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ORIGINAL RESEARCH article
Sec. Antimicrobials, Resistance and Chemotherapy
Volume 7 - 2016 | https://doi.org/10.3389/fmicb.2016.01985
Expanding the Repertoire of Carbapenem-Hydrolyzing Metallo-ß-Lactamases by Functional Metagenomic Analysis of Soil Microbiota
- 1Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark
- 2Department of Medical Biotechnology, University of Siena, Siena, Italy
- 3Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
- 4Clinical Microbiology and Virology Unit, Florence Careggi University Hospital, Florence, Italy
- 5Don Carlo Gnocchi Foundation, Florence, Italy
- 6School of Life Sciences, University of Warwick, Coventry, UK
- 7Department of Biomedical Sciences, Ross University School of Veterinary Medicine, St. Kitts, West Indies
Carbapenemases are bacterial enzymes that hydrolyze carbapenems, a group of last-resort β-lactam antibiotics used for treatment of severe bacterial infections. They belong to three β-lactamase classes based amino acid sequence (A, B, and D). The aim of this study was to elucidate occurrence, diversity and functionality of carbapenemase-encoding genes in soil microbiota by functional metagenomics. Ten plasmid libraries were generated by cloning metagenomic DNA from agricultural (n = 6) and grassland (n = 4) soil into Escherichia coli. The libraries were cultured on amoxicillin-containing agar and up to 100 colonies per library were screened for carbapenemase production by CarbaNP test. Presumptive carbapenemases were characterized with regard to DNA sequence, minimum inhibitory concentration (MIC) of β-lactams, and imipenem hydrolysis. Nine distinct class B carbapenemases, also known as metallo-beta-lactamases (MBLs), were identified in six soil samples, including two subclass B1 (GRD23-1 and SPN79-1) and seven subclass B3 (CRD3-1, PEDO-1, GRD33-1, ESP-2, ALG6-1, ALG11-1, and DHT2-1). Except PEDO-1 and ESP-2, these enzymes were distantly related to any previously described MBLs (33 to 59% identity). RAIphy analysis indicated that six enzymes (CRD3-1, GRD23-1, DHT2-1, SPN79-1, ALG6-1, and ALG11-1) originated from Proteobacteria, two (PEDO-1 and ESP-2) from Bacteroidetes and one (GRD33-1) from Gemmatimonadetes. All MBLs detected in soil microbiota were functional when expressed in E. coli, resulting in detectable imipenem-hydrolyzing activity and significantly increased MICs of clinically relevant ß-lactams. Interestingly, the MBLs yielded by functional metagenomics generally differed from those detected in the same soil samples by antibiotic selective culture, showing that the two approaches targeted different subpopulations in soil microbiota.
ß-lactams were introduced in clinical practice in the 1940s and currently account for more than half of all antibiotic prescriptions for human use (Tahlan and Jensen, 2013). Among the β-lactams, carbapenems are last resort drugs for treatment of life-threatening nosocomial infections caused by multidrug-resistant Gram-negative pathogens. Carbapenem resistance mediated by the production of carbapenem-hydrolizing enzymes (carbapenemases) has spread globally in Enterobacteriaceae (Nordmann et al., 2011). Carbapenemases are classified into classes A, B, and D according to the Ambler classification of β-lactamases based on amino acid sequence similarity (Ambler, 1980). Classes A and D comprise serine-utilizing enzymes, whereas enzymes belonging to class B, also called metallo-ß-lactamases (MBLs), require zinc as an essential co-factor (Walsh et al., 2005). MBLs are inhibited by metal chelators such as EDTA and are not susceptible to the currently available ß-lactam inhibitors such as clavulanate, sulbactam, tazobactam, and avibactam (Walsh et al., 2005; Cornaglia et al., 2011). Based on sequence similarity, MBLs are classified into subclasses B1, B2, and B3 (Galleni et al., 2001). Subclass B1 comprises most of the MBLs acquired by bacteria of clinical relevance, such as IMP, VIM, and NDM (Nordmann et al., 2011). Subclasses B2 and B3 have mostly been described in environmental bacteria as resident (i.e., not associated with mobile genetic elements) resistance determinants (Rossolini et al., 2001; Saavedra et al., 2003; Stoczko et al., 2006). The only exceptions are AIM-1 and SMB-1, which have been reported in clinical isolates of Pseudomonas aeruginosa and Serratia marcescens, respectively (Wachino et al., 2011; Yong et al., 2012). B1 and B3 enzymes generally exhibit broad β-lactam substrate profiles, whereas B2 enzymes preferentially hydrolyze carbapenems (Bebrone et al., 2009; Fonseca et al., 2011).
The origins of the carbapenemases found in clinically relevant bacteria are unknown. Soil bacteria represent an important reservoir of antibiotic resistance determinants, including β-lactamases (Allen et al., 2009; Forsberg et al., 2012; Nesme and Simonet, 2015), and therefore a possible reservoir of carbapenemases in nature. However, little information is available about the occurrence and types of carbapenemases in soil microbiota. In a previous study, we have shown that subclass B3 MBLs are widespread in different genera of the culturable fraction of soil bacteria (Gudeta et al., 2016a). Since only a small fraction of the soil microbiota is culturable, the actual diversity of carbapenemases is likely greater than what it could be appreciated by culture-dependent methods. The aim of this study was to elucidate occurrence, diversity, and functionality of carbapenem-hydrolyzing enzymes in soil microbiota using a functional metagenomics approach. We identified new types of carbapenemases, demonstrated their functionality in Escherichia coli, and predicted the most likely bacterial taxa from which they originated.
Materials and Methods
Ten soil samples were selected from a collection of 25 samples that were previously screened for the occurrence of carbapenemase-producing bacteria by antibiotic selective culture (Gudeta et al., 2016a). We selected one sample per soil type and per geographical origin (Table 1). If more than one soil sample met these criteria, we selected a sample not yielding any carbapenemase-producing strains by selective culture.
Table 1. The 10 soil samples and corresponding yields of genes encoding metallo-β-lactamases obtained by functional metagenomics (this study) and by antibiotic selective culture (Gudeta et al., 2016a).
Metagenomic Library Construction and Screening
Metagenomic DNA was purified from one gram of soil by PowerSoil® DNA Isolation Kit (MO BIO Laboratories, CA, USA) using four power-bead tubes per sample. After verifying the quality of the DNA yield by gel electrophoresis, DNA was sheared by Ultrasonic homogenizer (BANDELIN electronic GmbH & Co. KG) to approximately 0.5–7 kb fragments as previously described (Gudeta et al., 2016a). Fragments with size ranging 1.5–5 kb were separated by electrophoresis and purified by GeneJET Gel Extraction Kit (Thermo Scientific, Vilnius, Lithuania). Gel-purified DNA was blunted in a standard 50-μl reaction by End-It™ DNA End-Repair Kit (Epicentre, Madison, USA). Blunted DNA was purified and ligated into HincII-linearized pZE21MCS vector as previously described (Gudeta et al., 2016a). The ligation mix was dialyzed for 2 h using 0.025 μm DNA filter paper (Millipore, Massachusetts, USA) and distilled water. A 4-μl aliquot of the ligation mix was used to transform One Shot® TOP10 Electrocomp™ Escherichia coli (Invitrogen, Carlsbad, USA) using Gene Pulser II (Bio-Rad, California, USA). One milliliter of SOC medium was added to the transformation mix and incubated at 37°C with shaking. After 1 h incubation, 400 μl were plated on Luria Bertani (LB) agar (Difco, Le Pont-de-Claix, France) containing 50 μg/ml of kanamycin and 30 μg/ml of amoxicillin (Sigma-Aldrich, Steinheim, Germany). Library coverage was estimated by plating a ten-fold diluted suspension of the recovered cells on LB agar containing 50 μg/ml of kanamycin according to the procedure described by Sommer et al. (2009). The remaining library was enriched overnight in LB broth containing 50 μg/ml of kanamycin followed by subculture on LB agar (Difco, Le Pont-de-Claix, France) containing 50 μg/ml of kanamycin and 30 μg/ml of amoxicillin. Amoxicillin was used for initial screening to facilitate detection of carbapenemases, as previously described by various authors (Poirel et al., 2000; Bellais et al., 2002; Girlich et al., 2010).
Detection of E. coli Recombinant Clones Expressing Carbapenemase
Up to 100 randomly selected amoxicillin-resistant colonies per sample were screened for carbapenemase production by CarbaNP test (Dortet et al., 2014). Briefly, cells were lysed in 100 μl of Tris-HCl buffer (Thermo Scientific, Rockford, Il, USA) and the lysate was mixed with 100 μl of phenol red solution containing 6 mg/ml imipenem/cilastatin (Fresenius Kabi, Bad Homburg, Germany). Phenol red solution without imipenem was included in the test as a negative control. After incubating at 37°C for a maximum of 2 h, red to orange/yellow color shift in the test vial and no color change in the negative control were interpreted as imipenem hydrolysis. Plasmid inserts of the carbapenemase-producing clones were sequenced using the primers described in Table 2. Sequences displaying less than 70% amino acid sequence identity to known MBLs were defined as new MBLs, as suggested by Cornaglia et al. (2007).
Determination of Minimum Inhibitory Concentration (MIC) and Carbapenemase Activity
The MICs of selected ß-lactams were measured in carbapenemase-producing recombinant E. coli TOP10-derived clones by broth microdilution using Sensititre ESBL plates (Trek Diagnostic Systems, OH, USA). The MICs of third-generation cephalosporins, cefepime, imipenem, and meropenem that fell outside the range of concentrations included in these commercial plates were determined by the broth microdilution method according to the Clinical Laboratory Standards Institute (CLSI) guidelines (Clinical Laboratory Standards Institute, 2015). Carbapenemase activity in bacterial crude extracts was determined by UV spectrophotometry as described previously (Lauretti et al., 1999), using 150 μM imipenem as the substrate in a Cary 100 UV-Vis spectrophotometer (Varian, Walnut Creek, CA).
The sequences of carbapenemase-encoding genes were used as queries in BLASTX in the NCBI database (default parameters). Hits showing maximum identity to the query sequence, except putative homologous proteins with unknown function, were downloaded into a local database for sequence alignment and amino acid comparison. MBLs with previously determined 3-D structure were used for structural alignments to ascertain if the known metal-binding amino acids were conserved in the new MBLs detected in this study. Additional sequences of previously described MBLs were obtained from published studies and added to the local database for phylogenetic tree construction. Amino acid sequence alignment was performed by MUSCLE (http://www.phylogeny.fr/one_task.cgi?task_type=muscle). Maximum likelihood analysis was performed by raxmlGUI 1.5b (Silvestro and Michalak, 2012) using the WAG amino acid substitution model, which was selected using Akaike Information Criterion implemented in PROTTEST 3 (Darriba et al., 2011). The data were analyzed using rapid bootstrap algorithm with 1000 bootstrap replicates. The phylogenetic tree was visualized by FigTree v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/) and the possible bacterial hosts of MBL-encoding genes were predicted by RAIphy based on comparison of relative abundance of unique 7-mers in the query sequence with reference genomes (Nalbantoglu et al., 2011; Forsberg et al., 2014).
The nine MBL nucleotide sequences described in this study have been submitted to GenBank and assigned accession numbers KU167035 to KU167043.
Library Coverage and Carbapenemase Activity of Recombinant Clones
The 10 constructed plasmid libraries, each containing DNA fragments of 1.5 kb on average, collectively covered an estimated size of 16 Gb, corresponding to over 3500 E. coli genome equivalents. Only three libraries yielded amoxicillin-resistant colonies after direct plating onto LB agar containing kanamycin and amoxicillin (3–84 colonies per library), whereas all libraries yielded amoxicillin-resistant colonies (17 to > 500 colonies per library) when plating onto selective agar was preceded by overnight enrichment in LB containing kanamycin, proving the usefulness of this enrichment step for detection of amoxicillin-resistant recombinant clones. Carbapenemase-producing recombinant clones were detected by CarbaNP in six of the 10 libraries (Table 1). The overall rate of carbapenemase-positive colonies was 10.6% (94/890).
Identification and Phylogeny of Carbapenemases Detected by Functional Metagenomics
Among the 94 carbapenemase-positive colonies, nine recombinant clones originating from six soil samples were shown to contain a unique insert (Table 1). All the inserts were genes encoding for MBLs, including two subclass B1 (GRD23-1 and SPN79-1) and seven subclass B3 (CRD3-1, PEDO-1, GRD33-1, ESP-2, ALG6-1, ALG11-1, and DHT2-1). The MBLs identified in three of the libraries by direct plating on selective LB agar corresponded to those detected after overnight enrichment of the same libraries. However, four MBLs (ALG6-1, ALG11-1, DHT2-1, and SPN79-1) were only detected by enrichment. PEDO-1and ESP-2 were identical or closely related to MBLs detected in the previous study by selective culture of soil (100% and 99.7% identity, respectively) (Gudeta et al., 2016a). The seven remaining enzymes were regarded as new MBLs since they showed only 33–59% amino acid identity to known carbapenem-hydrolyzing MBLs (Table 3). All known zinc-binding amino acids in subclasses B1 and B3 enzymes were conserved (Figure 1). The mid-point rooted phylogenetic tree confirmed that the new MBLs belonged to subclasses B1and B3, as supported by high bootstrap values (100%). Three of them (CRD3-1, SPN79-1, and GRD23-1) shared a common ancestor with transferrable MBLs but none was closely related to the main MBLs found in pathogenic bacteria. Although phylogenetic analysis showed a likely evolutionary relationship between SPN79-1 and VIM-1 (Figure 2), SPN79-1 displayed low amino acid identity (<40%) to all VIM-types.
Table 3. Predicted bacterial host and closest homologs of the nine metallo-β-lactamases (MBLs) detected in soil microbiota by functional metagenomics.
Figure 1. Amino acid alignment of the new subclass B3 (A) and B1 (B) metallo-ß-lactamases (MBLs) detected in this study with known MBLs whose 3-D structure has been resolved by X-ray crystallography. Zinc-binding amino acids are indicated by a star.
Figure 2. Mid-point rooted phylogenetic tree of subclass B1 and B3 metallo-ß-lactamases (MBLs). The evolutionary distances are expressed in unit of amino acid substitution per site. Bootstrapping support using 1000 replicates was applied and is indicated on branches where values are >70%. The new MBLs detected in this study are indicated in black while previously described acquired and resident MBLs are written in red and blue colors, respectively. The MBLs detected in clinical and environmental bacteria are indicated by a dot and a star, respectively.
β-Lactam Susceptibility and Functional Characterization of MBL-Producing E. coli Clones
Expression of the nine MBLs recovered from soil microbiota increased the MIC values of ampicillin and cefoxitin above the clinical resistance breakpoints in E. coli TOP10. The MICs of 1st and 3rd generation cephalosporins were increased according to various enzyme- and compound-dependent profiles (Table 4). None of the MBL-producing clones exhibited decreased susceptibility to cefepime, a 4th generation cephalosporin. As for carbapenems, expression of all MBLs determined a 2- to 16-fold increase in the MIC of imipenem compared to the wild type strain, and all except SPN79-1 resulted in 2- to 64-fold higher MICs of meropenem (Table 4). However, for none of the MBLs, the MIC of imipenem was increased above the CLSI clinical resistance breakpoint (R ≥ 4 μg/ml), and clinical resistance to meropenem was only achieved through expression of ALG11-1, CRD3-1, and GRD23-1.
Table 4. Imipenem hydrolytic activity and minimum inhibitory concentration (MIC) of selected ß-lactams in Escherichia coli TOP10 expressing nine distinct metallo-ß-lactamases (MBLs) recovered from soil microbiota by functional metagenomics.
Carbapenemase Activity of MBLs
The enzymatic assays showed a variable amount of imipenem-hydrolyzing activity in the various crude extracts (Table 4). Crude extract from the CRD3-1-producing strain showed the highest specific activity (1120 ± 125 nmol/min·mg of protein). Notable imipenem-hydrolyzing activities were also detected in crude extracts of the strains producing ALG11-1, GRD33-1, PEDO-1, DHT2-1, SPN79-1, ESP-2, and GRD23-1 (37–620 nmol/min·mg of protein). Only the crude extract from the ALG6-1-producing strain exhibited low imipenem-hydrolyzing activity, which was consistent with the low imipenem MIC observed in this strain. It is interesting to note that the production of ALG6-1 in E. coli yielded a phenotype notably different from that mediated by the other MBLs, with high-level resistance to ceftazidime and relatively limited activity on carbapenems (Table 4). Thus, the substrate profile of ALG6-1 was unusual, resembling that of CAR-1 from Erwinia carotovora (Stoczko et al., 2008).
Culture-Based Method vs. Functional Metagenomics
We compared the carbapenemase yield obtained by functional metagenomics to that obtained by antibiotic selective culture of the ten soil samples analyzed in this study (Gudeta et al., 2016a). Three samples (ID 6, 7, and 10) did not yield any carbapenemase-encoding gene by both methods. The metagenomics approach detected at least one MBL gene from four samples that did not yield any carbapenemase producers by selective culture (Table 1). On the contrary, in one sample (ID 4), carbapenemase-producing bacteria were detected by culture but no carbapenemase-encoding genes were detected by functional metagenomics. Finally, in two samples (ID 3 and 23) both methods allowed detection of carbapenemase-encoding genes but different types were detected depending on the method used (Table 1). Three soil samples yielded simultaneously two MBL-encoding genes by functional metagenomics and one of these samples (ID 23) was also the only soil yielding more than one (n = 4) MBL producers by antibiotic selective culture.
Prediction of Bacterial Hosts
According to prediction analysis using RAIphy, six enzymes (CRD3-1, GRD23-1, DHT2-1, SPN79-1, ALG6-1, and ALG11-1) originated from Proteobacteria, two (PEDO-1 and ESP-2) from Bacteroidetes, and one (GRD33-1) from Gemmatimonadetes. The two former phyla (Proteobacteria and Bacteroidetes) were also represented among the MBL producers isolated by selective culture in the previous study (Gudeta et al., 2016a), whereas the third one (Gemmatimonadetes) was only detected by functional metagenomics. The likely genus from which blaPEDO−1 originated could not be determined.
Nine carbapenem-hydrolyzing MBLs, including seven novel enzymes, were identified in six out of ten soil samples using a functional metagenomics approach. All these enzymes showed hydrolytic activity toward imipenem (as measured in bacterial crude extracts) and increased the MICs of different ß-lactam agents in E. coli, thus confirming their functionality. Based on sequence and phylogenetic analyses, two enzymes belonged to subclass B1, and the remaining seven belonged to B3. Previous studies have highlighted the relative abundance of subclass B3 enzymes in the soil resistome (Rossolini et al., 2001; Docquier et al., 2002; Stoczko et al., 2006; Allen et al., 2009; Gibson et al., 2015). However, carbapenemase activity was not examined in presumptive MBLs reported by previous functional metagenomics studies (Allen et al., 2009; Gibson et al., 2015). Neither subclass B2 nor serine-based (class A and D) enzymes were detected. The lack of subclass B2 enzymes may be due to the screening approach used since amoxicillin is a poor substrate for this MBL subclass. Differently from zinc-based carbapenemases, serine-based carbapenemases have mainly been reported in aquatic bacterial isolates (Henriques et al., 2004; Poirel et al., 2004; Aubron et al., 2005; Girlich et al., 2010; Gudeta et al., 2016b), suggesting that yet unknown ecological factors may play a role in the distribution of these two groups of enzymes in nature. Interestingly, none of the MBLs detected in soil was closely related to common MBLs in clinical bacteria. It will be interesting to see if any of these MBLs will emerge in clinical bacteria in the future.
The new MBLs described in this study showed significant sequence heterogeneity in comparison with previously described MBLs (Table 3). Our phylogenetic analysis shows that within the three subclasses B1 and B3 MBLs do not cluster according to the origin (clinical vs. environmental) of the bacterial hosts (Figure 2), underlining a huge diversity within this family of enzymes. Although transferrable MBLs mainly belong to subclass B1 (Cornaglia et al., 2007; Nordmann et al., 2012), transfer of subclass B3 MBLs such as AIM-1 and SMB-1 from environmental bacteria to human pathogens has been hypothesized (Wachino et al., 2011; Yong et al., 2012). Most of the MBLs identified in soil microbiota by functional metagenomics were predicted to originate from Proteobacteria, which comprise the majority of human pathogenic bacterial species. This taxonomic relationship may ease acquisition of new MBL-encoding genes from soil microbiota by pathogenic bacteria.
When expressed in E. coli, the MBLs from soil microbiota conferred resistance to different classes of β-lactams, including penicillins and several cephalosporins. None of these enzymes affected the MIC of cefepime, which is in line with the poor catalytic efficiency of MBLs for this substrate (Bellais et al., 2002; Naas et al., 2003; Stoczko et al., 2006). Heterologous expression resulted in higher MICs of carbapenems (Table 4), even though in most cases did not confer resistance according to the CLSI clinical breakpoint (R ≥ 4 μg/ml). Other authors have reported low-level resistance to carbapenems as a result of heterologous MBL expression in laboratory E. coli strains, regardless if the expressed MBL originated from environmental (Rossolini et al., 2001; Docquier et al., 2002; Stoczko et al., 2006; Gudeta et al., 2016a) or clinical bacteria (Lauretti et al., 1999; Poirel et al., 2000; Gibb et al., 2002; Yong et al., 2012). This apparent incongruity may be explained by the very high permeability of the E. coli outer membrane to carbapenems, which results in higher intracellular drug concentrations despite of MBL activity (Matsumura et al., 1999).
The discovery of new MBL-encoding genes from samples that did not yield MBL producers by culture suggests that those genes may originate from the unculturable fraction of the soil microbiota or from bacteria non-culturable under the conditions used in the previous study (Gudeta et al., 2016a). On the other hand, none of the MBLs previously identified by culture were re-detected by functional metagenomics in the 10 samples that were analyzed by both methods (Table 1). The lack of detection of these MBLs by functional metagenomics may be due to various factors, including low concentration of culturable MBL-producers in the sample or loss/dilution of DNA during the cloning procedure. Although this study was not designed to compare the occurrence of MBLs in agricultural and grassland soil, it is interesting to notice that MBLs were detected more frequently and displayed higher diversity in grassland samples than in agricultural samples irrespective of the method used (Table 1).
Even though the study failed in detecting any close ancestors of the main carbapenemases observed in clinical bacteria, it has improved the current knowledge of the diversity of MBLs in soil microbiota by describing seven new enzymes belonging to subclasses B1 and B3. All these MBLs were functional in E. coli, resulting in detectable imipenem-hydrolyzing activity and significantly higher MICs of clinically relevant ß-lactams. The MBLs detected by functional metagenomics differed from those detected in same soils samples by selective culture, showing that the two approaches targeted different subpopulations in soil microbiota. This evidence supports the notion that functional metagenomics and culture may be complementary tools for detection of antibiotic resistance determinants in soil.
DG performed all the bioinformatic analyses and the laboratory work except for the enzyme activity assay by UV-spectrophotometry, which was carried out by the group at the University of Siena (SP, JD, and GR). VB supervised study design and laboratory work at the University of Copenhagen. EW and GA trained DG in metagenomic library construction at the University of Warwick, the UK. LG conceived the idea of the study, raised the necessary funds and contributed to discussion of the results and writing of the manuscript together with all authors.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors thank Geir Wing Gabrielsen and Kristian Koefoed Brandt for providing soil samples. This study was supported by grant HEALTH-F3-2011-282004 (EvoTAR) from the European Union and by the University of Copenhagen.
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Poirel, L., Naas, T., Nicolas, D., Collet, L., Bellais, S., Cavallo, J. D., et al. (2000). Characterization of VIM-2, a carbapenem-hydrolyzing metallo-β-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob. Agents Chemother. 44, 891–897. doi: 10.1128/AAC.44.4.891-897.2000
Rossolini, G. M., Condemi, M. A., Pantanella, F., Docquier, J. D., Amicosante, G., and Thaller, M. C. (2001). Metallo-β-lactamase producers in environmental microbiota: new molecular class B enzyme in Janthinobacterium lividum. Antimicrob. Agents Chemother. 45, 837–844. doi: 10.1128/AAC.45.3.837-844.2001
Saavedra, M. J., Peixe, L., Sousa, J. C., Henriques, I., Alves, A., and Correia, A. (2003). Sfh-I, a subclass B2 metallo-β-lactamase from a Serratia fonticola environmental isolate. Antimicrob. Agents Chemother. 47, 2330–2333. doi: 10.1128/AAC.47.7.2330-2333.2003
Stoczko, M., Frère, J.-M., Rossolini, G. M., and Docquier, J.-D. (2006). Postgenomic scan of metallo-β-lactamase homologues in Rhizobacteria: identification and characterization of BJP-1, a subclass B3 ortholog from Bradyrhizobium japonicum. Antimicrob. Agents Chemother. 50, 1973–1981. doi: 10.1128/AAC.01551-05
Stoczko, M., Frère, J.-M., Rossolini, G. M., and Docquier, J.-D. (2008). Functional diversity among metallo-ß-lactamases: characterization of the CAR-1 enzyme of Erwinia carotovora. Antimicrob. Agents Chemother. 52, 2473–2479. doi: 10.1128/AAC.01062-07
Wachino, J., Yoshida, H., Yamane, K., Suzuki, S., Matsui, M., Yamagishi, T., et al. (2011). SMB-1, a novel subclass B3 metallo-β-lactamase, associated with ISCR1 and a class 1 integron, from a carbapenem-resistant Serratia marcescens clinical isolate. Antimicrob. Agents Chemother. 55, 5143–5149. doi: 10.1128/AAC.05045-11
Yong, D., Toleman, M. A., Bell, J., Ritchie, B., Pratt, R., Ryley, H., et al. (2012). Genetic and biochemical characterization of an acquired subgroup B3 metallo-β-lactamase gene, blaAIM−1, and its unique genetic context in Pseudomonas aeruginosa from Australia. Antimicrob. Agents Chemother. 56, 6154–6159. doi: 10.1128/AAC.05654-11
Keywords: carbapenems, antibiotic resistance, soil, functional metagenomics, metallo-ß-lactamases
Citation: Gudeta DD, Bortolaia V, Pollini S, Docquier J-D, Rossolini GM, Amos GCA, Wellington EMH and Guardabassi L (2016) Expanding the Repertoire of Carbapenem-Hydrolyzing Metallo-ß-Lactamases by Functional Metagenomic Analysis of Soil Microbiota. Front. Microbiol. 7:1985. doi: 10.3389/fmicb.2016.01985
Received: 17 July 2016; Accepted: 28 November 2016;
Published: 26 December 2016.
Edited by:Manuela Caniça, National Institute of Health Dr. Ricardo Jorge, Portugal
Reviewed by:Heather K. Allen, National Animal Disease Center (USDA), USA
Robert Kourist, Ruhr University Bochum, Germany
Copyright © 2016 Gudeta, Bortolaia, Pollini, Docquier, Rossolini, Amos, Wellington and Guardabassi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Luca Guardabassi, email@example.com
†Present Address: Dereje D. Gudeta, Department of Microbiology and Immunology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, USA
Valeria Bortolaia, National Food Institute, Technical University of Denmark, Kgs. Lyngby, Denmark
Gregory C. A. Amos, Scripps Institution of Oceanography, University of California, San Diego, San Diego, CA, USA

domain: biology


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The deep blue waters of the Mediterranean Sea were shrouded in dust from North African deserts on January 29, 2005. A dusty haze stretches across the center and top parts of the scene from the Gulf of Sirte (left center edge), past the Nile Delta (right of center), all the way to Turkey (top right). The Red Sea (lower right corner) appears to be dust free. Between the knob of land in Libya just east of the Gulf of Sirte and the green fan of the Nile Delta lies a large desert called the Great Sand Sea. Dust storms are a serious natural hazard in the region, particularly for aircraft. This image was captured by the Moderate Resolution Imaging Spectroradiometer (MODIS) on NASA’s Aqua satellite.
NASA image created by Jesse Allen, Earth Observatory, using data obtained from the MODIS Rapid Response team.

domain: geography


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22 January 2018 | jwwalrath-227-85487
Nice, but lightweight
Mary and the Witch's Flower is a fun film that should delight the kiddies, but is not a perfect film.
The animation is excellent, not to mention the character and set design. Has a very unique old-timey fantasy feel to it. Mary is a strong likable character. The conclusion is both action and emotionally strong.
However, the first act lags. Running at a little over two hours, the film could have cut this down somewhat. The biggest flaw is that besides Mary, most of the film is very one-dimensional. They don't explain the world of magic and the magical university really well. (After the university is introduced, they never even bother showing the staff or students again except for three characters.) The world is more of a plot device than a truly living world.

domain: artistic


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DJ Zinhle is one of the South African DJ, Producer, and Media personalities, apart from this, she is also a businesswoman. AT present she has been the headline of the news, her real name is Ntombezinhle Jiyane, but most of the time she is recognized by her stage name DJ Zinhle. She has gained lots of fan following over the social media platforms. If you are also one of his fans then do read the post, we will let you know every single detail about her such as DJ Zinhle’s wiki, boyfriend, net worth, age, and more.
DJ Zinhle Wiki
DJ is professionally a DJ, producer, and media personality, she was born on December 30, in the year 1983, at present her age is 37 years old as of now. DJ Zinhle’s real name is NtombeZinhle Jiyane, her hometown is Dannhauser, KwaZulu-Natal, she is holding a South African Nationality, her marital status is married, even she has a child.
DJ Zinhle Age
Name: DJ Zinhle
Date Of Birth: December 30, 1983
Birthplace: Dannhauser, KwaZulu-Natal
Age: 37 years old as of now
Marital Status: Married
Profession: DJ, Producer, and Media Personality
Net Worth: Under Review
DJ Zinhle Early Life
Talking about her early life then Jiyane was born on the 30 of December in 1983, at present she has been 37 years old as of now, she belongs to Dannhauser, KwaZulu, Natal. After she left her hometown, she settled in Newcastle, DJ Zinhle’s family is not revealed yet her mother’s and father’s names and professions are hidden yet.
DJ Zinhle Career
DJ has begun her career in the year 2004, in the beginning, she wanted to work as a TV presenter, she has never thought to be a DJ and producer, her brother has a passion for music and she got fascinated by him, and took so much interested in that. In her career, she has worked with a number of artists such as Dr. Malinga, Khanyi Mbau, Mphoza, and many more. Even DJ associated with South African Music icon Boom Shaka on the album I Put It In.
DJ Zinhle Wife
Let’s delve into her personal life, we would like to tell you that she has been in some relationships, previously she was in a long relationship with the South African Rapper AKA, unfortunately, they have been separated in the year 2020 in Jan, she has a child from her past relationship, DJ Zinhle’s child’s name is Kairo Owethu

domain: celebrity


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Exploring the Shropshire Hills by Mountain Bike
The Shropshire Hills boast some really great mountain bike trails, not least on land managed by the Forestry Commission, where you’ll find everything from family fun rides to cross-country and serious downhill challenges. The Commission publishes its own well illustrated guides to bike trails in Hopton Woods, which you can download. Eastridge Woods are also a popular destination look online for details.
But you don’t have to cycle only in forests – the Long Mynd is criss-crossed by bridleways that provide excellent mountain bike enjoyment and the National Trust publishes a map of suitable routes that you can also download.
If you are planning a longer ride in the Shropshire Hills and doing your own route finding, remember these basic rules:
- You can cycle on bridleways, but always be respectful of other users
- You can cycle on Byways, but you may encounter motor vehicles on Byways Open to All Traffic (BOATs)
- You can very often cycle on forestry tracks, but should stick to waymarked routes
- You can ride on green lanes, or unmade roads
- You very often have the right to cycle on roads that are marked white on the Ordnance Survey map
- You are not allowed to ride on footpaths or disused railway lines, unless the latter have been specifically designated for cycle use
- You should always ride responsibly, braking carefully to help avoid erosion.
Accommodation in and near Ludlow
The Feathers – historic hotel in central Ludlow
Accommodation at Bucknell
Baron at Bucknell – country inn with rooms, including luxury garden rooms
Accommodation near the Long MyndThis product has been developed as part of the Shropshire Hills Tourism Cooperation Project, funded by the Department for Environment, Food and Rural Affairs under the auspices of the European Agricultural Fund for Rural Development. The project is delivered by Gravity Consulting Ltd on behalf of Shropshire Hills and Ludlow Destination Management Partnership.

domain: travel


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We can all live our best lives by incorporating resolutions into our everyday routines. Everyone has a home, and everyone aspires to be elegant and attractive. Employing wonderful vintage art collections or designs is the ideal technique to enhance your home's decor's plain, blank walls.
We still have extra items and variety to advance and take advantage of the situation. The collection of old species or materials with historical significance is referred to as vintage art. But do you know what vintage means? If not, you have come to the proper place. This page will thoroughly review the artisans and vintage art creations.
They draw inspiration and ideas from numerous historical eras and vintage designs. Our vintage wall art offers beautiful framed prints, world atlases, floral prints, and more may be found in this vintage art collection. These ingenious and lovely suggestions provide your space with a vintage appearance and feel.
The secret to vintage art design is to keep things clean and uncluttered. You focus more on the people, activities, and experiences you can create over time than the things themselves. It simply suggests that we place a lot of value on worldly goods in the hopes that they will bring us complete joy and contentment.
What does Vintage Art mean?
"vintage art" broadly refers to the commercial artwork generated or produced from the 19th century to the middle of the 20th century. Watercolor and oil paintings on canvas are not to be in a state of disarray.
When asked to give a design or image a vintage appearance, vintage refers to various styles, protracted decades, and design disciplines. Technological advancements, innovations, and other conveniences have improved our decor. Having a home with a rustic and vintage feel has made people's lives much easier and more sentimental.
We'll provide you with enough details on this page's vintage or retro art designs to be helpful. There are many other qualities of vintage art. It features diverse graphical depictions of vintage flyers, postcards, prewar posters, labels for orange crates, propaganda posters, and wall art.
Vintage Wall Art for your home décor
This section will discuss some vintage art style design ideas that would give your home a lovely and cute look. Vintage art adds a timeless touch to your wall or home decor if you've decided to purchase antique items from flea markets or antique shops. Selecting colorful and ornately framed portraits, landscape paintings, and—more importantly—vintage posters or prints that offer a fleeting glimpse of historical animals and entities is the ideal approach to transforming your house or office into a stunning space.
When choosing wall hangings, be sure to consider your color scheme. You fall in love at first sight when vintage wall art transforms your home's atmosphere into something exotic and seductive. You may add the most excellent and effective antique art wall paintings. The use of vintage items creates a beautiful appearance and feel.
Any home or business would benefit significantly from including various art décor ideas. The most striking and bright vintage-style decorations include botanical art, antique displays, and stairway vintage art galleries. Except for the design you choose, your decor will always be unique.
Additionally, when choosing the ideal designs for your wall art, vintage art and antique prints should be taken into account. We also provide a variety of patterns and styles that you might need when designing the interior and exterior of your home.
You can also add particular items as decoration. The right antique or vintage art design may do wonders and enhance your home. A home's walls can be decorated with other accessories as well. Our collection of vintage wall art's gorgeous designs instantly transforms them into the room's focal point.
Are you prepared to add a dash of sophistication and vintage art to your living space? The first step is to beautify your walls with our vintage wall art!


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Maximizing Home Value and Comfort with Timely Window Upgrades
In the dynamic world of home improvement, the role of windows transcends mere aesthetics. They are pivotal in ensuring energy efficiency, security, and enhancing the overall ambiance of your living space. MasterRoof Contracting, renowned for its expertise in home renovations, underscores the importance of replacing windows at the right time to elevate your home’s functionality and comfort.
Identifying the Need for New Windows
Windows generally offer a functional lifespan of two decades or more. However, certain indicators necessitate an earlier replacement:
- Energy Inefficiency Signs: Drafts or fluctuating room temperatures suggest your windows are failing to insulate properly.
- Operational Challenges: Difficulty in opening or closing windows points towards potential structural issues.
- Moisture Between Glass Panes: This indicates a compromised seal, diminishing the window’s insulative effectiveness.
Selecting Ideal Windows for Your Residence
When exploring options for Dayton windows and doors, it’s crucial to choose types that align with your home’s architecture and your personal needs. MasterRoof Contracting provides a diverse range of styles, including:
- Double-Hung Windows: Perfect for classic home designs, they offer easy maintenance and superior ventilation.
- Casement Windows: Excellent for energy conservation, these windows are ideal in windy regions due to their enhanced sealing capability when closed.
- Picture Windows: Best suited for rooms with picturesque views, they allow abundant natural light and offer expansive outdoor views.
The Installation Journey: Precision and Expertise
The caliber of window installation in Dayton, Ohio, is as significant as the windows themselves. MasterRoof Contracting’s installation process is meticulous and thorough, involving:
- Personalized Consultation: Tailoring window solutions to fit your specific requirements and style preferences.
- Precise Measurement: Guaranteeing a perfect fit for your new windows in the existing spaces.
- Expert Installation: Our team of proficient window installers in Dayton, Ohio, employs state-of-the-art techniques for a flawless installation.
- Quality Assurance Check: A thorough inspection post-installation to ensure optimal performance and aesthetics.
Advantages of Expert Window Replacement
Choosing professional Dayton window replacement brings several benefits:
- Boosted Energy Efficiency: Modern windows significantly cut down on energy costs by providing superior insulation.
- Enhanced Property Value: New windows improve both the external and internal appeal of your home, potentially increasing its market value.
- Augmented Comfort and Safety: Upgraded windows offer better noise reduction and advanced security features, contributing to a serene and secure living environment.
Timely and professional window replacement is a crucial aspect of home renovation. Opting for MasterRoof Contracting’s services in window companies in Dayton, OH, ensures that your home not only gains aesthetically but also becomes more energy-efficient and comfortable. Elevate your living experience with our expert window solutions, tailored to meet and exceed your expectations.

domain: design


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In a trading update last week, the major bank said its decision is also designed to bring operations closer to its customer businesses and continue operational efficiency gains.
The changes follow a planned move for group chief operating officer Alistair Currie to step down from ANZ later this year reflecting Mr Currie’s ambition to continue his international banking career.
In the coming months a number of key changes will occur in ANZ’s technology, operations and shared services divisions. CIO Scott Collary will report to Mr Currie until the transition of the group technology reporting line to CEO Shayne Elliott later this year, reinforcing technology’s strategic role alongside digital banking. Other key changes include:
- Divisional chief operating officers will report directly into the Australia, New Zealand, institutional and wealth businesses, providing greater end-to-end control of customer processes as well as driving the next phase of productivity gains.
- Deputy CEO Graham Hodges will assume responsibility for group hubs, enterprise services and group property. Craig Sims has been appointed to a new role as group general manager of operations and services reporting to Mr Hodges. Mr Sims will have responsibility for group hubs and enterprise services, and will work with divisional COOs to build on the momentum established in operational efficiency.
- General manager of transformation projects Nigel Dobson will assume responsibility for wholesale digital strategy and payments transformation, reporting to group executive digital banking Maile Carnegie.
ANZ chief Shayne Elliott said the changes to the group’s operating model support ANZ’s strategic priorities better and allow the bank to build on the significant progress that has been made in recent years in technology, operations and shared services.
“This progress highlights the importance of Alistair’s contribution to ANZ. He joined us in 2008 and led the creation of our regional transaction banking business,” Mr Elliott said.
“He became group chief operating officer in 2011 and has built a strong regional team and an approach to technology and operations which has significantly improved service quality for customers as well as delivering consistent productivity gains. During this time, Alistair also played a key role in setting the early direction for our digital transformation.
“I understand Alistair’s desire to further his international banking career, and I thank him for his contribution and wish him every success in the future.”
[Related: ANZ names new CEO]

domain: news


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Total Calories: 337
Combine rotini, chicken, and dressing in a bowl, and mix well. Combine mayonnaise, lemon juice, mustard, onion, olives, cucumber, celery, pepper, and artichoke hearts, and mix well. Add to pasta mixture, and mix well. Season with salt. Chill, covered, 1–2 hours.
"I must say this is the best recipe software I have ever owned."
"Your DVO cookbook software saves me time and money!"
"I saw lots of recipe software for PC computers but I was having a hard time finding really good mac recipe software. I'm so glad I discovered Cook'n! It's so nice to have all my recipes in a computer recipe organizer. Cook'n has saved me so much time with meal planning and the recipe nutrition calculator is amazing!!!
My favorite is the Cook'n Recipe App.

domain: food


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Unbiased.Co.Uk is an online platform that provides financial advisors, mortgage brokers, accountants and solicitors.Read more
VouchedFor is perceived as one of unbiased.co.uk's biggest rivals. VouchedFor was founded in 2011 in London, England. VouchedFor is in the Systems Software field. VouchedFor generates $1M less revenue than unbiased.co.uk.
Group IFA is unbiased.co.uk's #2 rival. Group IFA is a Private company that was founded in 2009 in Lancashire, England. Group IFA competes in the industry. Compared to unbiased.co.uk, Group IFA has NaN fewer employees.
No recent acquisitions found related to unbiased.co.uk
No recent funding data found related to unbiased.co.uk
No recent investments found related to unbiased.co.uk

domain: economics


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a poem written by Paige Lavay
The sky was a perfect shade of blue.
My bare feet against the cool grass.
Dandelions blowing in the breeze.
I wonder why we have it so good
when so many others don't.
And I feel so blessed.
Looking at the full moon,
I know He is there, He has a plan.
A plan He wants me to fulfill.
Everything around me echos His Glory.
Those evenings that no one,
not the best poet- no one,
could ever put into words.
Because God is like that.
*nervous laugh* ...
That was my first ever poem that I wrote just a few days ago. *cringes* I KNOW, it's awful and I'm not sure if I like the ending. plus it's so common, like a poem that anyone can write. Mom inspired me to write a little poem, at least give it a try, cause she thought I could do it.
okay but really! Some nights are really like that!! Those peaceful wonderful, amazing evenings when you just feel God all around you. They are wordless. and I'm so greatful for this wonderful world He gave us!!
p.s. I know I said my next post was a surprise, but I haven't gotten everything together yet, so just a little while longer!! Ash needs to get pictures of ..*something*... and then I'll upload them and get it together. But Ash is barely ever here, so it's kinda hard working around her work hours. But soon peeps!! soon.


YARDAGE PRO Scout Laser Rangefinder
Item No. 200001
Reg. Price: $319.59
Sale Price: $225.00
You Save: $94.59
Only one left at this price!
Take your best shot with confidence with help from the Bushnell Yardage Pro Laser Rangefinder. Using advanced digital technology, the Bushnell Yardage Pro Rangefinder takes the guesswork out of measuring distances. This rangefinder delivers superb range performance to targets from 10 to 700 yards. The Bushnell Yardage Pro Rangefinder is accurate to within one yard even if you're measuring in rain, snow or through foreground clutter like brush and branches.
Bushnell Yardage Pro Laser Rangefinder is a precision instrument designed to provide many years of enjoyment. The Bushnell Yardage Pro Rangefinder emits invisible, eyesafe, infrared energy pulses. Sophisticated circuitry and a high speed clock are used to instantaneously calculate distances, by measuring the time it takes for each pulse to travel from the rangefinder, to the target, and back.
The ranging accuracy of the Bushnell Yardage Pro Laser Rangefinder is plus or minus two yards / meters under most circumstances. The unit will only display even numbers. The maximum range of the instrument depends on the reflectivity of the target.
Note: You will get both longer and shorter maximum distances depending on the reflective properties of the particular target and the environmental conditions at the time the object is being distanced to. The colour, surface finish, size and shape of the target all effect reflectivity and range. The brighter the colour, the longer the range.
What's in the Box:


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Croatian national team pitcher Ernesto Pereira, who was born in Venezuela, is going to join the Bern Cardinals for the 2014 season. The 31-year-old left-hander has been pitching for and in Croatia since 2005. He played for Zagreb last year. The Cardinals have won nine national championships in Switzerland since 2001. In 2013 they became the first Swiss club to play in the A-Pool European Cup.
Jan '14 21


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A whole summer of blueberries! Each collection contains three blueberry plants — 1 Patriot, 1 Pink Lemonade, and 1 Elliott. Patriot ripens in June, with Pink Lemonade in late July, followed by Elliott in August.
Agricultural regulations prohibit shipment of certain types of trees and plants. We do our best to provide a selection that gives everyone a chance to grow their own. This item cannot be shipped to all of GA, OR, and WA.

domain: agronomy


The Annabelle mattress is part of the new, exciting Sophia Briar-Rose collection. A range of mattresses designed to promote well being and improve sleep, made by reputable UK bed manufacturer Sleep Revolution.
The mattress is 27cm thick and contains sumptuous layers of selected quality fillings, making it the perfect option if you prefer a medium comfort support with a luxurious feel.
Featuring 2,000 pocket springs, it has been specially designed to mould to the body and balance weight distribution, whilst also minimising pressure points to improve blood circulation.
Finished in a traditional high grade, double jacquard fabric with air ventilators to help the mattress to breathe, flag stitched handles for easy movability, hand tufted for durability and 4 rows of hand stitched side borders.


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Jacopo Tintoretto (1518–94) is an ambiguous figure in the history of art. His radically unorthodox paintings are not readily classifiable, and although he was Venetian by birth, his claim to be truly a member of the Venetian School has often been doubted. As a youth, he was rejected early on from the workshop of the great Titian, who was accepted then, as now, as the quintessential Venetian painter. In the long career that nonetheless followed, Tintoretto abandoned the humanist narratives and sensual color values typical of Titian’s work in favor of a renewed concentration on core Christian subjects. He painted these in a chiaroscuro-based style using a rough and abbreviated technique.
Writers such as Giorgio Vasari and John Ruskin interpreted Tintoretto’s opposition to the artistic practice of his time as an aspect of personal eccentricity or spirituality. Jean-Paul Sartre saw the painter as “the son of an artisan . . . attacking the patrician aesthetics of fixity and being.” These oversimplified and a-historical interpretations mean that Tom Nichols’s re-assessment of Tintoretto’s place in the history of art is long overdue.
This generously illustrated book, , and featuring 16 added illustrations and a new Afterword by the author, charts the artist’s life and work in the context of Venetian art and the culture of the Cinquecento. The book deals extensively with Tintoretto’s greatest works, including the paintings at the Scuola di San Rocco in Venice.


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Jeff Pierce Jr., Karen Starich, & Chris Ebert
Karen Starich has been using her astrology skills to predict market trends since 2009. She then became a contributing writer on Zentrader.ca, writing financial astrology articles and trend analysis for gold and silver. Her accuracy for predicting the market direction combined with stock analysis grew a large following that led to her partnering with Jeff Pierce to launch Astrology Traders, a subscription newsletter focused on trends and stock analysis in 2011. Her trading style is focused on targeting the sectors and stocks that are poised to benefit from astrological cycles.
Jeff Pierce has been trading since 1999 when the tech bubble was in full swing. Having seen both extremes with bull & bear markets he applies his form of technical analysis to momentum charts and times entries & exits in conjunction with his market timing system. His trading style can best be summed up as buy high and sell higher.
Chris Ebert has been trading and investing for over 25 years, with a focus on options for 10 years. His book "Show Me Your Options!" has been among the most popular options books at Amazon. Having worked closely with both Karen and Jeff for several years, he has developed a unique type of market "barometer" based on option performance, that tends to correlate with future movements of stock prices, and broad market indices such as the S&P 500. His option trades are intended to minimize the risk of being right about a trade, but wrong about the timing. No form of stock-market analysis, whether through astrology, technical analysis, or an options barometer, can provide its fullest benefit unless it enables the trades to be held to fruition, even if the timing of the trade was not ideal. Chris uses options to shift the risk of a trade, so that exact timing is not necessary. Options allow profitable trades to be held through minor setbacks, without the risk of triggering a stop-loss prematurely, and without subjecting a trading account to excessive losses.

domain: finance


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What is Assisted Living and When To Consider It For A Loved One?
In the US, nearly 70% of all retired citizens will need some form of long-term care in their senior years. Whether that care is in the form of assisted living, memory care, or nursing home care will depend on the depth of need.
Many seniors will start needing assisted living.
You might wonder what services are offered through an assisted living arrangement. More importantly, if you have a loved one in their senior years, you might wonder if it’s time to consider assisted living for them.
Read on to learn more about assisted living and how you know if it’s time to consider this type of care for your loved one.
What Is Assisted Living?
Your loved one may have a preconceived assisted living definition that they equate to a nursing home or being ‘put in a home.’
In fact, assisted living homes have become so much more, and many seniors quickly realize it’s like moving to a new home instead of being put in a home.
Many assisted living locations offer separate apartments or suites for residents that can be set up with their own furniture and items from home, making this space feel like home.
Signs Your Senior May Need Assisted Living
While many seniors will resist assisted living, paying attention to the clues that it is time is essential.
In many cases, subtle and not-so-subtle ways indicate your loved one needs more help. They show signs that they’re struggling to live on their own.
Difficulty With the Activities of Daily Living
There are two terms often used in evaluating how a person manages living independently: basic Activities of Daily Living (ADL) and Instrumental Activities of Daily Living (IADLs).
Many of the warning signs on this list are connected to ADLs.
Basic ADLs include:
- Personal hygiene
IADLs include things like:
- Transportation and shopping
- Managing finances
- Shopping and meal preparation
- House cleaning and home maintenance
- Managing communication
- Managing medications
If your loved one starts to show signs of struggle with the items that are connected to activities of daily life, they almost always need more help, and it’s time to consider assisted living.
Decline in Health
One sure sign your loved one needs more help is a decline in health. It might be an age issue, and their health is just getting worse.
It might also be that they’re recovering from something and need more help.
As their health declines, taking care of many other daily living things becomes too much to handle, and more involved care is needed.
As many seniors age, their secure ability to move around decreases. Falling as a senior can cause other significant risks, like a bone break.
You should be concerned if your loved one shows signs of instability. If your loved one has had a recent fall, you have a genuine reason for concern.
You certainly don’t want your loved one to take a fall and be left lying on the floor without help.
As an assisted living resident, you know your loved one will be cared for to avoid the risk of a fall and further injury.
If you notice your senior living on their own and struggling to keep up with their medication, it’s a warning sign they may need more help.
Not taking medication can be dangerous. Or worse yet, taking it incorrectly can be another danger to their health.
In assisted living, your loved one will have the support needed to ensure they take their medication at the right time and dosage.
Many seniors face struggles with food. They either have a hard time shopping for food or preparing food for themselves. Some seniors even forget to eat.
If you have a loved one with mobility issues, they may be struggling with food preparation or even the ability to prepare single servings of food.
In assisted living, not only do meals get prepared for them, the caregivers will also make sure they’re eating.
It’s not uncommon for many seniors to struggle with self-care, including things like bathing and brushing their teeth. You may notice that your loved one isn’t clean or has body odor that they didn’t use to have.
Some will simply forget to bathe and wash up, and others won’t do it because it’s too hard to do on their own. Whether they struggle to get into and out of the shower or bath or can’t safely reach their own body parts for cleaning, it’s time to get them some help.
Being clean is important to maintaining a healthy lifestyle. You don’t want your loved one to be dirty because they can’t handle caring for their own hygiene.
For many seniors, mobility issues lead to other problems. In addition to worrying about them falling, you should worry if they’re getting enough active movement in their day.
Can they safely get up and down from a chair? Should they be still driving? Sometimes mobility issues mean they can’t get themselves to the supermarket or doctor’s appointments.
Mobility issues might also just present themselves as inactivity. Some seniors will compensate for their struggles to move about by saying they just want to stay still.
Overwhelming Home Maintenance
If your loved one lives on their own, you may notice a decline in the condition of their living environment. Maintaining a yard can be overwhelming and even dangerous if they still own their own home.
They may struggle to keep the place clean, whether it’s their own home or a condo or apartment. It can be challenging to bend and clean a bathroom or keep floors clean.
While they may be struggling, the loved one is often resistant to outside help coming into their home.
Need More Socialization
As your loved one ages, they may have fewer opportunities for socializing when they live on their own.
Social interactions are important to help stall the aging process and encourage activity. It helps keep the brain active and engaged and generally makes a person feel better when they’re around friends.
In assisted living, there are ample opportunities to interact and do activities with other residents.
Messy Living Conditions
Do you walk into your loved one’s home and think, “Oh my, what a mess!”? This is a sure sign they need more help.
It might be that the place is just cluttered with newspapers and mail. It might be that dishes are piling up in the sink.
If they aren’t keeping their living environment clean, especially if they used to be very fastidious about such things, then they need more help.
Changes in Behavior
Many families will begin to notice their loved one is displaying concerning changes in behavior from their usual personality.
They may come across as more angry or even aggressive. They become defensive or mean when asked about how they’re doing.
Sometimes, a loved one will just seem unhappy. Aging can be a difficult life stage to navigate, and it can make many seniors feel blue or frustrated.
About 22% of seniors between the ages of 84 and 89 have dementia. Changes in behavior can indicate they may be suffering from some form of dementia.
Feelings of Isolation or Depression
You may notice that your once very social parent or loved one now stays alone for days on end. They choose not to leave their home. They avoid participating in social activities they used to enjoy.
You’ll need to consider what’s driving their isolation. Can they not get to those activities, or do they not feel well enough to go?
Many seniors even suffer from some types of depression.
Assisted living provided the social interactions needed. It also provides the check-ins by staff to ensure your loved one is feeling okay.
Concerns from Physician
If your loved one has a long-standing relationship with their medical provider, the doctor may notice some concerning changes.
There may be a health condition that means they should no longer live on their own.
Take the advice of a doctor seriously if they have concerns about your loved one living on their own.
Current Caregiver Can’t Handle the Load
You may already have a caregiver who comes into the home of your loved one to help them out there.
If the caregiver finds they can no longer manage the amount of care your loved one needs, it’s time to make a change.
Some families will hire a caregiver to come in and check on their loved ones once a day. If medication or meals are being missed because that is no longer enough care, it’s time to consider assisted living.
Get the Help Your Loved One Needs
Sometimes deciding what’s best for your loved one can be a real challenge. Many seniors resist and want to stay in the comforts of their own home.
Yet, their safety and well-being should be paramount in your decision-making about what they need to live safely.
If you’re considering an assisted living option for your loved one, we can help. Visit one of our locations, book a tour, and check out our many services through our assisted living programs.

domain: sociology


Early this year we featured the AR drones – lightweight hover crafts that are essentially straight from Terminator – and we’ve featured them a few more times with the incredible things people have done with them. Now, we’re featuring them again, as a French inventor has turned them into a toy/game for kids using augmented reality. The drones are equipped with a camera so you can see what they see through your phone, and with an augmented reality overlay, you can shoot lasers and missiles at your opponents while trying to evade theirs. If you remember laser tag from the 90s, it kind of works like that, and the kid inside me wants one really bad – Santa, I’m looking at you. This guy programmed his drone to track a ball.


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Displaying Items in:
Designer Signed Costume Jewelry
Vintage Van Dell Rose & Yellow Gold Filled Sterling Flower Brooch: This vintage Van Dell flower brooch from the 1940's features classic retro styling. The graceful flower has both rose and yellow gold fill over sterling silver. The flower is centered with a large red rhinestone. The front of the brooch is in fabulous condition, with light normal age wear to the back. the brooch measures 3 1/4 by 1 1/2" and is signed Van Dell Sterling, 1/20th 12k gold fill.
Vintage Signed Ambassador Molded Glass Buddha Brooch: This vintage brooch from Ambassador features a molded jade green glass Buddha in a seated position. The antique gold tone background is accented with small faux pearls. The brooch is shown in Marcia Brown's book "Rhinestone Jewelry Figural, Animals, and Whimsicals" on page 39. The brooch measures 1 3/4 by 1 1/8" and is signed Ambassador. Excellent condition.
Vintage Benedikt NY Star & Crown Topaz Rhinestone BRooch/Pendant: This stunning design from the hard to find Benedikt NY company features a multi-dimensional star design accented with crowns. The center is a blazing deep topaz dentelle rhinestone, securely set in prongs. The textured gold tone metal is in excellent condition. the brooch also features a hook to wear it on a chain as a pendant. it measures 2 1/2 by 2 1/2" and is signed Benedikt NY(c).
Vintage Signed Vogue Faux Pearl & AB Rhinestone Earrings: These stunning vintage clip back earrings from Vogue feature three rings of AB rhinestones, each centered with a large faux pearl. All stones and pearls are prong set. The large earrings are beautifully constructed, they are reinforced by a metal ring on the back side. They are in excellent condition, signed Vogue and measure 1 1/4".
Vintage H. Pomerantz Black Diamond Rhinestone Crescent Brooch: This fabulous vintage brooch from the hard to find H.Pomerantz jewelry company is crafted in silver tone pot metal and features blazingly beautiful black diamond rhinestones set into a crescent shaped brooch. The teardrop and chaton black diamond rhinestones simply sparkle with color! Like most of their pieces, this is a larger brooch measuring 2 3/4 by 2 1/4". It is signed H. Pomerantz.
Huge Alexis Bittar Faceted Flawed Emerald Glass Two Tone Ring: This fabulous ring from Alexis Bittar is huge and gorgeous! Crafted in gold plated metal with silver accents in an organic, open design, the ring features a large faceted flawed emerald glass center stone. The ring sits up very high on your hand, having a rise of 3/4". The top of the ring is 1 5/8" long. This ring measures a size 7 on my mandrel. It is not signed, but I have the matching bracelet. This ring is from the estate of the late Jo Ann Christian, wife of LBJ's press secretary.
Vintage Danecraft Sterling Lily Panel Bracelet: This stunning vintage sterling silver bracelet from Danecraft ( circa 1940's) features four detailed panels with a lily and leaf motif, joined by heavy ribbed links. The patina is lovely! Each panel is marked sterling, and the plaque reads "sterling, Pat.-D, 121374. This patent number is attributed to Danecraft, and they used this same design later with signed pieces. The bracelet is in wonderful condition and measures 7" long by 1" wide.
Vintage Laurel Burch Enamel Snake Brooch: This adorable snake brooch from Laurel Burch features a wonderful, whimsical pattern of multi-color enamels. The little snake has a smile on his face as he slithers on his way. He is in excellent condition, measures 3 by 1/4" and is signed Laurel Burch, Snake.
Vintage Signed JJ Wizard with Sword Brooch: This mystical wizard brooch from JJ ( Jonette) is a favorite of mine! This large brooch has amazing details. The wizard is crafted in pewter colored metal, the sword is gold plated and accented with rhinestones, and the blade even has chasing. A lucite teardrop "crystal" drops from below the beard. In excellent condition, the brooch measures 4 1/2" long by 3 1/2" wide. Signed JJ (c).
Vintage Erwin Pearl Byzantine Link Gold Tone Necklace: This classic and elegant Byzantine link necklace from Erwin Pearl is a beauty! substantial and extremely well made, the collar necklace lays fluidly around the neck. It measures approximately 16" and is 7/8" wide. The heavy plating is in excellent condition and the fold over link is tight and secure. It is signed E.Pearl(c).
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Nigeria’s number 1 street music act, Olamide Badoo has gotten a new tattoo, but this time on his right leg.
Olamide leg tattoo is of a guiter.
The YBNL boss took to his Instagram to share the newly acquired ink and it looks cool.
See some of the photos below:


2017 Ford Raptor Owners Manual – Virtually all folks get a new Ford after which they simply experience in with out even searching on the 2017 Ford Raptor Owners Manual. Even so, this typical book may possibly supply outstanding knowledge on vehicle’s internal operations and assistance about ownership. Although it could be quite bored to study an owner’s manual book, you can find substantial details to reinforce your Ford ownership knowledge. Properly, it includes the starting-up from the new car, fix tips, strategies, and preparation for emergencies.
2017 Ford Raptor Owners Manual: What Data Will it Cover
Here’s an illustration of how relevance your 2017 Ford Raptor Owners Manual will probably be. Whenever your individual Ford has the vehicle maintenance gentle, this usually indicates which you must set up an oil modify. Usually the glow remains to be on following servicing by an impartial maintenance workshop. Typically, the mechanic forgets to reset the light once the provider. Fortunately, the owner’s handbook offers instructions on how to try this, so that you do not need to return to the workshop. This is just one of a great deal of valuable tidbits from your crucial book.
Here are several useful subjects within an owner’s handbook. First is new vehicle configuration. A vast variety of exclusive configurations are available through owners manual for brand spanking new automobiles, such as the keyless entry seem, delays in lights, the amount cap for the audio method, along with other knowledge shows. The training and adjustment from the choices available can improve the driving expertise.
2nd is head restraints adjustment. The pinnacle restraints of a vehicle are crucial to forestall wounds to the whiplash from the neck usually associated with a rear-end collision. Limits need to be sufficiently substantial to coat the head just above spine. Head restraints for several automobiles are height-adjustable. For Ford, people who do not study the 2017 Ford Raptor Owners Manual are likely to possess a difficult time changing it.
Up coming is timetable of maintenance. Your manual consists of details as to when and what to inspect in your car. This is the rule, so your local provider retailer or perhaps plant distributor may well not try to buy your profit-driven timetable in your vehicle.
There’s also details about elimination from the substitute tire and jack operation. It seems like a thing that is simple to work on when happens, nonetheless it may well not perform in case you are along a crowded highway making an attempt to substitute a tire throughout a wet night. You much better not just take this option, and skim the 2017 Ford Raptor Owners Manual rather, so that you are well prepared whenever something like that occurs.
Furthermore, you are able to also locate about strain of pneumatics. Newer automobiles have a very tire pneumatic tension inform plan that signifies a low-pressure tire. Discover the proper tire strain, how warnings ought to be interpreted, and the way to add strain. Additionally, constantly sustain the correct strain in your tires. Do not get to the tire’s maximum inflation tension blindly, rather utilize the inflation pressures usually observed inside the driver’s jamb doorway, as indicated through the car maker.
Additionally, once the tires have stood down for 3 or more several hours inspect the inflation pressures. Normally, heat tires may have a substantial strain. That’s all of your 2017 Ford Raptor Owners Manual must include.
2017 Ford Raptor Owners Manual
Get HERE 2017 Ford Raptor Owners Manual.


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You want to go on a Maine vacation, but putting Fido in a kennel is a pain! Good news, lots of Maine resorts and lodging properties are now pet-friendly. You can have your trip and bring your four-legged friend too. There are many friendly Maine bed and breakfasts, inns, hotels and motels that allow dogs. There are pet friendly campgrounds and cottages that will also accommodate your family dog or cat during your camping trip to Maine.
Some even have pet friendly packages that include special treats and maybe even a dog bed for your pet.
Those dog days of summer in Maine are even better when you can bring man's best friend to these dog friendly hotels throughout the state. There are several dog-friendly areas and dog parks in Maine, where your pet can run and play in the fresh Maine air. Area beaches usually allow dogs year-round, except in summer months when there may be certain time restrictions on when they are allowed on the sand. Dogs will love running along the soft sand or fetching a ball in the cool waters.
Many hotels and innkeepers are happy to provide pet owners with maps and names of local pet-friendly businesses, beaches and walking areas. Pets are a welcome addition to many Maine towns, and many store fronts and shop owners will even leave water or food bowls outside their door for those long, hot summer days.
Here are several Maine lodging properties that are happy to accommodate you and your pets! Be sure to read the fine print before you make your paw prints as some pet restrictions may apply (some inns may accept one pet or only small dogs, etc.).

domain: pet


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FIST OF AWESOME gets a new trailer
Retro styled woodland creature brawler Fist of Awesome has received a new trailer that shows off the sort of bear punching action you can expect when the game arrives on the App Store in May.
The game, which has been developed by Nicol Hunt, whose previous employers include Codemasters and Realtime Worlds, had a successful Kickstarter funding push last year, and is set to see the light of day in a couple of months.
Thanks to the Kickstarter cash - the game earned over £11,000, more than double what was asked for - Hunt has been able to bring in more help to get the game finished, which is good news for anyone who's longing for an iPad or iPhone game where knocking out bears and deer is par for the course.
You can check out the new trailer below, and head over to the game's website for some more information by clicking right here


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At first, KMS California Artistic Director, Paula Daddario, wasn’t even considering a career in hairstyling. “When I was 16, my mother offered me a summer job at her salon and I was like, ‘No way.’ But once I started working there, I thought, ‘This is not work, this is amazing.’ I just fell in love with it,” she says. Her enthusiasm for her craft continues to this day. A native of St. Catharine’s, Ontario, Paula trained at Toronto’s Bruno School of Hair Design and supplemented her industry acumen by taking college degree business classes.
Following in her mother’s entrepreneurial footsteps, Paula has owned her own salon since the early stages of her career and chose KMS as the brand she would work with exclusively. She has become an expert in the hair extension industry and her salon focuses on extension services. Paula has also developed an extension program for the FUSION ACADEMY that she runs with the help of her team. She is very enthusiastic about training for KMS. “I definitely feel great when I’ve shown somebody a new method and you see the light go on in their eyes. And it doesn’t just happen with ‘newbies,’ but sometimes with stylists who have been in the industry for years. It’s wonderful to encourage people and it’s a great industry in which to work.”


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Here’s the Fire Department’s scanner traffic.
A three-alarm fire torched several buildings near 3rd and Main Tuesday night.
The cause of the blaze was unknown. Several residences and business were without power when the fire brokeout. The blaze apparently started in a condo or apartment complex that is under construction.
Several other buildings appeared to catch fire as well.
Firefighters from Alhambra, South Pasadena and Los Angeles County responded to the thre-alarm blaze, officials said.
The amount of damage caused by the blaze was unknown. It is unknown if there were any injuries.
— Frank C. Girardot
Just saw KCBS/KCAL reporter Serene Branson do a story on this video. Thought I’d share it with Crime Scene readers:
Reporter Maritza Velazquez tracked down a young woman who lost everything in Hurricane Katrina only to face the similar prospect as a resident of Diamond Bar Saturday. Here’s the top of her story:
DIAMOND BAR — Three years ago Danita Clark lost everything.
She came close to experiencing a similar loss Sunday when she and nearly 1,800 others were evacuated from their neighborhoods in Diamond Bar ahead of the raging Triangle Complex Fire.
“I took everything I could. I’ve been through Hurricane Katrina, so I know how it is to come back and not have anything,” Clark said.
In August 2005, the 22-year-old was forced to evacuate her Slidell, Louisiana home when Hurricane Katrina hit and ultimately devastated the Gulf Coast.
After living in a hotel and eating from fast food restaurants everyday for a month, she returned. But her former home was in ruins. The furniture was severely molded and the home was flooded with water that reached six-feet in height.
This comes from blogger Bear Chick:
We’re smack in the middle of Brea/Anaheim Hills/Yorba Linda, then being called the Freeway Complex fire. Last night Carbon Canyon was added and this morning Chino Hills and Diamond Bar came into the mix and now it’s called the Triangle Complex fire. I don’t think we’ve ever been this close to a fire this big. We’ve seen fire in the hills in Yorba Linda but the problem with this weekend’s fire was the Santa Ana winds kicking up. The Brea fire is about 2 miles from us, which seems like a safe distance I suppose but the fire crossed a freeway in Yorba Linda! For those of you not familiar with freeways in Cali, the 91 probably has at least 8 lanes where the fire jumped it. So 2 miles away doesn’t seem that far in view of Mother Nature’s leaping ability!
From the Associated Press:
Nick Carone, left, and Daniel Brooks of the San Bernardino County Fire Dept. Grand Terrace unit fight a wildfire in Chino Hills , Calif., Sunday, Nov. 16, 2008. (AP Photo/Marcio Jose Sanchez)
An exclusive enclave of multi-million homes in Diamond Bar is being evacuated, according to officials at the Walnut Sheriff’s substation. The estates are home to rapper Snoop Doggy Dogg and U.S. Rep Gary Miller as well as several other celebrities and sports figures.
View Larger Map
Again from the Associated Press:
AP Radio News plans to carry a news conference with California Governor Arnold Schwarzenegger regarding the wildfires. It’s expected to start around 12:45 pes but this is not a hard start time.


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An examination of the dissertation and the sources it cites identified around more than a dozen sections of text that have been lifted, with little to no changes, from other scholarly works without proper attribution. In some instances, Crowley footnoted her source but did not identify with quotation marks the text she was copying directly. In other instances, she copied text or heavily paraphrased with no attribution at all.
By checking passages in the document against the sources Crowley cites, focusing on paragraphs that come before and after footnotes of key sources in her bibliography, we found numerous structural and syntactic similarities. She lifted passages from her footnoted texts, occasionally making slight wording changes but rarely using quotation marks. Sometimes she didn’t footnote at all.
Parts of Crowley’s dissertation appear to violate Columbia’s definition of “Unintentional Plagiarism” for “failure to ‘quote’ or block quote author’s exact words, even if documented” or “failure to paraphrase in your own words, even if documented.” In other cases, her writing appears to violate types I and II of Columbia’s definition of “Intentional Plagiarism,” which are, respectively, “direct copy and paste” and “small modification by word switch,” “without quotation or reference to the source.”
This is not surprising, given revelations about her book.

domain: library


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U.S.-based Myriad Pictures has come on board Belladonna Prods.’ Cold War thriller “Divided.”
“Divided” is a political thriller based on the building of the Berlin Wall in the early 1960s and a young couple’s struggle, determination and ultimate sacrifice to stay together amidst the devastation.
Myriad will handle all worldwide sales.
In the lead roles are Diane Kruger, who has just been named Mistress of Ceremonies at the upcoming Cannes Film Festival, and Thomas Kretschmann.
Belladonna’s producers Rene Bastian and Linda Moran are in negotiation to cast an American solider who plays a key role in the story.
Pic will be directed by animation director Michael Johnson in his live-action directorial debut. The story is loosely based on his parents’ ordeal in Germany during the Cold War.
Both Johnson and Kretschmann are of German descent with similar childhood memories of the times. The script was written by Theresa Alto and Michael Johnson.
Pic will shoot in Germany, beginning with Berlin in the summer.

domain: entertainment


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Louis I. Kahn (1901-1974)
Posted by: Squirreleen
N 39° 56.977 W 075° 09.966
18S E 485811 N 4422178
Quick Description: A marker at the location of architect Louis Kahn's offices in Philadelphia.
Location: Pennsylvania, United States
Date Posted: 10/6/2008 6:00:36 PM
Waymark Code: WM4WNR
Among the foremost architects of the late 20th century. Trained in the Beaux-Arts method at the University of Pennsylvania, Kahn redefined architecture through his extraordinary use of mass, light, and structure. A Penn professor, his office was here.
Note: A recent film, "My Architect", gives a very personal look into the life of this man.
Marker Name: Louis I. Kahn (1901-1974)
Date Dedicated: 06/24/2004
Marker Type: City
Location: 1501 Walnut St., Philadelphia
Category: Buildings, Education, Professions & Vocations
Website: [Web Link]
In your log, please say if you learned something new, and if you took any extra time to explore the area once you stopped at the historic marker waymark.
Please post a photo of you OR your GPS at the marker location.

domain: history


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Work directly in Revit with Point cloud instances to create topo, floor and roof elements that accurately match the original point cloud data. Offers a decimation slider so that you can choose how many points are used to make the new surface element, for either high accuracy or high speed depending on your requirements.
PointCloud to Surface has been designed for surveyors, architects, builders and developers working on as-built projects. It reads laser scan data captured onsite and imported into Revit, automating the tedious process of manual point entry needed to create complex surfaces in a BIM model.
The app allows you to draw a selection box around the points of interest then select whether they should be mapped as either a topo, floor or roof element. For roofs and floors, you can select the build type and level for the new element.
The section box can be used to filter out points on a broad scale, so by working in 3d view the section box in conjuction with the user drawn selection box allows very specific filtering of points to be added to each new element.
Works with both structured and unstructured point cloud instances. Supports shared co-ordinate models.
Instructions For Use
1. Insert pointcloud into Revit via the pointcloud insert tab. We suggest working in 3D view and activating the section box to focus on specific structures in the scan.
2. Run the plugin with the PointCloud to Surface button on the add-ins tab.
3. Select the pointcloud instance you wish to select points from.
4. Draw a selection box around the points you wish to convert to a surface element
5. You will be prompted to choose whether to create a topo, floor or roof element.
6. Choose the maximum number of points with the decimate slider, and for floor and roof, you will be able to designate a type and level to the new element.
7. The app will convert your selected point data to a new element that accurately matches the original scan data.


Chattisgarh, India, February 2015:
Saurabh Agrawal an activist and journalist from Raigarh, posses for a portrait at the public hearing meeting. He expressed concerns over the environmental standards not being met and lack of compensation.
Photographs for a story on land allocation for coal mines in Chattisgarh. Modi's new government in the centre has relaxed the environmental regulations so the land can be allocated to both public and private sector companies easily.
Photo by Sami Siva for Al Jazeera America.


text: I have been having palputations since I was little, but I would only have one maybe once a month, and it was only one! now it seems like I am getting a heart rate that speeds up huge when I stand up, and slows quite significantly when I sit or lay down! I am also experiencing my childhood flutters more often then not! what could cause this? how do I get rid of it? if it is stress or anxiety how do I deal with that ? I am begging for some help, its like I can constantley feel my heart beating! also I had blood work done, and an ekg which came back all normal, I swear I am feeling this and it is ruining my life!

domain: health