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Identification and validation is the first phase of target-based antimicrobial development. BfmR (RstA), a response regulator in a two-component signal transduction system (TCS) in Acinetobacter baumannii, is an intriguing potential antimicrobial target. A unique characteristic of BfmR is that its inhibition would have the dual benefit of significantly decreasing in vivo survival and increasing sensitivity to selected antimicrobials. Studies on the clinically relevant strain AB307-0294 have shown BfmR to be essential in vivo. Here, we demonstrate that this phenotype in strains AB307-0294 and AB908 is mediated, in part, by enabling growth in human ascites fluid and serum. Further, BfmR conferred resistance to complement-mediated bactericidal activity that was independent of capsular polysaccharide. Importantly, BfmR also increased resistance to the clinically important antimicrobials meropenem and colistin. BfmR was highly conserved among A. baumannii strains. The crystal structure of the receiver domain of BfmR was determined, lending insight into putative ligand binding sites. This enabled an in silico ligand binding analysis and a blind docking strategy to assess use as a potential druggable target. Predicted binding hot spots exist at the homodimer interface and the phosphorylation site. These data support pursuing the next step in the development process, which includes determining the degree of inhibition needed to impact growth/survival and the development a BfmR activity assay amenable to high-throughput screening for the identification of inhibitors. Such agents would represent a new class of antimicrobials active against A. baumannii which could be active against other Gram-negative bacilli that possess a TCS with shared homology.
IMPORTANCE Increasing antibiotic resistance in bacteria, particularly Gram-negative bacilli, has significantly affected the ability of physicians to treat infections, with resultant increased morbidity, mortality, and health care costs. In fact, some strains of bacteria are resistant to all available antibiotics, such as Acinetobacter baumannii, which is the focus of this report. Therefore, the development of new antibiotics active against these resistant strains is urgently needed. In this study, BfmR is further validated as an intriguing target for a novel class of antibiotics. Successful inactivation of BfmR would confer the multiple benefits of a decreased ability of A. baumannii to survive in human body fluids, increased sensitivity to complement-mediated bactericidal activity and, importantly, increased sensitivity to other antibiotics. Structural studies support the potential for this "druggable" target, as they identify the potential for small-molecule binding at functionally relevant sites. Next-phase high-throughput screening studies utilizing BfmR are warranted.
The incidence of infections due to multidrug-resistant, extensively resistant, and pan-drug-resistant (MDR, XDR, and PDR) Gram-negative bacilli (GNB) is increasing, with Acinetobacter baumannii one of the most problematic species (1–8). Treatment of infections due to these strains has become challenging, with resultant increased morbidity, mortality, and health care costs (9–11). The promise of a postantibiotic era is on the cusp of being fulfilled for A. baumannii (1–8, 12, 13), and a true PDR strain has been reported (14). This reality has been appreciated for a number of years, with the 2004 report by the Infectious Diseases Society of America, Bad Bugs, No Drugs: As Antibiotic R&D Stagnates, a Public Health Crisis Brews, being one of several attempts to increase exposure and enable solutions (15–20). However, development of new antimicrobials is a long, arduous, and expensive process. As a result, many major pharmaceutical companies left the antimicrobial research and development (R&D) business (19, 21–23). The burden of early-stage development has fallen in part to academia and to "start-up" companies. The term ESKAPE pathogens was coined to advertise the major culprits, with Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter spp. being the initial GNB implicated. A. baumannii, with its propensity of being XDR and PDR, has been termed the "poster child" for this ever-increasing threat to health care. The need to identify new antimicrobials active against A. baumannii is more pressing than ever. Therefore, as a first step, our group identified A. baumannii essential genes in vivo (i.e., those required for bacterial growth and survival in a host) which represent potential novel drug targets (24), with the long-term goal to develop a new class of antimicrobials. In support of such efforts, new regulatory paths may make the development of focused treatments for XDR pathogens, such as A. baumannii, commercially viable (25).
One of the potential targets identified as essential in vivo was BfmR (24). BfmR is the response regulator of a two-component signal transduction system (TCS) which propagates the signal from its corresponding sensor histidine kinase BfmS and acts as a transcriptional regulator (26). The environmental stimuli that the BfmR/BfmS (also referred to RstAB) system responds to are unknown, other than a direct or indirect effect of sub-MICs of chloramphenicol in the strain ATCC 17978 (27). Such systems have been considered potential drug targets (28), and the histidine kinase QseC has been successfully targeted by using the small organic molecule LED209 (29). Although LED209 inhibited virulence in vivo, it did not inhibit pathogen growth. Since inactivation of BfmR resulted in loss of viability and was essential in vivo (24) and important for in vivo pathogenicity in a murine pneumonia model (30), its successful targeting may result in even greater efficacy than that observed for antimicrobials targeting QseC. Phenotypes conferred by BfmR include increased resistance to complement-mediated bactericidal activity, biofilm formation, and scaffolding of the outer membrane protein OmpA, as well as increased resistance to selected antimicrobials (e.g., carbapenems) (27, 31, 32). In this report, we extend our initial biological observations on BfmR and add structural insights focused on its receiver domain (residues 1 to 130; BfmR1‑130) that further support this protein as a potential drug target in A. baumannii.
Disruption of BfmR synthesis affects growth/survival in human ascites fluid and serum ex vivo, but not in rich laboratory medium.A critical criterion for a potential drug target is essentiality. We have previously published data that demonstrated BfmR as being essential in vivo in a rat infection model (24). To extend this observation, we performed quantitative growth/survival studies in human serum and ascites fluid samples. AB307.70 (ΔbfmR) underwent a 2-log complement-mediated decrease in growth/survival over 24 h in serum (Fig. 1A). This loss in growth/survival was restored to wild-type (wt) levels when AB307.70 was complemented in trans (AB307.70/pNLAC1[bfmR]). Surprisingly, when AB307.70 was grown in serum in which complement activity had been inactivated (Δ56°C), growth was not restored to wild-type levels, but instead an approximate 1-log loss in growth/survival was observed (Fig. 1A). Importantly, the decrease in growth/survival of AB307.70 was significantly greater in untreated serum than in serum in which complement was inactivated. Likewise, AB307.70 underwent a greater-than-3-log decrease in growth/survival when grown in human ascites fluid, and this loss in growth/survival was again complemented in trans (AB307.70/pNLAC1[bfmR]) (Fig. 1B). These data further support an essential role for BfmR for optimal growth/survival of AB307-0294 in these clinically relevant environments. In contrast and, importantly, AB307.70 is capable of growth in rich laboratory medium, with a slight decrease in log-phase growth but a similar plateau for cell density as with AB307-0294, thereby excluding a generalized growth defect (Fig. 1C). These data demonstrate that BfmR mediates (directly or indirectly) resistance to complement-mediated bactericidal activity. These data also demonstrate that BrmR regulates bacterial factors needed for optimal growth, presumably via nutrient acquisition, since decreased growth of AB307.70 was observed in serum compared to growth of its wild-type parent when complement was inactivated.
Disruption of BfmR synthesis has no effect on capsule production.Previously published work by our group demonstrated that in A. baumannii the capsular polysaccharide was a major factor contributing to resistance of complement-mediated bactericidal activity (33). Therefore, as a first step in examining the mechanism by which BfmR confers serum resistance, the production of the K1 capsular polysaccharide in AB307-0294 (wt) and AB307.70 (ΔbfmR) was assessed. Both strains were grown in LB medium at either 21°C (room temperature) or 37°C, and cell lysates or supernatant fractions underwent Western blot analysis using the monoclonal antibody (MAb) 13D6, which has been shown to recognize the K1 capsule present on AB307-0294 (33). Surprisingly, capsule was produced in roughly equal amounts by both AB307-0294 and AB307.70 (Fig. 2A). Further, the majority of capsular polysaccharide was cell associated. Next, Western blot analysis was performed to determine if growth at 37°C in minimal medium and human ascites fluid (in which complement was inactivated to enable the growth of AB307.70) affected the production of the K1 capsular polysaccharide in AB307-0294 and AB307.70 (Fig. 2B). These data clearly demonstrated that the loss of BfmR does not decrease capsule production. Taken together, these data do not support BfmR as a positive regulator of capsule production. Further, the mechanism by which BfmR confers resistance to complement-mediated bactericidal activity appears to be independent of capsule production.
BfmR increases resistance to the antimicrobials meropenem and colistin.Previous studies had suggested that BfmR caused increased resistance to colistin (polymyxin E), rifampin, erythromycin, and imipenem (27, 31). However, these studies used the A. baumannii type strain ATCC 17978, which is of unclear clinical significance (34). Therefore, we assessed the sensitivities of AB307-0294 (wt), AB307.70 (ΔbfmR), and AB307.70/pNLAC1[bfmR] to the carbapenem antimicrobials meropenem, colistin, and tigecycline. BfmR did not affect the activity of tigecycline (Fig. 3C). In contrast and consistent with prior studies (27, 31), BfmR significantly increased resistance to meropenem and colistin (Fig. 3A and B). The MIC for meropenem decreased from 1.0 µg/ml to 0.125 µg/ml, and for colistin it decreased from 2.0 µg/ml to 1.0 µg/ml with the loss of BfmR. These data demonstrate that BfmR mediates an increased level of resistance to two important classes of antimicrobials.
Disruption of BfmR synthesis in a second A. baumannii clinical isolate (AB908ΔbfmR) does not affect capsule production and decreases growth/survival in human ascites fluid and serum ex vivo.To confirm that the phenotypic effect of BfmR was not unique to AB307-0294, a site-directed disruption of bfmR was created in the contemporary clinical isolate AB908, resulting in the construct AB908ΔbfmR. As was observed for AB307-0294, disruption of bfmR did not affect capsule production (Fig. 4A), resulted in decreased growth/survival in human ascites fluid and serum (Fig. 4B and C), and demonstrated similar growth in rich laboratory medium compared to its wild-type parent (Fig. 4D). Likewise, as observed for AB307.70, when AB908ΔbfmR was grown in serum in which complement activity was inactivated (Δ56°C), growth was not restored to wild-type levels (Fig. 4B). These data support that the phenotypic effects of the disruption of BfmR synthesis were similar in 2 clinically relevant strains of A. baumannii.
BfmR is not homologous to human proteins and is conserved among isolates of A. baumannii.A desirable trait of a potential antimicrobial drug target is a lack of homology to human proteins in order to enable target specificity. An amino acid sequence similarity search against the human proteome revealed a lack of any human proteins homologous to BfmR. This was an expected result, as TCSs are not known to be present in humans and other mammals (35, 36).
A second desirable trait of an antimicrobial target is overall protein conservation, and perhaps more importantly, conservation of the targeted drug binding site across bacterial species and strains of interest. BfmR consists of 238 amino acids, has a predicted molecular mass of 27.1 kDa, and has a predicted pI of 4.98. A BLASTp search of all nonredundant A. baumannii protein sequences (14 July 2015 data set) identified 908 proteins that were ≥99% identical (majority, 100%; over ≥99% coverage) to AB307-0294 BfmR. This analysis was complemented using the OMA orthologue database (September 2014 release) (37), which employs only high-quality complete genomic sequences. BfmR was 100% prevalent with fully identical sequences in the eight A. baumannii strains annotated in the OMA (Orthologous MAtrix) database (AB307-0294, ATCC 17978, 0057, AYE, ACICU, SDF, 1656-2, and TCDC-AB0715) plus the two A. baumannii strains sequenced by our group in addition to AB307-0294: AB853 (blood isolate from Iraq) and AB979 (environmental isolate from Iraq) (unpublished data). Additionally, two other Acinetobacter species present within OMA, A. baylyi ADP1 (a nonpathogenic soil bacterium) and A. calcoaceticus PHEA-2 (an industrial wastewater isolate) both contained a BfmR orthologue with fully identical amino acid sequences. These data support that BfmR is conserved across A. baumannii strains.
BfmR1-130 crystal structure.The ability to determine a crystal structure is an important tool for characterizing ligand-protein interactions during downstream drug development. A. baumannii BfmR is predicted to belong to the OmpR/PhoB response regulator family based on sequence analysis, and it contains a receiver domain (BfmR1-130) and a DNA binding effector domain (BfmR131-238). BfmR1-130 was selected for structural studies, as conformational changes within the receiver domain dictate DNA binding activity of the effector domain. BfmR1-130 was a promiscuous crystallizer, with multiple hits observed in the automated 1536 condition crystallization screen. A selection of chemically diverse crystallization conditions were evaluated for optimization, but in each case the resulting BfmR1-130 crystals were relatively small. Evaluation of X-ray diffraction by crystals grown under a condition that was inherently cryoprotective demonstrated that the rod-like crystals of 0.15 to 0.20 mm length but only ~0.03 mm in diameter, with a hexagonal cross-section, produced data usable for the structure determination (Table 1).
The BfmR1-130 crystal structure was readily determined by molecular replacement, using the PhoP receiver domain from Bacillus subtilis (PDB 1MVO ; protein sequence 50% identical and 73% similar over 119 residues) as the search model. The BfmR1-130 crystal structure asymmetric unit contained two protomers, associated as a dimer by a noncrystallographic 2-fold rotation (root mean square [RMS] deviation for the C-α atom superposition of the two protomers is 0.18 Å). The space group was determined to be P65, with symmetry-related reflections scaling much poorer in the higher-symmetry P6522 space group (Rmerge, 8.6% versus 35.2%, respectively, to 2.10-Å resolution). The final refined model possessed a defining electron density for Lys6-Thr125 for both protomers, plus 181 modeled solvent molecules. The fold of an individual BfmR1-130 protomer is highly similar to the α5-β5 doubly wound motif observed for other OmpR/PhoB family receiver domains, as expected (38).
BfmR1-130 homodimerization.The OmpR/PhoB family of response regulators is activated by autophosphorylation of a conserved aspartate residue in the receiver domain (Asp58 in BfmR). Phosphorylation induces an equilibrium shift favoring the active conformation, which includes formation of a 2-fold symmetrical homodimer involving the α4-β5-α5 face of the receiver domain. This homodimeric conformation facilitates the effector domain to bind DNA and modulate transcription (39, 40). A small molecule that interrupts this activation in BfmR would be a potential drug-lead candidate. In the BfmR1-130 crystal structure described here, Asp58 was not subjected to phosphorylation or the presence of a phosphorylation mimic (e.g., BeF3−). Moreover, the divalent cation (Mg2+ or Mn2+) required for autophosphorylation was not observed in the electron density in the cleft neighboring Asp58. Thus, BfmR1-130 is expected to be in an inactive state (i.e., a monomer or a nonproductive homodimer complex) within this crystal structure.
The BfmR1-130 crystal structure exhibited a noncrystallographic 2-fold symmetric homodimer in the asymmetric unit, formed by the α1-β2-α2 face of each protomer (Fig. 5). This α1-β2-α2 interface buried a total of 906 Å2 (6.7% of the total surface area). A search of the PDB revealed that this specific homodimer interface has not been previously observed for response regulator receiver domains. Moreover, the BfmR residues that mediate this interface are poorly to modestly conserved in other OmpR/PhoB family receiver domains (Fig. 6). A second BfmR1-130 noncrystallographic 2-fold symmetric homodimer was also present, unexpectedly exhibiting the α4-β5-α5 interface typical of OmpR/PhoB family receiver domains in the active phosphorylated conformation. This homodimer was comprised of a protomer A and a protomer B from two neighboring asymmetric units. This interface buried a total of 2,051 Å2 (or 15.3% of the total surface area), similar to that observed in crystal structures of receiver domains activated using BeF3− (41, 42). Furthermore, the amino acid composition of the BfmR1-130 α4-β5-α5 interface was highly similar to that present in other OmpR/PhoB family receiver domains (Fig. 6). For example, comparison of the BfmR1-130 structure with the analogous interface in BeF3−-activated Escherichia coli PhoB (PDB 1ZES ) revealed that of the 21 interface residues per protomer, 12 (57%) are identical and 5 (24%) are similar in PhoB. By comparison, the overall residue identity between these two receiver domains is 39%. The four residue positions that are significantly different at this interface are BfmR His78, Gln79, Gln93, and Val113 versus PhoB Asp76, Ile77, Arg91, and Glu111, respectively. The BfmR His78-Gln79/PhoB Asp76-Ile77 residues participate at their respective dimer interfaces through formation of hydrogen bonds between their main chain carbonyl oxygens to the guanidinium group of BfmR Arg124′/PhoB Arg122′, respectively, where the prime symbol indicates a residue belongs to the partner protomer within the dimer. In PhoB, a salt bridge is formed across the dimer interface by Arg91 and Glu111′. The positionally equivalent residues in BfmR, Gln93 and Val113′, are incapable of participating in an analogous interfacial salt bridge. However, to partially compensate for the loss of this stabilizing interaction in BfmR, Gln93 participates in a hydrogen bond with the guanidinium group of Arg117′ across the interface.
The BfmR1-130 α4-β5-α5 dimer interface is also three-dimensionally similar to the typical OmpR/PhoB family receiver domain active homodimer structure. The RMS deviations for superimposed C-α atoms from BfmR1-130 and E. coli PhoB (PDB 1ZES ) individual protomers range between 0.96 and 1.08 Å, versus 1.12 Å for the superimposed α4-β5-α5 homodimers. In addition to homodimerization, the canonical activation mechanism involves a characteristic shift of a serine or threonine switch residue toward the phosphorylated aspartate, such that the side chain hydroxyl group forms a hydrogen bond to the phosphate (42). An aromatic residue (tyrosine or phenylalanine) on the β5 strand undergoes a rotamer change to shift its bulky side chain away from the α4-β5-α5 dimerization interface and into the volume previously occupied by the Ser/Thr side chain. The BfmR1-130 crystal structure exhibits the active conformation of these two residues (Thr85 and Tyr104), further suggesting that this structure represents the active form of the BfmR receiver domain, despite the absence of phosphorylation or a phosphorylation mimic (Fig. 7).
Prediction of small-molecule binding hot spots.Computational predictions (via the FTMap server) of small-molecule binding hot spots (i.e., surface regions with major contributions to ligand binding free energy) on BfmR1-130 were performed to estimate the likelihood that small-molecule inhibitors acting specifically through the BfmR receiver domain can be identified (43). This analysis predicted 11 binding hot spots on the BfmR1-130 protomer surface, which could be classified as very strong (1 predicted hot spot), strong (3 predicted hot spots), medium (3 predicted hot spots), or weak (4 predicted hot spots). It was hypothesized that a BfmR inhibitor would likely bind at the α4-β5-α5 face, thereby preventing productive formation of the active BfmR homodimer, or it would bind near Asp58 to inhibit phosphorylation. Six binding hot spots were identified at these locations (Fig. 8A). A medium binding hot spot was present in the cleft near Asp58, occupying the volume where phosphorylation would occur. A single weak hot spot was predicted on the middle of the α4-β5-α5 face, in a shallow cleft between strand β5 and helix α5, and one medium and two strong hot spots were predicted on the periphery of this dimerization face. A sixth predicted binding hot spot was located neighboring the phosphorylation site, between helix α1 and the N terminus of helix α5.
Screening of drug and drug-like virtual libraries.The binding hot spot predictions were based upon docking of chemically simple probes composed of 2 to 8 nonhydrogen atoms. The potential for BfmR to bind more elaborate drug-like small molecules was further evaluated through a blind docking strategy. This in silico analysis was performed by docking virtual libraries representative of FDA-approved, NCI diversity 3 and LOPAC1280 compounds over the entire BfmR receptor domain protomer surface to predict preferential binding sites. Two clusters of high-scoring docked molecules were present about a subset of the FTMap-predicted binding hot spots at functionally important locations, indicative of locations likely to be suitable for binding drug-like molecules (Fig. 8B). Specifically, one cluster was at the dimer interface occupying both the binding hot spot at the cleft formed by the N terminus of strand β5 and the C terminus of α5 (red mesh in Fig. 8B) and the hot spot at the center of the α4-β5-α5 face (blue mesh in Fig. 8B). Binding interactions for this cluster also extended under the C-terminal ends of helices α3 and α4. A second cluster occupied a binding hot spot in the cleft between helix α1 and the N terminus of helix α5 (gray mesh in Fig. 8B). Several molecules in this docking cluster extended to also occupy the binding hot spot located in the phosphorylation site (cyan mesh in Fig. 8B).
Findings from this report further support BfmR as a potential drug target of A. baumannii. An important and highly appealing characteristic of BfmR is that its inhibition would have the dual benefit of significantly decreasing in vivo survival as well as increasing the sensitivity of A. baumannii to other antimicrobials.
A biological property that supports this concept is the previous demonstration that BfmR is essential for survival in vivo (24). Here, we have demonstrated a mechanism responsible for this phenotype. BfmR enhances growth/survival in human ascites fluid and serum, which was mediated, at least in part, by conferring resistance to complement-mediated bactericidal activity (Fig. 1A and B and 4B and C). A recent report that studied A. baumannii strain ATCC 17978 also demonstrated that BfmR increased resistance to complement-mediated bactericidal activity and that this phenotype was mediated by decreased capsule production (27). However, the mechanism by which BfmR mediates resistance to complement-mediated bactericidal activity in AB307-0294 and AB908, the strains assessed in this study, appears to be different. Although our group previously demonstrated that capsular polysaccharide confers resistance to complement-mediated bactericidal activity (33), in AB307-0294 BfmR does not affect K1 capsule production (Fig. 2). An alternate explanation would be the production of a second, non-K1 capsular polysaccharide. However, surface polysaccharide was purified by extraction with pyridine acetate (44) from AB307-0294 (wt) and AB307::Tn17 (both capsule positive) and mutant constructs with disruptions in either a K1 nucleotide-sugar biosynthetic gene (gna; AB307::Tn69) or a K1 capsule export gene (wzc; AB307.30) (both K1 capsule negative) (33, 45), resolved by SDS–8% polyacrylamide gel electrophoresis and subjected to alcian blue staining. A capsule was observed for AB307-0294 and AB307::Tn17, but not AB307::Tn69 or AB307.30, thereby excluding a cryptic second capsule (data not shown). Therefore, the mechanism by which BfmR confers resistance to complement-mediated bactericidal activity is independent of capsule production. This difference is likely strain dependent. AB908 is a contemporary clinical isolate, and the model pathogen AB307-0294 is a blood isolate that was frozen from the primary clinical microbiology laboratory culture, thus maintaining its genetic integrity. It possesses a complete capsule, is resistant to 90% human serum, and is virulent in rat pneumonia and soft tissue infection models and a mouse systemic infection model (46). Disruption of the capsule transport gene wzc results in the loss of capsule but is not lethal (33), and capsule is primarily cell associated (this study). In contrast, the ATCC 17978 strain produces a thin capsule (27), is relatively sensitive to complement-mediated bactericidal activity (34), disruption of wzc appears to be a lethal mutation (27), significant amounts of capsule are detected in culture supernatant (27), and it is less virulent in both mouse and Galleria mellonella infection models (34, 47). Therefore, ATCC 17978 does not appear to be an optimal clinical representative of A. baumannii, making the translational implications of studies using this strain unclear.
Although the effect of BfmR on capsule production was discordant between AB307-0294 and ATCC 17978, its effect on conferring increased resistance to selected antimicrobials was similar (27, 31). Previous published studies demonstrated that BfmR increased resistance to colistin, imipenem, rifampin, and erythromycin, but not chloramphenicol, ceftazidime, amikacin, or ciprofloxacin (27, 31). These data are consistent with the concept that BfmR confers increased resistance to certain antimicrobials. Limitations of those studies were the use of ATCC 17978, which is not an optimal representative of A. baumannii, and the fact that erythromycin and chloramphenicol are not antimicrobials usually considered for the treatment of infections due to A. baumannii. In this report, we used a clinically relevant strain and antimicrobials. The mechanism that mediates this effect is unclear; however, a previous report demonstrated that BfmR affected the scaffolding of OmpA (31). It is tempting to speculate that alterations in outer membrane proteins could affect the sensitivity to certain antimicrobials. Regardless of the mechanism, these data add further support to BfmR as an antimicrobial target, since its inactivation would also have the additional beneficial effect of increasing its sensitivity to other antimicrobials.
Lastly, although we did not assess the effect of BfmR on biofilm formation, other investigators have demonstrated that it mediates increased biofilm production (32). Biofilms can directly contribute to infection (e.g., intravascular access or implanted devices). Therefore, targeting BfmR may have further benefit for these difficult-to-treat infections.
Understanding the structure of a potential drug target may provide valuable information for guiding drug development. For TCS response regulators, structure-function insights may identify possible ligand binding sights that will modulate formation of the active homodimer. BfmR exhibited several interesting structural features. The active homodimeric conformation of BfmR1-130 was observed despite not undergoing phosphorylation or phosphorylation mimicked by BeF3− or other means. This unexpected active form has been observed previously for other OmpR/PhoB family receiver domains. It is thought that the high protein concentration present during crystallization may shift the equilibrium from monomer (or a nonproductive dimer) to the active homodimeric form (41, 42, 48, 49). Conversely, the crystallization conditions and resulting crystal lattice packing may favor the active BfmR homodimer.
The computational prediction of small-molecule binding hot spots on BfmR1-130 supports the possibility of identifying small-molecule inhibitors of BfmR activity that act by specific binding to the receiver domain. Binding hot spots on the BfmR1-130 protomer were predicted on the protomer face that participates in the active homodimer interface and at the phosphorylation site. It is possible that ligand binding at other hot spots outside these two regions may inhibit BfmR activity through an allosteric mechanism, but that is beyond the scope of this study, as is consideration of the possibility of inhibitor binding to the DNA-binding domain.
A predicted strong binding hot spot (red mesh in Fig. 8A) lies between the N terminus of strand β5 and the C terminus of α5, and the Arg124 side chain fits into this niche on the partner protomer of the homodimer. A small molecule bound in this niche would likely interrupt the Arg124 hydrogen bond to the His78′ carbonyl oxygen and the salt bridge to the Asp102′ carboxyl group. A second predicted strong hot spot (orange mesh in Fig. 8A) and the predicted weak hot spot (blue mesh in Fig. 8A) neighbor the Arg117 side chain, and a small molecule bound in either location would likely interfere with formation of the Arg117-Asp103′ salt bridge across the interface. Given the conserved nature of the OmpR/PhoB family receiver domain α4-β5-α5 face, it may be possible to develop a small molecule that inhibits activation of a broad range of response regulators. Interestingly, an in silico ligand-screening study for the E. coli PhoP receiver domain focused upon binding sites near PhoP residues Arg111 (analogous to BfmR Arg117) and Arg118 (analogous to BfmR Arg124), as these residues are key determinants of PhoP homodimerization and activation (50). Importantly, ligand binding hot spot prediction has been demonstrated to be less sensitive to protein conformational changes than the in silico prediction of a specific binding site for a specific ligand (43).
Blind docking results supported that several but not all predicted binding hot spots may be suitable for participating in binding interactions with drug-like molecules that will interfere with BfmR activation. However, this analysis also suggested that successful binding of drug-like molecules likely requires interactions with two binding hot spots or neighboring regions. For example, the phosphorylation site by itself was not a promising drug-like molecule binding site. Rather, docked drug-like molecules bound at the phosphorylation site also required binding interactions at the neighboring binding hot spot between helix α1 and the N terminus of helix α5. The phosphorylation site pocket is comprised primarily of charged and polar residues. A general feature of drug-protein complexes is the presence of hydrophobic in addition to charged or polar amino acids at the binding site (51), and so the phosphorylation site is not optimal for interacting with drug-like molecules but may participate in a larger binding site.
Computational methods predicted that BfmR is likely a druggable target. However, development of robust BfmR activity assays are required prior to further experimental exploration to identify and characterize hit or lead molecules for therapeutic development. Cell permeability and efflux pumps are major barriers in the identification of tractable Gram-negative antimicrobial lead molecules (52). Thus, an assay to evaluate efficacy of a compound with a BfmR-specific mechanism of action in the context of live A. baumannii is required. Furthermore, an assay to determine the threshold of BfmR inhibition necessary to result in therapeutically relevant antimicrobial activity is also needed, as a small-molecule drug may not completely inhibit the BfmRS signaling pathway in vivo. Previous efforts to identify small-molecule inhibitors of response regulator activation lacked assays to evaluate if targeted TCSs were effectively and specifically downregulated and thereby conferred significant biological effects (50, 53).
A. baumannii BfmR is an appealing antimicrobial target, because its inhibition would significantly decrease in vivo survival and increase sensitivity to antimicrobials. In addition, it has a lack of significant sequence similarity to any human protein, and BfmR belongs to a protein family absent from humans and other mammals. Lastly, the BfmR sequence is highly conserved across A. baumannii strains. However, several important questions remain to be addressed before BfmR can be classified as an established target. Thus far, no receiver domain from the OmpR/PhoB family has been demonstrated as a druggable target (i.e., activity can be modulated by a drug-like small molecule). Likewise, determining the degree of BfmR inhibition needed to impact growth/survival, an efficient BfmR activity assay for high-throughput screening BfmR activity against a small-molecule library, and an assay for determining specificity of the mechanism of action are needed for progress of BfmR as a potential new antimicrobial target. Additionally, major impediments for development of an antimicrobial effective against GNB are permeability and efflux, and a small-molecule inhibiting BfmR activity must overcome these barriers. Despite these challenges, an intriguing aspect of TCS response regulators as antimicrobial targets is the possibility of developing drugs effective against either a narrow or broad spectrum of bacteria. Additionally, the development of small-molecule tool compounds that modulate activities of response regulators will allow chemical biology dissection of bacterial signaling pathways responsive to environmental stimuli. The OmpR/PhoB family receiver domains in response regulators are widely present in bacterial pathogens and possess a degree of shared homology at the dimer interface and phosphorylation site. Therefore, it is possible that a drug inhibiting BfmR activity through its receiver domain will also inhibit the activity of additional TCS response regulators. This multitarget approach may increase durability and overcome a major concern in drug development, the development of resistance. Taken together, data presented in this report support further studies designed to develop BfmR as a drug target, including development of assays required for further target validation.
Bacterial strains. A. baumannii strain AB307-0294 (blood isolate; sequence type 15, clonal group 1, based on the methods of Ecker et al. [54]) has a K1 capsular serotype (55) and was isolated from a patient hospitalized at Erie County Medical Center, Buffalo, NY, in 1994 (56). The genome of AB307-0294 has been fully sequenced, and it contains 3.76 Mbp with 3,531 predicted open reading frames (57). Previous comparisons with A. baumannii strains in the public domain support AB307-0294 as being clinically appropriate, representative of contemporary A. baumannii strains, and an ideal background to identify new or unrecognized antimicrobial targets of interest (24).
AB307.70 is an isogenic BfmR-minus derivative of AB307-0294 generated by transposon mutagenesis, and it possesses a disruption in bfmR (nucleotides 1 to 717) due to the transposon insertion after nucleotide 592. bfmR putatively encodes a response regulator protein, BfmR (24). Polar effects were excluded, as described previously (24), and the complemented derivative, AB307.70/pNLAC[bfmR], was generated for this study to confirm that the observed phenotypic differences between AB307-0294 and AB307.70 were due to BfmR. A second BfmR-minus derivative was created by site-directed mutagenesis of the clinically relevant isolate AB908 (obtained from Walter Reed Medical Hospital) by using a one-step chromosomal gene inactivation method described elsewhere (58). In brief, a linear PCR-generated amplicon (forward primer, 5′-ATGAGCCAAGAAGAAAAGTTACCAAAGATTCTGATCGTTGAAGACGACGAGCGTTTAGCGCGATTAACTCAAGAATATTTAATCCGTAATGGTTTGGAAGTTGGTGTAGAAACCGATGGTAACCGTAAAGCCACGTTGTGTCTCAAAATC-3′; reverse primer, 5′-TTACAATCCATTGGTTTCTTTAACAAACAAGTAACCTTTACTACGTACAGTTTTAATACGTTTTGGATTTTCAGGATCATCGCCAATTTTTGGACGAATACGTGAAATACGTACGTCAATTGAACGCATTATTCCCTCCAGGTATTAGAA-3′; bold sequence portions are homologous to the kanamycin resistance gene sequence) that contained a kanamycin resistance cassette flanked by the first and last 125 bp of bfmR was electroporated into AB908/pAT02 (58), and recombinants were selected on Luria-Bertani (LB) plates containing kanamycin. The successful disruption of bfmR in AB908ΔbfmR was confirmed by sequence analysis, and a derivative cured of pAT02 was used for subsequent studies. AB307.30 and AB307::Tn69 are isogenic derivatives generated by transposon mutagenesis that possess disruptions in wzc (a capsule transport gene) and gna (a capsule biosynthetic gene), respectively (33, 45). AB307.30 and AB307::Tn69 were used in studies to exclude the presence of a second, non-K1 capsule. AB307-0294 (wt) and AB307::Tn17 (ΔlpsB) (59), which possesses a disruption in a glycosyltransferase involved in lipopolysaccharide core biosynthesis, are both capsule positive and served as positive controls. Strains were maintained at −80°C in 50% LB medium (5 g of yeast extract, 10 g of tryptone, and 10 g of NaCl per liter) and 50% glycerol.
Media.The procedures for obtaining human ascites fluid and human serum were reviewed and approved by the Western New York Veterans Administration Institutional Review Board. Informed consent was obtained from healthy volunteers for blood collection. Blood underwent coagulation at room temperature for 15 min, followed by refrigeration at 4°C for 60 min to enable clot retraction. Serum was obtained from the blood after subsequent centrifugation at 4°C for 15 min at 3,000 × g. For the collection of ascites fluid, the Western New York Veterans Administration Institutional Review Board waived informed consent because the ascites fluid was collected from deidentified patients who were undergoing therapeutic paracentesis for symptoms due to abdominal distension. These individuals were not being treated with antimicrobials and were not infected with human immunodeficiency virus, hepatitis B virus, or hepatitis C virus. The ascites fluid was cultured to confirm sterility, divided into aliquots, and stored at −80°C. Each batch was obtained from a different patient and was designated by the date of removal. A single ascites fluid batch (obtained on 18 October 2012) was used for growth studies. For some experiments, human serum and ascites fluid were incubated at 56°C for 30 min to inactivate complement-mediated bactericidal activity (Δ56°C). BBL Müeller-Hinton II cation-adjusted (MH) broth consisted of 22 g of powder per liter (Becton, Dickinson). Minimal medium consisted of 200 ml of solution A [2.0 g (NH4)2SO4, 6.0 g Na2HPO4, 3.0 g KH2PO4, 3.0 g NaCl, 0.011 g Na2SO4], 800 ml of solution B (0.2 g MgCl2, 0.0132 g CaCl2 ⋅ 2H2O, 0.0005 g FeCl3 ⋅ 7H2O, 2.9241 g citrate [trisodium salt dehydrate]), and 3 g Casamino Acids. AB307.70 was grown in the presence of 40 µg/ml of kanamycin, and AB307.70/pNLAC[bfmR] was grown in the presence of 40 µg/ml of kanamycin and 200 µg/ml of carbenicillin.
In vitro and ex vivo growth in LB medium, human ascites fluid, and human serum.Growth experiments in LB medium, human ascites fluid, and human serum were performed as described elsewhere (56), with aliquots removed for bacterial enumeration at various times. For growth studies, 100% LB medium, 90% ascites fluid–10% 1× phosphate-buffered saline (PBS; pH 7.4), and 90% serum–10% 1× PBS were used.
Analysis of capsule production.AB307-0294 (wt) and AB307.70 (ΔbfmR) cells were grown overnight in LB medium, minimal medium, or human ascites fluid. The next day, an aliquot was removed for enumeration and bacterial cells were concentrated by centrifugation and resuspended in 50 µl/1 ml of culture lysis buffer (2% SDS, 10% glycerol in 1 M Tris [pH 6.8], to which fresh 4% β-mercaptoethanol was added). Cells in lysis buffer were boiled for 10 min. Next, the lysate was treated with 20 µl/50 µl of lysate with proteinase K (1.25 mg/ml) for 120 min at 60°C. Supernatant was generated by precipitation with 5 volumes of ice-cold 75% ethanol, which was placed at 4°C for 18 h. The precipitant was recovered by centrifugation at 8,000 × g for 30 min at 4°C. The supernatant was discarded and the tubes were air dried. The dried precipitates were resuspended in lysis buffer at the corresponding volume of the concordant pellet, boiled for 10 min, and treated with proteinase K in the same manner as the pellets. The cell pellet and supernatant volumes used for gel loading were normalized to a bacterial titer of 3.9 × 106 CFU by dilution in lysis buffer. Capsular polysaccharide was resolved by SDS–8% polyacrylamide gel electrophoresis and subjected to Western blot analysis with MAb 13D6, which is directed against the AB307-0294 capsule (33), or were stained with alcian blue.
Antimicrobial susceptibility testing. A. baumannii strains were grown for 18 h in MH broth at 37°C. Bacteria were diluted 1:100 in the same medium, and titers were determined by serial 10-fold dilutions and enumerated on LB plates with or without appropriate antibiotics. Assays were performed in 96-well microtiter plates. Wells contained either 0 µg/ml, 0.0078 µg/ml, 0.0156 µg/ml, 0.0312 µg/ml, 0.0625 µg/ml, 0.125 µg/ml, 0.25 µg/ml, 0.5 µg/ml, 1.0 µg/ml, or 2.0 µg/ml of meropenem or 0 µg/ml, 0.0312 µg/ml, 0.0625 µg/ml, 0.125 µg/ml, 0.25 µg/ml, 0.5 µg/ml, 1.0 µg/ml, 2.0 µg/ml, 4.0 µg/ml, or 8.0 µg/ml of colistin or tigecycline in 100 µl of MH broth to which 100 µl of the same medium containing bacteria was added. Final concentrations of bacteria were 2.4 × 105 CFU/ml for AB307-0294, 5.0 × 105 CFU/ml for AB307.70, and 1.8 × 105 CFU/ml for AB307.70/pNLAC[bfmR] for meropenem studies; 6.2 × 105 CFU/ml for AB307-0294, 8.9 × 105 CFU/ml for AB307.70, and 6.8 × 105 CFU/ml for AB307.70/pNLAC[bfmR] for colistin studies; and 7.1 × 105 CFU/ml for AB307-0294, 7.9 × 105 CFU/ml for AB307.70, and 1.3 × 106 CFU/ml for AB307.70/pNLAC[bfmR] for tigecycline studies. Control wells contained MH broth only. The plates were incubated for 18 h at 37°C. The optical density at 600 nm (OD600) of each well was measured in a microplate spectrophotometer (SpectraMax 190; Molecular Devices) at 26°C. Two independent experiments, each with an n of 6, were performed for each strain and antimicrobial concentration.
Bioinformatic analysis of BfmR sequence and structure.The protein sequences for BfmR orthologues were generated from respective bfmR gene sequences. The OMA orthologue database (September 2014 release) was searched to evaluate BfmR conservation across A. baumannii strains and additional Acinetobacter species (37). BLASTp (60), as implemented on the NCBI server (http://blast.ncbi.nlm.nih.gov ), and Clustal Omega (61) were used to align sequences, and ESPript 3.0 (62) was used to display protein sequences. FTMap was used to predict locations of small-molecule binding hot spots on the exposed surface of the BfmR receiver domain, using the default set of small organic molecular probes (16 chemicals; mass range, 30.1 to 106.1 Da) (43). AutoDock Vina v. 1.1.2 and PyRx v. 0.8 were used for in silico screening of the BfmR receiver domain against a virtual library of drug and drug-like molecules (63, 64). The virtual molecular libraries represented small-molecule chemicals approved for clinical trials or clinical use (3,180 chemicals; mass range, 60.1 to 829.0 Da), the NCI Diversity 3 collection (1,880 chemicals; mass range, 114.1 to 697.1 Da), and the LOPAC1280 collection (1,280 chemicals; mass range, 59.1 to 836.4 Da), with molecular descriptor files obtained from the ZINC database (65).
Recombinant protein expression and purification. E. coli strain DH5α (Invitrogen) was used during construction of the bfmR expression vector, and strain BL21(DE3) (Novagen) was used for recombinant protein expression. PCR amplification of the portion of the bfmR open reading frame (NCBI accession number ACJ59134 ), coding for the N-terminal receiver domain (BfmR1-130), was acccomplished using purified AB307-0294 chromosomal DNA (RefSeq NC_011595.1 ) as the template and the primers forward, 5′-GCGCGACATATGATGAGCCAAGAAGAAAAGTTA-3′; and reverse, 5′-ATATATCTCGAGTTATTCAACAGTTTTATCCGTACG-3′, which included an NdeI and an XhoI cleavage site, respectively. Gel-purified PCR products were restriction enzyme digested and ligated using T4 DNA ligase (Promga) into a linearized customized pET15b-TEV (tobacco etch virus) vector containing an N-terminal His5 affinity tag plus a TEV protease cleavage site replacing the usual thrombin site (pET15b-TEV-bfmR_rec). The integrity of the expression cassette was verified by DNA sequencing (Roswell Park Cancer Institute Sequencing Facility).
E. coli BL21(DE3) cells transformed with pET15b-TEV-bfmR_rec were grown to an OD600 of 0.8 at 30°C in LB medium supplemented with 100 µg/ml ampicillin. Protein expression was induced with 1 mM β-d-1-thiogalactopyranoside (IPTG). The culture was incubated for an additional 5 h prior to harvesting by centrifugation. Frozen cell pellets were thawed and resuspended in lysis buffer (20 mM Tris-HCl [pH 8.0], 200 mM NaCl, 10 mM imidazole, and 1 mM β-mercaptoethanol). Cells were lysed using a Microfluidizer (Microfluidics). Filtered lysate supernatant was loaded onto a HisTrap immobilized metal ion affinity chromatography (IMAC) column (GE) and eluted with a 10 mM-to-300 mM imidazole gradient plus lysis buffer. Elution fractions containing His5-BfmR1-130 were dialyzed into lysis buffer plus 60 mM imidazole. TEV protease was added to the fusion protein at a 1:100 mass ratio, and the mixture was incubated overnight at 4°C (66). The sample was reapplied to a HisTrap IMAC column to separate the cleaved His5 affinity tag from BfmR1-130. Eluted BfmR1-130 was dialyzed against 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 1 mM dithiothreitol and then was concentrated (final concentration, 11.25 mg/ml) by using Centricon-3 centrifugal concentrators (Amicon). Small aliquots were flash-frozen in liquid nitrogen and stored at −80°C. Protein purity was verified via SDS-PAGE.
Crystallization.Crystallization screening via microbatch under oil in 1,536-well microtiter plates was conducted using the high-throughput robotics at the Hauptman-Woodward Institute (67). Selected hits were optimized via manual sitting-drop vapor diffusion. Crystals used for diffraction data collected were obtained by equilibration at 20°C of a crystallization drop containing 2 µl of purified BfmR1-130 at 11.25 mg/ml plus 2 µl of reservoir solution against 1 ml of reservoir (20% [vol/vol] polyethylene glycol 400, 20% [vol/vol/] glycerol, 100 mM Tris-HCl [pH 8.5], and 150 mM NaCl).
X-ray diffraction data collection and processing.Crystals were harvested using a nylon loop and then subsequently flash-cooled in liquid nitrogen. No additional cryoprotectant was required due to the composition of the reservoir solution. Crystals were shipped to the Stanford Synchrotron Radiation Lightsource (SSRL), where diffraction data were collected remotely at −173°C by using a MARmosaic 325 charge-coupled-device detector on beamline 9-2. Data were collected using an X-ray wavelength of 0.97946 Å, a crystal-to-detector distance of 330 mm, a φ range of 180.4° by 0.55° increments, with 5-s exposures per frame. Data were indexed, integrated, and scaled using HKL2000 (68).
Statistical analyses.Continuous data were assessed for normality and are presented as means ± standard errors of the means (SEM.) P values of 0.05/n (n = the number of comparisons) were considered statistically significant based on the Bonferroni correction for multiple comparisons. To normalize ex vivo growth/survival data (Fig. 1), log10-transformed values were utilized, the area under each curve was calculated, and these areas were compared using two-tailed unpaired t tests (Prism 4 for MacIntosh; GraphPad Software, Inc.).
Structure solution and refinement.The receiver domain from the response regulator PhoP from Bacillus subtilis (PDB 1MVO ) was used as the search model in molecular replacement, following removal of associated solvent and ions. The Auto-Rickshaw server (69) was used to determine the molecular replacement solution, which was automatically fed to Arp/wARP (70) to produce an initial BfmR model. Refinement was conducted using Phenix, including TLS refinement to model anisotropic displacements and noncrystallographic symmetry (NCS) restraints (71). Manual model building and analysis were performed using Coot (72). Structure analysis and validation made use of PyMol (Schrödinger, LLC), Phenix (71), PISA (73), and the validation tools present in the wwPDB deposition tool (http://deposit.wwpdb.org/deposition ).
Protein structure accession number.The refined coordinates and scaled diffraction data have been deposited in the PDB (PDB code 5E3J ).
We thank Leah Herzog and Wendy Franke for assistance in expression vector and protein production and L. Wayne Schultz (HWI; presently with QuaDPharma) for helpful discussions. The TEV protease expression vector was a kind gift of David Waugh (NCI).
This research is based in part upon work supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (IBX000984A, T.A.R.). This work was also supported in part by Telemedicine and Advance Technical Research Center (TATRC) cooperative agreement W23RYX1055N607 and an interdisciplinary grant from the University at Buffalo (T.A.R. and T.C.U.). Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (including grant P41GM103393).
The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.
- Received March 28, 2016.
- Accepted April 12, 2016.
- Copyright © 2016 Russo et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license .


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Bartolomeo makes school safety recommendations
HARTFORD >> Following a meeting Wednesday of the legislature's Bipartisan Task Force on Gun Violence Prevention and Children's Safety, state Sen. Dante Bartolomeo, D-Meriden, issued a brief list of some of the school safety recommendations she compiled after meeting with parents, educators, city officials, public safety officers and others in her district over the past several weeks.
Bartolomeo, who serves on the School Security subcommittee of the Bipartisan Task Force, met with residents and town officials from Cheshire, Meriden and Middletown to hear their input on what they believe would enhance security at the schools following the massacre at the Sandy Hook Elementary School in Newtown this past December.
Bartolomeo visited Farm Hill School at the end of last month to gather information from school and city officials there.
"Parents, school staff and police officers have the best knowledge of what is working and what is not in their local schools, and they have some of the best suggestions as to where Connecticut can take action to improve student safety," Bartolomeo said. "I'm thankful for their input, which I found insightful and very helpful... I am forwarding their suggestions to our subcommittee for consideration as the legislature puts together proposals to address school safety, gun safety and mental health."
The school safety proposals received by Sen. Bartolomeo include including school resource officers at every school, practicing active intruder drills regularly, providing mental health evaluation and treatment to students, more cameras and crisis response buttons at schools and setting statewide security standard for new school construction or renovation.

subset: commoncrawl


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Sandalwood Market The global Sandalwood market was valued at million US$ in 2018 and will reach million US$ by the end of 2025, growing at a CAGR of during 2019-2025. This report focuses on Sandalwood volume and value at global level, regional level and company level. From a global perspective, this report represents overall Sandalwood market size by analyzing historical data and future prospect.
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text: I have a weldment with over 100 parts made of plate steel. I am modeling it primarily as surfaces so that I can mesh it with shell elements, always with the surface on one side of the shell. How can I tell which way the shell offset is going in the shell element?


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Last April I was making some pretty awesome progress in making puzzle boxes. Crafting puzzle boxes was relaxing and very gratifying at the same time. Spending time in my workshop is always great, but time spent on something relatively small that will be a gift just makes that workshop time even better.
Over the past few months I've spent most of my free workshop time on insulating it. My intent was to get that done first and then jump back into making boxes. After yesterday it just felt right to start making them again. Dad had always wanted to know how I made them and after as close to him as I felt yesterday I got the urge and gave in.
No major progress was made, but just the act of making the blanks felt great. When I went to cut the first one I just laughed at how rusty I was. I totally screwed up the first cut and all I could do was chuckle. Time to get back into the swing of it!
While that was going on Dominic was at a Boy Scout merit badge day and was able to work on earning two merit badges. Gavin spent much of the afternoon working on his Game Design activity pin for Weblos. I got out den meetings mapped out for the rest of the school year and have it worked out so the boys will have the opportunity to earn their Weblos badge. Lots of time and work spent on scouting, and it was pretty awesome. Both of the boys had a great sense of accomplishment after their hard work, I was proud of both of them for focusing on reaching the goals they set for themselves.
This was a great day spent mainly at home and it was awesome. Working on the puzzle boxes and making progress on scouts made it all the better.


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Nowadays municipalities in the Netherlands work together more intensively with other municipalities in the region. Also cooperation with companies, institutions and societal organizations is more often looked for at the regional level. In practice this brings along many problems and difficulties. For several reasons it appears not to be easy to combine the implementation strengths of municipalities and societal partners. This article presents a new approach (based on the theory of 'new regionalism') to regional implementation strength. This approach is not only about designing regional administrations, but is mainly about the factors that induce administrations as well as companies and institutions to commit themselves jointly for the region. To increase the regional implementation strength more is needed than the formation of a regional administrative structure in which municipalities do not cooperate in a non-committal manner. To induce municipalities and societal partners to commit themselves jointly to handling new tasks or new challenges it is also necessary to have a clear strategic vision on these issues that binds parties and makes them enthusiastic and that regional cooperation is rooted in a societal breeding ground. It also asks for an administrative structure that does justice to the contribution every municipality and societal partner makes to the realization of the strategy and for a democratic involvement of municipal councils and sector-based interest groups.
Strategie, structuur en samenleving: drie dimensies van regionale uitvoeringskracht
|Bestuurswetenschappen, Aflevering 2 2014
|Marcel Boogers
Intergemeentelijk samenwerken: het kan ook licht
Een verkenning van lichte vormen van intergemeentelijke samenwerking
|Bestuurskunde, Aflevering 1 2014
|inter-municipal cooperation, light forms of cooperation, modes of cooperation
|Leon van den Dool en Linze Schaap
Many tasks will be decentralized to municipalities in the Netherlands in the coming years. To deal with these challenges, central government encourages municipal mergers, while municipalities often prefer a light form of cooperation. Since municipal boarders are converging less and less with the boarders inhabitants experience in their daily lives, municipalities feel free to cooperate in a variety of ways with other partners. This poses new challenges to democratic legitimacy, effectiveness and the role local authorities play. Local governments therefore do not need new regulations or legal forms of co-operations, but rather a repertoire fitting their role. We argue that local governments need to analyse their tasks, choose the form of cooperation that fits best and develop a repertoire for their cooperation. Light forms of cooperation are very important for developing a variety of cooperative forms and roles local governments need to play.
To PPS or not to PPS? (Publiek-)private samenwerking rond groen gas
|Bestuurswetenschappen, Aflevering 1 2014
|Maurits Sanders en Michiel Heldeweg
In policy practice sometimes organizational arrangements appear that at first glance manifest itself as cooperative relations between private organizations, but about which on second thoughts the question can be asked if after all there is an active input from the side of the government. This is for instance the case in the construction of biogas infrastructures. In this article the authors discuss if we can talk about PPC after all. In the debate on governance this question is important because in the design of PPC the public interest involved must be sufficiently guaranteed in terms of control and accountability. On the basis of a confrontation between the results of a literature review and an empirical study of the case of a Green Gas pipeline in North-East Friesland ('Biogasleiding Noordoost Fryslân') in the Netherlands, the authors conclude that public steering in practice can take a form in disguise. Using 'intermediate' civil law legal persons, governmental influence indeed can be and is exercised during the cooperation. Especially law poses specific demands on control and accountability to take care of public interests, like the promotion of the use of renewable energy. Likewise in this kind of projects, especially in comparison with pure private-private cooperation, the public and if possible even the public law regulation must be safeguarded, for instance by transparency of form and content of steering. Of course this has to be done with preservation of the cooperative nature that is typical of PPC.
Herwaardering van herindeling: een evaluatie van 10 jaar gemeentelijke herindelingen in Gelderland, Limburg en Overijssel
|Bestuurswetenschappen, Aflevering 1 2014
|Jony Ferket, Martin Schulz, Mark van Twist e.a.
This article presents the effects of an evaluation study of different municipal amalgamations in the past ten years in the Dutch provinces Gelderland, Limburg and Overijssel. It is an evaluation that passes through two tracks; we investigate by written sources and evaluation studies of specific amalgamations its gains, but we also by the method of a survey-feedback have asked the opinions on the amalgamation of a considerable group of people involved in the amalgamation. Would they do it again this way years after the amalgamation and they do have a positive or negative assessment of the amalgamation as a whole afterwards? The answer to this question is surprising: a lot of people involved are quite positive on a municipal amalgamation and would choose for it again in the same circumstances. They also think it is an alternative to be preferred over piling up arrangements of municipal cooperation. There is also a remarkable small difference between the assessment afterwards of a voluntary or a 'forced' amalgamation. That difference of assessment can be felt intensively in the process before and during the amalgamation, but afterwards the respondents are also positive about amalgamations that have been imposed 'top-down'. This result suggests that the proverb of a 'bottom-up amalgamation' needs relativisation and the provinces and the central government can play a more active part in the process of amalgamation.


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Nnoshé earned the name step mother because of her motherly nature. The first spring she was here she did not lay an egg but wanted to incubate an egg that had been laid by another female who showed no interest in her egg. She was very defensive and guarded the egg as if it were her own. After the egg was removed she focused her attention to Mskwabimesh when he arrived. Being a juvenile, he was very inquisitive and playful. He would often take food from the other eagles. Nnoshé seemed to tolerate him more than the others. She would often become very vocal when the other eagles resisted when he tried to take their food like any good mother would.
She is an early riser and regularly greets the morning sun with a series of short high pitched whistles. On cleaning days after the waterfalls and streams are refilled she is the first to come back to the water to inspect the aviary staffs cleaning job. So far she approves.


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Top attractions in Fort Lauderdale
Everglades: Sawgrass Park Airboat Tour with Exhibit Entrance
Go Miami Explorer Pass: Over 28 Tours And Attractions
Miami/Fort Lauderdale: Sightseeing Pass for 35+ Attractions
Miami: Halloween Horrorland
5-Hour Miami City Tour and Speedboat Ride
Everglades and Miami: 5-Hour Shore Excursion
Private Sedan-MIA Apt↔Port Everglades/FLL Apt↔Port of Miami
Ft. Lauderdale: Private Airport Shuttle to the Port of Miami
Fort Lauderdale: Mai-Kai Dinner and Polynesian Show
Everglades Eco Adventure: 2-Hour Sawgrass Recreation Park
Key West: Day Trip from Fort Lauderdale w/ Activity Options
Things to Do in Fort Lauderdale
Top Attractions in Fort Lauderdale
Cities in United States
Other Sightseeing Options in Fort Lauderdale
Want to discover all there is to do in Fort Lauderdale? Click here for a full list.
What people are saying about Fort Lauderdale
What a great one day introduction to the Everglades and Cypress Swamp!
Starting with an easy pickup at our hotel, our guide Zach took us on an amazing adventure. We saw lots of birds, identified by Zach, and many alligators. The airboat ride in the 'Glades was a hoot! Then an easy nature walk on a raised walkway in the Cypress Swamp. A great lunch at a local, non-touristy restaurant. Next a boat ride among the coastal islands. I was expecting something sedate, but those boats can move! We saw dolphins, two types of pelicans, and much more. Then we wandered through a museum that was formerly a trading post and local store with post office. On the ride back to our hotel, Zach regaled us with stories from his youth in a somewhat different Florida. Altogether a wonderful day!
Good value for money tour. Great show and boat ride.
We booked this and were there early. But the bus had already left - we saw it leave 7-8 minutes early as we were approaching the pick up point! We contacted the company and got rebooked for the next day. We caught this bus and it was a great day. The show was entertaining and the boat ride both fun and great for seeing the wildlife in the Everglades. Saw a few gators up close which was awesome!
Get up close to the gators in their natural habitat
We saw 4 alligators during the trip. Bob our captain told us all about the history and environmental issues of the Everglades and its inhabitants. Amazing trip and you get the chance to hold a baby gator in the exhibit area for a small donation. Ear defenders not supplied like other tours but earplugs are provided.
Zoo part great boat too long stop talk, more boat ride .
We enjoyed the boat ride when we were moving, a short, brief stop is fine, but the stop was waaaaay to look long. Got very hot and stinky and boring. Should have been more racing across the seagrass. The little zoo was great my grandsons loved the gator holding. All the animals were interesting.
A brilliant experience - well-organised and great fun!
Sawgrass Recreation Park provided a great Airboat experience. We managed to spot 2 alligators even though the high water level made it more difficult. There is also a small collection of animals here - all rescue animals who could not survive in the wild - very intetesting.


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Montreal-based Parta Dialogue has completed its acquisition of all of the issued and outstanding shares of Sense Fab, who developed MyTwatch, a private Paris-based e-intelligence and social marketing technology company.
"This acquisition is an important step for us in building out the most robust offering in the market for brands and companies to support their entire social marketing cycle, from the data they want, to the insights they need leading to conversions they crave. MyTwatch's award winning proprietary listening technology and semantic analysis – currently utilized by numerous clients, round out our strengths in creating, distributing and measuring social media marketing programs. With the capability to execute in 6 languages, their platform will become a key aspect in our effort to attract customers globally," explained Paul Allard, CEO of Parta.
MyTwatch was founded by two internet and marketing experts, Philippe Gablain and Edith Nuss, capitalizing more than 25 years of e-marketing and internet services experience. The company has developed an innovative social media crawler capable of gathering large volumes of data across anything digital (all social media platform, forums, blogs etc.) and through semantic analysis, can determine important critical outcomes such as topic identification, influence and lead generation.


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Charles H. "Chuck" Embs, AIF® (email@example.com)
Principal/Investment Advisor
President/Chief Executive Officer
Chief Compliance Officer
BS, United States Air Force Academy, 1991
MS, Air Force Institute of Technology, 1995
Squadron Officer School, 2003
Air Command and Staff College, 2010
Air War College, 2016
Employee since 2003
Chuck founded Chesme Capital Management in 2003 on the basic principle of providing prudent investment advice and fiduciary counsel that is free from the many conflicts-of-interest inherent to the financial services industry. Today, Chuck continues to set the strategic direction of the firm and oversees all operations and compliance efforts.
Chuck continues to serve as an Officer in the United States Air Force Reserves and he is the Co-Founder of Zero Day, a not-for-profit organization serving military Veterans through education, vocational training, counseling, and housing assistance.
Stephen M. "Steve" Erb, AIF® (email@example.com)
Principal/Investment Advisor
BA, Michigan State University, 1991
Employee since 2005
Steve has more than 20 years of experience in the financial services industry. He is a leading authority in retirement plan services and prudent investment practices. Steve manages all institutional relationships at Chesme and is committed to comprehensive due diligence on all investment-related matters and the application of a fiduciary standard of care in all client relationships.
Douglas J. "Doug" Vaughn, CFP® (email@example.com)
Principal/Investment Advisor
BS, University of Florida, 1991
Employee since 2005
Doug has over 15 years of experience in the financial services industry. He is responsible for individual wealth management relationships at Chesme where his combined expertise in comprehensive financial planning and investment management offer the firm's clients an appropriate level of service based on the client's goals.
Doug served in the United States Navy for 12 years and actively supports the local community through membership in the DeWitt Lions Club and as a board member for the YMCA of Metropolitan Lansing.
Michael J Voorheis (firstname.lastname@example.com)
Investment Advisor
BS, Spring Arbor College, 1989
Employee since 2018
Mike has more than 30 years of experience in the financial services industry. He started as a financial advisor in 2009, bringing more than 20 years of banking and management experience to wealth management and retirement planning. Mike was previously a CEO & Senior Vice President for a Michigan-based credit union.
Angela Chisman (email@example.com)
Business Operations Manager
Deputy Chief Compliance Officer
Accredited Advisor of Insurance, Olivet College, 2002
Laurel Oaks Career Development Center, 1988
Employee since 2007
Angela has over 10 years of experience in the financial services industry. As a former small business owner herself, she brings a wealth of knowledge to the management and oversight of all business operations at Chesme. Angela also assists the Chief Compliance Officer in the maintenance of the firm's compliance program to ensure Chesme's advisory operations remain aligned with all regulatory requirements and responsibilities.
Jessica Noyce (email@example.com)
Advisory Operations Manager
Associates Degree, Lansing Community College, 2015
Employed since 2010
Jessica has nearly 10 years of experience in the financial services industry. She is responsible for the management and oversight of all advisory operations at Chesme. Jessica works closely with the firm's investment advisors to ensure the highest quality service delivered to the firm's individual and institutional clients.
Miranda Wellman
Client Services Administrator
Dale Carnegie, 2016
Employed since 2018
Miranda joined our team in 2018. She works closely with the firm's investment advisors to provide client-facing service and support to the firm's individual clients.


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A Look Inside Kanye's 'Pablo' Pop-Up Shop in NYC
The pop-up will be open March 18 until 8 p.m. and from noon to 8 p.m. on both Saturday March 19 and Sunday March 20.
The pop-up will be open March 18 until 8 p.m. and from noon to 8 p.m. on both Saturday March 19 and Sunday March 20.
Kanye's upcoming album SWISH will premiere at Madison Square Garden (and select theaters around the world) on Feb 11.
If you struck out trying to score the Adidas Yeezy Boost 350 "Pirate Black" this past weekend, your faith has been restored.
The latest offering from the Adidas and Kanye collaboration finally gets a release date. Adidas took to twitter earlier to announce the Yeezy 350 Boost will drop worldwide on June 27th. Details on where they will be stocked is still sketchy but sources suggest this could be a much more widespread release compared to the 750 earlier this year.
After the initial release of the Adidas Yeezy Boosts in NYC earlier this month, the Boosts will finally see an international release beginning tomorrow (February 28th) with more outlets stocking them on March 26th. Kanye plans to release the shoes in multiple waves to give as many people as possible the chance to get them.
Check here to see the full list of stores that will be carrying the Boosts.
Kanye's creative agency, DONDA, has just launched an official online store for GOOD Music clothing.
The all black/white clothing line includes the GOOD Angel Hoodie—recently seen worn by Kanye West himself—along with shirts, caps, socks and more. Prices range from $34–$160.
Check it out at http://GOODMusicMerch.com
Kanye West is back in the fashion scene with his second collection, and will show it off at Paris Fashion Week on March 6th.
Is this not what you have been waiting for? Last night Complex hit you with what was the clearest look of the Nike Yeezy 2 but this morning we get even more detailed shots of the most hyped shoe since — well the Nike Air Yeezy.


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Table of Contents
Living along a river, the mountains and the forest, Darchelle never was lacking in entertainment. Spending time in any of the three places was something she found she adored. The mountains were considered dangerous from her family, as were the forest and the river, but nothing could keep the growing girl away.
As a young girl, she ventured off with her older or younger brothers and the twin sister Mari, off to the forest to find mischief, climb trees, avoid the rain. The hot summer months were spent swimming and the winter was spent mostly at the manor. The mountains were a call to her though, the majestic beauty, and so when her family would make their rounds to visit family in Roundway, where her mother was from, she and her siblings would go exploring.
Eventually, Mari decided to be a knight and she squired in Roundway, with mother's family, grandfather, a well known and notable knight from there, Sir Derec de Roundway. He taught Mari, trained her and until she was knighted and returned home. These years were the most stressing to Darchelle. She was really attached to her twin sister and it looked as if the time stopped without Mari. Darchelle herself was too fragile to use heavy swords or axes, or spears, lances. So, it was decided by the Gods, that she will never be a knight.
So, Darchelle decided to improve her knowledge in Chirurgery instead, in order to be able to follow her knight sister in battles. Maybe some people do not believe in tight attachment of twins, but that was what Darchelle felt. She honestly believed that even each time Mari was hurt, Elle felt it. The young redhead lady was a quick lerner and made proud not just her mother, but the local healers, who shared their knowledge with the girl.
Because she had a good time management, Elle found time to improve some of the hobbies and one of the most important ones was hunting. Another one was talking to the people, and the third one was following mother at first, and oldest brother Hydd's wife Sarah later, who were responsible for family's treasury. Elle always loved numbers and she saw mother and Sarah as a perfect ladies to whom she would love to be similar, when she will grow up.
When I was 10 years old I always needed to be told
In the daylight I could cope, because the sun seemed full of hope
Isn't it too hard to conceive
«Kyla La Grange - The River»
On the Grid
Known Associates
We all learn to make mistakes
'Cause I'm just one of those ghosts
And now I'm told that this is life
Would someone care to classify?
«Paramore - Misguided Ghosts»
Recent Logs
(515-05-23) Second Impressions
Summary: Sir Gilchrist de Woodford pays a call to Woodborough Manor, to present himself to his...
(515-04-20) A Bunny Saved
Summary: Darchelle goes hunting, Iago hunts whoever is shooting those arrows, and a drunk Anwir...
(515-04-15) The Failed Virgin Ploy
Summary: On Easter Eve, a group of pagans go on a hunt, and find a Lesser Unicorn. Their methods to...
(515-04-09) Performance Round 2: Darchelle vs Leodwen
Summary: Darchelle de Woodborough is facing Leodwen de Falt in the second round of performance...
(515-04-02) Lovely Challenges - Round 1
Summary: In the first round of the performance contests, Leodwen faces Zaira, and Darchelle competes...
(514-04-24) A Musical Offense
Summary: or the return of a knight who has been long gone from Salisbury. Date: April 24th, 1515...


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