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Context-specific inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase center
Edited by Peter B. Moore, Yale University, New Haven, CT, and approved September 2, 2016 (received for review August 5, 2016)
Chloramphenicol and linezolid interfere with translation by targeting the ribosomal catalytic center and are viewed as universal inhibitors of peptide bond formation. We show that, contrary to this view, the activity of these antibiotics critically depends on the nature of specific amino acids of the nascent chain carried by the ribosome and by the identity of the residue entering the A site. These findings indicate that the nascent protein modulates the properties of the ribosomal catalytic center and affects binding of its ligands. Understanding the principles of context specificity of ribosomal drugs may help develop better antibiotics.
The first broad-spectrum antibiotic chloramphenicol and one of the newest clinically important antibacterials, linezolid, inhibit protein synthesis by targeting the peptidyl transferase center of the bacterial ribosome. Because antibiotic binding should prevent the placement of aminoacyl-tRNA in the catalytic site, it is commonly assumed that these drugs are universal inhibitors of peptidyl transfer and should readily block the formation of every peptide bond. However, our in vitro experiments showed that chloramphenicol and linezolid stall ribosomes at specific mRNA locations. Treatment of bacterial cells with high concentrations of these antibiotics leads to preferential arrest of translation at defined sites, resulting in redistribution of the ribosomes on mRNA. Antibiotic-mediated inhibition of protein synthesis is most efficient when the nascent peptide in the ribosome carries an alanine residue and, to a lesser extent, serine or threonine in its penultimate position. In contrast, the inhibitory action of the drugs is counteracted by glycine when it is either at the nascent-chain C terminus or at the incoming aminoacyl-tRNA. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center.
The key chemical reaction catalyzed by the ribosome is peptide bond formation. This reaction occurs in the peptidyl transferase center (PTC) where the transfer of the nascent peptide from the P-site–bound peptidyl-tRNA (pept-tRNA) to the A-site–bound aminoacyl tRNA (aa-tRNA) leads to elongation of the growing protein chain by one amino acid at each round of translation (reviewed in ref. 1). Although the peptidyl transfer reaction is potentially assisted by the chemical environment of the PTC (2⇓–4), the main contribution of the ribosome to peptide bond formation is entropic, as the major acceleration of the reaction stems from the proper orientation of the donor and acceptor substrates within the catalytic center (5⇓–7).
The PTC is a primary target for antibiotics that inhibit protein synthesis (see ref. 8 for review), such as chloramphenicol (CHL), one of the first broad-spectrum antibacterials in clinical use (9). CHL binds to the PTC A site, preventing proper placement of the aminoacyl moiety of aa-tRNA (Fig. 1) (10⇓–12). Because the key interactions with the ribosome in the A site are shared by all aa-tRNAs, CHL is viewed as a universal inhibitor of peptide bond formation (reviewed in ref. 13). However, this commonly accepted model of CHL action fails to explain several experimental observations such as the differential inhibition of translation of specific mRNA templates (14, 15) or only partial inhibition of puromycin-mediated release of nascent chains in polysomes even at saturating CHL concentrations (16). The perceived ability of CHL to indiscriminately inhibit formation of any peptide bond also conflicts with its role as an inducer of resistance genes. Activation of a number of CHL resistance genes relies on the antibiotic-promoted arrest of translation at a specific codon within the regulatory leader ORF (reviewed in ref. 17). Thus, to activate the expression of the resistance locus in response to the antibiotic assault, the ribosome should be able to progress through several leader ORF codons to reach the site of the programmed translation arrest. How the ribosome can polymerize a segment of the leader peptide, if CHL indiscriminately inhibits formation of any peptide bond, remained unclear.
Although CHL is one of the oldest known antibiotics, linezolid (LZD) belongs to one of the newest classes of clinically important PTC-targeting protein synthesis inhibitors, the oxazolidinones (Fig. 1). Genetic, biochemical, and crystallographic evidence showed that LZD binds in the PTC A site, at a location overlapping with the CHL binding pocket. Similar to CHL, LZD directly clashes with the placement of the aminoacyl moiety of the aa-tRNA, suggesting that the drug should indiscriminately inhibit peptidyl transfer reaction (Fig. 1) (18⇓⇓–21). It remained unexplained, however, why LZD, which readily interfered with in vivo protein synthesis or cell-free translation (22), failed to inhibit peptide bond formation between fMet-tRNA and puromycin (18, 23).
Because of the accumulating biochemical evidence that contradicted the conventional view of the mode of action of CHL and LZD, we questioned whether these antibiotics universally block the formation of every peptide bond during protein synthesis. Here, we show that CHL and LZD do not act as global inhibitors of peptide bond formation, but rather block translation at specific locations within the mRNA in a context-specific manner. Furthermore, we present evidence that the specificity of action of these drugs is defined by the nature of the penultimate residue of the nascent peptide as well as by the amino acid residues directly participating in peptide bond formation. Our findings unveiled an important functional influence of the nascent peptide on the properties of the PTC and expand the concept of the context-specific action of ribosomal antibiotics.
Toeprinting Analysis Reveals Site Specificity of CHL and LZD Action.
To assess the mode of action of CHL and LZD in vitro, we used toeprinting analysis, which determines the position of the antibiotic-stalled ribosome on an mRNA (24, 25). If, as alleged, CHL and LZD act as global inhibitors of the PTC catalytic function, preincubation of the ribosome with these antibiotics should prevent the formation of the very first peptide bond. However, consistent with our previous observations (25), even at very high concentrations of CHL or LZD, ribosomes were not arrested at the initiator codon of any of the tested mRNA templates (Fig. S1A). Instead, translation progressed through few codons until the ribosome was stalled, with varying efficiency, at specific downstream sites. In an attempt to understand the requirement for the preferential action of CHL and LZD at specific mRNA locations, we aligned the encoded protein sequences leading up to the experimentally identified sites of CHL- or LZD-induced arrest and examined the nature of the P- or A-site amino acids directly participating in peptide bond formation (Fig. S1B). Nonetheless, no clear conservation of the donor or acceptor residues was immediately obvious. We realized that, to search for the motifs conducive to the action of CHL and LZD, we needed to operate with a broader array of sites of the preferred action of these drugs. Therefore, we used ribosome profiling, which allows analyzing the mode of action of protein synthesis inhibitors at the genome-wide level (26⇓⇓–29).
Identity of the Penultimate Residue of the Nascent Peptide Is Critical for the Action of CHL and LZD.
The ribosome-profiling experiments were carried out with the Escherichia coli strain BWDK, a descendant of the WT E. coli K-12 strain, where the absence of the tolC gene (a key component of the multidrug efflux pump) renders the cells hypersusceptible to antibiotics. Exponentially growing cells were exposed to a 100-fold excess over the minimal inhibitory concentration of CHL or LZD for 2.5 min, a time period sufficient to reach maximum inhibition of translation (Fig. S2). The ribosome-protected mRNA fragments were then prepared, sequenced, and mapped to the genome using established procedures (30, 31). Treatment with any of the two inhibitors caused a modest redistribution of ribosome density along the genes relative to the untreated control (Fig. S3). Thus, it became evident that exposure to the antibiotic does not immediately "freeze" translation. Instead, ribosomes can still polymerize a few peptide bonds before pausing at particular codons. This observation is consistent with our in vitro toeprinting results, which showed that CHL and LZD stall translation at a number of specific locations within the protein-coding sequences (Fig. S1).
We identified the preferential sites of antibiotic action by computing changes in ribosome occupancy at ∼60,000 individual codons between the antibiotic-treated and untreated cells and ranking all of the analyzed codons by the magnitude of the change (Fig. 2) (see SI Materials and Methods for detail). For each antibiotic, we then selected the top 1,000 codons, where the strongest drug-induced translation arrest was observed. Within these sites, we searched for a specific sequence signature among amino acids encoded within the nine codons preceding the arrest site (positions −1 to −9), the arrest codon (position 0), which occupies the P site of the stalled ribosome, and the following codon (position +1), corresponding to the A-site codon (Fig. 2). Remarkably, the preferential CHL arrest sites showed significant enrichment in Ala (38.1%) and, to a lesser extent, of Ser (14.8%) or Thr (6.3%) codons, in the −1 position compared with the expected random occurrence of these residues (15.2%, 7.8%, and 5.5%, respectively) (Fig. 2A and Fig. S4). The sites of LZD-induced arrest exhibited an even stronger preference for Ala codons (69.9%) in the same position (Fig. 2B and Fig. S4). Although Ala and Thr can be defined by four codons each and Ser is defined by six codons, no preference for any specific Ala, Ser, or Thr codon at the sites of arrest was apparent. This lack of codon bias argues that the specificity of antibiotic action is defined by the nature of the encoded amino acids rather than the mRNA sequence or tRNA structure. The occurrence of Ala, Ser, or Thr in the penultimate peptide position strongly correlated with the drug-induced translation stalling throughout the entire range of the analyzed locations, and their presence progressively decreased toward the end of the spectrum where codons with the least pronounced ribosome stalling were grouped (Fig. S5A). Furthermore, when all of the ∼60,000 analyzed sites were grouped into 20 bins, according to the nature of the nascent-chain penultimate residue, the sites with Ala, Ser, or Thr in position (−1) showed a significant increase of the cumulative ribosome occupancy across all of the analyzed codons (Fig. S5B). All of these data strongly argue that presence of Ala (and, to some degree, Thr or Ser) as the penultimate amino acid in the nascent chain stimulates the inhibitory action of CHL and LZD.
To further examine the influence of the sequence context on drug-induced arrest, we selected a well-defined model system appealingly suited for cell-free translation experiments (32). According to the ribosome-profiling data, the drug-induced ribosome stalling at the Leu5 codon of the hns gene represents one of the 10 strongest arrest sites common for both CHL and LZD (Fig. 3A). Drug-induced ribosome stalling at the hns Leu5 codon was readily reproduced in vitro in the toeprinting assay (Fig. 3B). By comparing the fraction of the ribosomes arrested at the Leu5 codon with those that bypassed the site of drug-induced translation stalling, we compared the efficiency of translation inhibition for the H-NS mutants containing every possible amino acid substitutions of Ala4 (Fig. 3 C and D). The results of the in vitro experiments confirmed that the presence of Ala in the penultimate position of the H-NS N-terminal pentapeptide significantly enhanced the action of CHL and LZD. Replacing Ala4 with any other amino acid greatly reduced the efficiency of drug-induced ribosome stalling. Furthermore, in agreement with the profiling data (Fig. S4), Ser and, in the case of LZD, also Thr at position −1 were more conducive to the antibiotic action than any other substitution of Ala4 (Fig. 3 C and D).
Having verified the importance of the penultimate peptide residue in the mechanism of LZD and CHL action, we reexamined the collection of the arrest sites identified by toeprinting in our initial experiments (Fig. S1B). Consistent with the trend observed in vivo, more than 80% of the drug-induced arrests detected in those experiments occurred when the penultimate amino acid of the nascent peptide was Ala, Ser, or Thr (marked by arrowheads in Fig. S1B).
Collectively, the results of the genome-wide analysis and of the in vitro testing converge on the key stimulatory role of Ala, Ser, or Thr in the penultimate position of the nascent peptide in the mechanism of action of the PTC-targeting antibiotics CHL and LZD.
CHL and LZD Poorly Inhibit Peptide Bond Formation When Glycine Residues Are Involved.
The codons with the maximally increased ribosomal occupancy in drug-treated cells represent the sites where antibiotic action is most pronounced (red frames in Fig. 2). Conversely, the codons whose occupancy significantly decreases in the presence of the drug are those where the antibiotic fails to hinder peptide bond formation. Despite the presence of the inhibitor, the ribosomes proceed with translation, traversing these codons until they encounter the nearest "strong" arrest location. In contrast to our finding for the top arrest sites, the analysis of the 1,000 codons characterized by the most dramatic decrease in ribosome occupancy in the drug-treated cells (green frames in Fig. 2) did not show a dramatic bias in favor of any particular amino acid in the position (−1) of the peptide. Instead, it revealed a strong prevalence for the presence of glycine residues in either the P site (position 0) or the A site (position +1) of the translating ribosome. These data suggest that the peptidyl transfer reaction involving a Gly residue either as a donor or as an acceptor is poorly inhibited by CHL or LZD.
The occurrence of Gly codons in either the P or A sites negatively correlated with the efficiency of drug-inflicted stalling throughout the entire range of analyzed sites (Fig. S5A). Consistently, the presence of glycine codons in the P or A sites correlated with the most pronounced decrease of the cumulative ribosomal density at the corresponding codons in antibiotic-treated cells (Fig. S5B). These findings derived from the ribosome-profiling analysis were in keeping with the results of the subsequent in vitro testing with the hns gene: presence of a Gly residue in the P or the A site made the action of CHL or LZD inefficient (Fig. 3 D and E).
Thus, we concluded that the amino acid sequence context is capable of either stimulating or counteracting the inhibition of peptide bond formation by CHL and LZD.
Induction of CHL Resistance Genes Relies on the Stimulatory Effect of the Penultimate Residue of the Nascent Chain on Drug Action.
Our newly gained understanding of the site specificity of CHL action prompted us to reevaluate the mechanism of inducible CHL resistance. We analyzed CHL-dependent programmed translation arrest at the leader ORFs of two resistance genes, catA86 (originated in Gram-positive bacteria) and cmlA (common to Gram-negative species) (33) (Fig. 4A). Toeprinting analysis showed that, in the presence of the antibiotic, translation driven by E. coli ribosomes stalls when the fifth codon of catA86L or the eighth codon of the cmlAL ORF enter the ribosomal P site (Fig. 4 B and C). The same stalling site was observed when translation of cat86AL was catalyzed by ribosomes isolated from Gram-positive Bacillus subtilis (Fig. S6A). These results are in line with the conclusions of the previous genetic testing, which suggested that the N-terminal pentapeptide MVKTD of Cat86AL or the octapeptide MSTSKNAD of CmlAL are synthesized in the presence of CHL (33, 34). Remarkably, translation arrest at the leader ORFs occurs when the nascent chain carries in the penultimate positions amino acid residues conducive to CHL action: Ala (in the case of CmlAL) or Thr (Cat86AL) (Fig. 4 B and C). Thus, the mechanism of induction of CHL resistance simply exploits the site specificity rules that have emerged from the profiling and toeprinting analyses. In agreement with this assertion, in the alanine-scanning mutagenesis experiment carried out with cat86AL and cmlAL, we observed the appearance of new ribosome stalling sites consistently occurring at the codon subsequent to the engineered Ala mutation (asterisks in Fig. 4 D and E). In addition, replacement of Thr4 of the wt Cat86AL with Ala significantly enhanced CHL-mediated stalling at the "native site" (compare lanes MVKTDK and MVKADK in Fig. 4D), corroborating our conclusion that an Ala residue in the penultimate peptide position is the most stimulatory for drug activity.
Codons 2–4 of the cmlAL ORF specify Ser and Thr, which are expected to moderately stimulate CHL action (Fig. 3 and Fig. S4). Curiously, however, translation of the ORF in the presence of the antibiotic proceeds fairly efficiently until the eighth codon (large black triangle in Fig. 4C), with only a moderate arrest at the codon 5 (small black triangle in Fig. 4C). One possible explanation is that efficient CHL-induced stalling requires the nascent chain to reach a certain length. Indeed, sequential 5′-terminal truncations of the cmlAL or cat86AL ORFs diminished the efficiency of drug-induced arrest at the native site when the nascent chain became shorter than 4 (Cat86AL) or 5 (CmlAL) aa long (Fig. 4 F and G).
Although the regulatory ORFs of the CHL resistance genes have been evolutionarily selected to respond specifically to the presence of natural CHL, the similarity in the context specificity of action of CHL and LZD suggested that the latter, a synthetic antibiotic, could also act as an inducer. Indeed, LZD and even the newest oxazolidinone antibiotic, tedizolid, were able to stimulate ribosome stalling at the fifth codon of cat86AL or the eighth codon of cmlAL, although less efficiently than CHL (Fig. S6). Thus, synthetic oxazolidinones likely would be able to induce the natural CHL resistance genes.
SI Materials and Methods
Kinetics of Translation Inhibition by CHL and LZD.
Kinetics of inhibition of protein synthesis in Escherichia coli was followed as previously described (32) by monitoring incorporation of l-[35S]methionine into the trichloracetic acid-precipitable material after exposure of cells to 100-fold minimum inhibitory concentration (MIC) of CHL (100 µg/mL) or 50-fold MIC of LZD (400 µg/mL).
pLogo Analysis of the Amino Acid Biases at the Strongest and Weakest Arrest Sites.
The total number of reads assigned to the three nucleotides of a codon was normalized to the total reads within the gene, generating the relative codon density (RCD). For analysis of the changes in the relative codon occupancy, only genes that had a gene score greater than 100 in drug-treated and control samples were used, and for those, only the codons with at least five assigned reads were included. The gene score was calculated according to the following equation:
The relative fold difference values (RFDVs) for individual codons were calculated as the ratio of RCD in the drug-treated sample relative to the control. The sites located at a distance of less than 10 codons from the beginning of the gene and those followed immediately by the stop codon were excluded.
The RFDVs (61,250 for CHL vs. control or 56,251 for LZD vs. control) were then arranged in a descending order. For each of the sites, the encoded peptide sequence was extracted; this included the 10-aa C-terminal sequence of the nascent chain ending with the codon specifying the residue at the P site to which the ribosome density was assigned, and the following A-site amino acid. pLogo analysis (47) was carried out using this 11-aa sequence. The pLogo foreground was the amino acid sequences of either the 1,000 strongest or weakest arrest sites. The background was amino acid sequences of all analyzed sites (61,250 for CHL or 56,251 for LZD).
Computing the Changes of Occurrence of Individual Amino Acids at Specific Sites as a Function of Efficiency of CHL or LZD Action.
A 1,000-site sliding window was used to compute the occurrence of each of the 20 aa in positions −1 (the penultimate peptide residue), 0 (P site), or +1 (A site). The occurrence was calculated as the total number of sites within a window with a specific amino acid in the corresponding position relative to the total count of that amino acid in that position in all sites. For example, the first 1,000-site window for the CHL sample contains 298 sites with Ala in the position (−1) relative to a total of 9,370 sites with Ala in position (−1) among all of the analyzed 61,250 sites. After plotting the obtained values the curves were smoothed by using the best-fit quadratic slope algorithm of Prism 7 (GraphPad).
Estimation of the Cumulative Differences in Relative Codon Occupancy.
For computing cumulative change in ribosomal occupancy, all of the RFDVs were split into 20 bins according to the chemical nature of the amino acid at the penultimate (−1) position in the nascent chain, the P site or the A site, and the average fold difference was computed for every bin. Those sites within nine codons of the start site or having a stop codon in the A site were ignored. In a similar fashion, the average RFDVs for all possible 2-aa combinations (400 bins) found the in the pairs −1 and P, −1 and A, or P and A sites were calculated.
Generation of Templates for in Vitro Translation and Toeprinting Analysis.
All DNA templates for toeprinting were produced using AccuPrime DNA Polymerase (Thermo Fisher). The names and sequences of all of the primers are listed in Table S1. The osmC, cspA, hns, and mipA templates were generated by PCR amplification of E. coli genomic DNA. All of the templates contained T7 RNA polymerase promoter and initiation region of the ermCL gene at their 5′-end and the sequence complementary to the toeprinting primer NV1 at the 3′-end (43). The cat86L, cmlAL, the WT, and −1 and P-site position mutant hns templates, as well as the alanine scanning mutagenesis or N-terminal truncations of H-NS, were generated by four-primer PCRs (44), using the pair of template-specific "long" primers at 10 µM and the pair of "short" primers (T7 and NV1, or T7 and a short template-specific reverse primer) at 100 µM. The hns templates with mutant A sites were generated by a five-primer PCR. For this, each reaction contained 10 µM of one of 19 primers designed to introduce the mutant A-site codon, in combination with 10 µM of hns_trunc_rev primer corresponding to the 3′-end of the template. After the first three PCR cycles, 10 µM of primer T7_hns_fwd, corresponding to the 5′-end of the template, and 100 µM of primers T7 and NV1 were added, and the PCR continued for 27 more cycles.
We have presented evidence that antibiotics CHL and LZD, belonging to two different classes of ribosomal PTC inhibitors, do not actively block formation of every peptide bond, but instead interfere with translation in a context-specific manner. The nature of the two C-terminal nascent peptide residues as well as of the A-site acceptor strongly influence the ability of these drugs to inhibit peptidyl transfer. The presence of Ala, and to a lesser extent of Ser and Thr, in the penultimate position of the peptide stimulates the action of the drugs. In contrast, Gly in the P or A sites of the PTC strongly counteracts the antibiotics' inhibitory effect.
Our findings reveal the most extreme manifestation of the dependency of CHL or LZD action on the nature of the PTC donor and acceptor substrates. It is very likely that the action of these antibiotics is affected by a more extended context. Thus, despite a strong preference for the presence of Ala at position (−1) in the sites of the strongest arrest, this amino acid can be found in the penultimate peptide position throughout the entire spectrum of ∼60,000 codons that were analyzed in the CHL and LZD samples (Fig. S5A). Of note, the positive effect of Ala (−1) on CHL-dependent stalling at the fifth codon of the hns gene seems to be alleviated by the Leu5 to Ala mutation (Fig. 3D), an effect that was not observed at other examined sites. Therefore, the context in which Ala (−1) appears affects the strength of the drug-dependent translation arrest. For example, the stimulatory effect of Ala (−1) upon CHL or LZD action can be completely negated when Gly is present at the peptide's C terminus (Fig. S7A), whereas C-terminal Asp seems to additionally boost the effect of penultimate Ala, Ser, or Thr on CHL-dependent translation arrest (Fig. S7E). A more comprehensive analysis of the cumulative changes in codon-specific ribosome occupancy showed that both the stimulatory effect of Ala (−1) and the counteracting influence of Gly (0) and Gly (+1) may be modulated by the identity of the other amino acid residues in these three positions (Fig. S7 A–D).
Because CHL has been traditionally viewed as a universal inhibitor of peptide bond formation, it has been used to "freeze" the ribosomes on mRNA in some profiling experiments (30, 35), an approach challenged by several studies where trends similar to the ones we presented here were noted (29, 31, 36, 37). We show that exposure of the cells to high concentrations of CHL (or LZD) does not in fact freeze the ribosomes on mRNA, but allows them to redistribute from the sites less favorable for the drug-dependent arrest to the downstream strong-arrest codons. This effect, for example, can be readily observed near the sites of translation initiation. A peak of ribosome density is prominent at the start codons of many genes in the untreated cells (30) and can be visualized by the metagene analysis (Fig. S8, gray plot). In the CHL- or LZD-treated cells, the relative overall start codon occupancy is dramatically decreased, indicative of poor ability of LZD or CHL to inhibit the first peptide bond formation and the resulting redistribution of the ribosomes from the translation initiation site to the downstream codons (Fig. S8, blue and orange plots). This observation is in line with the results of toeprinting experiments where both antibiotics failed to arrest translation at the start codon (Fig. 3 and Fig. S1). Altogether, our results strongly argue against the use of CHL in the ribosome-profiling experiments if precise position of the ribosomes on mRNA is being analyzed.
Our finding that CHL and LZD act at the defined locations within the gene extends the concept of context-specific action of ribosome-targeting inhibitors (32). Macrolides, which bind to the nascent peptide exit tunnel, arrest translation at a limited number of codons within the ORF also depending on the nature of the nascent peptide and of the incoming amino acid (27, 28, 32, 38). The context requirements for macrolides, however, are principally different from those for the PTC-targeting antibiotics. The locations where LZD or CHL would arrest translation are defined primarily by individual amino acids, but the sites of macrolide action are delineated by short amino acid sequences (27, 28). As a result, PTC inhibitors halt translation at multiple locations along the gene, whereas macrolides arrest ribosome only at a limited number of codons within the ORF, and synthesis of the proteins lacking the "problematic" sequences may not be inhibited at all (32, 39). For this reason, cells treated with excess of macrolide antibiotics continue to synthesize a limited subset of polypeptides (32), whereas protein translation in cells exposed to CHL or LZD is essentially abolished (Fig. S2). Nevertheless, our data suggest that CHL and LZD should be viewed as "poor" inhibitors of peptide bond formation even within the preferred context. At high drugs concentration, translation is only transiently paused at specific codons,, leading to the appearance of a number of "arrest bands" in toeprinting gels, rather than at a unique stalling site (Fig. S1A and Fig. 3B).
Similar to the inducible macrolide resistance genes, induction of CHL resistance requires programmed ribosome stalling within the leader ORFs (40). Our findings revealed the principles of antibiotic-induced site-specific arrest of translation of the regulatory ORFs of two model CHL resistance genes. Translation of the cmlAL ORF is stalled by CHL after Ala, the amino acid most conducive to antibiotic action, appears in the penultimate position of the nascent peptide (Fig. 4C). Furthermore, synthesis of the CmlAL peptide is halted within the sequence ADK, one of the best motifs for CHL-dependent arrest (Fig. S7E). Arrest of translation of the Cat86AL leader peptide occurs within a similar context, except that Ala is replaced with Thr, another amino acid conducive to CHL action (Fig. 4C and Fig. S7E). Thus, programmed ribosome stalling responsible for induction of CHL resistance simply exploits the sequence context most favorable for the action of this antibiotic. Although the cmlA and catA86 resistance genes come from evolutionarily distant bacterial species, the sites of CHL-induced ribosome stalling within the leader ORFs of these genes conform to the contexts that we identified when exploring the CHL and LZD action in E. coli. Therefore, we believe that the uncovered trends in the specificity of LZD and CHL action are universal, rather than strain or species specific.
We still lack the understanding of the mechanistic principles of context specificity of CHL and LZD action. Crystallographic reconstructions have uncovered how these drugs bind to the PTC of the vacant bacterial ribosome (10⇓–12, 19, 20). However, the gained structural and functional implications are likely incomplete or possibly even misleading because our data clearly show that the key aspects of the drug binding and action must critically depend on the properties of the peptidyl nascent chain and the aminoacyl-acceptor. The effects of the PTC substrates could be direct, involving immediate interactions between the key amino acid residues and the drug, or indirect, when the properties of the drug-binding pocket are allosterically affected by the ribosomal ligands.
The context-specific action of the PTC inhibitors reflects the functional interplay between the nascent chain and the PTC, which has been demonstrated in a range of ribosomal functions: from programmed translation arrest, to termination and recoding (reviewed in ref. 41). Our previous studies (32) and the findings presented here strongly suggest that many antibiotics that directly or indirectly influence peptide bond formation, translocation of the substrates through the PTC, or egress of the nascent protein chain will be influenced by the sequence context. Improving our understanding of these principles will pave the way for the knowledge-based discovery of better protein synthesis inhibitors.
Materials and Methods
Ribosome Profiling.
Ribosome profiling was carried out using the antibiotic hypersusceptible E. coli strain BWDK, a derivative of the strain BW25113 with inactivated tolC gene [F−, DE(araD-araB)567, lacZ4787(del)::rrnB-3, λ−, rph-1, DE(rhaD-rhaB)568, hsdR514, tolC] (32). The overnight culture grown in LB medium was diluted 100-fold into three conical baffled 1-L flasks with 100 mL of LB supplemented with 0.2% glucose and grown with vigorous shaking at 37 °C. When the culture density reached OD600 of ∼0.5, CHL or LZD were added to a final concentration of 100 or 800 µg/mL (100-fold minimum inhibitory concentration) and incubation continued for 2.5 min. The control culture did not receive a drug treatment. Cells were harvested by rapid filtration, frozen in liquid nitrogen, and processed for ribosome profiling as described (30). The library for Illumina sequencing was prepared using RNA fragments ranging in size between 28 and 42 nt. Analysis of ribosome profiling was carried out using the GALAXY pipeline (28, 42). The first position of the P-site codon was assigned at 15 nt away from the 3′-end of the read (37). Details of analysis of the ribosome-profiling data are presented in SI Materials and Methods.
Generation of Templates for in Vitro Translation and Toeprinting Analysis.
Preparation of DNA templates for toeprinting is described in SI Materials and Methods, and the primers used are listed in Table S1. Toeprinting reactions were carried out in 5 µL of PURExpress transcription–translation system (New England Biolabs) as previously described (43, 44). The final concentrations of LZD, CHL, mupirocin, borrelidin, or thiostrepton in the reactions were 50 µM, unless otherwise indicated. Gels were quantified using ImageJ (45), and relative stalling efficiency was calculated using the following equation:
where Ab is the intensity of the band representing CHL- or LZD-dependent stalling, Cc is the intensity of the band representing ribosome stalling at the downstream "catch" codon, and Bd is the background.
We thank Joseph Dang for carrying out early experiments, Jiyoung Lee for the invaluable assistance with bioinformatics analysis, Yury Polikanov for fruitful discussions and help with preparation of Fig. 1, and Alejandro Mankin for proofreading the manuscript. This work was supported by National Institutes of Health Grant AI125518.
↵1J.M. and K.K. contributed equally to this work.
↵2Present address: Synthetic Genomics, Inc., La Jolla, CA 92037.
↵3Present address: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92307.
↵4Present address: Institut de Biologie et Chimie des Protéines, UMR5086 CNRS/Université Lyon 1, 7 passage du Vercors, 69367 Lyon, France.
- ↵5To whom correspondence may be addressed. Email: shura{at}uic.edu or nvazquez{at}uic.edu.
Author contributions: J.M., K.K., N.V.-L., and A.S.M. designed research; J.M., K.K., E.J.R., D.K., and C.O. performed research; J.M., K.K., A.K., C.O., N.V.-L., and A.S.M. analyzed data; and J.M., N.V.-L., and A.S.M. wrote the paper.
Conflict of interest statement: The research in the laboratory was supported by, among other sources, grants from the pharmaceutical companies Melinta Therapeutics and Cempra Pharmaceuticals.
This article is a PNAS Direct Submission.
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE86536).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1613055113/-/DCSupplemental.
Freely available online through the PNAS open access option.
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To keep your employees happy and productive – what HR people refer to as "engaged", you need to focus on driving employee performance.
Some business owners think of this as screaming and tearing their hair out each time something goes wrong. We think of it as using proven techniques and tools in performance management.
Another important clarification that business owners and organizational leaders need to be aware of is that the primary purpose of performance management is not necessarily to reward or sanction employees; but rather, it is to get your employees to perform.
Understanding this is critical to designing an effective performance management system.
Here is list of Top 7 powerful things that you can do to improve employee performance in your organization:
#1: Set Clear Performance Expectations:
Driven by your organizational strategy and goals, and linked to the Job roles and descriptions for each individual, you should be able to establish clear performance expectations for each of your employees.
These expectations will align with your overall strategy and goals in a manner that you can see your strategic goals "trickle down" to individual job expectations.
Say for example, a school has a strategic goal of "increasing enrolment through referrals from parents", you will find performance expectations like – Develop and Implement Referral Card Programme by 02 June" as a performance expectation for the School Administrator and "Host 2 Classroom events with 90% participation from parents" as a performance expectation for a Class Teacher.
In technical HR language these expectations are often referred to as "Key Result Areas (KRAs)" or "Performance Objectives". Effective KRAs will consist of the goals, timelines, and the measures and trackers to evaluate them. Good KRAs should be Specific,
Measurable, Achievable, Relevant and Time-bound – SMART.
Some business owners also set behaviour related expectations that usually focus on the "how" of performance and are linked to the company's values.
This will involve describing in more details the specific behaviours expected under each of your values, and measuring this over time.
Behaviour based performance expectations are more subjective than the results-based ones, and a number of business owners combine both results and behaviour expectations in their performance management systems.
Having set these expectations, the business owner must take time to clearly communicate them to employees and get their buy-in. Some companies create Performance Contracts which employees sign each year, capturing these performance expectations or targets.
#2: Provide Tools and Training:
You should then provide tools and training to enable your employees achieve their performance goals. Again, using the example of the school – you will ensure that there is a budget for each Classroom Event as well as for designing and printing the materials needed for the Referral Cards and for purchasing of bulk sms to contact parents and follow up with them to attend the events.
You may also need training in some areas related to the delivery of an employee's performance expectations, especially where the employee has no prior experience with that specific type of work. Failing to provide adequate training may ultimately impede the employee's performance.
#3: Monitor And Track Performance:
You must also design systems to monitor and track performance on all the performance objectives or KRAs that have been set.
Using our school example again, you will have a notebook where each parent signs in at the Classroom Events – this is how you will determine the actual attendance of parents viz-a-viz the target of 90%.
Continuous monitoring, tracking and reporting is good for a healthy Performance Management System.
The use of dashboards is also encouraged, wherein you create a pictorial log of everyone's performance and have it displayed, so that everyone can see what they have achieved so far.
Again in the school, you will have a notice board in the staff room where a display of the actual percentage attendance at Classroom Events will be placed and updated from time to time.
It can even be designed as a leader-board ranking all the teachers in a manner that promotes healthy competition.
#4: Measure Performance:
Usually, forward-thinking businesses will formally measure performance twice a year or in the worst case, once in a year.
At these performance appraisals, the performance expectations that were set earlier will be discussed, and the actual performance on each KRA will be reviewed. Usually the evidence of performance: trackers and dashboards from previous months will be brought together to enrich the discussion.
A well-structured and implemented system will reduce possible emotional outbursts and confrontations that happen at some performance appraisal sessions.
If your KRAs were well set and communicated, tracked and monitored effectively, you should have a tears-free performance appraisal.
#5: Give Performance Feedback:
As the year progresses especially at those points when you are publishing performance updates and results, ensure that you give feedback to employees in an effective manner – no shouting or screaming, just good old professional feedback that is balanced, observed, owned, specific and timely – BOOST.
You should also give feedback in this manner at the mid-year and year-end appraisals as applicable, but the all-year-round feedback that happens each day is much more important in line with the philosophical objective of "getting employees to perform".
#6: Reward Performance:
At the end of the performance cycle, business owners should identify employees who did well in achieving the performance targets and provide them performance rewards and incentives based on their existing reward mechanisms.
This will also be an opportunity to differentiate employees, providing lower rewards and incentives to middle and bottom performers. Incentives could include salary increments, performance bonuses, and promotions.
#7: Manage Poor Performance:
As a business owner, you should also pay attention to managing poor performance by providing performance counselling to your bottom performers as well as establishing a performance improvement plan with clear timelines to ensure that they have a clear focus on what improvements are required, the support systems to aid their performance, the timelines for improvements and the consequences of continued under-performance.
Well-structured and implemented performance improvement systems can turn around almost 85% of poor performers, leaving the balance with the options to resign voluntarily or be exited in line with your policies.
With a clear and workable performance management system like we have captured above, you do not have to tear your hair out or scream to manage performance any longer. With these practices and tools you can truly place your business on the path to greatness!

subset: commoncrawl


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How have I not alighted on this band previously? World War II (loosely) themed Canadian black metallers Panzerfaust have been kicking around for a while now but, with only three albums before this, it's perhaps understandable they could have flown under my radar despite the obvious attraction of what they're trying to patch together here. The band's name may be taken from a German anti-tank weapon but (and it pains me to have to add this these days) the band is pretty clear that that's where its interest in the Third Reich ends and that its members reject extremism in all its forms and 'stands for the free expression of all people'. The band is more interested in the terrors of war and combines that with religious – or perhaps more appropriately anti-religious – imagery and sentiment. Not so much the machine gun fire of Marduk, as might have been expected from the name alone, but a far more distinctly French depth to their sound that combines darkly delivered fervour. Dipping into their back catalogue just out of curiosity suggests the band's first couple of albums were very much in the early Marduk or Enthroned mould but things really started to shape up into something far more interesting during the band's last album 2013's Jehovah-Jireh: The Divine Anti-Logos where things took a different turn. The album, which would later attract the attention of Eisenwald who unsurprisingly snapped them up, serves up some instant gems with and …And Jesus Wept and The First Con Man, The First Fool delivering some powerful black metal.
The Suns of Perdition kicks off firmly where that left off in that Panzerfaust are clearly determined to set a course into less predictable, slightly more progressive black metal territory. Searing, nose-bleed riffs combine with the more occult-ish, progressive approach while the band use sound effects sparsely and to maximum effect, such as with the excellent strafing Stuka sounds to emphasise the sweeping percussion-driven and sinister melody on the second track 'Stalingrad, Massengrab'. The effect is a soul-destroying rapid fire dirge straight out of the Antaeus/ VI/ Aosoth/ Temple of Baal/ Order of Apollyon handbook. The Decapitator's Prayer encapsulates it all nicely in a brutal six minute assault and a piercing use of tremolo guitar that is impressive to pull off and gets better as the track reaches its climax. After three tracks that are aimed squarely between the eyes like silver, crossbow-fired crucifixes into the sheet white forehead of a vampire, it's a little disappointing to find we've already reached the final track. But what the hell. The Men of No Man's Land 13-minuter only makes you wonder what Panzerfaust could have done with another 20 minutes – and this album is billed as the first part of four after all.
The giant closer pulls just about every trick in the book and then some. The track is a glorious ode to the pointlessness of war backed by floating, rhythmic backing vocals, intermittent excerpts of sound and a bassline so thundering it at times makes you wonder whether we've crossed genres into Bolt Thrower territory. The lyrics are based largely on In Flanders Fields by World War 1 Canadian war poet John McCrae. The deluge of sound does indeed borrow some from death metal but the finished result is a feat that could only have been delivered by a black metal band incorporating such a mass of aural patterns to such effect that time loses all meaning. A great return from Panzerfaust – a band name that probably now does them a disservice, since its likely to put some more sensitive types off even checking them out, even though it does also aptly describes some of their most powerful moments. Listening to this I had a twinge of regret for missing them at their recent London date. Next time maybe… Meanwhile, here's looking forward to the next three parts of this ambitious project.
(8/10 Reverend Darkstanley)


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Best Of,United,AAdvantage,Delta,US Airways
You Don't Need to Be a Hub Captive When Collecting Miles
August 9, 2013
I think it's a big mistake to be a hub-captive. There's no reason that the credit cards you open have to be related to the dominant carrier at your airport.
Your life will be better if you instead open up the best card for the trip you want regardless of the airline that dominates your home airport.
Why should you ignore your hubby?
Imagine someone in Atlanta who wants to visit Buenos Aires.
Delta is the dominant carrier at ATL, and the only one with a direct flight to Buenos Aires.
United,AAdvantage,Award Booking
Summer 2014 Award Space Picture to Europe Right Now
August 7, 2013
Early planners are looking to book their summer trips to Europe
Rapid Rewards,United,Avios,AAdvantage,Delta,US Airways
What I'd Least Like to See Changed about Each Frequent Flyer Program
July 13, 2013
United scared me a lot on Friday when it tweeted that changes were coming to MileagePlus.
United still hasn't told us what those changes are, but insists we'll like them.
It's very unlikely I'll like them much. It could be a minor enhancement, but there are very few things I would change about United MileagePlus that United would also change.
Rapid Rewards,United,Avios,AAdvantage,Delta,US Airways
What I'd Least Like to See Changed about Each Frequent Flyer Program
July 13, 2013
United scared me a lot on Friday when it tweeted that changes were coming to MileagePlus.
United still hasn't told us what those changes are, but insists we'll like them.
It's very unlikely I'll like them much. It could be a minor enhancement, but there are very few things I would change about United MileagePlus that United would also change.
United,AAdvantage,Award Booking,Free Oneway
A Letter in My Outbox
April 7, 2013
There are a lot of reasons to use an Award Booking Service, like the MileValue Award Booking Service. One great reason is because you have miles in several accounts and want to use the most efficient type for the trip you have in mind. Different miles have different best uses.
Recently I was contacted by a friend who had mid-six-figure account balances in his American, United, and Delta accounts. He wanted a simple open jaw trip to Europe in economy class. From Washington-Dulles to Nice and returning from London to Dulles.
Below is the email I sent to him.
How to Exploit the American Airlines/US Airways Merger
February 14, 2013
The offers for the cards listed in this post have expired. Click here for the top current credit card sign up bonuses.
The merger of American Airlines and US Airways, announced today, is no news in the near term, fantastic news in the medium term, and bad news in the long term for me and similarly situated frequent flyer mile hackers.
No News in the Near Term
Your US Airways Dividend Miles are still there.
15,000 to Hawaii? We can do better!
February 13, 2013
American Airlines is offering 15,000 miles per direction awards to Hawaii. (Hat tip Lucky)
My first thought: we can do better!
1. American Airlines Award Free Oneway- On any award returning from Europe, you should be able to get a free or cheap oneway to Hawaii. Zero miles or 9,000 Avios is a better deal than 15,000 AA miles.
2. Allegiant Air $358+ roundtrip- Allegiant Air is a super-low cost carrier that announced flights to Hawaii that will start in November.
United,AAdvantage,Award Booking
Anatomy of an Award: 12,500 Miles to Asia in First Class
February 10, 2013
I recently booked a fascinating award that shows just how simply you can turn a good itinerary into a great itinerary using the the concept I laid out in a post titled 7,500 Miles to Europe and Negative 7,500 Miles to South America.
The basic premise is the inverse of a free oneway. If you are booking an award within the US on American Airlines, consider adding on an international leg to the domestic leg for very little extra.
My client Mark came to me with 105,000 American Airlines miles and 103,000 Ultimate Rewards.
American Airlines & Virgin America: Complete Buy Miles Analysis
January 7, 2013
American Airlines Buy Miles Promotion
American Airlines is offering bonus miles on all AAdvantage miles purchases through January 31. As with past promotions, the bonus miles awarded are scaled. Buying 40,000 (or more) miles will yield a 15k bonus. Buying lesser quantities will result in fewer bonus miles being awarded.
The full promotion chart can be found below.
American seems to be running these deals fairly regularly.
AAdvantage,Hawaiian Airlines,Forum Buzz
Hawaiian Airlines Exciting New Route to Taipei, Taiwan
December 17, 2012
According to this thread on FlyerTalk, Hawaiian Airlines will be adding an interesting new route to their network in July of 2013. Hawaiian will add nonstop service between Honolulu and Taipei, Taiwan three times weekly. They will be flying this route with their 294 seat A330-200 aircraft.
Scott actually just wrote a mini review of Hawaiian's A330 product in economy. You can read it in this post, 20k Points Roundtrip to Hawaii, 45k Roundtrip to Asia, 55k Roundtrip to Australia. The economy seat looks really solid with personal TVs and decent food offerings.


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To be yourself in a world that is constantly trying to make you something else is the
Member since Jul 2012
Blogging for 4 years in October since discovering my love (obsession) for makeup. Dolce Vanity consists of all things makeup, skincare (homemade, natural, organic) hauls, reviews and whatever pops into my mind that I would love to share.
- Location: Luton
- Birthday: 28 Apr
- Age: 25
- Eye Colour: Brown
- Hair Colour: Brunette
- Skin type: Combination
- Skin tone: Medium


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The purpose of the first post on this subject was to try and ascertain the interest in the frame building community in constructing their own carbon fibre frames using tube to tube construction. I have only dabbled in adding carbon to my steel frames, but the techniques for building carbon tube to tube frames appears to be much the same, in as much as they can be cut and mitred and assembled and glued in a standard frame building jig. I have been put off by the subsequent wrapping of the joints to finish off and strengthen the build, feeling this has to be done properly. I am given to understand that it can be completed without the use of an oven afterwards, presumably by using the correct type of epoxy and that wrapping of the joints is not as difficult as it may appear. My own perusal of the internet has only come across projects to produce moulded frames, which as a steel frame builder, is a completely different way of working. It also would require more space than I have available. I have not found any books on the subject and there seems to be one teaching course in the USA, in Tucson, Arizona (https://www.framebuildingschool.com/carbon). There is however some useful information on several manufacturer websites, albeit rarely related to bicycle construction per se.
I was, however, contacted by a third party frame building enthusiast who has built carbon and carbon/ titanium frames in a restricted environment and is keen to offer some help and instruction in using the complementary tube sets he has had made. These have been produced with matching dropouts in aluminium, head tubes and bottom bracket inserts, examples being below. The Tubing is Toray T700 unidirectional.
Early size options are below: Seat tube 600mm x 34.9mm O.D x 31.6mm I.D for 31.6mm seat post. Top tube 600mm x 34.9mm O.D x 31mm Down tube 660mm x 41mm O.D x 38mm Head tube 200mm x 48mm O.D x 44mm I.D for EC44/ZS44 Head set BB sleeve for T47 Bottom Bracket Seat Stays 508mm x 16.5mm O.D x 14mm I.D Chain Stays 500mm x 20mm O.D 16mm I.D Round to Round. (Tapered Chain stays will be available soon if there is sufficient interest). Apparently data sheets would be available or all the tubing. Manufacture of the final chain stay pattern is on hold depending on interest. The proposed round tapered chain stays should produce a round to round 24mm O.D tapering to 15mm O.D over 350mm – 400mm. This should be ok for tyre clearance up to 28mm using chain stay length of around 410mm.
In the previous post I asked for some feedback on the proposals and thank the 31 people who completed the short questionnaire the results of which are as follows:
35.5% Are already interested in Carbon fibre tube to tube framebuilding and are ready to go.
12.9% Are interested and are happy to research the process themselves.
54.8% Would be interested if they had some detailed guidance on the actual building process – and that includes me!
12.9% Would be interested if they had some detailed guidance AND access to a demonstration build process
3.2% Had no interest at all (that is in fact one person).
I also asked whether respondents would prefer a round or oval chain stay design for the proposed tapered stays and this was 16 to 15 in favour of round stays.
David Protheroe
David is the frame builder and prospective supplier of the carbon frame sets described above and can be contacted directly for purchase or more information. At present he is only wishing to supply complete tube kits for a bike rather than individual components. His business goes under the name of Revolucion Frames and the tubes are made in Taiwan. I understand data sheets are available for all the tubes. David has made some frames using his tubing and some using a flax/carbon tubing in titanium lugs as well. He can be contacted by email at: email@example.com, or directly on 0755141474.
An example of his carbon frame can be viewed on the Revolucionframes instagram account, though unfortunately this is not a medium I seem able to access without signing up.
I hope I may be able to post some detailed information on Carbon Framebuilding in the future, or, if there is anyone out there conversant with it that would like to send me their own documented frame build I would be happy to hear from you. In the meantime I add link to an article about the Parlee Cycles factory methods of tube to tube building which lacks detail, but you get the idea.


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By Mark A. Taylor
Everyone serving the Lord struggles sometimes with tension between external actions and internal motives.
Am I singing or preaching or teaching because I love to be in front of people, or because I love to communicate God's Word?
Do I give out of guilt or out of gratitude?
Do I approach Bible study, prayer, or weekly worship solely out of duty, or are they a delight to me?
And when it comes to ministers who lead growing churches, the tensions multiply. Am I seeking church growth to build the kingdom or to build my ego? Am I more concerned about my reputation or the needs of those we're serving? Who's glorified most because of our ministry, those leading it or God?
Ministers with the three largest Christian churches/churches of Christ in America talked about this in my Beyond the Standard discussion with them May 15. I asked them, "How do we balance concern for numerical growth with attention to spiritual growth?"
Dave Stone, minister with Southeast Christian Church in Louisville, Kentucky, said, "I prefer to talk about church health as opposed to church growth. When an organism is healthy, growth is a natural by-product." But he pointed out that seasons of plateau or times for pruning are sometimes natural or necessary. And he admitted that he had his own struggle with ego when church attendance dropped by 1,500 in the first year of his ministry after longtime Southeast minister Bob Russell retired.
Don Wilson, who serves Christ's Church of the Valley in Peoria, Arizona, looks at three external indicators of internal spiritual growth for any Christian: Are you generous? Are you serving in ministry? Are you sharing your faith?
And Jud Wilhite, minister with Central Christian Church in Henderson, Nevada, referred to 2 Corinthians 5:12, where Paul speaks about external success contrasted with internal sincerity:
Are we commending ourselves to you again? No, we are giving you a reason to be proud of us, so you can answer those who brag about having a spectacular ministry rather than having a sincere heart (New Living Translation).
Wilhite's interpretation: "Paul's alluding to the fact that people in his day were bragging about numbers, if you will. If you just look at the numbers, he wouldn't have a spectacular ministry. But he has a sincere heart."
Each of these three fellows has what could be called a "spectacular ministry." (Their three congregations are reaching a total of more than 60,000 worshippers every weekend!) And not everyone is comfortable with their success. Questions from those who called into the program all hinted at skepticism or concern about why and how megachurches achieve their numbers.
But as each of these leaders offered gracious answers to those questioners, I heard indications of humility and concern that underlie every ministry led by someone with a sincere heart.
Listen to the whole discussion with Wilhite, Stone, and Wilson, including how ministry has stayed the same for them, no matter the size of their congregations. Find the hour-long program here.
1 Comment
How many Paid Spiritual Leaders shoot for Growth because they are thinking about their salary and not thinking about what God's Will for them might be?
Just because your Fellowship is "Healthy" does not mean the numbers will grow. Noah preached for over 100 years and yet only 8 were saved. Noah's family was "Healthy" but the Numbers Growth was not there. Yet Noah trusted God for the needs of Himself and his family. He was not selling tickets or recruiting "passengers" who would then "tithe" to him.
I think American Spiritual Leaders focus too much on the Rich. That's who they generally target as they are "eyeballing" the salaries of such people and in their minds have already planned how they wish to spend the "tithe" that these Rich Members will bring to them. This is counting your chicks before they have hatched.
On the opposite end I see very little compassion for the poor who live abroad. I see little compassion for the poor in Christ abroad. American Fellowships spend Billions of Dollars on themselves to "attract" Rich people but they do very little real "Missional" work.
I myself earn my living as an Engineer. I do not ask others to give money to Rich Churches so they can build Elaborate Worship Centers. Instead I ask that others to give to the truly poor as our Vietnamese sister in Christ below herself does.
Vietnam – Kim Phuc – 2014, The Napalmed Girl who came to Christ
Jesus requires the Rich to share with the poor. This is what I strongly suggest that we American believers do.


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11 Jan 2019 13:22

Social Media And you - Advertising and marketing Data You could Know

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Admission Procedure
Admission Criteria
The school has the full responsibility for placing the pupils and students in the proper class after examination. Successful applicants will then be issued with :
* A Registration Form
* The School Brochure
* Medical Certificate Form
Admission Policy
For parents that have up to 4 children in the school, the fourth child will not pay development fee and will only pay 10% of tuition.
Application Process
The Pupil/Student returns the completed form and a copy of the teller showing evidence of payment accompanied with the following:
* 1 Recent Passport photograph
* Entrance Exam
* Testimonial
* Medical Certificate (if any)
* Letter of undertaking by Parents/Guardian
School Fees
School Fees are paid first week of resumption. Fees can be paid termly or for the whole session at once. Cash allowed but cheque payments are not allowed; all monies are paid into the bank or School.
Extra Requirements
* Black shoes with White socks (Secondary)
* Black shoes with white socks (Primary/Nursery)
* Black belt for boys(Secondary)
* School Bag
* Mathematical Set(Secondary)
* A pair of canvas for sports


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text:
- Year:
- 2017
- 3 Views
Shush! Be quiet.
I had a busy last month,
so my cards are all maxed out.
Go talk to your sister!
Bro, we're better than this.
Talk to your sister!
Send it to my account, got it?
What's going on here?
Did you see his face? He must be overworked.
Be good to him, I feel terrible for him.
You must want something.
Hey, hey, wake up.
Hey, dumbass, aren't you going to work?
What's with you all today?!
I'm off duty today!
- It's chief pyo.
- I don't care!
I'll take it then, hello?
- He won't answer...
- Give it!
I knew you'd take it.
Come eat breakfast!
Don't want any.
Good morning.
So much trash!
- Why are you here?
- Let me help.
Forget it.
Are you rested?
Like hell, I need to look for a part-time job.
15 years of partnership means shit.
This is what loyalty is all about?
I took easy on you because of our loyalty.
Remember how you lost
And the missing $5,000 after the ssangmun raid.
You came to tell me that?
Don't worry, really.
I made sure there's no strikes on your record.
- Bullshit.
- Come on!
Joint investigation?
Heard about next week's ministerial talk?
Their delegation will include a detective.
Anyway, there was a criminal
who defected to the south,
and they want to catch him themselves.
What crime did he commit?
I dunno.
It's an order from the commissioner.
Everyone's going rabid to take this case.
Cover their detective, you'll get my support.
You want me to babysit a commie?
- Don't call them that.
- Forget it!
I worked towards a just society,
but I'm just a corrupt cop.
But now you want me to do what?
How long must I sacrifice for my country?
You're hopeless, you really are.
You don't get it, do you?
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text:
Assalaam alaykum wa rahmatullahi wa barakaatuh. If a person converts to Islam during the day does he/she have to make up for the prayers that he/she has lost of that day? For example if a person converts to Islam when it is asr time. Do he have to make up fajr and dhuhr? Jazakallahu khayran.
All perfect praise be to Allaah, The Lord of the Worlds. I testify that there is none worthy of worship except Allaah, and that Muhammad is His slave and Messenger.
If a non-Muslim embraces Islam, then he is obliged to pray the prayer that is presently due and he is not obliged to make up for the prayers whose time have already expired.
However, the question remains as to whether a new Muslim is obliged to perform the first prayer of the two prayers that are permissible to combine at the time of one of them. For example, if someone accepts Islam at the time of 'Asr, is he obliged to perform the Thuhr prayer, or if he accepts Islam at the time of 'Ishaa, is he obliged to perform the Maghrib prayer? The scholars have two different opinions regarding the obligation to perform those previous prayers; some of them said that it is obligatory to perform them, and others said that it is only obligatory to perform the present prayer.
Ibn Qudaamah said in his book Al-Mughni: "If a menstruating woman becomes pure, and a non-Muslim embraces Islam, and a child reaches puberty before sunset, then they should pray Thuhr and then 'Asr, and if a child reaches puberty, and a non-Muslim embraces Islam and a menstruating woman becomes pure before dawn, then they should pray Maghrib and 'Ishaa'. This view about a menstruating woman who becomes pure was traced back to 'Abdur-Rahmaan ibn 'Awf, Ibn 'Abbaas, Taawoos, Mujaahid, An-Nakha'i, Az-Zuhri, Rabee'ah, Maalik, Al-Layth, Ash-Shaafi'i, Is-haaq, and Abu Thawr.
Imaam Ahmad said: 'All the Taabi'is are of this view, except Al-Hasan who said: She is only obliged to perform the prayer during the time of which she became pure.' This is the view of Ath-Thawri and the view of the scholars of the Hanafi School. The basis is that the time of the first prayer expired at the time when she had a sound excuse, so this prayer is not an obligation on her, in the same way that if she caught nothing from the time of the second prayer. It was also reported from Maalik that if one has time to perform five Rak'ahs in the time of the second prayer, then the first prayer becomes an obligation, because the time enough for the first Rak'ah of the five Rak'ahs is a valid time for praying the first prayer when one is excused, so it (the first prayer) is an obligation because there is time to pray do so (to pray one Rak'ah of the first prayer) just as if one had that much time from its preferred time, contrary to having less time than this.
Our evidence is what Al-Athram, Ibn Al-Munthir and others narrated with their chain of narrators on the authority of 'Abdur-Rahmaan ibn 'Awf and 'Abdullah ibn 'Abbaas that they said about a menstruating woman who becomes pure one 'Rak'ah before the Fajr prayer: she has to pray Maghrib and 'Ishaa', and if she becomes pure before sunset, then she has to pray Thuhr and 'Asr altogether.
Another basis that the time of the second prayer is a time for the first prayer in situations where one is excused, so if a person who is excused [has a sound reason] catches this time (of the second prayer), he is obliged to pray the first prayer in the same manner that he is obliged to pray the second prayer."
Allaah Knows best.
You can search for fatwa through many choices


text:
Where to Find Cryptomining Farm
HashFlare would like to make it simple. Since that time, several cryptocurrencies have been made. To begin with, there are two types of fees.
About cloud mining companies There is a lot of cloud mining providers on the market which provide a wide variety of services. Sure it will take some opportunity to setup each one of the different wallets and mining software but when all is said and done, you will delight in a bigger profit margin for your mining venture. If a cloud mining provider accepts bitcoins then there's an excellent chance it's a scam.
The profit calculator on Cryptomining Farm helps you choose which plan is the one which is suitable for your needs to the very best. So when the hiring company hasn't provided a salary for work, we look at salary data from related businesses and locations to think of a sensible estimate for what you may expect. Fantastic condition performance of finance indicates the potency of management in deal with the financial in company.
The site is neatly laid out and also provides a fee estimator to permit you to calculate daily profits in exchange for the hashpower you get. Secondly, among the only parts of information that they offer their potential investors is that their profits are ensured. Any company offering completely free trials, particularly if they require payment info, is most likely a scam.
There's much more to learn about Bitcoin and associated technologies. You pay the business and rent out a number of the hardware. Software is vital because it lets you direct your hardware's hash power to the pool you want.
How to Choose Cryptomining Farm
This difficulty is reset every fourteen days and is founded on the normal network hash power (how many attempts per second the range of miners connected to the network can make to locate an appropriate hash). There's no more info about what are GHS exactly if they're running a private blockchain in the background or the way that it is connected to bitcoin. Quite simply, acquiring lots of hashes alone does not guarantee a reward, it simply increases the odds of locating a block.
How to Find Cryptomining Farm on the Web
Ohhh wait, it is not so accurate. If you enjoy enthusiasm, try out the Bitcoin brokers and check your trading abilities. All the UFOMiners' equipment undergoes a two-stage test procedure to be able to fulfill the maximum quality standards.
Bitcoin has plenty of unique parts you might benefit from understanding and knowing more about. The very first thing you're likely to have to do is to choose the hardware which will be utilized to mine Bitcoin. Let's look at the most well-known algorithms today.
Ok, I Think I Understand Cryptomining Farm, Now Tell Me About mingergate Cryptomining Farm!
With offices all over the world, Bitfury is manned by professionals from all types of fields and all sorts of places, all working with each other to preserve the accessibility and transparency of the Blockchain, along with creating tools to streamline its usage. As crypto-currency has grown in popularity around the world, the demand for cloud mining programs has grown too, and customers ought to be able to locate a superior alternate for their needs so long as they look into companies which specialize in their selection of crypto-currency. HashFlare is an internet website, launched in the start of 2015, offering a variety of cloud mining services.
Introducing Cryptomining Farm
If somebody wants to join the mining industry of crypto-currency, but it does not need to have to manage the noise and heat due to the mining equipment. You may mine all on your own with just a single mining client. Everyone can buy exclusive mining equipment.
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Why Almost Everything You've Learned About Cryptomining Farm Is Wrong
Central bank representatives have stated that the adoption of cryptocurrencies like bitcoin pose a considerable challenge to central banks' capacity to influence the cost of credit for the entire economy. In Russia, though cryptocurrencies are legal, it's illegal to really purchase goods who have any currency apart from the Russian ruble. Therefore, they ought to guarantee that their existing assets are ever sufficient to cover the recent liabilities.
With mining, it's important to know different kinds of blocks that arrive with it as a result of the effect it can have on your expected income. The approach takes just a few minutes to hours. Rather than hooking up your Bitcoin miner which you have bought you can outsource it further by purchasing a mining contract that provides you rights to a specific amount of hash power for a specific period of time.
Bitcoin might be the absolute most popular cryptocurrency thus far, but it doesn't indicate it's the very best crypto to mine. The absolute most advanced mining hardware and just freshly mined Bitcoins, Ethereum and Monero straight from the block rewards are a few clicks away. KanoPool has existed since 2014.
What Needs to be Done About Cryptomining Farm Before You Miss Your Chance
If you're feeling exactly the same way since I was before, don't hesitate to use my spreadsheet info. There's additionally a brief explanation about the cryptocurrency mining workflow, for people who would love to get more knowledge and an in-depth comprehension of its working mechanics. The degree of complexity may also increase through time, therefore it can become more difficult to mine them.
The Fundamentals of Cryptomining Farm Revealed
If this is the case, you might want to read our extensive Hashflare review. This decision has resulted in outrage among users. There are two issues with this claim alone, the first being that if a solution or service delivers a guarantee, they have to provide you some kind of a bonus or incentive should they don't meet their guarantee.
The Chronicles of Cryptomining Farm
Therefore, if you're a high roller which wants to make the most of the new cryptocurrency highway, Bitfury may be your very first stop. If your searching for a different type of investment with faster returns it is possible to try Bitconnect. At the exact same time, Bitcoin is growing more and more popular, and a growing number of individuals are getting into mining for the same quantity of reward.
Cryptomining Farm Help!
It is possible to use a mining calculation app to look at the rates. It's a disruptive and bold undertaking, but one that's simple as a solution, and additionally quite necessary in the time of information privacy. Quite often, free VPN services only allot a limited quantity of data usage per month.


text:
I said the other day that I couldn't wait to see Total War : Warhammer in action. We've seen a lot of promo screens and the like but alas, how the game will work from minute to minute is lost on us.
While I don't have what the actual game will look to play, perhaps this is the second best thing. An in-engine trailer has been released by the Creative Assembly showing off a huge battlefield full of men, orcs, trolls, dragons and more.
Take a look:
I really am excited for this iteration of the Total War franchise. I just want to see what it looks like to play now.


text:
The Daily Mail are attempting to crush Tottenham Hotspur by breaking two stories which would horrify Spurs fans. After they revealed that Modric may be on his way to Manchester United for £25 million, they inserted another nail into the coffin of Tottenham's potential transfer list by revealing Gonzalo Higuain's ambition to stay at Real Madrid.
They have reported that Higuain has stated that he wants to stay at the home of the La Liga champions despite his lack of first team football in the past season. The 24 year old has apparently told his agent and the club that he wants to stay and fight for his place in the team.
But, his scoring record clearly shows why he is being targeted by clubs such as Manchester City and Chelsea. Playing 54 games in total (including coming on as a substitute) in all competitions, the striker scored 26 goals and assisted a further 10. As a result of this, Higuain was responsible for approximately 22% of Real Madrid's goals in all competitions last season.
But what about Modric? The newspaper have alleged that Manchester United have offered Tottenham a £25 million initial fee with a further £5 million being spent on future clauses. With a rumoured £125,000 being offered to the Croatian on a 4 year deal, the Tottenham chairman would struggle to fight off the Red Devils if they match previous offers for the Tottenham playmaker.
But as the offer stands, it is extremely unlikely that Spurs will accept £25 million. With 3 years still remaining on Modric's contract, it feels that an offer of the higher £30 millions will be required for Levy to be persuaded to see farewell to Luka Modric.
But as it stands, it doesn't look like Modric will be on his way out. With a stubborn chairman and the need to reinvest into younger talent with world class funds, it will take a tough negotiation process before Modric may have the opportunity to leave the Lane for good.
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Bruce AllenBruce Allen Attorney
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I'm trying to create a click-once deployment of a desktop app using the .NET SDK 100.0.0. When the application is installed and run it throws an error saying 'Error initializing ArcGISRuntimeEnvironment. Unable to find ArcGISRuntime deployment folder.' Everything runs fine within the development platform.
It appears the ArcGISRuntime100.0 folder is created in the deployment folder, but it is not present in the app.publish folder.