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The recently launched veterinary anthelmintic drench for sheep (Novartis Animal Health Inc., Switzerland) containing the nematocide monepantel represents a new class of anthelmintics: the amino-acetonitrile derivatives (AADs), much needed in view of widespread resistance to the classical drugs. Recently, it was shown that the ACR-23 protein in Caenorhabditis elegans and a homologous protein, MPTL-1 in Haemonchus contortus, are potential targets for AAD action. Both proteins belong to the DEG-3 subfamily of acetylcholine receptors, which are thought to be nematode-specific, and different from those targeted by the imidazothiazoles (e.g. levamisole). Here we provide further evidence that Cel-ACR-23 and Hco-MPTL-1-like subunits are involved in the monepantel-sensitive phenotype. We performed comparative genomics of ligand-gated ion channel genes from several nematodes and subsequently assessed their sensitivity to anthelmintics. The nematode species in the Caenorhabditis genus, equipped with ACR-23/MPTL-1-like receptor subunits, are sensitive to monepantel (EC50<1.25 µM), whereas the related nematodes Pristionchus pacificus and Strongyloides ratti, which lack an ACR-23/MPTL-1 homolog, are insensitive (EC50>43 µM). Genome sequence information has long been used to identify putative targets for therapeutic intervention. We show how comparative genomics can be applied to predict drug sensitivity when molecular targets of a compound are known or suspected.
Author Summary
Increased use of anthelmintics has contributed to the emergence of drug-resistant nematodes, causing serious problems for more than one billion sheep worldwide. The last class of compounds indicated for livestock was introduced 28 years ago. Recently, however, Novartis AH developed a new anthelmintic active against drug-resistant nematodes of sheep, the amino-acetonitrile derivative (AAD) monepantel. We have previously indirectly shown that the AADs have a novel mode of action involving acetylcholine receptor subunits: the ACR-23 protein in Caenorhabditis elegans and a homologous protein, MPTL-1 in Haemonchus contortus. To better understand the mode of action of the AADs, we performed comparative genomics of all ligand-gated ion channel genes from a range of organisms, including members from all nematode clades. We confirmed that MPTL-1 belongs to a unique, nematode-specific sub-family of receptor subunits. We also found that some nematode species lack ACR-23/MPTL-1 and predicted them to be monepantel insensitive. We challenged this hypothesis in a panel of drug tests: several species of Caenorhabditis nematodes equipped with ACR-23/MPTL-1-like receptor subunits were found susceptible to monepantel, whereas Pristionchus pacificus, closely related to these worms but lacking an ACR-23/MPTL-1 homolog, was tolerant. The parasitic nematode Strongyloides ratti, which has only a remote homolog of DES-2 and ACR-23/MPTL-1, was also tolerant to monepantel. This confirms our prediction and highlights how comparative genomic data can be used to predict a drug effect.
Citation: Rufener L, Keiser J, Kaminsky R, Mäser P, Nilsson D (2010) Phylogenomics of Ligand-Gated Ion Channels Predicts Monepantel Effect. PLoS Pathog 6(9): e1001091. doi:10.1371/journal.ppat.1001091
Editor: Edward J. Pearce, Trudeau Institute, United States of America
Received: December 24, 2009; Accepted: August 6, 2010; Published: September 9, 2010
Copyright: © 2010 Rufener et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a Novartis PhD fellowship (to L.R.), a Swiss National Science Foundation grant (to D.N. and P.M.) and a Swedish Science Council/Stiftelsen Olle Engqvist Byggare post-doctoral scholarship (to D.N.). Novartis AH did participate in the design and conduct of the study, and in the collection, analysis, and interpretation of the data, and in the preparation, review, and approval of the manuscript since L.R. and R.K. are Novartis employees. The other funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: L.R. and R.K. are employed by Novartis AH, which has developed monepantel. The use of AADs for veterinary purposes is patented (WO2002049641).
Nematode parasites of sheep represent one of the major constraints in the wool, meat and milk industries world wide [1]. The gastro-intestinal parasite Haemonchus contortus, in particular, causes substantial losses. In the mid-1950s, the existence of anthelmintic resistant worm populations came to light with the failure of phenothiazine against Haemonchus [2]. Since then, nematode populations resistant to the three classical groups of anthelmintics, i.e. the benzimidazoles, imidazothiazoles and macrocyclic lactones, and combinations of these have been described [3]–[7]. Recently, the amino-acetonitrile derivatives (AADs) have been reported as a new class of synthetic anthelmintics active against gastro-intestinal nematodes of sheep [8] and a first drug from this family, monepantel, was, at the time of writing licensed to market in Australasia, Europe and Latin America (ZOLVIX; Novartis Animal Health Inc., Switzerland; [9]). Investigations to understand the mode of action of the AADs in Caenorhabditis elegans have been performed using chemical mutagenesis and gene mapping via a genetic recombination approach. Out of 44 isolated resistant alleles, 36 were mapped to a single gene, acr-23, designating it as a major contributor to the AAD response in C. elegans [8]. A further study on mutant H. contortus isolates identified the gene monepantel-1 (Hco-mptl-1) as a major target candidate for AADs action in this species [8], [10]. Both Cel-acr-23 and Hco-mptl-1 are predicted to encode a nicotinic acetylcholine receptor (nAChR) subunit. These belong to the superfamily of ligand-gated ion channel (LGIC) subunits. These are the modular components with the ability to form a large number of channels with different properties through heteromultimerisation (see e.g. [11]), as all characterised LGIC function as penta- or tetramers. They provide many important drug and toxin targets: levamisole and pyrantel act as agonists of nAChR [12], paraherquamide as a competitive antagonist of these channels [13] and ivermectin modulates glutamate-gated chloride channels [12], [14], [15]. ACR-23 and MPTL-1 are members of the DEG-3 subfamily of acetylcholine receptor subunit genes and distinct from those targeted by imidazothiazoles [16], [17]. Members of this subfamily have so far only been found in nematodes and no cross-resistance between the AADs and the imidazothiazoles have been documented [8].
All animals appear to have about the same number of nAChR α subunits (around 16), with exception of the nematodes [18]. Among the completely sequenced genomes from animals, those with the highest (C. elegans) and smallest (Brugia malayi) numbers of such genes are both from the nematodes [19]. The reason for the variation in numbers of nAChR α subunits and other LGIC subunits is not clear. However, certain LGIC subunits form heteromultimeric channels that provide prominent anthelmintic-specific drug targets (highlighted in Figure 1), in particular for ivermectin (AVR-14, AVR-15, GLC-1), levamisole (UNC-38, UNC-29, UNC-63, LEV-1, LEV-8) and monepantel (MPTL-1). Several new, draft nematode genomes as well as pre-publication quality assemblies are now available from ongoing or recently finished sequencing projects (see Table 1).
NJ tree (clustalw) from the LBD region of conceptually translated putative LGIC genes as detected with Genewise after an initial Blast screen with the 210 seeds (see Materials and Methods) - 1426 sequences in total. Thousand bootstrap iterations were performed and branches below 50% bootstrap support were collapsed. Nematode sequences are shown in shades of green, platyhelminthes yellow, insect purple and vertebrate red. Some C. elegans and human subunits are labelled, and the labels for proteins involved in drug susceptibility are coloured: levamisole - cyan, monepantel - orange and ivermectin – blue. Green asterisks indicate branches that similar to these latter appear broad and nematode specific and could be attractive for further investigation as targets for other compounds.
To learn what parts of the LGIC superfamily are unique to nematodes and in consideration of much new sequence information, we constructed a simple phylogenomic pipeline to further understand the mechanisms behind the action of monepantel. We explored the LGIC superfamily by in silico searches, and while we found a considerable number of tentative new family members since the last such survey was made [19], the DEG-3-subfamily remains nematode specific. In an in vitro drug assay we further show that susceptibility to the AADs directly follows the presence of ACR-23 or MPTL-1 homologs in the genomes from the nematodes investigated.
Materials and Methods
Genome analysis
Genome data in the form of contig or supercontig DNA sequence fasta files were downloaded from GenBank (NCBI), Ensembl, WTSI, nematode.net, GSC/WUSTL, Broad and WormBase (all attributed and referenced in Table 1). Sequences from genomes with long contiguous sequences were artificially divided into 100 kb segments (indicated by asterisks in Table 1). Seed sequences were obtained as peptide fasta files from WormBase [20] and Uniprot [21].
A Blast [22] screen with the seed sequences as queries against the genomic sequence databases was performed. Only contigs with hits (E<0.1) were searched by Genewise [23] with the PFAM [24] motifs LGIC_LBD (PF02931.15) and LGIC_MEMB (PF02932.8) for global scoring (ls). Splice sites were considered using the Genewise-provided worm gene model. The seed peptide sequences were searched using the same PFAM profiles but with hmmsearch from the hmmer2 package (by Eddy, http://hmmer.janelia.org). The protein domains, conceptually translated from DNA or directly from the seed proteins, that exhibited E-values below the trusted E-value cut-off were aligned and an nj tree (bootstrap 1000 iterations) was constructed with clustalw [25]. These steps were automated in bash and Perl using tools from the EMBOSS package [26] and executed on LINUX computers using less than 1.5Gb RAM. Trees were visualised with Dendroscope [27] and HyperTree [28]. For Figure 1, furcations with bootstrap support below 50% were fused in jtreeview (Frickey, Lupas http://www.eb.tuebingen.mpg.de/departments/1-protein-evolution/software/jtreeview/). Co-segregation with known named seed sequences in the bootstrapped tree was used for assigning putative identity to homologous genes. Trees based on available, confirmed or predicted, full-length protein sequences (WormBase WS195 C. brenneri, C. elegans, C. briggsae, C. japonica, C. remanei, Pristionchus pacificus and B. malayi) were also constructed. The same seed sequences were used to pick predicted genes with a blastp similarity (E<0.01) for inclusion in a profile search and tree construction using the aforementioned methods.
Nematode strains
Nematode strains C. briggsae AF16, C. brenneri PB2801, C. remanei PB4641, C. japonica DF5081 and P. pacificus PS312 as well as the mutants VC1598: Cel-acr-20(ok1849)/mT1 II; +/mT1[dpy-10(e128)] III, NC293: Cel-acr-5(ok180) III, TU1803: Cel-deg-3(u662) Cel-des-2(u695) V and RB1226: Cel-acr-18(ok1285) V were obtained from the Caenorhabditis Genetics Center (CGC), Minneapolis, USA, which is funded by the NIH National Center for Research Resources (NCRR). Caenorhabditis elegans Bristol N2 and AP134: Cel-acr-23 (cb27) V [8] were kind gifts from Prof. Alessandro Puoti, University of Fribourg. Nematodes were maintained at 20°C on Nematode Growth Medium (NGM) plates (3 g NaCl, 17 g Agar, 2.5 g peptone in 975 ml H2O, autoclaved, added 1 ml cholesterol (Sigma) prepared to 5 mg/ml in EtOH, 1 ml M CaCl2, 1 ml M MgSO4 and 25 ml KPO4-buffer), and inoculated with E. coli OP50, and transferred every 3 days.
Strongyloides ratti L3 were obtained from the feces of infected rats following standardized procedures based on the Baermann technique at the Swiss Tropical and Public Health Institute. Freshly harvested S. ratti L3 were washed 3 times with PBS buffer and used immediately for in vitro drug testing. The species of nematodes used in the in vitro drug test was confirmed by a PCR targeting the 18S rRNA region of C. elegans, C. briggsae, C. remanei, C. brenneri, C. japonica and P. pacificus. Using the forward primer SSU18A and the reverse primer SSU26R [29] (Supplementary Table S1), a ∼950 bp fragment was amplified using FastStart High Fidelity PCR system (Roche). The reaction conditions were: 95°C for 10 min without Taq polymerase; 95°C for 2 min; 35 cycles of [95°C for 30 sec; 52°C for 30 sec; 72°C for 1 min 10 sec]; 72°C for 10 min. PCR products were purified using the Wizard SV PCR Clean-Up kit (Promega) and sequenced in both directions with SSU18A and SSU26R at Microsynth AG (http://www.microsynth.ch). Sequence quality check and assembly was done using 4Peaks (by A. Griekspoor and T. Groothuis; http://mekentosj.com) and a nucleotide blast was made on-line (NCBI) against the nucleotide collection (nt).
Drug test
Ivermectin and AAD-1566 were provided by Novartis Animal Health, Centre de Recherche Santé Animale, Fribourg, Switzerland. The compounds were diluted in pure DMSO to 10 mM and 250 mM, respectively. Appropriate dilutions of drugs were placed at the bottom of wells in 24-well plates and 1 ml NGM was added per well. The first well in each row served as a control with 1% DMSO. The plates were well shaken, allowed to dry at RT for several days, then inoculated with 10 µL E. coli OP50 and incubated at 37°C overnight. Eggs were purified from adults of the different species as follows: plates were washed with 3.5 ml water and incubated with 1.5 ml 5% bleach mixed 1∶1 with 5M NaOH for 10 min at room temperature. The eggs were washed with water and counted. A volume corresponding to 100–200 eggs, depending on the species, was added to each well of the drug plates. Plates were incubated at 20°C and scored microscopically for adults after 3 days and inspected again after 6 days for potential F2 generation of L1-larvae.
For the S. ratti in vitro tests 25 L3 larvae were incubated in 96-well plates containing 30 µl PBS buffer supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen) and appropriate drug dilutions. Control wells contained the highest percentage of solvent (2% DMSO). At each examination point (24, 48 and 72 h post-incubation) 15–20 µl of hot water (80°C) was added to each well, the larval movement observed and live worms counted using a dissecting microscope. All water stimulated wells were excluded from further reading. Half maximal effective concentration (EC50) values were calculated by non-linear regression of the sensitivity data, expressed as the percentage of surviving worms/larvae compared to the untreated control, to a sigmoidal dose-response curve of variable slope using Prism (GraphPad Prism version 5.00 for Mac OS X, GraphPad Software, San Diego California USA, http://www.graphpad.com).
Discovery of new Haemonchus contortus DEG-3 subfamily members
A previously described procedure was followed for RNA extraction, cDNA synthesis and rapid amplification of cDNA ends by PCR (RACE-PCR) for H. contortus [10]. Briefly, total RNA was extracted from a pool of approximately 50 adult nematodes. To generate cDNA, 1 µg of total RNA was reverse transcribed to cDNA using a d(T)30 primer and a SuperScript II Reverse Transcriptase (Invitrogen). For RACE-PCR, an internal reverse primer (Supplementary Table S1) was combined with splice leader sequence (1 or 2) to obtain the 5′ untranslated region (UTR), or an internal forward primer combined with a poly-dT primer for the 3′ UTR of the transcript. We cloned and sequenced the full-length Hco-acr-5, Hco-acr-17 and Hco-acr-24 coding sequence from H. contortus cDNA (GenBank accessions GU109271-GU109279) using primer pairs NheI_acr-5_frw2.1 and NotI_acr-5_rev2.1, NheI_acr-24_frw1 and XhoI_acr-24_rev1, NheI_acr-17_frw1 and XhoI_acr-17_rev1 (Supplementary Table S1). PCR products were gel purified using the Wizard SV PCR Clean-Up kit (Promega) and cloned into pCRII-TOPO (Invitrogen). Plasmid DNA was purified using the QIAprep Spin Miniprep kit (Qiagen) and three clones of each gene were sequenced using the standard primers M13 forward and reverse. The reported sequences in Supplementary Figures S1, S2 and S3 are each from one of the nearly identical single clones.
Accession numbers
Hco-acr-5, Hco-acr-17 and Hco-acr-24 coding sequence from H. contortus cDNA have been deposited with GenBank accessions GU109271-GU109279. MPTL-1 ACO48330 (GenBank).
SwissProt entries for mentioned proteins: ACC-1 Q21005_CAEEL, ACR-16 ACH1_CAEEL, ACR-17 P91320_CAEEL, ACR-20 B1Q281_CAEEL, ACR-21 Q9N5U8_CAEEL, ACR-23 O61884_CAEEL, ACR-5 ACR5_CAEEL, ACR-8 Q23355_CAEEL, AVR-14 Q95Q96_CAEEL, AVR-15 Q95PJ6_CAEEL, CUP-4 CUP4_CAEEL, DEG-3 ACH3_CAEEL, DES-2 ACH4_CAEEL, EXP-1 Q9TZI5_CAEEL, GAB-1 GBRB_CAEEL, GGR-2 Q2WF64_CAEEL, GLC-1 O17793_CAEEL, LEV-1 ACH7_CAEEL, LEV-8 Q93329_CAEEL, MOD-1 Q58AT9_CAEEL, PBO-5 Q67X94_CAEEL, UNC-29 ACH2_CAEEL, UNC-38 ACH5_CAEEL, UNC-49 Q0PDK2_CAEEL, UNC-63 ACH6_CAEEL, ZACN ZACN_HUMAN.
A phylogenomic pipeline
Caenorhabditis elegans peptide sequences annotated with the Gene Ontology term GO:0005230, 'extracellular ligand-gated ion channel activity', were retrieved from WormBase. These together with a similarly extracted set of human genes from uniprot and six H. contortus LGICs of the DEG-3 subfamily ([10] and Supplementary Figure S1, S2 and S3) were used as seed sequences for a Blast search against contiguous sequences from the abundant nematode, vertebrate and insect genome projects (Table 1). Caenorhabditis elegans is arguably the only finished eukaryote genome, but the genomes published as drafts are essentially complete, and several of the ongoing projects are well underway in terms of sequencing and assembly, only so far lacking in gene annotation. In this survey, we included data from 10 more nematode genomes, ranging from early shotgun stages to mature assemblies in annotation. Gene finding and annotation has become a major bottleneck, after next generation sequencing techniques accelerated sequence generation. By using Genewise to search the genome sequences directly we could also make use of unannotated genes. To assess nematode specificity of the herein predicted LGIC genes and to obtain more phylogenetic information, we also included three platyhelminth projects, four insect and nine vertebrate genomes (Table 1). No LGIC_LBD (from ligand binding domain, LBD) was found in eight plant species searched (www.gramene.org), which is in agreement with previous efforts [30]. The closest LGIC relatives in plants are highly diverged glutamate receptors [31]. Many plant toxins act on animal LGICs (e.g. curare, extracted from the plant Strychnos toxifera [32]). Due to the lack of LGICs, the toxic compounds pose little risk to the plants themselves.
Method recall ability
The automated approach identified 84 out of 102 annotated C. elegans LGICs using the LGIC_LBD profile alone. Only one additional LGIC was identified when the 39 membrane binding domain hits, from the LGIC_MEMB profile, were also included. The recall of the profile itself from full-length peptides was nearly complete. Using hmmer2, all 102 were found with the LBD profile and 98 with the MEMB profile. The lower complexity of the trans-membrane domains and a presumed lower need for conserved sequence specificity, together with the often extensive and variable internal loop between trans-membrane domain 3 (TMD3) and TMD4, all complicated by a slightly larger number of introns, apparently makes the LGIC_MEMB Hidden Markov Model profile less successful for finding family members directly from genomic nucleotide data.
A domain centric approach, as used here, is highly useful to compare the whole spectrum of LGICs. The domain approach is straightforward, can be applied directly on sequence data without prior exon prediction and gives alignments where the aligned positions are largely comparable. It would also be much more challenging to align the protein family meaningfully over the full length. Inclusion of more variable regions e.g. the internal loop between TMD3 and TMD4 would make the interpretation more difficult. While the recall of C. elegans receptor subunit genes by the identification of the LBD domain directly from the genome is not complete (80%), it is reassuring that the full-length peptide results for genomes, where such are available, are similar to the ones obtained through searches on the genomic DNA level, in particular in the DEG-3 subfamily.
Effects of genome project completeness
If genome sequence coverage is lacking altogether or if other problems keep the assembled contig size small, the number of LGICs predicted from our pipeline will be low. If the contigs with LGIC genes are too short so that they do not encompass the introns and exons for the LBD, they will not be detected by Genewise with a global (ls) type PFAM LGIC_LBD motif, even if fragments were detected by the initial BLAST screening.
To help assess the reliability of the number of genes found in the face of incompleteness of the ongoing projects, we measured the average contiguous sequence length (Table 1). Such a central measure can however be somewhat misleading for mature projects with a very high contig size variance. Indeed, the B. malayi and L. scapularis genome sequences show low sequence unit average length (<5 kbp), although the longest few contiguous sequences have considerable size (≫100 kbp; marked in Table 1). Gene counts for the genomes with average contig sizes below 3 kbp (Ascaris suum, Nippostrongylus brasiliensis and Teladorsagia circumcincta) in particular should not be taken for final.
Additional DEG-3 homologs
The full length coding sequences of H. contortus genes Hc-acr-17 (1590 bp), Hc-acr-5 (1833 bp) and Hc-acr-24 (1698 bp) were cloned by RACE PCR (see Materials and methods) and sequenced (Supplementary Figures S1, S2 and S3), helping to complete the understanding of individual DEG-3 subfamily members roles in monepantel drug action (Figure 2 and Table 2). Both Hco-acr-5 and Hco-acr-24 carried a spliced leader 2 (SL2) sequence at their 5′ end while Hco-acr-17 had a spliced leader 1 (SL1) sequence. The predicted LGIC proteins possess motifs typical for Cys-loop ligand-gated ion channels, including an N-terminal signal peptide, with the exception of Hco-acr-24 (as determined with Phobius [33], [34]), four transmembrane domains and the Cys-loop (two cysteines separated by 13 amino acids). Loops A to F, which are involved in ligand binding [35] are also present in the proteins. These loops are not annotated for Hco-acr-17 as the alignment with other related nAChRs were the loops location are known is poor. In loop C, there are two adjacent cysteines, defining Hco-acr-5, Hco-acr-17 and Hco-acr-24 as nAChR α subunits. Hco-acr-5 and Hco-acr-24 have the characteristic FxCC pattern, conserved among other ACR-5 and ACR-24 homologs, in contrast to Hco-acr-17 bearing the most common YxCC α subunit signature in loop C.
Detailed view of the DEG-3 sub-family from the LBD region NJ tree, with branches below 50% support after 1000 bootstrap iterations joined. A few related subfamilies are shown as collapsed branches. CEL, Caenorhabditis elegans, CBN, C. brenneri, CBR, C. briggsae, CRE, C. remanei, CJA, C. japonica, PPA, Pristionchus pacificus, HCO, Haemonchus contortus, HBA, Heterorhabditis bacteriophora, MHA, Meloidogyne hapla, NM3MIC/MIN, M. incognitia, NB3AYC Ancylostoma ceylanicum, SRA, Strongyloides ratti, BMA, Brugia malayi, TSP, Trichinella spiralis, SME, Schmidtea mediterranea. Pristionchus pacificus lacks a close MPTL-1 homolog and was predicted to be less sensitive to AAD-1566 than species such as C. japonica, H. contortus and H. bacteriophora.
Detecting LGICs
We used our phylogenomic pipeline on 33 genomes of varying levels of completeness, detecting 1273 putative genes bearing the PFAM LGIC_LBD motif (Table 1). The average number found in nematode genomes with an average sequence unit larger than 3 kbp was 56.1, whereas the same number was 41.7 for vertebrates, 18.0 for insects and 0 for plants. We also searched the nembase3 and nematode.org expressed sequence tag sets, finding a total of 27 LGICs with the LBD motif. An average of 31 and 57 LGICs were found in parasitic and non-parasitic organisms, respectively. The trend among the nematodes is clearly in agreement with the hypothesis that parasites have a reduced number of LGICs. It has been suggested that this could be a consequence of the less variable environment they encounter in comparison with their free-living relatives [19].
There is also considerable variation in LGIC number among the vertebrates (Table 1). The teleost genomes show a larger set of LGIC, in comparison to, for example, Bos taurus and Homo sapiens. The teleost repertoire appears to consist largely of multiple closely related variants of the terrestrial vertebrate LGIC types. The nematodes show a larger repertoire (Figure 1).
While the platyhelminthes included in the survey showed a smaller overall number of LGICs, they did have several unique types. LGIC subunits that are known to constitute part of drug target receptors are labelled in Figure 1. It is interesting to see how these drug target subunit genes form rather broad, i.e. member rich, yet nematode specific sub-branches of the superfamily tree. Importantly, the DEG-3 family appears nematode specific. In an optimistic outlook, several other such broad nematode specific branches exist in the tree, which could potentially be exploited as new anthelmintic targets.
Co-segregation with known named seed sequences in the bootstrapped tree was used for assigning putative identity to homologous genes. Interestingly, we found that neither P. pacificus nor S. ratti carries an ortholog of Hco-mptl-1 (Figure 2). Based on a single drug target model we thus predict P. pacificus and S. ratti to be insensitive to monepantel. We proceeded to test this hypothesis in vitro.
In vitro assay
An in vitro assay was established. Nematodes of one species were grown on 24 well NGM plates where each four well column was treated with a different drug concentration. An equal amount of eggs was added to each well, and the nematodes were scored microscopically after 3 and 6 days. A final concentration of 1% DMSO was used in all wells for the drug tests, including the no-drug controls (Figure 3, Supplementary Figure S4). All species tested (C. elegans, C. japonica, C. briggsae, C. brenneri, C. remanei, P. pacificus) tolerated up to 1% DMSO (Supplementary Figure S4 and Supplementary Table S2). For S. ratti the highest percentage of solvent (2% DMSO) found in the plate was used in the control wells and well tolerated.
The percentage of the average number of adult worms present after 3 days exposure relative to a control is plotted versus drug concentration for Caenorhabditis elegans, mutated C. elegans strain acr-23 (cb27), C. japonica, C. briggsae, C. remanei, C. brenneri, Pristionchus pacificus and S. ratti. Sigmoid dose-response curve fit was performed in Prism. EC50 values with standard errors, estimated from data points with four replicates, are shown inset.
All Clade V species tested (C. elegans, C. japonica, C. briggsae, C. brenneri, C. remanei, P. pacificus) exhibited a similar sensitivity to ivermectin (EC50≥10 nM; Supplementary Figure S7 and Supplementary Table S3). S. ratti L3 exposed to ivermectin at concentrations of 10 µM and above showed decreased survival rates 24–72 h post-incubation (EC50 of 13.6 µM 72 h post-incubation) (Supplementary Figure S7). This served as an additional positive control for the methods. It appears likely that drug sensitivity can be consistently determined for all strains. In a similar experiment the assayed nematode species showed varying degrees of concentration-dependent sensitivity towards DMSO, used as a solvent for the drugs. Even a compound with a small effect in this in vitro test could still be of therapeutic value, as levamisole clearly demonstrates. Levamisole does not directly kill the parasitic nematodes but creates a short term reversible paralysis, sufficient to allow the host to e.g. expulse the worms [12]. Up to mmolar concentrations of levamisole did not produce any effect detectable by our test readout (data not shown).
Hco-mptl-1 ortholog detection predicts AAD-1566 sensitivity - an MPTL-1/ACR-23 ortholog is the primary target
The present study tests the hypothesis that MPTL-1 is a major target of monepantel, since a nematocide effect on P. pacificus or S. ratti, which lacks an MPTL-1 homolog, would negate this. The P. pacificus genome has been published in a draft state, and as the assembly is nearly complete it is unlikely, but not impossible, that an eventual acr-23 ortholog could have been missed. The S. ratti genome is still in progress, but we were able to detect a subunit that appears branched prior to the ACR-23/MPTL-1/ACR-20 and the DES-2 split (see Figure 2 and Table 2). This species arguably helps us narrow down how deep the sensitivity to monepantel reaches in the tree.
Among the species C. elegans, C. japonica, C. briggsae, C. brenneri, C. remanei, P. pacificus as well as the mutated C. elegans strain acr-23 (cb27), C. japonica was the most sensitive to monepantel with EC50 values in the low nM range (Figure 3 and Table 1). This is comparable to the results previously obtained for H. contortus [10]. C. elegans was strongly affected at 1 µM, with an estimated EC50 of 0.19 µM. C. remanei, C. briggsae and C. brenneri showed similar EC50 values, but we found a comparatively large number of adult C. brenneri even at higher µM concentrations, e.g. 7.6% at 100 µM (Supplementary Table S4). C. brenneri has the largest assembly of LGICs in the study, and also possesses an extra DES-2 paralog and an additional ACR-23. Closer examination of the sequences of these copies did not present a convincing explanation of the diminished phenotype. One explanation may possibly lie in the gene doses of the channel subunits, leading to different stochiometries of the assembled channels, as has been observed in vitro [36].
The difference in EC50 value between C. japonica and to the other sensitive worms in the Caenorhabditis genera is already large. While we would not venture a molecular correlate, it is interesting to observe that the both the Cjp-ACR-23 and Cjp-ACR-20 seem to have diverged somewhat from the other sensitive Caenorhabditis worms, branching prior to them, possibly retaining more of an element important for high sensitivity, common with the earlier branched Hco-MPTL-1.
Pristionchus pacificus is rather insensitive to monepantel with an EC50 of 43 µM (Table 2, Figure 3). Furthermore, our in vitro test with S. ratti, bearing an early branching relative of ACR-20/ACR-23/MPTL-1, showed that monepantel lacks activity against S. ratti. A survival rate of 69% was observed after 72 h even with the highest concentration (250 µM) tested (Figure 3). A direct molecular mechanism is beyond the scope of the present investigation. However, we found that the phylogenomic detection of the ortholog of Hco-mptl-1, previously found mutated in strains insensitive to AAD-1566 [10], coincides with sensitivity to AAD-1566. This in agreement with our hypothesis that MPTL-1 is a major target of the drug.
Caenorhabditis elegans mutated in the DEG-3 family
acr-23 (cb27), a strain of C. elegans exhibiting a large deletion in Cel-acr-23 [8], was much less sensitive than wild type (genome strain N2), with an EC50 of 25 µM (Table 2, Figure 3). The difference in growth was marked and clearly visible to the naked eye (Supplementary Figure S5). This test can naturally not rule out the involvement of other LGIC subunits or indeed other genes in the susceptibility to AAD-1566. However, a set of C. elegans strains mutant only in other genes of the DEG-3 family (DEG-3/DES-2, ACR-5, ACR-18, and ACR-20) showed no loss of sensitivity towards AAD-1566 (Supplementary Figure S6 and Supplementary Table S5). This further strengthens the hypothesis that a subunit orthologous to MPTL-1/ACR-23 is required for the observed effect.
Detailed phenotype suggests additional secondary target – sensitivity stays in the family
For species that possess an MPTL-1 ortholog (e.g. C. elegans with Cel-ACR-23), AAD-1566 is lethal in vitro at nM concentrations, and a concentration-dependent retardation of development was observed. The strains without an Hco-MPTL-1 ortholog (P. pacificus and C. elegans acr-23 (cb27)) also exhibited a drug concentration-dependent developmental retardation. However, the substance was not lethal to them at the tested concentrations, as growth could still be observed after 6 days. Also in the case of S. ratti the survival rate of the larvae was slightly affected at high drug concentration (69% at 250 µM) and less at lower concentration. This suggests that there is at least one additional target.
One candidate is DES-2. In nematode strains selected for loss of sensitivity to AAD-1566, mutations in addition to those affecting Hco-mptl-1 were found in the Hco-des-2 gene 5′ UTR, introducing two novel upstream open reading frames, possibly reducing protein expression [10]. All tested species possess the DES-2 ortholog that bears the highest similarity to the established target outside the Cel-ACR-20/Cel-ACR-23 branch. If MPTL-1 is a primary target, causing high nematode lethality from AAD-1566, strains with modulations in the expression of a second target, DES-2, would only be selected for once MPTL-1 sensitivity was lost. It was noted in proof that in a recent study [37] Rufener et al. have expressed a functional H. contortus DES-2/DEG-3 channel in Xenopus oocytes that shows monepantel sensitivity. Though active against a range of clade V gastrointestinal nematodes, monepantel was reported to have only limited efficacy against Trichuris ovis (clade I) [38]. Genomic information to correlate this result with the absence of MPTL-1/ACR-23/DES-2 homologous subunits would be interesting.
There are a number of nematodes that, based on their complement of predicted nAChR genes, would be interesting to test for their sensitivity to AADs, but this would require other test methods. Two Meloidogyne species bear no close MPTL-1 homologs but have an ACR-5, homolog, which P. pacificus lacks. Heterorhabditis bacteriophora carries a DEG-3 family complement, which is highly similar to H. contortus, and we would thus predict a similar drug effect. Some important human parasitic nematodes of the clade I (Trichinella spiralis) and III (Brugia malayi) have more distant DES-2/DEG-3 homologs, much like Schmidtea mediteranea. A conjecture would be that they would show sensitivity only at a higher concentration. Tests on them could perhaps show what level of sequence identity is required, or what regions of the subunit need to be conserved, for any paralysis effect to be seen.
The family of LGIC provides many important drug and toxin targets, with nematodes bearing several unique subfamilies well diverged from those of other eukaryotes. We have constructed a simple phylogenomic pipeline to detect LGIC subunit genes. We survey the gene family in the many complete and ongoing sequencing projects in the nematode phylum and contrast these to genomes from some other relevant phyla to establish that the DEG-3 family indeed appears nematode specific to date. The survey also establishes the detection of drug sensitivity groups.
Given the hypothesis that an MPTL-1 homolog is the primary target of monepantel, the phylogenomic information gathered predicts P. pacificus and S. ratti to be insensitive to the drug, while four other model nematode species were predicted to be sensitive. These conjectures were tested experimentally. The in vitro effect of AAD-1566 on the panel of nematodes was found consistent with the hypothesis. All data point towards MPTL-1 as a primary target, in agreement with previous studies. We further hypothesise an additional secondary target for AAD-1566, possibly DES-2. This would explain a dose dependent growth retardation effect that is largely masked by the stronger, MPTL-1 mediated response.
Supporting Information
Figure S1.
Haemonchus contortus Hco-acr-5 cDNA sequence. Nucleotide sequence from Haemonchus contortus Hco-acr-5 cDNA with conceptual peptide translation. Putative transmembrane domains TMD1-TMD4 are highlighted in grey. Prominent conserved LGIC α subunit loops are highlighted in green. Note the C-loop motif FxCC, typical for ACR-5. Export signals, as predicted by Phobius, are highlighted blue.
Figure S2.
Haemonchus contortus Hco-acr-17 cDNA sequence. Nucleotide sequence from Haemonchus contortus Hco-acr-17 cDNA with conceptual peptide translation. Putative transmembrane domains TMD1-TMD4 are highlighted in grey. Prominent conserved LGIC α subunit loops are highlighted in green. Export signals, as predicted by Phobius, are highlighted blue.
Figure S3.
Haemonchus contortus Hco-acr-24 cDNA sequence. Nucleotide sequence from Haemonchus contortus Hco-acr-24 cDNA with conceptual peptide translation. Putative transmembrane domains TMD1-TMD4 are highlighted in grey. Prominent conserved LGIC α subunit loops are highlighted in green. Export signals, as predicted by Phobius, are highlighted blue.
Figure S4.
In vitro test of DMSO tolerance. Live adults on NGM wells after 3 days from egg deposition, in percent compared to control (0% DMSO) average, was plotted against DMSO concentration for Caenorhabditis elegans, C. japonica, C. briggsae, C. remanei, C. brenneri and Pristionchus pacificus. Green field background denotes presence of progeny after 6 days, indicating the ability to complete a whole life cycle. Error bars represent the SEM from three replicates. All species tolerated the typical dose of 1% DMSO. 2.5% leads to a marked development retardation.
Figure S5.
Comparison of phenotype between Cel N2 (wild type) and acr-23 (cb27) after AAD-1566 exposure. N2 worms were more sensitive, with the bacterial lawn intact down to drug doses of 1 µM, whereas feeding activity was noticeable up to 100 µM, with 1 µM being visually no more affected than the control after 6 days.
Figure S6.
Sensitivity to AAD-1566 determined in vitro. The percentage of the average number of adult worms present after 3 days exposure relative to a control is plotted versus drug concentration for Ceanorhabditis elegans, and mutant strains VC1598, TU1803, NC293 and RB1226. Error bars represent the SEM from four replicates. Sigmoid dose-response curve fit was performed in Prism.
Figure S7.
Sensitivity to ivermectin determined in vitro. The percentage of the average number of adult worms present after 3 days exposure relative to a control is plotted versus drug concentration for Caenorhabditis elegans, C. japonica, C. briggsae, C. remanei, C. brenneri and Pristionchus pacificus. Sigmoid dose-response curve fit was performed in Prism. EC50 values with standard errors, estimated from data points with four replicates, are shown inset.
Table S1.
Primers used for PCR amplification. Primers used for PCR amplification of 18s rRNA of Caenorhabditis sp. or Pristionchus pacificus and deg-3 subfamily genes from Haemonchus contortus.
Table S2.
Sensitivity to DMSO determined in vitro. Number of adult worms present after 3 days exposure for C. elegans, C. japonica, C. briggsae, C. remanei, C. brenneri and P. pacificus. Green field background denotes presence of progeny after 6 days, indicating the ability to complete a whole life cycle. Yellow fields in t-test rows indicate that the hypothesis of the counts of that concentration being drawn from a normal distribution with the same average as the control (0%) could not be rejected at a 95% confidence level (two-tailed heteroscedastic t-test).
Table S3.
Sensitivity to ivermectin determined in vitro. Number of adult worms present after 3 days exposure for C. elegans, C. japonica, C. briggsae, C. remanei, C. brenneri and P. pacificus. Yellow fields in t-test rows indicate that the hypothesis of the counts of that concentration being drawn from a normal distribution with the same average as the control (0%) could not be rejected at a 95% confidence level (two-tailed heteroscedastic t-test).
Table S4.
Sensitivity to AAD-1566 determined in vitro. Number of adult worms present after 3 days exposure for C. elegans, C. japonica, C. briggsae, C. remanei, C. brenneri and P. pacificus. Green field background denotes presence of progeny after 6 days, indicating the ability to complete a whole life cycle. Yellow fields in t-test rows indicate that the hypothesis of the counts of that concentration being drawn from a normal distribution with the same average as the control (0%) could not be rejected at a 95% confidence level (two-tailed heteroscedastic t-test).
Table S5.
Sensitivity to AAD-1566 determined in vitro for mutant C. elegans isolates VC1598: Cel-acr-20(ok1849)/mT1 II; +/mT1[dpy-10(e128)] III, NC293: Cel-acr-5(ok180) III, TU1803: Cel-deg-3(u662) Cel-des-2(u695) V and RB1226: Cel-acr-18(ok1285) V. Green field background denotes presence of progeny after 6 days, indicating the ability to complete a whole life cycle. Yellow fields in t-test rows indicate that the hypothesis of the counts of that concentration being drawn from a normal distribution with the same average as the control (0%) could not be rejected at a 95% confidence level (two-tailed heteroscedastic t-test).
We are grateful to Alessandro Puoti for helpful advice and for the kind gift of C. elegans N2 and acr-23 (cb27) strains. Several strains and species were obtained from the Caenorhabditis Genetics Center (CGC), Minneapolis, USA, which is funded by the NIH National Center for Research Resources (NCRR). We thank Adrian Streit, Barry Hosking, Andrew Little and Arthur Redpath for critical review of the draft manuscript. We thank Mireille Vargas for excellent technical assistance.
Author Contributions
Conceived and designed the experiments: LR JK RK PM DN. Performed the experiments: LR JK DN. Analyzed the data: LR JK RK PM DN. Contributed reagents/materials/analysis tools: LR JK DN. Wrote the paper: LR JK RK PM DN.
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text:
'splaining to the cars
No, the young man on our left is not flipping this guy off. Really. Zoom in and see!
This mass featured more pissed off drivers than any I can remember except the legendary ride down 99 in June this year. At least nobody got nailed this time.
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Critical Essays
Gay and Lesbian Writers of Color
By (author) John DeCecco Emmanuel S. Nelson
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Critical Essays by John DeCecco
Book DescriptionThis pioneering work is the first book to systematically explore the literature of gay and lesbian writers of color in the United States. Critical Essays challenges the marginalization and tokenization of gay men and lesbians of color in the dominant academic discourses by focusing exclusively on the imaginative work of representative Native-American, Asian-American, Latino(a), and African-American gay and lesbian writers. As the first book offering a scholarly assessment of ethnic gay and lesbian writing in the U.S., Critical Essays simultaneously defies ethnic and mainstream homophobia as well as straight and gay/lesbian racism. This deliberate counter to the dominant white discourse of gay and lesbian literature offers a lively contribution to the debate on the intersections of race, ethnicity, gender/sexuality and class in American literature. A wide range of critical approaches, including historical readings, cultural analysis, and deconstructive criticism, is employed to the works of such major literary figures as Audre Lorde, James Baldwin, John Rechy, Paula Gunn Allen, and Gloria Anzaldua. These thought-provoking chapters disrupt the complacent notion of a unified gay/lesbian community by questioning the presumed similarities of persons who share sexual identity. Some of the specific topics explored in Critical Essays include: * post-coloniality and gay/lesbian identities * emerging Asian-American gay and lesbian writers * redefining the Harlem Renaissance from gay perspectives * contemporary African-American gay male performance art * relocating the gay Filipino This groundbreaking volume will be of immense interest to undergraduate, graduate, and advanced scholars in Gay and Lesbian studies, Women's studies, African-American studies, Asian-American studies, Latino(a) studies and Native-American studies. It will also serve students and scholars as a valuable introduction to the diversity of authors that comprise twentieth-century American literature.
Buy Critical Essays book by John DeCecco from Australia's Online Bookstore, Boomerang Books.
Book DetailsISBN: 9781560244820
(216mm x 156mm x 23mm)
Pages: 254
Imprint: Routledge Member of the Taylor and Francis Group
Publisher: Taylor & Francis Inc
Publish Date: 20-Dec-1993
Country of Publication: United States
Other Editions...
Books By Author John DeCecco
Behind the Mask of the Mattachine, Hardback (November 2006)
The Mattachine is the origin of the contemporary American gay movement. This work travels back to the times before Stonewall and its aftermath, to the beginnings of the modern homosexual movement and the lesser-known individuals who started it. This chronicle gives the history of the activists who organized homosexuals.
Men, Homosexuality, and the Gods, Hardback (August 2004)» View all books by John DeCecco
An eye opening look at the traditions of particular religions and their edicts concerning gay sex. Draws a parallel between homosexuality and the idea of religion, suggesting that gay rights can be understood as a freedom of religion issue.
» Have you read this book? We'd like to know what you think about it - write a review about Critical Essays book by John DeCecco and you'll earn 50c in Boomerang Bucks loyalty dollars (you must be a member - it's free to sign up!)
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"Small Town Security" is returning to AMC on May 6 with its third season, and Zap2it has an exclusive first look at the upcoming episode. In the above promo, the folks at JJK Security celebrate the returning season with — what else? — a foot massage day.
In Season 3, new "out-of-this-world" characters will join the cast of "Small Town Security." Meanwhile Chief Joan Koplan, Dennis, Irwin, Christa, Brian and Joan's beloved dog Lambchop are "forced to contend with a series of unexpected shocks and developments — from dwindling business and serious health issues to Dennis' continued gender transformation and Joan's 'speecy spicy' meatball musical performance."
"Small Town Security" Season 3 premieres Tuesday, May 6 at 11 p.m. ET/PT on AMC.


text: David Hepfinger has resigned as Weis Markets' president and CEO, as well as from the board of directors, "to pursue other interests," the company says. Hepfinger, promoted to CEO in 2009, is being replaced temporarily by Vice Chairman Jonathan Weis. Kurt Schertle, executive vice president of sale and merchandising, is assuming responsibility for store operations.


text:
Originally published March 29, 2014 at 1:08 AM | Page modified March 29, 2014 at 3:08 AM
Astrological Forecasts for 03/30/2014
Aries (March 21-April 19): Today is an 8 -- Make it a low risk day. Avoid travel, expense or gambling. You're even more powerful than usual. Opportunity comes pounding on the door. If you open the wrong door, try again. Persistence pays, and love triumphs.
Taurus (April 20-May 20): Today is a 7 -- One phase of a project closes and another opens. Take notes on the previous stage. Repair, organize and clean up. A creative idea doesn't work. Don't make assumptions. Keep at it. Follow the rules. Keep tuning and refining.
Gemini (May 21-June 20): Today is a 6 -- Discover a time-saving tool at work. Guard against impetuous decisions. Keep to basics, using tested tricks. A female handles chores and obligations. Your family and friends offer support. Don't blow all your cash partying. Keep it frugal.
Cancer (June 21-July 22): Today is a 6 -- There's a change in plans, a disruption to the routine. Work it out with the woman in charge. You know exactly where your work will take you. Intuition gives you a nudge. Graduate to the next level.
Leo (July 23-Aug. 22): Today is a 7 -- You want to take action in your studies, travels and exploration. Be practical, not whimsical. Rebellions could flare up. Dedicate energy and attention to your work, and minimize expenses. Use what you're learning to expand your perspective.
Virgo (Aug. 23-Sept. 22): Today is a 6 -- Don't run away from a problem. Plan how you'd like it to go. Tackle the details, with persistence and determination. Show respect. Test your work before presenting. Take actions to grow your nest egg.
Libra (Sept. 23-Oct. 22): Today is a 7 -- An upsetting discovery could rock the boat. Movement inspires your team... walk and talk together. Don't argue, or give up if one method doesn't work. Negotiate your way through minor adjustments. The basis of your strength is love.
Scorpio (Oct. 23-Nov. 21): Today is an 8 -- Your workload's getting more intense, and the excellent job you've been doing reflects well on you. Use gentle pressure, rather than force. Work with quality materials. Close deals and make new contacts. Keep a secret. Unleash your creativity.
Sagittarius (Nov. 22-Dec. 21): Today is a 6 -- Work takes precedence over travel. You're almost to spring your trap. Fun comes from unpredictable directions. Don't try to force an outcome. It could feel chaotic. Keep the big picture in mind. The truth comes out.
Capricorn (Dec. 22-Jan. 19): Today is a 6 -- Distractions and frustrations could slow the action at home. Propose a change, gently. A female provides support, discipline, and practical questions. Wait until this stress is past. Stick to your budget. Cultivate compassion for others.
Aquarius (Jan. 20-Feb. 18): Today is a 6 -- An opportunity arises. Avoid risks, gambling and disagreements. Focus on the practical plan. List actions to take, and talk with the team to choose and schedule tasks. Things don't go as planned, and communication provides the key.
Pisces (Feb. 19-March 20): Today is an 8 -- Take care of business. Focus on bringing in the money, despite roadblocks. Work with a colleague to your mutual benefit. Hold hands, taking two steps forward, and three steps back. Chaos and surprises change the route. Keep steady.


text:
Chris-after my visit to Plant Delights last week your place sounds like just
the right spot for a nice piece of art. have no idea what you like, but
maybe a fountain or some statuary. The green glazed pot sounds wonderful!
Take pictures.
Andrea H
Beaufort, SC
----- Original Message -----
From: <email@example.com>
To: <email@example.com>
Sent: Friday, August 06, 2004 9:35 PM
Subject: [CHAT] Garden Projects
> Dear All,
> I've been trying to put some finishing touches on some of my gardens. Some
> of the areas needed something to unify or define them. For instance, I
> a wide brick pathway leading to my house that is straight on one side and
> curved on the other. One side gets a lot of shade and is planted with
> hellebores, hosta, fringed bleeding heart, anemones, HAKONECHOLA MACRA
> AUREOLA, Liriope, tiarella, Japanese painted ferns, columbines,
> etc. There are shade loving shrubs and red cedars in this garden as well.
> It has a meandering pathway going through it that was just shredded mulch.
> Today I placed some flat rocks, that I had left over from dismantling a
> small rock wall, along the path in an asymmetrical pattern. Then I added
> big deep green glazed pot that I am going to plant with ferns, variegated
> grasses, and maybe a silvery begonia or pulmonaria. What a difference it
> made to the garden! It feels like a "destination" now!
> I have other shady gardens with wide pathways that just have leaf mulch
> walkways. I was thinking of cutting up some red cedar logs that we have
> the property, after cutting down some trees, and making pavers out of them
> to define another pathway. These are informal areas with naturalistic
> plantings, so edgings don't really fit. Does anyone have any other ideas?
> I also spent hours in the last few days trying to clean Houttiana cordata,
> Chameleon vine, out of a small potion of another bed. I've done this
> in another area and the roots of this plant must go down to China and
> off as soon as you try to put them out, always leaving a piece of root
> behind to start a new plant! I can honestly say that I will never get rid
> of that plant in my lifetime!
> I spruced up another pathway by weeding it and covering it with layers of
> newspaper and mulch. I have to renew the mulch every few years, but it's
> worth it!
> This must be the year of the pathway, I guess! I have a load of gray-blue
> irregular wall rock to go in the white garden. I have to limb up some
> yews before that walkway goes in. My son was supposed to help with that
> project, but he's been busy working this summer as a camp counselor. He
> 5th grade boys and comes home exhausted! I have 12 special education
> preschoolers every day during the school year, so I know about being wiped
> out!
> Does anyone know how to get rid of ivy that's invading my grass? I tried
> pulling it out, but there's just too much! I'm thinking Roundup might be
> only answer.
> Chris
> Long Island, NY
> Zone 7
> ---------------------------------------------------------------------
> Support hort.net -- join the hort.net fund drive!
> http://www.hort.net/funds/
Support hort.net -- join the hort.net fund drive!


text:
Blog: Hiring Revolution
Who Writes It: Folks at Talent Zoo, an employment mecca for the ad industry
Why You Should Read It: Sassy tips, best practices, book recommendations, and news-worthy items to arm HR professionals for the "war on talent." Sample offering: A job offer made early in the week is more likely to be accepted than one made Friday. Plus a step-by-step guide on the "art of the offer."
Blog: George's Employment Blawg
Who Writes It: Attorney George Lenard and consultant Michael Harris
Why You Should Read It: To keep up with gnarly employment law issues. Lenard and Harris steer clear of legal jargon, making for a blog that's surprisingly conversational, easy to follow, and relevant even for non-lawyers (e.g., "Everything you ever wanted to know about employment testing—but didn't know where to ask").
Blog: Cheezhead
Who Writes It: Joel Cheesman of HRSEO, an online-recruiting consultant
Why You Should Read It: "Online Recruitment's Most Dangerous Blog," Cheezhead chronicles how technology is reshaping the HR function. Cheesman loathes "the Trumpasaurus" (i.e., Monster.com) and likens Google's plans to integrate job content from Google Base into Web search to "readying the troops for Normandy."
A version of this article appeared in the June 2006 issue of Fast Company magazine.


text:
Notify me when this product is available:
The largest lavender we have on sale. Great for filling large spaces with its tall, wide spreading foliage. Gross is also the variety that we have found to be most fragrant. Expect bees to pay a regular visit through the summer!
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© 2017 LavenderWorld.
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More Videos...
1969 Chevrolet Camaro Racing the Streets!
This is a video of a 1969 Chevrolet Camaro racing the streets of Gander Newfoundland for the 2010 annual Targa Newfoundland. Tags: 1969 chevrolet Camaro the trailer park boys ricky julian rob wells tremblay bubbles 2010 porsche cayman 911 c4 1971 dart demon fast race car racing streets awesome video nascar indy formula one crash wreck fire jeff gordon dale earnhardt jr big michael schumacher gander newfoundland airport airplanes taking off landing war ufo ghost stories automobiles prank motorcycle accident home cars showoff stunts
Nani Roma MINI John Cooper Works Monster Rally Season 2013 [Passats de canto]
www.naniroma.com Facebook / Nani Roma Fans Page www.passatsdecanto.com facebook / Passats de Canto email@example.com Nani Roma - Pilar Barceló / Winner Rallisprint Les Corbes 2013 / Peu a Baix / PCR Sport / MINI John Cooper Works / Facebook Nani Roma Fans page / KH7 Racing / Monster Energy Drink / VISION Advisors / Michelin Tyres / Rallye Season 2013 / Passats de canto!
Scottish Mini Racing - Knockhill 26/08/2012
Scottish Mini Cooper Cup - BTCC Supporting them at Knockhill 26/08/2012
Mini Cooper S vs 2 Corvettes
After seeing Steve Dinitz take on a Corvette with his Mini I have been inspired. This is run 1 in the Blue group at Sebring 2010-05-01 with Chin Motorsports. I was very happy to be able to keep these guys in sight. Handling vs horsepower. I always enjoy the match-up. You might also like https://www.youtube.com/watch?v=44rTAsRcg04 and http://www.youtube.com/watch?v=o3WH3BYQ0Xw


text:
My DNA extraction are using NaOH and a kitt of DNAzol. However when I measure the concentration of the DNA using spec at 260/280 i've got 3.57 for NaOH and 2.89 for DNAzol. My friend said it may be due to protein contamination.
So my question is: how to remove all the protein and obtain pure DNA concentration?
Submit your paper to J Biol Methods today!
Protein contamination - How to remove all protein from my DNA?
Started by syazmin, Sep 22 2011 06:03 AMprotein contamination how to DNA
3 replies to this topic
Posted 22 September 2011 - 06:03 AM
Posted 22 September 2011 - 08:11 AM
Perhaps a proteinase K treatment before DNA extraction could be helpful.
Nature magazine. Do you qualify for a free subscription?
Posted 22 September 2011 - 09:26 AM
A phenol choroform extraction removes all proteins. Make sure to do a chloroform only extract to remove traces of phenol from the final product.
Posted 22 September 2011 - 05:19 PM
Thanks for the suggestions. However, actually i don't want to use phenol-chloroform since i'm doing a research of comparing 3 different methods of DNA extraction. Besides NaOH and DNAzol, i'm also using method extraction of phenol-chloroform & isoamylic acohol. So i don't want to mix these method cause i'm afraid it will affect my results of comparison.
So is there any other suggestion of how to remove the contaminant which i assume proteins?
So is there any other suggestion of how to remove the contaminant which i assume proteins?
Also tagged with one or more of these keywords: protein, contamination, how to, DNA
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Wego.com recently found 5 hotels in Bartow, United States. Book last minute hotel or find great hotel deals such as Holiday Inn Express & Suites BARTOW for US$136 per night. Save more on cheap hotels with rates from as low as US$99 per night.
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Sports Illustrated is taking a look back at the movie Major League in this week's issue. It is part of the annual project, where are they now?
Here is a link to their story about the movie and the Indians.
Yahoo Sports! made a top 10 list about the movie.
What is your favorite scene?
I will always remember JoBu and the locker room scene and his rituals with his bat.


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Perfect for orchestral recordings – the MKH 8090 wide cardioid condenser microphone
Sennheiser is expanding its MKH studio microphone series with the addition of the MKH 8090, whose wide cardioid pick-up pattern makes it the ideal microphone for orchestral recordings.
"The MKH 8090 combines omni-directional and cardioid pick-up patterns to produce an impressive orchestral microphone,"
Meticulous sound tuning
One of those responsible for fine-tuning the microphone was tonmeister Gregor Zielinsky, International Recording Applications Manager at Sennheiser: "During the development of the MKH 8090, we focused on ensuring that the sound perception of the microphone is precisely between that of the omni-directional MKH 8020 and the cardioid MKH 8040. Through further fine-tuning, we succeeded in creating a microphone with a sound that seems to 'shine', and which has great presence and musicality."
Accessories for (almost) every application
The MKH 8090 benefits from the wide range of accessories available for the 8000 series, such as microphone stands with different heights, various microphone clips, a shock mount, remote cables with different lengths, windshields and accessories for ceiling mounting.
Also available as an accessory is the MZD 8000 digital module, which converts the audio signal of the MKH 8090 into a digital signal according to the AES42 standard (Mode 2) directly at the microphone head, thus ensuring a lifelike, natural sound entirely without cable losses or interference from other sources.
Sennheiser's MKH series works according to the RF principle, which Sennheiser has been using for more than 50 years and has developed to absolute perfection, for example through the use of symmetrical transducers.
The MKH 8090 will be available in October and will cost $1,199.95.
About Sennheiser
Sennheiser is a world-leading manufacturer of microphones, headphones and wireless transmission systems. Established in 1945 in Wedemark, Germany, Sennheiser is now a global brand represented in 60 countries around the world with U.S. headquarters in Old Lyme, Connecticut. Sennheiser's pioneering excellence in technology has rewarded the company with numerous awards and accolades including an Emmy, a Grammy, and the Scientific and Engineering Award of the Academy of Motion Picture Arts and Sciences.
You can find all the latest information on Sennheiser by visiting our website at http://www.sennheiserusa.com or by contacting:
Rachel Smolin
Public Relations
Sennheiser Electronic Corporation
(860) 434-9190, Ext. 180
Jeff Touzeau
Public Relations
Hummingbird Media, Inc.
(914) 602-2913
MKH 8090.jpg:
Technical Data MKH 8090
Pick-up pattern
Frequency response
Sensitivity -34 dBV/Pa (20 mV/Pa)
Max. sound pressure level 142 dB SPL
Equivalent noise level 13 dB(A) (DIN-IEC 651), 23 dB (CCIR 268-3)
Nominal impedance
Min. terminating impedance
Phantom powering
Current consumption 3.3 mA
Weight 25 g (55 g with XLR module)
Operating temperature -10 °C to +60 °C
The MKH 8000 series at a glance
MKH 8020 omni-directional
MKH 8040 cardioid
MKH 8050 super-cardioid
MKH 8060 short shotgun
MKH 8070 long shotgun
MKH 8090 wide cardioid
MKH 800 Twin
The principle of the RF condenser microphone
RF condenser microphones are a special type of condenser microphone. Instead of the high polarisation voltage that is usually required, a relatively low RF voltage generated by a low-noise oscillator is applied to the capsule. This voltage is modulated by capacitance changes produced by the sound waves moving the capsule diaphragm. After demodulation, a low-noise audio frequency signal with very low source impedance is available. This signal can be used to directly drive 'ordinary'
Symmetrical capsule design
RF condenser microphones from Sennheiser have a special symmetrical 'push-pull'
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Purity comes from a direct connection with Nature…
Rainier Distillers Photo Slideshow
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|General Chat Almost anything goes, have a look and drop in a few lines. Think of it as a campfire chat with the kids around.
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94 Rangies?
hi all
just was cruising the trading post and looking at 94 range rovers. Not really interested in buying, but just wondered what these things were like. Am i right in saying these cars preceded the p38? How did they compare to discos of the mid 90s? are they a good thing? Did they all have airbag suspension through the variant range?
And finally how are the variants graded, ie which is the top of the line and which is bottom?
sorry for all the questions, but I am curious about them
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The Vogue SE soft-dash classic rangie's came out around mid '94. They were used as a trials platform for some of the p38 goodies - dual airbags, new 'soft-dash', traction control, air suspension, abs, etc.with the 3.9 donk and with perhaps less of the electrical head-aches of the p38. In my opinion, ride and finish is significantly better than a 90's disco (I've owned both), however there are more things to go wrong in the rangie. I love the self levelling suspension thou - especially the little dance it does when you stop at the lights after and extended period of running at 80km/hr (when it lowers itself) and then it restores normal hieght when u stop. PEople used to think air-suspension was a liability. Not any more - it's not as expensive to maintain as it once was.
A soft-dash LSE would be my pick, a bit heavier but much more passenger leg room. Perhaps some collector value also.
Last edited by Taz; 28th August 2007 at 11:16 PM.
I have one and i think its great, its a vogue se, i believe 94, you could only get a Vogue SE or LSE, there wernt any other models. Mine has coils now, if i had the money i would get another mint cond one with the airbags. (there is a nice silver one for sale atm for $11k but it has high km totaly std) for a road car, for me atm springs are a good thing as it is used off road.
However, with more techonolgy than the models it superceeded comes more problems, for instance, my SRS airbag light likes to come on and stay on until reset by a dealer or specialist, it'll be right for a while but bump it the wrong way and it will come on. TC and ABS have a mind of its own, sometimes itll be fine, most of the time the lights will come on and it wont work, i suspect i will need to have the wabco unit ovehauled and the bands replaced, and checking the wheel bearings may help as it can throw the signal. BHSS rebuilds the wabco unti for approx. $400 My memory functions for the seats and mirrors need to be recalibrated, and the cruise doesnt work which i suspect is the rotatry coupling behing the driver airbag. i dont have remotes for the locks/alarm either which can be annoying sometimes but you get that. IN the not to distant future though ill be having all the problems rectified as im looking at spending a considerable about of $$$ on it for mods and other stuff soon.
So would i get another one - if only there wernt more of them, I'd like a completly std one with some nice mags and road tires for a nice cruiser and what i have for a 4by - but then again, there comes a cost to it all, but no more than a old cruiser of the same vintage and u ge alot more for your money.
Hey Ryan,
re cruise control not working - have you checked the vaccum hoses for leaks? It was a common problem and very easily fixed.
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i noted the nice dash layout. im assuming that the post 93 disco dashes were inspired by them?
Yes - the second series D1 shared the same center section of the dash (twin temp controlls etc) which I read some where was a honda decendent(?) The disco binnacle was not integrated like the Rangie, however they share the same instrument assembly and passenger air-bag arrangement.
BTW - I think these two vehicles, the Vogue SE and the Disco, were the first 4wd vehicles in the world to have factory airbags.
Last edited by Taz; 29th August 2007 at 11:01 PM.
hi guys, might chuck my ore in here, ive had my 94 soft dash for about 18 months now. had a few dramas when i got it, new engine after two weeks, but i have just put it on lpg and put a set of extractors and a hi flow exhaust and what a car, i love it pure luxury around town and very capable off road, there are plenty of niggles with them but if your prepared to keep them maintained then there unreal, i dunno but range rovers seem to have a nicer ride to me, love discos but i saw a program on the queen the other night and she drives a rangey, so did her mam, phil was drivin a lovely hse d2. the ones ive seen lately for sale look great, if you dont mind travelling a bit to get the one you want then your getting a lovely car for very little dollars. just go and get one you know you want to
I'd really like a lwb version.
I have had a hankering for owning one for a loong time, but my beef with thm is that you can't fit a bullbar as they are equipped with SRS air bags. As a town and 'normal' off-road vehicle (not places where I go ) they're a superb unit.
I've Seen a couple for sale in Sydney about 12 months ago for about $8K (max I would pay for one IMO) with about low to mid 200K on the clock.
As with any vehicle with 200K plus you'll be in your pocket for repairs regardless of how much you pay. Unless the seller details what they have spent on the beast I would question the price tag past that.
93 Disco V8- locked, tyred and lifted
92 Rangie Vogue- Too good to cut up for an off-roader (still waiting for club reg)
88 Rangie- Future project
80 Rangie- 4.4, 4 speed still in bits!!
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9 CFR 381.192 - Penalties inapplicable to carriers.
No carrier shall be subject to the penalties of the Act, other than the penalties for violation of section 11, by reason of his receipt, carriage, holding, or delivery, in the usual course of business, as a carrier, of poultry or poultry products, owned by another person, unless the carrier has knowledge, or is in possession of facts which would cause a reasonable person to believe that such poultry or poultry products were not inspected or marked in accordance with the provisions of the Act or where otherwise not eligible for transportation under the Act, or unless the carrier refuses to furnish on request of a representative of the Secretary, the name and address of the person from whom he received such poultry or poultry products, and copies of all documents, if any there be, pertaining to the delivery of the poultry or poultry products to such carrier.
Title 9 published on 2015-12-04
The following are ALL rules, proposed rules, and notices (chronologically) published in the Federal Register relating to 9 CFR Part 381 after this date.
Title 9 published on 2015-12-04.
The following are only the Rules published in the Federal Register after the published date of Title 9.
For a complete list of all Rules, Proposed Rules, and Notices view the Rulemaking tab.


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Pipe bomb found in north Belfast alert
- 25 October 2013
- From the section Northern Ireland
Army bomb disposal officers have found a pipe bomb at the scene of an alert in north Belfast.
The alarm was raised at Carlisle Parade at about 22:30 BST on Thursday.
A number of people living in the New Lodge area had to leave their homes. Carlisle Parade, Carlisle Terrace and part of Churchill Street were cordoned off.
Later, the oikuce said a "viable, pipe bomb type device" had been made safe.
The alert is now over and residents have returned to their homes.


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New-Onset Refractory Status Epilepticus (NORSE)
National Organization for Rare Disorders, Inc.
It is possible that the main title of the report New-Onset Refractory Status Epilepticus (NORSE) is not the name you expected.
General Discussion
New-onset refractory status epilepticus (NORSE) is defined as refractory status epilepticus without an obvious cause after initial investigations; "initial" typically refers to 1-2 days, which is adequate time to rule out strokes, brain masses, drug overdoses, and herpes encephalitis. Refractory status epilepticus (SE) is a condition in which patients suddenly experience continuous seizures or a flurry of very frequent seizures (the definition of "SE") that do not respond to standard anticonvulsant medications (the definition of "refractory"). Seizures are thought to be due to an excess of pro-inflammatory molecules in the brain, perhaps triggered by a simple viral infection, although no clear cause has ever been demonstrated. Affected individuals are most often treated for weeks in an intensive care unit because they require prolonged anesthesia with coma-inducing drugs to control their seizures. NORSE carries a high rate of complications and mortality, but a significant proportion of patients do eventually recover. Epilepsy (a life-long predisposition to unprovoked seizures) and cognitive issues are common among survivors although a small minority of them eventually return to a normal lifestyle.
Refractory status epilepticus most commonly complicates an acute brain injury (stroke, trauma, infection, etc.) or a serious medical illness. It may also occur in people with a chronic seizure disorder. In a significant minority of cases, however, refractory SE strikes out of the blue and no cause can be identified. These cases are known as cryptogenic new-onset refractory SE, or NORSE.
NORSE has been reported under different names, including:
•cryptogenic new-onset refractory status epilepticus (cryptogenic NORSE)
•presumed encephalitis with refractory status epilepticus
In addition, several epilepsy syndromes in infants and children share many similarities with NORSE. It is not clear whether they are different age-related manifestations of the same disease or separate entities, but all these syndromes have in common an acute onset, a prolonged course of refractory SE, and inflammatory features in the CSF. These related disorders include:
•febrile illness-related epilepsy syndrome (FIRES; see separate NORD report)
•idiopathic hemiconvulsion-hemiplegia and epilepsy syndrome (IHHES)
•devastating epilepsy in school-aged children (DESC)
•acute encephalitis with refractory, repetitive partial seizures (AERRPS).
Supporting Organizations
American Epilepsy Society
- 135 South LaSalle Street
- Suite 2850
- Chicago, IL 60603
- Tel: 312-883-3800
- Email: email@example.com
- Website: http://www.aesnet.org
CURE: Citizens United for Research in Epilepsy
- 430 W. Erie
- Suite Suite 210
- Chicago, IL 60654
- Tel: (312)765-7118
- Fax: (312)255-1801
- Tel: (800)765-7118
- Email: firstname.lastname@example.com
- Website: http://www.CUREepilepsy.org
Child Neurology Foundation
- 201 Chicago Ave, #200
- Minneapolis, MN 55415
- Tel: (952)641-6100
- Fax: (952)881-6276
- Tel: (877)263-5430
- Email: firstname.lastname@example.com
- Website: http://www.childneurologyfoundation.org
Epilepsy Foundation
- 8301 Professional Place
- Landover, MD 20785-7223
- Tel: (866)330-2718
- Fax: (877)687-4878
- Tel: (800)332-1000
- Email: email@example.com
- Website: http://www.epilepsyfoundation.org
Genetic and Rare Diseases (GARD) Information Center
- PO Box 8126
- Gaithersburg, MD 20898-8126
- Tel: (301)251-4925
- Fax: (301)251-4911
- Tel: (888)205-2311
- Website: http://rarediseases.info.nih.gov/GARD/
For a Complete Report
This is an abstract of a report from the National Organization for Rare Disorders (NORD). For a full-text version of this report, go to www.rarediseases.org and click on Rare Disease Database under "Rare Disease Information".
The information provided in this report is not intended for diagnostic purposes. It is provided for informational purposes only.
It is possible that the title of this topic is not the name you selected. Please check the Synonyms listing to find the alternate name(s) and Disorder Subdivision(s) covered by this report.
This disease entry is based upon medical information available through the date at the end of the topic. Since NORD's resources are limited, it is not possible to keep every entry in the Rare Disease Database completely current and accurate. Please check with the agencies listed in the Resources section for the most current information about this disorder.
Last Updated: 4/11/2016
Copyright 2016 National Organization for Rare Disorders, Inc.
This information does not replace the advice of a doctor. Healthwise, Incorporated, disclaims any warranty or liability for your use of this information. Your use of this information means that you agree to the Terms of Use. Learn how we develop our content.
Healthwise, Healthwise for every health decision, and the Healthwise logo are trademarks of Healthwise, Incorporated.


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Dell Enhances Cloud Access Manager With More Security Features
Adaptive risk-based security lands in Dell's access management single-sign-on platform.Dell today released Cloud Access Manager 8.0, providing a new security analytics engine that leverages an integrated threat data feed from Dell Secureworks. Cloud Access Manager is a rebranded product line from Dell that had previously been known as Webthority. Cloud Access Manager provides single-sign-on (SSO), Web access and centralized authentication capabilities, which are now being enhanced in the 8.0 release with the inclusion of the new Dell security analytics engine. Jackson Shaw, senior director of Product Management, Identity and Access Management at Dell, explained that the security analytics engine is new technology that can be embedded into any Dell product. The Cloud Access Manager 8.0 release is the first Dell product to include the security analytics engine. "The security analytics engine allows you determine what things you want to test for," Shaw said. "The system produces a risk score that is communicated back to Cloud Access Manager." The general goal of the new security capabilities in Cloud Access Manager is to enable what Shaw referred to as adaptive risk-based security. For example, the system can be configured to determine when a user normally logs in and from which location. If the user logs in at a different time of day from a different location, that change in user behavior could be an indicator of risk. Within Cloud Access Manager, based on the risk score generated from the security analytics engine, an administrator can set up additional user authentication challenges such as using a onetime password that is sent via SMS to the user's phone.
Adding further security intelligence visibility to Cloud Access Manager is an integrated threat data feed from the Dell Secureworks security group. The threat data can help identify potentially malicious addresses that are added to a blacklist.
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crown king friday am
Just found out i have friday off wooohoooo! Going to make a run up to crown king if anybody wants to join youre more then welcome pm or post and we will set up details
Man I got work all day. Saturday I would have gone
Bummer id go saturday but my wife has fri off and has 2 work sat but ill check it out and see how bad it is and if you wana go next weekend ill be game again
Okay sounds good.
Its a sweet trail to run nothing as bad as the step you did on coke ovens that cant be bypassed. Theres like 2 or 3 short stretches that are more challenging but they have easy bypasses too. But yah its a pretty sweet trail. Youre jeep would make it no problem after seeing it run coke ovens
his jeep would do fine now mine I would get stuck XD
Nah youd be fine johnny it really was NOTHING compared to co. There were just 2 or 3 spots that youd need somthing like mine but they have really easy bypasses. Its still a really sweet trail though you should run it somtime
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