android as well for IOS devices. It is developed by All India ITR, authorized intermediately organization between the taxpayers and the government of India. It is a free app that can be downloaded from Google Play store (Android users) or from iOS App Store (Apple users). In this post, I will share key feature and usage of this app or ITR filing.
Easy e-filing – You can file income tax return with the click of a button. This app is suitable for both individual and businesses. This app will guide you through all stage of e-filing.
Income Tax Calculator – This app is launched with inbuilt Income Tax Calculator. This income tax calculator will help you to calculate and achieve maximum tax deduction.
HRA Exemption Calculator – Salariedpeople can calculate HRA exception from this app. The calculator given here will help you to obtain maximum HRA exemption. You need to provide HRA received from the employer, actual rent paid and metro or non-metro area as an input.
Refund Status – If you have already filed your return and you want to know refund status, you can do that by All India ITR Android App. A refund process information can be available after 10 days of filing income tax return.
Rent Receipt Generator – Rent receipt submission is mandatory if your rent is above Rs.8333 per month or HRA is above Rs.3000 per month. All India ITR mobile app allows you to generate rent receipt with a click of button. You need to fill up require information and click on Generate button to generate rent receipt. You can print and download rent receipt from this app.
How to use All India ITR Mobile App for ITR Filing?
Step -1 – Download and Install All India ITR app from Google Play Store or iTunes.
Step -2 – Open ITR app and click on Create New Account (First time user). Give your e-mail ID, mobile number and password to register.
Step -3 – It will generate OTP for verification. After verification, your account will be created. Login to All India ITR app with username and password.
Step -4 – Click on "Start e-filing" to file your income tax return. You have two option either you can upload Form 16 in pdf or submit documents. The second option of documents comes with cost.
E-filing of ITR gets easier. One can file return anytime, anywhere using mobile phone.
Form 16 upload facility gives benefit of auto fill ITR without any error.
This app saves time and enhances user experience.
Rent Receipt generator will help you to generate rent receipt easily.
You can also opt for assisted e-filing.
Internet access and web browser on mobile devices are required.
Difficult to upload form 16 and download rent receipt from mobile phone.
I have not used All India ITR app for filing income tax return. If you have used this app for filing income tax return or for calculation of tax, do share your feedback in the comment section given below.


Located in Covent Garden, the innovative hub hotel has impressed the tech world since its launch in late 2014, and is taking things one step further by embracing wearable technology, enabling guests to control their hotel room settings with a swipe and a tap from their wrists.
Using the Apple Watch, guests are able to control the lighting, heating and even the Smart TV via the hub app, developed in conjunction with London digital creative agency Monitise Create, making hub by Premier Inn one of the world's most connected hotels.
hub by Premier Inn launched in late 2014 from Whitbread, owners of the UK's most loved hotel chain – Premier Inn. The new hotel located in Covent Garden is the first of 14 hotels announced by the group who have ambitious growth plans for its new brand of contemporary, compact city centre hotels designed with comfort and convenience in mind.
hub's rooms include smart storage units, from a desk that folds into the end of the bed to luggage space under the bed. The stylish, modern design creates a calm, uncluttered environment for guests wanting a hassle free stay. As well as an array of carefully positioned connectivity and charge points, and a touch screen control panel built into the head of the bed, each room comes equipped a 40-inch smart TV, with access to the latest movies for free, games, the internet and catch-up TV. There is also super-fast, free Wi-Fi available throughout the hotel.
While hub by Premier Inn pushes many boundaries of a traditional hotel stay, it keeps the vitals central to the experience, a welcoming local team, available for guests 24/7 and a super comfortable Hypnos double mattress to ensure everyone enjoys a great night's sleep.
The travel sector is one that is likely to really embrace m-payments and combining this with new devices presents a massive opportunity. Find out more as to what mobile can do for your business at M-Payments Summit 2015.


text:
Q: Using forEach with Callbacks function with asyncawait im try to use asyncawait on a cronjob with callback, this callback are data from an API:
This is part of the code:
var sending_email = new CronJob({
    cronTime: time_hour,
    onTick: function(){
        console.log('Funciona cada: ', time_hour);

        sendService.GetSenderList(function(datos){
            console.log('a enviar: ', datos);
            var arrayInfo = [];

            await (
                datos.forEach(function(hash){
                //console.log('a enviar: ', hash.HASH_TOKEN);

                    var wait = sendService.GetMailContent(hash.HASH_TOKEN, function(content){
                        arrayInfo.push(content);
                        console.log(hash.HASH_TOKEN,content);
                    });
                })
            );

            console.log('array: ',arrayInfo);
        })
    },
    start: false,
    timeZone: 'Chile/Continental'
})

And my function to get api are:
exports.GetSenderList = async (function(callback){
    await (client.get(constant.API_URL_SENDER, function (data, response) {
        callback(data);
    }));
});

exports.GetMailContent = async.iterable (function(hash,callback){
    await (client.get(constant.API_URL_CONF+'/'+hash, function(data, response) {
        callback(data)
    }))
})

Could you helpme, i can't fixed, i try with return instead of callback, but still doen't work...
Thanks!
UPDATE
i change my code, thanks @jfriend00...
using "node-rest-client-promise"
var constant = require('./constant.json')
var mailConfig = require('./mailConfig.json')
// var forEach = require('async-foreach').forEach;
//var async = require('asyncawait/async');
//var await = require('asyncawait/await');

var nodemailer = require('nodemailer');

//var Client = require('node-rest-client').Client;
//var client = new Client();

var nodeRestClient = require('node-rest-client-promise');
var client = nodeRestClient.Client({
    requestConfig: {
        timeout: 30000, //request timeout in milliseconds
        noDelay: true, //Enable/disable the Nagle algorithm
        keepAlive: true, //Enable/disable keep-alive functionalityidle socket.
        keepAliveDelay: 100000 //and optionally set the initial delay before the first keepalive probe is sent
    },
    responseConfig: {
        timeout: 50000 //response timeout
    }
});

exports.GetSenderList = function() {
    let lista = client.getPromise(constant.API_URL_SENDER);
    //console.log('Get lista: ',lista)
    return lista
};

exports.GetMailContent = function(hash) {
    var normalHash = hash.replace(new RegExp("/", 'g'),"%2F");
    //console.log(normalHash);
    let lista = client.getPromise(constant.API_URL_CONF + '/' + normalHash);
    return lista
};

and on cron:
var sending_email = new CronJob({
    cronTime: time_hour,
    onTick: async function(){
        console.log('Funciona cada: ', time_hour);

        let arrayInfo = [];
        let arrayHash = [];
        let datos;

        try{
            datos = await sendService.GetSenderList();

            //console.log('datos: ', datos.data);

            for(let hash of datos.data){
              //console.log('hash', hash);
              let content = await sendService.GetMailContent(hash.HASH_TOKEN)
              arrayHash.push(hash.HASH_TOKEN);
              arrayInfo.push(content.data);
            }

            console.log('array: ', arrayInfo);
        }
        catch(e){
            console.log("onTick error ", e);
        }
    },
    start: false,
    timeZone: 'Chile/Continental'
})

A: await arr.forEach() doesn't work as you'd expect. In your case, you want to use map() to make it so you're awaiting on an array of promises.

await (Promise.all(datos.map(hash => sendService.GetMailContent(hash.HASH_TOKEN))));

I wrote a detailed blog post about making forEach() work with async/await, check it out if you want to learn more: http://thecodebarbarian.com/basic-functional-programming-with-async-await#motivation-and-foreach-

redpajama_set_name: RedPajamaStackExchange


text:
Boat crash off coast of France kills 31
Boat crash off coast of France kills 31 At least 31 people drowned in frigid waters off the coast of France on Wednesday, after a boat carrying migrants trying to reach Britain capsized in the English Channel
https://static.report.az/photo/944478ab-2e85-3308-8124-21c8ec75593c.jpg
At least 31 people drowned in frigid waters off the coast of France on Wednesday, after a boat carrying migrants trying to reach Britain capsized in the English Channel, one of the worst death tolls in recent years for migrants attempting the dangerous crossing, Report informs, citing New York Times.
Gérald Darmanin, France's interior minister, said that the dead, including five women and a little girl, were part of a group whose "extremely fragile" inflatable boat was found completely deflated by rescuers.
Two people were rescued but were hospitalized with severe hypothermia.
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Kanan Novruzov
https://static.report.az/photo/thunk_50.jpg
Azerbaijani version Fransada miqrantları daşıyan qayıq qəzaya uğrayıb, 31 nəfər ölüb


text:
World-famous Barber Motorsports Park gears up for Indy races
More stories in Enterprise
Barber Motorsports Park is preparing to welcome about 80,000 visitors this weekend for the Honda Indy Grand Prix of Alabama, the annual three-day event that showcases the Birmingham racing venue.
Many will take in the sights at the Barber Vintage Motorsports Museum, which houses the world's largest collection of motorcycles and is undergoing a major expansion.
Days ago, Barber officials and Birmingham-based contractor BL Harbert International held a topping out celebration for the project, which is expected to be complete this fall and is slightly ahead of schedule, said Jeff Ray, the museum's executive director.
The expansion will allow the Barber museum to display more vintage motorcycles. (Image: BL Harbert)
The expansion is adding 85,000 square feet to the current facility, bringing the total to 250,000 square feet.
That means the museum, which already houses a soaring glass and steel display of motorcycles, will have even more space to show off the collection of dairy magnate and park founder George Barber.
"It will be an even bigger wow," Ray said, adding that the extra room should allow for more multimedia exhibits and events at the facility. "Anything we can do to bring more people to Birmingham, that's what we want to do."
'WORLD-CLASS FACILITY'
Barber's collection, certified in 2014 by Guinness World Records, now tops 1,500 vintage and contemporary motorcycles. But the museum doesn't have enough room now to display them at the same time, so the exhibits are rotated in and out.
BL Harbert broke ground on the expansion in September 2015. Once it is complete this October, more than 5,800 cubic-yards of concrete and 435 tons of rebar will be used in the project. Williams Blackstock Architects is the designer.
"Mr. George Barber has created a world-class facility, spurring economic development, tourism and even a world record, right here in our backyard," said Parker Evans, senior project manager for BL Harbert. "BL Harbert is honored to be a part of the next chapter of growth for the Barber Vintage Motorsports Museum."
RECRUITING TOOL
Economic developers have been known to use the racing venue as a recruiting tool, entertaining prospects at private dinners among the museum's exhibits or during the festivals and races held on park grounds.
The park's centerpiece is a 17-turn, 2.38-mile road racing track. Set among lush landscaping and the rolling hills of Central Alabama, the scene has earned Barber the nickname, "the Augusta National of motorsports."
The park also is home to the Porsche Sport Driving School and has served as the backdrop for a number of automotive product launches. Another section, the Barber Proving Grounds, provides additional track space for events while the main track is in use.
New additions to the park over the past year include new garages for clients, a new classroom at the Proving Grounds and a second pedestrian bridge.
The Honda Indy Grand Prix of Alabama kicks off Friday and will feature 11 races throughout the three-day weekend.
Alabama mom's special needs shopping cart makes splash


Regardless of these matters, L. aurea is a lovely flowering bulb. The first time I saw one in north Florida I thought my eyes were deceiving me. It appeared that I was looking at a deciduous azalea, perhaps Rhododendon calendulaceum, on steroids. There were a couple problems with this – first of all, it was late summer, and the flowers were seemingly springing magically from the ground. Any person seeing Lycoris in flower for the first time has a similar experience, hence the common name surprise lily.
The flower trusses of Lycoris aurea remind me of a deciduous azalea on steroids!
Here in southern Japan plants usually flower in October, after L. radiata has finished or is finishing. I have two clones, an early flowering one and another that flowers a whole two weeks later. Of the two, the later flowering clone produces seed while the other doesn't. I have not tried to germinate these since it is said that Lycoris are ridiculously slow to flower from seed, with a minimum of 5 years needed. This season though, I'm giving them a try.
It is said that Lycoris aurea likes limey soils and a dry, hot summer. While this may be true, the plants I grow stay wet until early August at least. Around the second week of August and into late September the rains back off and the oven turns on in Kyushu with daily highs peaking in the mid 30's C (~94 F). This period of dry heat is supposed to enhance flowering in this species.
I do not add any lime to their bed either, and the local volcanic loam is definitely acidic in reaction. The plants do like a sunny location, perhaps even more so than L. radiata. Mine are in a spot that gets relatively little sun in the winter months and I've noticed they don't flower as well as plants I've seen in full sun here.
Lycoris aurea is the largest of all the Lycoris. In some vigorous specimens the leaves can reach nearly a meter in length. The leaves at the lower right are those of L. radiata.
New plants can be made through offsets, though recently planted bulbs often go through at least one year of sulking, particularly ones that have been dried and are rootless. This adds to the surprise element, because it is easy to forget you even planted a bulb two years earlier only to have it flower by seeming magic later on. They produce offsets fairly generously. The first bulb I planted has increased to a clump of at least 12 plants, and in a relatively short span of time – around 5 years.
In nature these are plants of subtropical grasslands and forest edges. In Japan they are grown well into warm temperate areas where the soil doesn't freeze in winter. Still, their soft, succulent leaves are not likely to take truly cold winters. It is likely that USDA cold hardiness zone 7 is their limit without protection.
In Japan L. traubii has been used as an emergency food in times of famine, in particular after WWII. Since Lycoris bulbs contain the toxic alkaloid lycorine, great care was needed to remove it before eating the starchy bulbs. Lycorine has also been used in Asian folk medicine as a diuretic and expectorant, but dosing is a serious problem. Too much of it leads to vomiting, diarrhea, convulsions, and possible death by paralyzing the central nervous system.
A far better use for the golden spider lily is as a garden subject. Few plants can give the startling display a well grown Lycoris can, and L. aurea is the king of this genus.
Hi, just wanted to say I found your webpage on Lycoris aurea extremely interesting. I have been trying to figure out for some years the conditions needed to get the best out of these bulbs and this year seemed to be exactly what they wanted. I am at 1340m above sea level in northern NSW, Australia, on basalt soils that are neutral to acidic. Normally we get two or three snowfalls each winter and about two days over 30degC in summer. Annual rainfall is about 900mm with much of this in summer. This year was a warm winter with only one snowfall and a record hot and dry summer. My Lycoris have been at the foot of a north west facing stone wall for three years and this year seems to have been exactly what they wanted. The previous year's flowering with a wetter summer was not nearly so good. Also, if not planted against a north facing stone wall, the leaves are burnt off by the frosts and they do not flower. In a normal winter we can get overnight minima down to -12degC, but mostly 30-40 nights down to -3degC.
Next summer, I am going to trial covering the bulbs from mid-December to mid-February with a strip of black plastic to try to re-create this year's dry hot summer.
Thanks for a very helpful webpage.
I'm glad I could be of some help. I didn't expect such low temperatures, even at 1340 meters, at your location. It is amazing how much altitude can change things. Here on Kyushu anything above 1500 meters has some pretty serious winters, while down in the lowlands where I live it is almost, but not quite, subtropical.
Lycoris aurea likes hot weather, as does L. radiata. Perhaps you would be better trying L. sanguinea since it is a true mountain plant even here, liking slightly cooler summers, while handling colder winters.
Hi botanty boy thank you so much for sharing details of Lycoris Aurea. I've got bulbs for this plant from a local nursery. I am in India and was wondering how much time will it take for the bulbs to bloom of I plant them mid April?
Im form argentina and dont wuite get when is that they flower (summer/spring/autum or winter, when to plant the bulbs (sping/summer/autum/winter)??
Since our seasons are reversed here in the north, I recommend simply planting the bulbs when you get them. They do not like being rootless and will take a season or more to settle in correctly. When they will flower for you is a mystery to me, but I'm guessing sometime in May or June?


text:
var circleModule = require('../../src/Circle');
var getCircumference = circleModule.getCircumference;

describe('Circle', function() {
  it('should have a circumference of 2PI when the radius is 1', function() {
    var radius = 1;
    var expectedCircumference = 2 * Math.PI;

    var circumference = getCircumference(radius);

    expect(circumference).to.be.equal(expectedCircumference);
  });

  it('should skip this test', function() {
    getCircumference(2);
    this.skip();
  });
});

redpajama_set_name: RedPajamaGithub


PT and PTA licenses expire on January 31st of even numbered years.
A maximum of 10 hours allowed from online courses. 4 hours of jurisprudence and professional ethics are required.
For more information visit the New Jersey State Board of Physical Therapy Examiners.


Welcome to Dave and Missy Photography! We specialize in real estate photography. We deliver excellent head shots that concentrate on branding your image and being able to stand out from the rest whether on social media or your website or business cards. We also specialize in interior and exterior photography that will showcase homes at their utmost potential. We work with real estate agents, and home owners. We are available for on-location sessions in your office or property for total convenience and flexibility. Clients have included realtors, home owners, interior designers and stagers. We welcome any questions! Click the contact link above to send us an email and we'll get right back to you!


text:
News, Press Releases
GGGolf partners with Tagmarshal to deliver better player experiences to golf courses
July 23, 2020 /by vusi
https://www.tagmarshal.com/wp-content/uploads/2020/07/GGGolf_Tagmarshal_golf_cart_gps.png 1000 1000 vusi https://www.tagmarshal.com/wp-content/uploads/2017/12/Tagmarshal_golf-course-intelligende_logo.png vusi2020-07-23 08:22:172020-07-23 08:22:17GGGolf partners with Tagmarshal to deliver better player experiences to golf courses
Tagmarshal launches golf industry's first social distancing monitor
May 14, 2020 /by vusi
https://www.tagmarshal.com/wp-content/uploads/2020/05/soc-dist.jpg 1000 1000 vusi https://www.tagmarshal.com/wp-content/uploads/2017/12/Tagmarshal_golf-course-intelligende_logo.png vusi2020-05-14 14:22:272020-05-22 14:00:28Tagmarshal launches golf industry's first social distancing monitor
#SafeGolfArticles, News, Press Releases
Golf Industry Collaborates to Develop Responsible 'Back2Golf' Guidelines and Operations Playbook
https://www.tagmarshal.com/wp-content/uploads/2020/05/Golf-Industry-Collaborates-to-Develop-Responsible-'Back2Golf'-Guidelines-and-Operations-Playbook.jpg 1000 1000 vusi https://www.tagmarshal.com/wp-content/uploads/2017/12/Tagmarshal_golf-course-intelligende_logo.png vusi2020-05-13 18:00:272020-09-08 14:41:54Golf Industry Collaborates to Develop Responsible 'Back2Golf' Guidelines and Operations Playbook


text:
Olivia Newton-John, AO, OBE is an English-born, Australian-raised singer and actress and philanthropist. With a career spanning over five decades with more than 100 million albums sold, Olivia's successes include four Grammys, numerous Country Music, American Music, Billboard and People's Choice Awards, an Emmy Award, ten #1 hits and more than fifteen top 10 singles. She also scored another #1 hit, this time on the Billboard Dance Club Songs chart, with 'You Have to Believe', a duet with her daughter Chloe Lattanzi – becoming the first mother/daughter duo to top this Billboard chart. In 1978, her co-starring role with John Travolta in Grease catapulted Olivia into super-stardom and to date it remains the most successful movie musical of all time. In 2018 the film celebrates its 40th anniversary. Olivia has been a long-time activist for environmental and animal rights issues. Her personal journeys with cancer led her to announce her partnership with Austin Health and the creation of the Olivia Newton-John Cancer Wellness & Research Centre (ONJCWC) on the Austin Campus in her hometown of Melbourne. She also co-owns the award-winning Gaia Retreat & Spa near Byron Bay
Books by Olivia Newton-John
Olivia Newton-John Articles
Hope, Sunshine, Recovery: Review of Don't Stop Believin' by Olivia Newton-John