Economically viable biobased production of bulk chemicals and biofuels typically requires high product titers. During microbial bioconversion this often leads to product toxicity, and tolerance is therefore a critical element in the engineering of production strains.
Here, a systems biology approach was employed to understand the chemical stress response of Escherichia coli, including a genome-wide screen for mutants with increased fitness during chemical stress. Twelve chemicals with significant production potential were selected, consisting of organic solvent-like chemicals (butanol, hydroxy-γ-butyrolactone, 1,4-butanediol, furfural), organic acids (acetate, itaconic acid, levulinic acid, succinic acid), amino acids (serine, threonine) and membrane-intercalating chemicals (decanoic acid, geraniol). The transcriptional response towards these chemicals revealed large overlaps of transcription changes within and between chemical groups, with functions such as energy metabolism, stress response, membrane modification, transporters and iron metabolism being affected. Regulon enrichment analysis identified key regulators likely mediating the transcriptional response, including CRP, RpoS, OmpR, ArcA, Fur and GadX. These regulators, the genes within their regulons and the above mentioned cellular functions therefore constitute potential targets for increasing E. coli chemical tolerance. Fitness determination of genome-wide transposon mutants (Tn-seq) subjected to the same chemical stress identified 294 enriched and 336 depleted mutants and experimental validation revealed up to 60 % increase in mutant growth rates. Mutants enriched in several conditions contained, among others, insertions in genes of the Mar-Sox-Rob regulon as well as transcription and translation related gene functions.
The combination of the transcriptional response and mutant screening provides general targets that can increase tolerance towards not only single, but multiple chemicals.
The finite supply of fossil fuels and their environmental impact has stimulated the development of alternative biobased solutions for production of chemicals derived from the petrochemical industry [1–3]. This industry relies on the conversion of fossil fuels into a limited number of chemicals that serve as building blocks for the production of a wealth of other chemicals. Likewise, a shift to a widespread biobased chemical production will result in the generation of building blocks employed as precursors for the production of other chemicals . Several potential building blocks have been proposed and include organic acids, amino acids, sugar alcohols, aldehydes and organic solvents, while commodity chemicals of interest include, among others, biofuels and polymer precursors [4–6]. The microbial production of such building blocks and commodity chemicals necessitates high titers to be economically sustainable. However, high titer poses a challenge for the microorganisms, as product toxicity is a likely consequence. Accordingly the development of efficient production strains has to be accompanied by engineering strains with high product tolerance [7, 8]. In addition to product toxicity, inhibitors present in biomass feedstock constitute another hindrance for microbial biomass conversion [9, 10].
Several strategies for engineering improved chemical tolerance exist that address the problem from different angles. One is improved chemical tolerance by rational engineering employing e.g. systems biology to understand the stress condition. Other strategies utilize mutant library screening, e.g. transposon or plasmid based, or natural selection by evolution to identify targets that improve tolerance [7, 11]. In E. coli these strategies have been employed towards a variety of chemicals, including but not limited to the investigation of the transcriptional response towards butanol , isobutanol , acetate , octanoic acid , produced free fatty acids (FFAs)  and furfural . Rational engineering attempts include improving tolerance towards butanol [18, 19], produced FFAs , and furfural [21, 22], while screening strategies have been employed towards butanol , acetate , and furfural stresses . Evolution based selection strategies have been implemented towards butanol [26–30], isobutanol [31–33], acetate [34, 35], octanoic acid , serine  and succinic acid stresses . Commonly, tolerance identification strategies are performed with exogenous addition of chemical with the rationale that most chemicals will have a cellular impact by diffusion, transport or membrane interaction. Moreover high extracellular titers are needed for an economically viable production.
In this work a combination of systems biology and screening approaches were employed to identify potential targets for improving chemical tolerance in E. coli. A thorough investigation of the chemical stress responses to 12 chemicals, including future building blocks and commodity chemicals, was undertaken to find shared features of the different transcriptional responses these chemicals impose on E. coli. To this end shared enrichments of functional and regulatory gene sets were identified. These genes are part of the chemical stress response and therefore candidates for tolerance engineering. In addition, a transposon mutant library sequencing analysis (Tn-seq) was carried out with the aim of identifying target genes conferring a tolerance improvement towards the selected chemicals. The inclusion of 12 chemicals in this study enabled the identification of targets that improve tolerance towards not just a single chemical but towards groups of chemicals with shared properties.
The E. coli transcriptome has been investigated under chemical stress conditions with 12 different compounds (Fig. 1a) in order to understand the cellular response and potentially infer the cellular effects of the imposed stress. Commonalities between transcriptional responses could provide understanding of general chemical stress as well as targets for tolerance engineering towards not just single compounds but groups of compounds with similar properties. A main criterion for the selection of chemicals was their potential for biobased production. Hence, most of the chosen chemicals were selected from the predicted top 30 chemical building blocks for future biobased production defined in a report by the US Department of Energy . The chemicals on this list were evaluated based on the criteria of having a sufficient level of inhibition and solubility, while avoiding more than two compounds with similar structures. Ultimately seven chemicals from this list were selected including hydroxy-γ-butyrolactone (Byr), furfural (Furf), itaconic acid (Ita), levulinic acid (Lev), serine (Ser), succinic acid (Suc), and threonine (Thre). Five other chemicals were selected based on other criteria including acetate (Ace), a common inhibitor during fermentation; butanol (But), a high-potential biofuel; 1,4-butanediol (Diol), a polyester precursor; geraniol (Ger), a biofuel and fragrance ingredient and decanoic acid (Deca). The latter belongs to the group of medium-chain fatty acids that can act as biofuel precursors [39, 40]. The selected chemicals have been grouped into four categories to reflect similarities in their chemical properties and expected cellular effects (Fig. 1a). Four chemicals (butanol, hydroxy-γ-butyrolactone, 1,4-butanediol, furfural) are to varying extents utilized as solvents and can therefore be described as organic solvent-like chemicals. Another four chemicals (acetate, itaconic acid, levulinic acid, succinic acid) are referred to as acids, two (l-serine, l-threonine) are amino acids, and two (decanoic acid, geraniol) are labeled membrane-intercalating based on their expected interaction with the bacterial membrane.
The study was designed to elucidate the inhibitory effects of the chemicals and investigate the chemical-specific transcriptional response without triggering a general severe stress response. To this end, concentrations were chosen that causes a 33 % reduction of the exponential growth rate, corresponding to a doubling time increase from 60 min to around 90 min. The resultant inhibitory concentrations of the chemicals span a wide range from just above 1 mM for geraniol to around 450 mM for 1,4-butanediol (Fig. 1b). The two membrane-intercalating chemicals have the lowest inhibitory concentrations. For the chemicals with higher inhibitory concentrations an osmotic stress is expected in addition to chemical stress. The cells were grown in minimal medium in triplicates for each condition and chemicals added during the exponential growth phase. Following 1 h of chemical stress, the cells were harvested for transcriptome profiling by RNA-seq.
The chemical stress conditions elicit varied and extensive transcriptional responses. An overview of the global transcriptional profile for each chemical (Fig. 2a) reveals that clustering of the conditions correlates well with the chemical properties of the compounds. The organic solvents constitute a subcluster as do the amino acids and membrane-intercalating chemicals, while the acids are clustered into two adjacent groups. Although the clustering suggests similarities within the chemical groups, examination of branch lengths indicates that differences also exist. For example, relatively long branch distances exist among the organic solvents. The acids cluster as dicarboxylic acids (itaconic acid and succinic acid) and monocarboxylic acids (levulinic acid and acetate), but this could also reflect the relatively high sodium ion concentration required for neutralization of the dicarboxylic acids.
The transcriptional response towards many of the chemicals is extensive (Fig. 2b) (Additional file 1). It is clear that both organic solvents and acids have an immense impact on the cell and the transcriptome even at a relatively low 33 % growth rate reduction and consequently have a broad effect on many cellular processes. In contrast, the amino acid and membrane-intercalating groups appear to have a more specific effect on the cell. For the latter group the cellular effects of the chemicals could be more immediate upon addition, possibly via rapid membrane interaction and destabilization, and therefore less pronounced after 1 h leading to a lower transcriptional response. For geraniol, evaporation and adherence of the chemical to the culture flask could also be issues.
On the basis of the clustering there is good agreement between the grouping of chemicals based on their chemical properties and their actual transcriptional profiles. Further inspection also reveals a large overlap of genes displaying differential expression in the same direction within the defined groups (Fig. 2c). There are 661 common differentially expressed genes between all the organic solvent conditions (either upregulated or downregulated in all), 402 between the acid conditions, 251 between the amino acid conditions and 121 for the membrane-intercalating conditions. Based on the average number of significant genes for each organic solvent condition one would expect 148 genes to be differentially expressed in all four conditions by chance (without taking expression direction into account) instead of 661, thus demonstrating a clear overrepresentation of overlapping genes. A substantial overlap is not only present within chemical groups but also between them. There are 206 common differentially expressed genes between the acids and organic solvents and 89 between organic solvents, acids and amino acids. This indicates that similar responses exist not only between chemicals with similar properties but also to some degree between chemicals with different properties.
Numerous gene expression changes are shared between multiple chemical stress conditions. Functional categories of many of the shared differentially expressed genes are presented in Tables 1 and 2. Many upregulated genes relate to energy metabolism, including genes involved in anaerobic respiration, fermentative pathways, glycolysis, the pentose phosphate pathway and the electron transport chain. The downregulation of motility and chemotaxis genes is a shared feature for all conditions and constitutes the majority of the 89 gene expression changes that are shared between the three chemical groups included in Fig. 2c.
The organic solvent and acid conditions display the most widespread changes in the functional categories presented in Tables 1 and 2. The genes associated with anaerobic energy generation are upregulated for organic solvents and acids, whereas the genes associated with general energy generation are only upregulated in the presence of organic solvents. The concomitant increases in glucose metabolism, electron transport chain and biosynthesis genes for the ubiquinol electron carrier are consistent. However, the increase in anaerobic energy metabolism is extensive and more surprising. Most anaerobic electron donor and acceptor genes are changed as are genes of fermentation enzymes and the electron carrier menaquinol. Taken together the data indicate that challenge with the organic solvent and acid chemicals, and especially the former, leads to a stress that generates a need for increased energy production. Another shared feature for organic solvents and acid conditions is the upregulation of genes involved in acid resistance including the acid fitness island, glutamate decarboxylase, amino acid antiporters and membrane modification genes. For the acid conditions this could be expected, but less so for the organic solvent conditions.
Genes of iron-sulfur cluster assembly systems (isc and suf) are upregulated for both acids and organic solvent conditions, indicating either oxidative stress or iron limitation. The upregulation of iron transport genes for organic solvent conditions however indicates the opposite; an excess of iron. Genes encoding other transporters such as potassium, copper and sugar transporters are also affected. Upregulation of genes involved in potassium uptake is present in both organic solvent and acid conditions and normally indicates an increase in osmotic stress. Sugar transporter genes are downregulated in both acid and organic solvent conditions including the primary glucose transporter gene (ptsG), a downregulation which opposes the upregulation of glycolytic genes. Other membrane modifications include genes involved in the synthesis of M antigen which is upregulated for most organic solvents and acid conditions, whereas genes involved in enterobacterial common antigen (ECA) and O antigen synthesis are downregulated for organic solvents. The M antigen, or colanic acid, is associated with biofilm formation and can be induced by osmotic and detergent stress, possibly explaining why it is upregulated here. The ECA is a glycolipid and has been related to resistance towards bile acids and short-chain fatty acids .
Many sigma factors have increased expression in multiple conditions. This is especially the case for the organic solvent conditions, where five of the main sigma factors (rpoS, rpoE, rpoH, rpoD, rpoN) exhibit significant increases in expression, providing an explanation for the vast transcriptional changes observed in these conditions. Some of the above mentioned expression changes during organic solvent stress are likely the effect of an increase in rpoS and rpoE expression (Table 1). However, upon examination of the transcriptional changes, footprints of rpoH and rpoN upregulation are less obvious. For instance, upregulation of some of the well-known chaperone genes regulated by RpoH (dnaJK, groEL) is not observed. For the acid conditions it is mainly rpoS and rpoE that are increased, matching well with the displayed expression changes. The observed expression changes for quorum sensing autoinducer related genes in the acid and organic solvent conditions could be a consequence of increased rpoS expression, although other explanations cannot be ruled out.
Metabolism and biosynthesis genes in particular are also affected in many conditions. Apart from those already mentioned, many biosynthesis genes are downregulated for organic solvents, including many encoding amino acid synthesis enzymes (glutamate, glutamine, tryptophan, methionine, arginine), nucleotide biosynthesis enzymes and polyamine biosynthesis and transport proteins. Accordingly, it seems that glucose is shunted towards energy generation rather than anabolism. However some biosynthesis genes are also upregulated, including ethanolamine for organic solvents, thiamine for most conditions and branched chain amino acid synthesis genes for various conditions. Phosphatidylethanolamine is one of the most abundant phospholipids in the membrane and the upregulation of ethanolamine metabolism genes could be a consequence of recycling of the phospholipid from within the cell or from other cells.
The response to a stimulus is mediated by the cellular regulatory network. In an attempt to elucidate the origins of the widespread transcriptional changes observed in response to chemical stress, the potential changes in regulator activity have been examined. These changes in activity may also provide clues to the direct cellular effects imposed by a chemical. Changes in regulator activities were determined by statistical analysis of gene enrichment of the regulon of each regulator using the R package piano . This approach is similar to standard gene enrichment analyses where functional categories are employed instead.
Many regulons are enriched for each condition as would be expected based on the vast transcriptional changes, and considerable overlaps within and between chemical groups are also present (Fig. 3). The regulons of flagellum transcription factors FlhDC and FliA are enriched for all chemicals and the regulon expression changes demonstrate a reduced activity of these two regulators leading to the downregulation of flagella and chemotaxis genes. The other universally enriched regulon for all chemical groups is that of catabolite repression protein (CRP), a major regulator of metabolism and many other processes in the cell. Out of the 509 genes in the regulon between 200 and 300 of these are differentially expressed in e.g. the organic solvent conditions. In Tables 1 and 2 the likely genes regulated by CRP are the sugar transport genes. The enrichment of the GatR and MalT regulons (Fig. 3) and therefore putative change in activity of these regulators are likely an effect of the changes in CRP activity.
The OmpR regulon is altered in all chemical groups except the membrane-intercalating group. OmpR activity is dependent on changes in osmolarity and it is likely that either the osmotic or the membrane interacting effect of the chemicals could elicit increased OmpR activation. OmpR positively regulates the flhC and flhD genes and it is therefore possible that the downregulation of flagellar genes are caused by OmpR activation. The fact that flagellar genes are downregulated in all conditions and the OmpR regulon is not enriched in all, does not exclude this, as ompR is upregulated transcriptionally in the two conditions (membrane-intercalating) where the regulon is not enriched. The absence of OmpR regulon enrichment in this group is due to genes in the csgDEFG operon being part of the regulon and not exhibiting differential expression here. CsgD activation is in turn a probable effect of OmpR activation.
ArcA and FNR, whose regulons are enriched for three groups (organic solvents, membrane intercalating and amino acids) and two groups (organic solvents and amino acids) of chemicals respectively, respond to anaerobic conditions and regulate genes involved in respiration and fermentation. Here, the majority of anaerobic respiration and fermentation genes presented in Tables 1 and 2 seem to be upregulated by these two regulators. Yet, the footprint of ArcA and FNR regulation is not pronounced for all genes within the regulons as quite a few display no differential expression or even expression in the opposite direction, possibly due to the effects of other regulators. One of the most enriched conditions is butanol while other conditions have a less pronounced enrichment in the correct directions.
This characteristic is also present in the regulons of Cra and Lrp as these genes are enriched in acid and organic solvent conditions. However for many of the genes, the direction of expression changes are not consistent with the effect of the regulator. Hence, these two regulators may not have changed activity. The differential expression of many of the genes in the regulons could rather be an effect of other regulators. The enrichment of the Fur regulon in the organic solvent conditions however corresponds well with the observed downregulation of iron transporters.
Understanding the cellular chemical stress response can indirectly provide information and clues towards improving tolerance. However, experimental approaches such as Tn-seq provide a more direct generation of targets for tolerance improvement. Tn-seq is a selection-based approach in which the distribution of a population of genome-wide transposon mutants is measured by sequencing and counting insertions in each gene before and after selective experimental conditions. It thereby provides a fitness estimate of each mutant in the experimental condition based on its pre- and post-selection abundance within the population. Here, a population of approximately 60,000 mutants were subjected to chemical stress at higher concentrations leading to doubling times of approximately 3 h, compared to a control with a doubling time of 1 h.
In all conditions there were mutants with increased and decreased fitness (Fig. 4a) and there is a slight trend towards more insertions conferring a fitness advantage rather than a fitness impairment. The organic solvents, apart from butyrolactone, again had a large number of genes differentially enriched or depleted in transposon insertions and the amino acid conditions likewise had a low number. By contrast, the membrane-intercalating conditions, especially decanoic acid, had relatively high numbers of genes with significant abundance changes in transposon insertions. The quantity of significant genes could be an indicator of the number of potential targets for tolerance improvement. Hence many targets could exist towards organic solvent and decanoic acid stresses while the potential low number of enriched genes for amino acids could reflect the specificity of this stress. Additional file 2 contains a list of all genes enriched and depleted in transposon insertions in all conditions.
The actual fold changes of transposon mutants are generally uniformly distributed within the range of ±tenfold (Fig. 4b). A fold change of ten would here on average correspond to a growth rate increase of 39 %. Specific enriched insertion mutants are presented in Table 3. These consist of the top-two genes for each condition as well as other significant genes from Tn-seq that were tested experimentally for improved tolerance towards the relevant condition. In order to investigate the validity of Tn-seq predictions, a total of 35 gene insertion mutants from the Keio collection of single gene knockout mutants  were tested. Eight insertion mutants (80 %) of the tested top-two genes with highest fold changes displayed improved tolerance while in total nineteen insertion mutants (54 %) displayed improved tolerance. Although there is not a direct correlation between the quantitative values of fold change and growth rate change, most beneficial insertions predicted by Tn-seq are true positives. Consequently the majority of Tn-seq predictions are expected to be correct and therefore provide a valuable list of targets for tolerance improvement.
The cellular function of the significant genes is varied not just for those in Table 3 but also among all significant genes. Some of the beneficial insertions could be expected, such as rob for butanol, marB for butyrolactone and acrR for geraniol. These are involved in the regulation of the mar-sox-rob regulon which is known to be involved in e.g. organic solvent tolerance . Many other insertions are not intuitively beneficial and many have unknown functions. Among the detrimental insertions are multiple genes encoding cytochrome bo (cyo), NADH oxidoreductase (nuo) and LPS synthesis (waa) enzymes in geraniol (cyo) and butanol and decanoic acid conditions (cyo, nuo and waa genes).
Optimally, gene deletions improving fitness towards not just one chemical stress, but multiple chemical stresses could be revealed using Tn-seq, and some transposon mutants also appear in several conditions within the top-two mutants with highest fold changes, i.e. marB (Byr, Ser, Suc), ydaE (Diol, Furf), acrR (Ger, Ace) and azuC (Ace, Lev). Even though marB and acrR repress genes related to e.g. organic solvent tolerance their inactivation is also beneficial in acid and amino acid conditions. Additionally the deletion of azuC, encoding an acid inducible protein, could be relevant for improving acid tolerance because its inactivation improves fitness in two acid conditions. Other genes were also found to be enriched in insertions across multiple conditions as well (Table 4). However, many of the genes presented in Table 4 generally have lower fold changes, as is displayed by the fact that only one, ydaE, is present among the genes with top-two fold changes. Consequently, these genes and their related processes could be examples of smaller yet general chemical tolerance mechanisms.
Tn-seq and RNA-seq are two different approaches for investigating the chemical stress in E. coli and it is therefore relevant to compare the results of these two methods. Comparison reveals that there is no general overrepresentation of Tn-seq significant genes among the RNA-seq significant genes. The average percentage of total RNA-seq significant genes is 26.7 % while among Tn-seq significant genes 27.3 % are also significant in RNA-seq, corresponding to a P value of 0.63 for a paired t test comparison. Furthermore, the fold change direction between the two methods is randomly distributed with 13.4 % of Tn-seq significant genes having the same direction in RNA-seq and 14 % with an opposite direction in RNA-seq (paired t test P value 0.85). The distributions are visualized by box-plots in Additional file 3. Consequently, there is no general trend of overlap in identified genes between these two methods.
In the present study two strategies were pursued to elucidate chemical tolerance mechanisms in E. coli. One entailed finding shared features of the transcriptional chemical stress response that can be applied as a guide for rational tolerance engineering towards multiple chemicals while the other involved transposon mutant library screening for genetic tolerance determinants. Understanding the transcriptional response of E. coli can provide clues to enable rational tolerance engineering.
Widely altered gene expression was observed when cells were exposed to the 12 selected chemicals. This was especially true for organic solvent (butanol, butyrolactone, 1,4-butanediol, furfural) and acid (acetate, itaconic, levulinic and succinic acid) chemical stresses and these conditions share expression changes in many general functional categories such as energy metabolism, iron metabolism as well as transport and stress mechanisms (Fig. 5). Notably, the upregulation of anaerobic respiration and fermentation genes constitute a general feature of organic solvent and acid conditions. The functional outcome of increased anaerobic respiration is periplasmic proton generation and thus a likely increase of the proton gradient for energy generation . The upregulation of electron transport chain components for organic solvent conditions should provide the same effect, while the upregulation of glycolysis and pentose phosphate pathway genes would also increase energy generation. This response during organic solvent stress could be due to an increased cellular maintenance energy caused by these chemicals.
Many of the genes of anaerobic respiration and energy metabolism are known to be regulated by ArcA and previous studies on butanol and isobutanol stress in E. coli [12, 13] also identified ArcA as involved in the stress response. Correspondingly, the regulatory footprint of changed ArcA activity was evident here during butanol stress, and while the ArcA regulon was enriched in the other organic solvent conditions, the footprint was less apparent. The physiological cause during isobutanol stress was hypothesized to be quinone malfunction due to membrane perturbation leading to ArcA activation . The same phenomenon appears to be present for the butanol condition based on the differential expression of the ArcA regulon. The other three organic solvent conditions also had increased expression of oxidative electron transport chain genes, but only few ArcA-regulated genes with differential expression in the expected direction. Thus the oxidative electron chain gene upregulations here are possibly not ArcA mediated and could instead be a more direct effect of membrane perturbation and possible quinone malfunction. The overall likely effect of the respiration and energy metabolism gene upregulations for both organic solvent and acid conditions is an increase in the proton gradient. For organic solvent conditions this could be a means to counter the reduced membrane potential caused by membrane perturbation while for acid conditions it could counter reduced proton motive force caused by cytoplasmic acidification . The presence of weak organic acids in the medium can lead to intracellular acidification by diffusion of undissociated acid  while the reduced or possibly lost proton gradient for organic solvents would correspond to a lower intracellular pH that is likely to mediate the observed acid stress response.
For organic solvent conditions iron metabolism is also affected (Fig. 5), where iron uptake systems are downregulated likely by Fur activation. This was also previously observed during isobutanol stress  and explained by a reduced occurrence of the Fenton reaction, as quinone failure causes less superoxide to be created through respiration. This ultimately leads to increased Fe2+ concentration and Fur activation resulting in iron uptake-system downregulation. Consequently reduced iron (Fe3+) availability is a likely effect of organic solvent stress. Fur activation could also provide an explanation for the upregulation of the isc iron–sulphur cluster assembly system. The suf iron–sulphur cluster assembly system which is upregulated in acid conditions can conversely be induced by oxidative stress . The observed upregulation of sodC and katE genes in these conditions supports the induction of oxidative stress in acid conditions.
Apart from iron transport, other transport mechanisms are affected, among these sugar transport and copper transport. CRP is a known regulator of the sugar transport genes and could mediate this. However, it could also be a growth rate related effect in the stress conditions, leading to a reduced glucose uptake demand, although this does not correspond well with the upregulation of glycolysis in organic solvent conditions. The cusABCF copper transport genes are downregulated in acid and amino acid conditions. CusR is the cognate regulator responding to copper concentration while the Cus transporter mediates periplasmic Cu+ efflux, having a role in alleviating copper toxicity under mainly anaerobic conditions . The underlying reason for the downregulation of the transporter here is unclear. Upregulation of colanic acid synthesis genes is present for organic solvent and acid conditions. Colanic acid is a negatively charged extracellular polysaccharide loosely associated with the outer membrane and is required for biofilm structure in E. coli [50, 51]. Apart from this, it is generally only produced during stressful conditions providing an explanation of its upregulation here. It has been shown to confer increased acid and osmotic stress resistance [52–54] and could thus contribute towards increased tolerance.
Altered activity of some of the functions shown in Fig. 5 was previously associated with increased fitness during butanol stress. Increased external iron concentration and increased expression of iron transport genes (fepA, feoA) or siderophore synthesis genes (entC, entD) improved E. coli butanol tolerance [26, 28, 55]. Consequently, increasing iron availability appears to improve fitness. As mentioned, the Fur-mediated downregulation of iron-uptake mechanisms likely leads to intracellular iron depletion. As this is the case for all organic solvent conditions, this strategy should therefore prove advantageous under conditions other than butanol stress. Genes related to another main function in Fig. 5, anaerobic respiration, have also been identified. Upregulation of nitrate reductase two genes were found in a CRP engineered strain with improved butanol tolerance  while overexpression of hyaF also improved fitness during isobutanol stress . Accordingly, a more systematic approach towards increasing respiration could increase organic solvent tolerance in E. coli, likely by increasing energy generation through an improved proton gradient. A potential risk is a lower yield in a production scenario, however this might be outweighed by improved survival or cellular state. Another strategy could be increasing membrane protein expression to account for the apparent membrane effects during many chemical stresses, analogous to the method of improving osmotic stress tolerance . Moreover, the CRP engineered strain with improved isobutanol tolerance displayed increased expression of acid stress resistance genes (glutamate decarboxylase and acid fitness island) , further implicating these in butanol tolerance. Overall, the above findings substantiate that several of the functions outlined in this study are targets for rational tolerance engineering. Apart from those mentioned it includes the identified significantly enriched regulators, their regulon members together with the presented functional gene categories.
The enriched and depleted genes obtained by Tn-seq provide a list of gene deletions that potentially confer either increased or reduced fitness in a given chemical stress. Although Tn-seq involves mutant generation by transposon insertion, the functional outcome is generally equivalent to a gene deletion as only insertions within protein coding regions are included during the analysis. There are in total 294 enriched gene-insertions and 336 depleted gene-insertions in the 12 conditions, and thus at least 294 gene deletions that potentially increase the fitness in one or more chemical stress conditions. A number of these have been experimentally verified and display a range of growth rate increases from 8 to 60 %. In general the genes belong to diverse functional classes and a large fraction have unknown or scarcely known function. Yet, some (marB, rob, acrR) relate to the Mar-Sox-Rob regulon that is involved in e.g. organic solvent tolerance and superoxide resistance and is regulated by the three transcription factors MarA, SoxS and Rob [44, 57, 58]. The enriched insertions in marB (Byr, Furf, Deca, Suc, Ser) would lead to upregulation of marA and hence the Mar-Sox-Rob regulon . Interestingly it is not the principal marA repressor, marR, which is enriched. In contrast, the enrichment of rob in the butanol condition abolishes any rob-mediated activation of the regulon, corresponding with the finding of increased isobutanol tolerance for acrAB and marCRAB deletion mutants . The acrAB and tolC genes encoding an efflux pump are part of the Mar-Sox-Rob regulon . Another transcriptional repressor of acrAB is AcrR which was found to be enriched in decanoic acid, geraniol and acetate conditions, indicating that upregulation of this efflux pump confers increased tolerance here. This was previously shown for geraniol  and loss-of-function of acrAB has also been implicated in reduced tolerance toward decanoic acid and other free fatty acids . Although the efflux pump is associated with increased organic solvent tolerance (e.g. hexane and octane), acrR is not enriched in any of the four organic solvent conditions here, likely a result of their much lower octanol–water partition coefficient as compared to n-alkanes, decanoic acid and geraniol.
Among the enriched genes with high fold changes or presence in multiple conditions are also many related to RNA or DNA processes. Examples of the enriched genes include srmB (helicase, ribosomal association), cspC (antiterminator activity, thought to stabilize rpoS mRNA), rraA (Rnase E inhibitor), dusB (5,6 dihydrouridine modification in tRNA, possible ROS and stress regulator), greA (release stalled polymerase), rpsT (S20 ribosomal protein), pnp (mRNA degradation, minimizes oxidatively damaged RNA) and rlmH (23S rRNA pseudouridine methyltransferase). For most, the beneficial effect is not immediately intuitive, however many of these gene deletions would be expected to result in broad effects on transcription and translation processes. It is likely that many chemicals in this study elicit defects in transcription and translation, as was found for ethanol stress . The inactivation of the brnQ gene provides the largest growth rate increase (60 %) and is enriched in threonine, succinic acid and butyrolactone conditions, although the latter could not be experimentally verified. It constitutes a branched chain amino acid transporter (LIV-II). Threonine is known to be a substrate of the LIV-I transport system  and based on the present findings possibly the LIV-II system as well, as its inactivation increases fitness during threonine stress. The most depleted gene insertion (ompA) is depleted in nine conditions (organic solvents, three acids and two membrane-intercalating conditions) revealing its relevance during chemical stress. Its overexpression could possibly provide a general tolerance mechanism towards many chemicals.
To be certain of the fitness effects of specific gene deletions, further experimental testing is required. Yet, by testing the fitness effect by measuring growth rate changes for 35 gene deletion mutants enriched in Tn-seq the majority of these conferred a fitness increase. In reality the proportion of fitness increasing gene deletions could be higher as a growth rate assay is a relatively insensitive assay. Employing a survival or competition assay might provide additional experimental verification. Further, in a production scenario a mere reduction of stress, without an effect on growth rate, could improve production. Additionally, as the Keio collection was employed for experimental verification, strain differences exist between the Tn-seq (K-12 MG1655) and growth rate experiments (K-12 BW25113). Some fitness enhancing mutations are likely not shared between these two genotypes.
A comparison of the RNA-seq and Tn-seq results showed no general overrepresentation of Tn-seq significant genes among the RNA-seq significant genes nor the presence of any direction-wise overrepresentation. This is not unexpected as each method provides information on different parameters, specifically transcriptional change or gene fitness. Further the chemical stresses represented here do not generally represent natural stresses encountered by E. coli in the environment, except for acetate and to some extent the acids. Although only 25 % of Tn-seq significant genes overlapped with corresponding RNA-seq significant genes for acetate conditions, most were enriched in the same direction. The general discrepancy does not imply that the methods provide conflicting results but rather that the two methods are complementary and each provide different levels of insight and targets for tolerance improvement. Consequently utilizing both approaches will increase the prospects for successful tolerance engineering.
The selection of 12 chemicals in this study enables the identification of general targets that can improve tolerance towards not just a single but a suite of chemicals. This potentially also includes chemicals with similar properties to those presented here. The transcriptional response of E. coli towards the 12 chemicals identified several functions that could be targeted by rational engineering, among these anaerobic respiration and fermentation genes for organic solvents and acids. In particular, improving the proton motive force for maintaining efficient energy utilization is an important parameter in both conditions. Further, engineering of stress responses such as oxidative stress and acid resistance systems could improve tolerance, while for organic solvents iron metabolism provides another engineering target. Some of the regulons with perturbed expression for several chemical stresses include ArcA, CRP, RpoS, OmpR, Fur and GadX and altering these could likewise enhance beneficial or eliminate detrimental expression changes. In the other approach of genome-wide screening of transposon mutants, the substantial number of enriched mutants (294) that were identified provides a hit-list for further study. Some of the target genes, such as those related to the Mar-Sox-Rob regulon, are known to be stress-related, however those associated with transcription and translation processes have so far received less attention in a chemical stress context and warrants further examination. Overall the two approaches have yielded different targets and therefore provide complementary information for engineering improved tolerance.
The Escherichia coli K-12 MG1655 strain was employed for transcriptional profiling while the strain background for Tn-seq experiments was Escherichia coli K-12 MG1655 ΔhdsR. The growth medium was M9 medium  with 0.2 % glucose with added trace elements and vitamins, as described previously . For transcriptome chemical stress experiments cells were grown overnight in M9 medium and diluted to an OD600 of 0.05 in 25 mL M9 medium in 250 mL baffled shake flasks. After growing to ~OD600 0.9, while the cells were still growing exponentially, 25 mL M9 medium with chemical stressor was added to the medium (in effect halving the cell density) and cells were grown for 1 h before harvest. Control cells without chemical stressor in the added 25 mL of M9 medium were grown for approximately 0.5 h to reach the same OD600, 0.6–0.7, as the chemically stressed conditions. Biological triplicates were performed for all conditions and the employed chemicals were sodium acetate (Sigma, S8750), butanol (Sigma, 281549), 3-hydroxy-butyrolactone (TCI Chemicals, H0939), 1,4-butanediol (Merck, 801534), decanoic acid (Sigma, W236403), furfural (Sigma, 185914), geraniol (TCI Chemicals G0027), itaconic acid (Sigma, I29204), levulinic acid (Sigma, L2009), l-serine (Sigma, S4311), succinic acid (Sigma, S9512) and l-threonine (Sigma, T8441). Where appropriate (itaconic acid, levulinic acid, succinic acid conditions) the medium was neutralized to pH 6.8 with NaOH.
RNA isolation and processing was performed as previously described . Briefly, cells were harvested by adding 10 mL of cell culture to 2 mL ethanol containing 5 % phenol, followed by pelleting of cells by centrifugation and freezing at −80 °C. Subsequently cell pellets were lysed with 1 mg/mL lysozyme for 5 min and RNA extracted using Trizol and chloroform. For DNA removal each sample was treated with 40 units DNase I for 30 min. The purity and quality of RNA was verified with spectrophotometer and Bioanalyzer (Agilent Technologies). Extracted RNA was depleted of 23S, 16S and 5S rRNAs by subtractive hybridization using the MICROBExpress kit (Life Technologies) and adding an HPLC purified custom Capture Oligo specific for 5S rRNA (5′-AAAAAAAAAAAAAAAAAAGCGTTTCACTTCTGAGTTCGGCA-3′). Transcript libraries were prepared using the TruSeq RNA Sample Preparation Kit v2 (Illumina) with minor modifications. The protocol was initiated at the Elute, Prime, Fragment step using 100–400 ng of rRNA-depleted RNA and followed until the completion of the Enrich DNA Fragments step. Libraries were quantified using a Qubit Fluorometer and size distribution assessed on a Bioanalyzer. Libraries were single-end sequenced on an Illumina Hi-Seq 2000 at BGI-Europe.
Obtained reads were trimmed employing Trimmomatic (default settings)  in order to remove low-quality read regions. Reads were mapped to the E. coli K-12 MG1655 genome (NC_000913.2) using CLC Genomics Workbench with default settings and read counts per gene extracted. Subsequently TMM normalization , a scaling normalization method employing weighted trimmed mean of the log expression ratios, was utilized. Differential expression analysis was performed with the edgeR R statistics package , regarding counts as a negative binomial distribution and data fitted to generalized linear models. Default parameters were employed and genes with FDR-values below 0.01 were defined as significantly differentially expressed. Heat map and hierarchical clustering employing the Pearson correlation as distance measure was performed with the R packages bioDist and gplots using normalized log(2)-transformed expression values. Regulon analysis was performed using the gene set analysis pipeline from the piano R package . The GSA method was set to median and fold change direction and P values from differential expression analysis were employed. Regulon classifications were obtained from RegulonDB . Regulon sets with non-directional adjusted P values below 0.05 were designated as significant.
The transposon (Tn5) mutant library was constructed as previously described . Briefly, E. coli K-12 MG1655 ΔhdsR was transformed with the transposase-transposon complex from the EZTn5 R6 Kγori/KAN-2_transposome kit (Epicentre, Madison, WI). Approximately 60,000 transposon mutans were generated in this library. For chemical stress selection, cryogenic library stock was grown at 37 °C in 50 mL M9 with 25 μg/mL kanamycin in a 250 mL shake flask. Before entry into stationary phase the culture was washed twice and inoculated in 50 mL M9 medium with or without chemical stressor to an OD600 of 0.05 and grown at 37 °C with shaking at 250 rpm. Concentrations of chemicals that lead to a doubling time of approximately 3 h, as compared to 1 h for the control, were chosen. Concentrations were: butanol 87 mM; butyrolactone 79 mM; butanediol 791 mM; furfural 12 mM; decanoic acid 16 mM, geraniol 3 mM; acetate 103 mM; itaconic acid 361 mM; levulinic acid 198 mM; succinic acid 381 mM; serine 67 mM; threonine 118 mM. To avoid entry into stationary phase, cultures were transferred at an OD600 of 1 into fresh M9 medium with or without stressor to an OD600 of 0.05. Cells were grown up to an OD600 of 1, harvested, centrifuged and cell pellets stored at −20 °C. Additionally, the preculture used for inoculation was subjected to the same procedure, serving as an initial mutant distribution reference.
DNA libraries were prepared for sequencing as previously described . Briefly, genomic DNA was extracted from cell pellets using the PureLink genomic DNA minikit (Life Technologies) and 2–3 μg DNA sheared in a Covaris E220 ultrasonicator. DNA fragments were subjected to end repair (NEBNext end repair module), dA-tailing (Klenow fragment exo−), ligated (NEBNext quick ligation module) with Illumina TruSeq adapters and PCR enriched using adapter and Tn5-specific biotinylated primers. Biotinylated PCR products were affinity captured using Dynal MyOne streptavidin C1 beads (Invitrogen). Single-stranded DNA was generated with 0.15 M sodium hydroxide, quantified by qPCR (Kapa Library Quantification kit) and size distribution determined (Agilent RNA 6000 Pico kit, Agilent 2100 Bioanalyzer). For each condition duplicate biological experiments were performed followed by sequencing, on an Illumina MiSeq. For itaconic acid only one sample was utilized in subsequent steps as one replicate had low sequencing depth. Reads were trimmed to 67 bp (containing 25 bp genomic sequence) and sequencing data analyzed using the ESSENTIALS pipeline  with the following parameters deviating from default settings: 150,000 library size; 3 nucl. barcode mismatch; 20 bp genomic sequence match. Gene-level insertion counts were normalized using TMM normalization and P values adjusted by Benjamini-Hochberg correction. Genbank version NC_000913.3 of E. coli K-12 MG1655 was employed. Selection pressure purely originating from the M9 medium was accounted for with the control condition (without chemical stressor). Fold changes of significantly enriched insertion mutants during stress (stress vs. control) were multiplied with the control condition fold change (control vs. initial pre-culture mutant distribution), if the gene was simultaneously significantly depleted in the control condition compared to the initial distribution. After division only insertion mutants with resulting fold change values above 1 were defined as significantly enriched during stress conditions (Eq. 1).
Thus only insertion mutants enriched compared to both the control condition and the initial distribution were included. The same procedure was applied for depleted insertion mutants.
Keio collection (K-12 BW25113)  gene deletion strains were employed for experimental verification of Tn-seq derived target deletions. Strains were grown overnight in M9 with trace elements and vitamins. An equivalent volume of fresh M9 medium was added to overnight culture and cells grown for at least 1 h to exit stationary phase, followed by inoculation into fresh M9 medium with trace elements, vitamins and with or without stressor to an OD600 of 0.05. A 1.4 mL culture volume was grown in Flowerplates with 1000 rpm shaking at 37 °C in a Biolector (m2p-labs). Growth rates were determined from at least triplicate cultures and significant differences between deletion strains and wildtype strain determined by one-tailed t test, P < 0.05.
MHR and ATN conceived the study. MHR and PV carried out experiments and RL provided experimental guidance. MHR performed data analysis. MHR, ATN and KL drafted the manuscript while RL and PV helped revise the manuscript. All authors read and approved the final manuscript.
We thank Anna Koza for providing expert Tn-seq sequencing, Pernille Smith for help with Keio collection propagation and Markus Herrgard for inspiration with RNA-seq analysis.
Complete differential gene expression data for each chemical stress condition compared to the control condition are available in Additional file 1. Complete data sets for enrichment or depletion of gene transposon mutants under chemical stress conditions are available in Additional file 2. Additional file 3 contains boxplots of Tn-seq and RNA-seq significant gene distributions.
The work was funded by the Novo Nordisk Foundation. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
12934_2016_577_MOESM1_ESM.xlsx Additional file 1. Differential gene expression values for each chemical stress condition compared to the control condition.
12934_2016_577_MOESM2_ESM.xlsx Additional file 2. Enrichment or depletion of gene transposon mutants under chemical stress conditions.
12934_2016_577_MOESM3_ESM.eps Additional file 3. Comparison of distributions of significant genes between RNA-seq and Tn-seq.


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Home / Breaking News / News / Story / New York City Wants Taxpayer Bailout for Low Wage Migrant Economy
New York City Wants Taxpayer Bailout for Low Wage Migrant Economy
Aprail Mathews March 29, 2020 Breaking News, News, Story
Taxpayers should bail out the low-wage migrant workers who support the city's elite economy during the coronavirus pandemic, according to a report by the city's comptroller, Scott Stringer.
Legal immigrants and illegal migrants are part of the city's one million population who are "too often ignored, underpaid, and overworked … [and] very often lack healthcare, have to travel long distances to get to work, and struggle with childcare," said Stringer's March 26 report.
But Stringer's plea for a taxpayer bailout of city's cheap labor economy ignores the reality that Americans are impoverished by the elite's support for mass migration, said Jessica Vaughan, policy director at the Center for Immigration Studies.
The report cites impoverished "Nurses, janitors, grocery clerks, childcare staff, bus and truck drivers … those in our social service, cleaning, delivery and warehouse, grocery, healthcare, and public transit industries."
"Back in the day when these workers were Americans and unionized, they were making more than a living wage," she said. Before elites encouraged millions of legal and illegal migrants to flood Americans' workplaces, she said:
People raised their families on those jobs — they were good jobs … They were jobs that people were glad to have, where they could support their families [and homes] on them. They had [medical] benefits. If that was still the system …. they would have good healthcare plans, and they would have sick leave.
Stringer is asking for "taxpayers to subsidize the city's cheap labor economy," and to preserve the city's policy of "corporate welfare through illegal immigration," Vaughan said.
The disguised plea for the illegal immigration bailout is spotlighted by Stringer's back-handed admission that "many in New York City are also undocumented, meaning they do all of the above while living in fear of deportation under the current federal administration."
Stringer's report touts the city's high poverty levels, noting that 34 percent of "all frontline workers" are at or below the poverty line. — including roughly 35 percent of rail clerks and 39 percent cleaning workers.
The report shows that 35 percent of cleaning workers and 27 percent of retail workers are either legal immigrants or illegal migrants:
This high-immigration low-wage economic policy is back by business groups that get more consumers, more renters, and more cheap workers. For example, Stringer's report was touted in a tweet shared by Todd Schulte, the director of FWD.us, an advocacy group that was formed by wealthy West Coast investors:
Eddie@EddieATaveras
.@NYCComptroller new study found 19% of "frontline workers" are non-citizens, "often placing them in a precarious and frightening position in this age of arbitrary ICE crackdowns." #COVID19 #NewYorkUnited https://www.nydailynews.com/coronavirus/ny-coronavirus-frontline-workers-non-citizens-scott-stringer-20200326-bzb6sg3ydbbxpkykhcozy5qspy-story.html …
Nearly 1 in 5 'frontline' NYC workers are non-citizens; comptroller calls for stronger support for...
Nearly one in five workers on the front lines of the city's coronavirus response are non-citizens, putting them in an especially precarious position, he said.
nydailynews.com
Stringer did not ask for an immigration reform that would create a tight labor market where Americans workers could bargain on equal terms with their employers.
Instead, Stringer asked for a series of narrow government programs that would ensure that Democrat city officials can claim credit for the taxpayer aid:
The City and State should help provide free protective equipment and gear—including gloves, masks, and hand sanitizer—to all businesses … The City and State should subsidize [coronavirus] hazard pay for small businesses in order to properly compensate workers and help recruit new employees in these high-risk and currently understaffed industries … The State and City should ensure access to free healthcare for all direct service workers in "essential industries"—either via Medicaid or otherwise … the State must expand its unemployment, healthcare, and other safety net programs to cover independent contractors … bus service should be preserved while regular off-peak schedules should be maintained on the subway and commuter rail … For the remainder of the COVID-19 crisis, Metro-North and LIRR should charge a $2.75 fare for any and all trips into New York City … the City should offer a subsidy to any frontline worker who is interested in purchasing a bike or e-bike … Free and Easily Accessible Child Care for all Frontline Workers.
Stringer's report also wants city officials to recruit migrants to vote for Democrat politicians:
Over half of frontline workers are foreign-born and nearly one-quarter are non-citizens … the City of New York must bolster pathways to citizenship and enable non-citizen voting in all municipal elections … It is only fair that those who pay taxes and provide essential frontline services to all New Yorkers should have a direct say in municipal affairs.
New York City's leaders "have institutionalized the use of illegal workers, and made them a fixture of the workforce in New York," responded Vaughan.
When this is exposed in an economic crisis, they want taxpayers and the government to bail them out with amnesty policies and cash assistance to these workers that they encouraged to settle there, while paying them substandard wages … The bill is coming due for the cheap labor policies that they encouraged [the city's] employers to adopt.
✔@NeilMunroDC
The public & GOP blocked the business push in Congress to keep 100K+ visa workers in US jobs.
Next, biz asks agencies for the labor giveaway, with deceptive op-eds & earnest pleas from elite professional trade associations.
OK, so who lobbies back?#H1Bhttps://bit.ly/3bsaaTk
Companies Lobby Trump's DHS to Preserve Huge Foreign Workforce
Immigration lawyers are urging President Donald Trump's deputies to extend the expiring work permits for an army of foreign contract workers.
8:16 PM - Mar 25, 2020
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redpajama_set_name: RedPajamaCommonCrawl


text:
Бур-де-Пеа́ж () — коммуна во Франции, в регионе Овернь-Рона-Альпы, департамент Дром. Население — 10 101 человек (2011).

Коммуна расположена на расстоянии около 480 км к юго-востоку от Парижа, 85 км к югу от Лиона, 18 км к северо-востоку от Валанса.

Примечания

Коммуны департамента Дром

redpajama_set_name: RedPajamaWikipedia


For more information on mountain activities in Les Contamines Montjoie please contact us.
To get a better idea and understanding of the ski resort of Les Contamines Montjoie and what else they can offer you for your holiday, we suggest you look up their website as every Tourist Office offers a fascinating and exciting insight into what they and the surrounding area can offer visitors. In addition many have a dedicated website or page for those who are in need of assistance, disabled, with young children and babies and those of us just getting on a bit in life!
All the news, feedback articles and press releases illustrate how Les Contamines Montjoie helps to enhance people's lives. Also, in many cases, showing how Ski 2 Freedom works in close association with Les Contamines Montjoie to help raise awareness of the empowering benefits of mountain activities such as skiing and promoting accessible, social and inclusive tourism.
If on reading our Les Contamines Montjoie page, you have your own experiences and feedback of the resort (winter or summer) that you would like to share or feel would be encouraging to others, please do not hesitate to send them to us for inclusion on both our website and social media – photos and film clips also welcome.


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You can settle any outstanding balances with us by paying, either on your portal or by contacting our team on 0330 024 17 77. For more information, see our Paying a bill article.
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text:
Абстра́ктный ме́тод (или чистый виртуальный метод (pure virtual method — часто неверно переводится как чИсто виртуальный метод)) — в объектно-ориентированном программировании, метод класса, реализация для которого отсутствует. Класс, содержащий абстрактные методы, также принято называть абстрактным (там же и пример). Абстрактные методы зачастую путают с виртуальными. Абстрактный метод подлежит определению в классах-наследниках, поэтому его можно отнести к виртуальным, но не каждый виртуальный метод является абстрактным.

Назначение
Абстрактный метод ничего не делает, но определяет параметры и возвращаемое значение.

Назначение абстрактных методов:
 описание абстракции, которая в более конкретизированном виде не может быть реализована;
 формальное (без реализации — есть или нет) удовлетворение требований о наличии статических методов при обращении к ним для прохождения проверки компилятора статической типизации, когда реализация их будет определена динамически (в процессе работы программы).

Примеры

С#
abstract type method_name(params);//определение в родительском классе

override method_name(params){...};//определение в дочернем классе

C++
virtual void Abstr () = 0; // Чистая (пустая) виртуальная функция.

Delphi
procedure AbstractProcedure; virtual; abstract;

Java
abstract void methodName();

PHP
abstract class AbstractClass
{
    abstract protected function methodName();
}

См. также
 Полиморфизм (программирование)
 Абстрактный класс

Примечания

Литература

Метод (информатика)


ROCHESTER — Young Lives, a branch of Young Life devoted to serving teen moms and pregnant teens, is launching in Strafford County.
The Young Lives program will include monthly events and supportive one-on-one mentors who will meet regularly with teen moms. Each monthly event will feature child care, a meal, games and a brief message from the Bible, while giving moms a chance to connect and have fun. Any teen mom or pregnant teenager is welcome, regardless of their hometown or the school they attend.
The first Young Lives event will be held at the Rochester United Methodist Church (34 South Main St. in downtown Rochester) on Saturday, May 31 from 4 to 6 p.m. For more information about Young Lives or Young Life in the Seacoast, visit www.younglife.org or call the Seacoast Young Life office at 778-1251.
ROCHESTER — The Rochester Republican City Committee will meet Wednesday, June 4 from 6 to 8 p.m. in the second floor Community Meeting Room at the Rochester Public Library, 65 South Main St.
Republicans registered in Rochester are cordially invited to attend this meeting and join the Rochester Republican City Committee. The members will adopt a set of bylaws, elect a treasurer and secretary, and form into working groups.
Republicans planning to run for election to the NH Legislature from Rochester are urgently invited to attend this meeting and introduce themselves to the committee with a brief address.
ALTON — How many people would just love to participate in Alton's townwide yard sale on Saturday, June 7, but can't or won't because they don't have the space, live too far out or just don't want the hassle of trying to find places for people to park?
The Masons have just the plan. For a $10 donation, why not put your table in their large, easy-to-find parking lot? No parking problems and everyone will be able to find you and all of your great stuff.
Give Richard Quindly a call at 875-2910 for all the details and to sign up to put your table at the best spot in town. The Masons will also have hamburgers and hot dogs available to keep the crowds coming.
ROCHESTER — Rochester Public Library will offer a June problem-solving knitting program facilitated by Mrs. Doris Breton. Sessions will be held on Monday evenings and Wednesday mornings and drop-ins are welcomed. All classes will meet in the Library's Community Room located on Level 2. Bring a current project that you may be stuck on or get a pattern from Doris.
To register, come into the Library and sign up at the main desk or call 335-7550. Class options are Monday evenings from 5:30 to 7:30 p.m., beginning on June 2, or Wednesday morning from 9:30 to 11:30 a.m., beginning on June 4. Additional information is available on their website at www.rpl.lib.nh.us. All are welcome and classes are free and open to the public.
Ample off-street parking is available behind the library. The Rochester Public Library is located at 65 South Main St.
DOVER — There will be a public supper on Saturday, May 31 from 4:30 to 6 p.m. at St. John's Methodist Church, 28 Cataract Ave., near McIntosh College.
The cost of the meatloaf dinner and all the fixings, beverage and Strafford County's best dessert bar is $7 for adults, $6 for seniors and $3 for children under 12. For more information, contact the church office at 742-3046 or visit www.stjohnsdover. org.
MILTON MILLS — Liberty Chapel will be sponsoring an acoustic music jam every other Saturday from 7 to 9:30 p.m. Musicians and listeners are welcome. Doors open at 6 p.m. and dates include May 31, June 14 and 28, July 12 and 26, Aug. 9 and 23, Sept. 6 and 20, and Oct. 4 and 18.
Join them at Liberty Hall (behind the church) on Highland Avenue in Milton Mills. There is free coffee and no admission charge, although donations are welcome. For more information, call 473-2522.
DURHAM — On Sunday, June 1 from 3:30 to 5 p.m., Emery Farm is inviting families to explore one of Durham's newest conservation lands and learn what it takes to make the place function. The walk will begin at 3:30 p.m. and will take place rain or shine. Participants are encouraged to bring binoculars for bird watching and to leave pets at home. The trails are easy, but appropriate footgear and insect repellent are recommended.
Call Cynthia Belowski at 868-5562 for more information. Participants will meet at the Emery Farm stand on Route 4 in Durham. This walk is sponsored by the Durham Land Protection Working Group and the Durham Conservation Commission.


When thinking about good ice cream, our minds immediately went to Italy and the memories of enjoying authentic gelato. With this in mind, when creating Coppa della Maga we started with an Italian recipe for home made ice cream. A traditional recipe used long before the invention of ice cream machines. We set about whisking our fresh milk, cream and butter (the longer, the better) and added in fruit, nuts, cocoa and sometimes citrus oils or other fragrant ingredients….and then something startled us – the sugar!
Every month, more and more research shows that sugar is the cause for a series of health problems. We found it outrageous that products claiming to be healthy or high quality have such a high content of sugar.
That is why, we decided no matter what, Coppa della Maga ice cream will be sugar free! Instead it will be sweetened with natural sweeteners.
Whilst the experts told us it isn't possible as traditionally sugar is used to help give ice cream its smooth texture, with the memory of Italy in our minds, we set out to prove them wrong.
Coppa della Maga, a very delicious 100% natural, gluten free and sugar free ice cream!
The experience was so inspiring, even though we were told it was impossible, we did it none-the-less and ended up with magic!
That is why we have named our brand "The Bowl Of the Magician" (Coppa della Maga).


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S.T.A.L.K.E.R.: Bundle is delivered digitally to your email address instantly after purchase, which means you can download and activate the product instantly.
S.T.A.L.K.E.R. Call of Pripyat $18.05
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S. T. A. L. K. E. R. : Call of Pripyat
S. T. A. L. K. E. R. : Clear Sky
S. T. A. L. K. E. R. : Clear Sky is set in 2011, one year prior to the events of the original S. T. A. L. K. E. R. game.
A group of stalkers, for the first time, reaches the very heart of the Zone-the Chernobyl Nuclear Power Plant-and triggers a cataclysm on the brink of a catastrophe. An immense blast of anomalous energy transforms the Zone: the once reliable and relatively safe roads are no longer so, the landscape is wiped clean by outbursts of anomalies, and previously unknown areas appear on the Zone map. Stalkers and expeditions perish or end up isolated within the lost territories.
S. T. A. L. K. E. R. : Shadow of Chernobyl
In 1986, the world's worst nuclear disaster occurred at the Chernobyl power station. Soviet authorities established a 30km 'Exclusion Zone' around this nuclear wasteland, but in 2006 a second explosion rocked the stricken reactor, obliterating all living things and causing the Zone's boundaries to ripple outwards. From this epicenter came waves of mutated creatures, deadly radiation, and a strange, anomalous energy. The Zone was cordoned off by the military, who would shoot on sight anyone foolish enough to brave the horrors within.
It is now 2012 - man has ventured further and further into the heart of the Zone driven by reports of strange 'artifacts' imbued with anomalous energy. Mercenaries and bounty hunters compete to recover these artifacts, which command extortionate prices on the black market. Others seek to find the truth behind the Zone, while some merely revel in the desolate lawlessness of the place. Whatever their motivation, over time these individuals - Scavengers, Trespassers, Adventurers, Loners, Killer, Explorers and Robbers - have become known as S. T. A. L. K. E. R. s.
Oppdag de beste Game Key-tilbudene, sammenlign priser og download S.T.A.L.K.E.R.: Bundle til den beste prisen. Aktiver CD-nøkkel på Steam-klienten for å laste ned spillet.
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Lightweight Aggregate, made from fly ash, heated to 1100 degrees to form hard pellets. Use for insulating fill under ground floor slabs (8/14mm), as a concreting aggregate (4/14mm) and used for floor and roof screeding (4/8mm). Up to 50% lighter than natural aggregate and reduces ground bearing loads on retaining structures by up to 65% in comparison to primary aggregate.


I get calls every single week from parents wanting our mental coaching for their son/daughter. I have to screen each parent, and one question I ask them, "Is this something your child wants?" Whatever the situation they have to want it, period. No matter the sport, the best athletes have that passion. They don't have to be asked to work at it, because they love it.
It's the gateway drug to specialization. Anything before late middle school is too early. A few travel tournaments or matches here and there is great, its fun! But even for young kids, the trips have become every single weekend. Here's the danger, it becomes expensive and once they start traveling, it's too easy to buy the idea that they now have to pick a sport and stay with it. Specialization isn't all that either because the specific movements with different sports actually transfer. Jumping, running, throwing, all transfer across sports! Playing a variety of sports achieves that goal of skill development. Plus, each sport offers a unique advantage, competitiveness. When they learn to compete in many different sports, they will eventually transfer that skill of competitiveness to their favorite!
Effort is everything. But, if we only emphasize the outcome, athletes will learn and internalize "all that matters is winning." Players that are good will win early and often, until they no longer win. If parents only emphasize rankings, final scores, and talent, then taking risks, addressing weaknesses, and competing become afterthoughts. At some point, they are no longer the best, and they can become stuck in limbo between past expectations and low confidence. Question for sports parents: shouldn't the best 12-year old in the nation almost always be one the best 18-year olds? Rarely happens because winning and outward appearance was rewarded instead.
I was sitting next to a parent of a future DI basketball player whose brother had made it to the NBA. This sport parent was miserable and every single play or refs call that did not go his son's way, was heard by everyone including his son. I cried on the inside, because there is no way that this kid was happy either. A little league coach once told me when he knew parents were talking about him because the kids would no longer look him in the eye. Sad…It's about progress not perfection. It's not your role to call or blame coach about playing time, change coaches or schools, or get a lesson every time they play bad.
There are good opportunities to talk about their performance and not good ones. During the game is NOT the appropriate time. However, all the time, parents are communicating with their son/daughter. Body language doesn't talk, it screams, and they can see your negative behavior. Also, the stands can be packed with hundreds or thousands of screaming people, and the ONE voice they will recognize is yours! Why are you trying to coach them during their performance?
This is really helpful … thanks for sharing!


Marcia Hovious is the principal and founder of Hovious Consultants, a firm that provides staff evaluation and recruitment, conducts fundraising assessments, and provides direction, coaching, mentoring and training for fundraising professionals, Executive Directors and Boards of Directors.
Established in 2011, Hovious Consultants has worked with schools, retirement communities, children services, health organizations, advocacy groups, animal protection agencies, and refugee services. Additionally, Marcia has helped to create and facilitate a Peer-to-Peer Development Learning Circle through the United Way in Houston TX. Hovious also taught Volunteer Management for Capital Campaigns as a part of Continuing Education program at Rice University.
Hovious began a professional fundraising career through her own personal philanthropic efforts with numerous non-profits in El Paso TX. After serving as a committed volunteer, Hovious was hired by the University of Texas at El Paso to be responsible for alumni relations, academic and athletic fundraising, special events, major donor relations, corporate sponsorships and a major endowment campaign.
Hovious' career then took her to Charlottesville VA as the regional development director for the University of Virginia. Her focus was a $1 billion capital campaign. This campaign, while exceeding expectations, made the University one of the first academic institution to execute a comprehensive fundraising campaign of this magnitude.
Following her tenure in Charlottesville, Hovious moved to Houston TX to work on Rice University's $500 million capital campaign. During the campaign, she was recruited to DePelchin Children's Center (a social service agency for adoption, foster care and residential treatment) to direct the nonprofit's $15 million capital campaign. The DePelchin campaign included funding for a new building as well as enlarging the development program to include Giving Societies and a Planned Giving program. Her next endeavor was Development Director for Theatre Under the Stars. Hovious was successful in erasing a $2 million budget deficit in less than one year and then doubling the company's annual contributed revenue.
Hovious and her husband, Brad, now reside in Nashville TN.


Like most of the Italian company's catalog, the Aprilia Concept RS660 is a stylish looking thing, and although it's focused on being a "fun bike" that's "thrilling for the road," Aprilia is enjoying the chance to experiment with aerodynamic ideas "unrestricted by any regulations" that might pop up in racing.
As such, it's a bit of an odd combination – the motor, a 660cc parallel twin that basically removes the two rear cylinders from Aprilia's existing RSV4 and Tuono 1100, is firmly street focused, as are the ergonomics, which feature a large seat, high semi-handlebars and comfy riding position. That collection of traits would make for a neat little streetbike.
On the other hand, the carbon fairings and wickedly racy tail give the impression of a track machine with a lap-time focus. And the idea of having active aerodynamics on board to add downforce when cornering and reduce drag on the straights isn't something that'll make much difference at legal road speeds.
In all, the Concept RS660 is probably most notable as our first look at a new platform. That aluminum frame and swingarm, plus the 660cc parallel twin, will form the basis of a new range of streetbikes, presumably including something sporty that doesn't look too dissimilar to this concept, and a naked machine. Aprilia says it plans to release the upcoming bikes in Europe, Asia and the United States.


text:
Q: AWS ACM PCA with KMS keys I'm building a service using MTLS for authentication with AWS ACM PCA as private Certificate Authority.
The certificate is stored in a password protected PKCS#12 file together with the private key in.
The private key is also used for signing data.
At the moment I'm creating key pairs programatically but want to switch into using KMS.
However, it seem like it's not possible to get the private key from KMS.
How am I supposed to allow for both client authentication and signing if I can't add the private key to the PKCS#12 key store?

A: You can use kms.generateDataKeyPair to create and export a public key and private key which can then be used locally.

redpajama_set_name: RedPajamaStackExchange


As with previous years there will be a Partners Program run in conjunction with the Conference Program. This year the program focuses around the attractions of Fiji and takes into consideration the different preferences of the attendees. We have selected a number of activities which will allow you to immerse yourself in the local culture as well as enjoy some relaxation.
You have a selection of activities below and you can choose three of these to fill the three mornings of the Conference Program. If you prefer you are also invited to attend the business sessions and this option is available for selection on the registration form. Spaces are limited on some activities so choose wisely and quickly so you don't miss out.
Please note lunch is provided for all attendees with the delegates.
Please note if minimum numbers for daily activities are not achieved by 27 June 2018 the activity will not be able to go ahead. In this situation the Secretariat will contact you and arrange to transfer you to another activity that has achieved minimum numbers on the selected day.
Sabeto is one of Fiji's hidden gems. Located halfway between Nadi and Lautoka, Sabeto is near the Sleeping Giants mountain formations. Continuing off the main highway down a dirt road, you will arrive at the Sabeto Hot Springs also known as the Sabeto Mud Baths.
Believed by locals to have healing properties because of the sulphur in the hot springs, making this a good social experience. The Sabeto Hot Springs has three pools set in lush natural surroundings and is ideal with travellers seeking for a unique experience. Immerse yourselves in the warm mud baths and have fun applying mud generously on yourself and your friends as well.
Get ready for the Gala Dinner!
Bollywood dancing is a combination of classical Indian dance, folk dancing such as Bhangra and sometimes with Latino and Arabic influence. It's fun and very expressive with dancers expressing what the music means through the graceful movements of the body, you will be unable to resist the urge to get yourself on and burning it up on the dance floor.
Bollywood dance classes are suitable for absolute beginners. As well as dancing, the Bollywood dance course will also draw out the inner actress/actor in you. Even if you do not know what the song means, the teacher will translate and allow you to express yourself through the Bollywood dance and develop performing skills.
What to wear in a Bollywood dancing class?
Soft comfortable clothing that you can move and exercise in. Suggested outfit can include tracksuits, leggings and a suitable top. In terms of footwear, barefoot or light slippers are suitable choices.
Enjoy a relaxing start to your day with a champagne breakfast at the Intercontinental Fiji on the Chapel Lawn. The morning will include a breakfast buffet with champagne so you can get your day off to a great start. There will be staff from the Intercontinental Spa who will provide relaxing head and neck massages for all those attending. A great way to start your day relaxed and rejuvenated.
Minimum: 40 people per day.
Maximum: 70 people per day.
Enjoy a one-hour session with professional florists learning the art of flower crafting, making your very own personal floral arrangement. The session will focus on making flora crowns with your own personal style and touch. You will enjoy some light refreshments to help bring out your creative side.
Minimum: 15 people per day.
Maximum: 20 people per day.
If you are interested in the business sessions on any given day you are welcome to select this option on the registration form. Simply select the day you wish to attend. This will include the sessions, morning tea and lunch.


text:
function [ simConfig ] = runSVMSimulation( simConfig )
%RUNSVMSIMULATION Summary of this function goes here
%   Detailed explanation goes here

% Allocate memory for all of the training data
trainingData = zeros(simConfig.NumPointsPerEpoch*simConfig.NumEpochs,2);
groupNames = cell(simConfig.NumPointsPerEpoch*simConfig.NumEpochs,1);
desiredOutput = zeros(simConfig.NumPointsPerEpoch*simConfig.NumEpochs,1);

% Train the algorithm for the number of epochs, on every iteration add in a
% new epoch to improve performance
for k=1:simConfig.NumEpochs
   disp(['Epoch ' num2str(k)]);
   % Generate the new training data for this epoch
   [newDataPoints,newGroupNames,newDesiredOutput] = ...
       genTrainingData(...
       simConfig.NumPointsPerEpoch,...
       simConfig.DesiredOutputValue);

   % Flip the generated data
   newDataPoints = newDataPoints';
   newDesiredOutput = newDesiredOutput';

   % Calculate the indexes
   startIndex = (k-1)*simConfig.NumPointsPerEpoch+1;
   endIndex = k*simConfig.NumPointsPerEpoch;

   % Add the new training data to the rest of the data
   trainingData(startIndex:endIndex,:) = newDataPoints;
   groupNames(startIndex:endIndex) = newGroupNames;
   desiredOutput(startIndex:endIndex) = newDesiredOutput;

   % Set optimization options, so we don't run into max iteration problems
   % Set the max number of iterations to 200000
   maxIterations = 200000;
%    options = optimset('quadprog');
%    options = optimset(options,'MaxIter',maxIterations);
    options = svmsmoset('MaxIter',maxIterations*200);

   % Get the starting time
   simConfig.StartTime = datestr(now);
   % Train the SVM
%    simConfig.SVMStruct = svmtrain(...
%        trainingData(1:endIndex,:),...
%        desiredOutput(1:endIndex),...
%        'Kernel_Function','rbf',...
%        'Method','QP',...
%        'QuadProg_Opts',options,...
%        'BoxConstraint',simConfig.BoxConstraint);
    simConfig.SVMStruct = svmtrain(...
       trainingData(1:endIndex,:),...
       desiredOutput(1:endIndex),...
       'Kernel_Function','rbf',...
       'Method','SMO',...
       'SMO_Opts',options,...
       'BoxConstraint',simConfig.BoxConstraint);
   % Get the ending time
   simConfig.EndTime = datestr(now);

   % Get the performance
   simConfig.Performance(k) = getPerformanceSVM(simConfig);
   disp(['Performance ' num2str(simConfig.Performance(k))]);

end

end

redpajama_set_name: RedPajamaGithub


text:
Ammyy Admin — система віддаленого доступу та адміністрування, розроблена компанією Ammyy Group. Дозволяє контролювати клавіатуру і мишу, запускати програми, передавати файли.

Ammyy Admin працює крізь NAT і не потребує зовнішніх IP адрес, встановлення додаткового ПЗ та налаштування переадресації портів. Усі дані, що передаються, шифруються за допомогою алгоритму Advanced Encryption Standard (AES) з різними ключами для кожної сесії. Ammyy Admin повністю сумісна з VNC, Terminal Server та іншими програмами віддаленого доступу.

Можливості
 Не потрібна установка програми.
 Не потрібне переналаштування мережевих екранів і Firewall.
 Опція тільки для перегляду.
 Конфігурування прав на основі computer ID.
 Сумісна з існуючими програмами віддаленого доступу: VNC, Terminal Server та іншими.
 Підтримка Microsoft Remote Desktop Protocol (RDP).
 Може працювати як системний сервіс.
 Дозволяє віддалене перезавантаження, вхід в систему, вихід, зміну користувачів.
 Передача файлів.
 Голосовий (аудіо) чат.

Безпека
В Ammyy Admin реалізовані такі функції безпеки:

 Всі дані шифруються за стандартом AES, і використовуються різні ключі для кожної сесії.
 Не відкриваються порти на локальному і віддаленому комп'ютері.
 Захист від атак перебору паролів (брутфорсу).

Див. також
 Virtual Network Computing
 Remote Desktop Protocol

Подібні програми
 Radmin
 TeamViewer

Посилання
 Офіційний сайт
 itc.ua
 Дистанційне обслуговування
 SHAREit

Інтернет-програми
Мережеве програмне забезпечення


The time-honoured tradition of having your shoes professionally shined, makes us feel confident, walk a little taller and from a very practical point-of-view, it makes tired old shoes look like new again. You see at Walter's, we've been in the business of caring for footwear since 1893. It's when our founder, Ernst Ferdinand Walter founded the company in Montreal, Canada. So when it comes to shoes, we've seen trends, fads and fashions come and go, but putting a shine on a fine pair of shoes is an art that will always be timeless.


Herbal pills are available that come in smaller sizes which make it easier to swallow. Herbal medicine can also be taken in the form of powders or granules which you can simply mix the recommended dosage with warm water to provide an instant herbal tea. At your consultation, you will have the opportunity to discuss your preferences in the administration of Chinese herbal medicine with Lily.


text:
"On the evening of June 7th, 2016, nine bicyclists, friends, fellow riders and cherished loved ones rode together as members of the Chain Gang bicycle club. The driver of a pickup truck crashed into the riders. Four cyclists were seriously injured in the crash. Five lost their lives in the tragedy. We mourn their loss."
I was approached about a year and a half after the crash by David Rachowicz, the director of Kalamazoo county parks, who had seen my work with the nearby Tom Harding Memorial and was curious if I could put together some designs. I'd already been approached once or twice by other groups– local political contenders hoping to boost their public image by spearheading a memorial, activist groups, one of which wanted to literally hammer together a memorial out of the still bloody bikes. The crash had made the international news, and that led to both an outpouring of sympathy and support, and a few more ghoulish proposals from people hoping to somehow turn the victims tragedy and literal blood into their own political opportunity or soapbox. I asked David who he was there on behalf of, and he said "We've been talking with the families of the victims.", which was what I needed to hear.
I met with the club once on a grey day in early fall, in a dark windowed room overlooking both the site of the crash and the eventual location of the sculpture, to talk about the process of how these kinds of public project generally come together. We built some trust, and a month or so later, I made models for 5 different designs- 3 of bronze figures with bikes, a more architectural shrine made of bike frames, and this simple wall. The group chose the wall immediately and unanimously.
The original design of the wall was very close to the final design– a slope with five empty bikes moving up it, placed to correspond the rough positions of each of the five slain riders before they were killed, with the wall curved to create a sheltering, protected circle. The bikes are riding toward the precipice at the highest point of the wall. The angle of the slope was adjusted to match the slope of the road, and the monument placed so that it's possible when standing in the middle of the circle to superimpose the ghost bikes onto the roadway where they rode moments before the tragedy.
The original plan was for one single, huge plate of stainless steel to be bent into a 12 foot diameter, but production constraints made it easier to split the piece into four individual panels, which was a better design anyway once we saw it. As the deadline loomed, Studio 431 of Landscape Forms Inc, a local company that manufactures benches, lampposts, and many other municipal fixtures as well as custom architectural elements, came on board and most of the physical production work was donated by both them and many of their suppliers.
This project had more media attention, more emotionally involved parties, and more stakeholders than anything I'd worked on previous to it.
Not only that, but the actual physical production was more cooperative, and shared between more hands than I've ever been a part of. As is almost always the case with big public art, I will get credit as the artist for the project and the design, but those who put in the (in this case, donated) hours to cut metal, roll huge sheets of steel, run structural models, make phone calls, hustle for donations, clear permits, coat stainless steel, pour concrete, and finish and weld, go unmentioned.
This design came to fruition through the work and material donations of so many craftsmen all over the state, and without their work,and the amazing coordination via Studio 431, there's simply no way we could have ended up with a piece this fine in such a short time. All credit due to Casey, David, Ron, John, Rich, Tom, Gene, Steve, Nate, Adam, and to everyone who put their hands and sweat onto the memorial.


text:
Home » Lifestyle » Virginia winter storm leaves 180,000 without power days after hitting area
Virginia winter storm leaves 180,000 without power days after hitting area
Fox Business Flash top headlines for January 5
More than 180,000 Virginians are still without power Wednesday morning after a winter storm brought heavy snowfall and freezing temperatures to the area earlier this week.
On Monday, power outages swept the state after up to 11 inches of snow fell in the area, according to the National Weather Service.
CLICK HERE TO READ MORE ON FOX BUSINESS
As of 10 a.m. ET on Wednesday, nearly 180,000 Virginians were out of power, according to Poweroutage.US.
Dominion Energy, the largest electric utility company in the state, tweeted that crews have been working through the night to restore power to residents after the storm "resulted in downed trees and tree limbs, and downed power lines, broken poles and cross arms at 6700+ work locations across Virginia."
GET FOX BUSINESS ON THE GO BY CLICKING HERE
However, crews have been faced icy roadways, road closures and downed trees.
"In some localities, the damage is so severe that some areas are not even accessible by foot, in those cases we are using drones to assess," Dominion Energy tweeted.
Crews are still making progress, with thousands of homes being restored over the past 24 hours.
While power is still being restored Wednesday, some parts of the state are facing a light freezing drizzle, according to the National Weather Service.
WINTER STORM IN VIRGINIA LEAVES OVER 250K WITHOUT POWER
Although rain and drizzle are expected to clear out by Wednesday afternoon, officials are still "watching chances for accumulating snow Thursday night into Friday," the National Weather Service tweeted.
COVID-19 testing won't be mandated before domestic flights for now, CDC says
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