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One Melbourne United veteran had a simple way to describe how tough it will be for his side to knock off Perth Wildcats at Hisense Arena on Sunday.
"It's never a good feeling when you come home on a Saturday and the team you play the next day is waiting for you," United guard Daryl Corletto mused this week.
Melbourne United arrived home on Saturday from an 80-72 loss to New Zealand Breakers in Auckland on Friday night and attention immediately turned to the reigning champions who recently beat United in Perth, albeit without co-captains Mark Worthington and Nate Tomlinson.
United had the worst possible start on Friday night, failing to establish themselves defensively then falling 20 points behind by half-time.
Despite a much-improved second half, United couldn't regain the lead.
The Wildcats are also coming off a Friday night road game, beating Adelaide 36ers 84-74 in Adelaide with import Jermaine Beal scoring a game-high 23 points.
Worthington said his side, despite Friday's result, would face the Wildcats with much more belief compared to their previous meeting.
"If you can't get up for a Perth game after a New Zealand game, there is something wrong with you," Worthington said.
"The Wildcats have had their struggles offensively but their defence is still outstanding, as is their offensive rebounding.
"It has won them a lot of games."
Even in the loss to the Breakers, United's pressure defence in the second half created some key turnovers. If they can do the same against Perth it could be a match-winning improvement.
"We have been clicking on the defence end ourselves and against Adelaide last round we clicked on the offensive end," Worthington said.
"If we can just built on what we did last weekend that would be great - we aren't going to connect on 22 three-pointers again. If we keep moving the ball like we have at training this week, we will be alright."
The match will also see the NBL's two NBA draft picks face-off with Perth forward DeAndre Daniels and Melbourne's Jordan McRae both key players.
Daniels' NBA rights are held by Toronto Raptors while McRae's are held by the Philadelphia 76ers.
They are very different players with McRae the NBL's leading scorer and Daniels' a freak athlete still learning how to dominate matches.
Melbourne United will also have to keep level heads to avoid foul trouble and unnecessary distractions should Perth try to coax a reaction from one of the home players.
United's rebounding has been much improved in recent matches but it remains a weakness, something Perth will look to exploit with players like Shawn Redhage, Jesse Wagstaff, Damian Martin and others known for grabbing key offensive rebounds.
Having Beal in form value-adds to the Wildcats' rebounding prowess as more often than not, the extra possessions gained from rebounds create his best scoring opportunities.
"We know with that team not much fazes them - especially on the defensive end," Worthington said.
"We have got to put our best foot forward. If we just put our head down and work our tails off we will be alright - half the battle is matching Perth's energy."
Worthington has also called on skilled reserves like Corletto, Lucas Walker, Chris Patton and Owen Odigie to stand up.
"Guys like Dazza [Corletto] and Waxy [Walker] are vital for us coming off the bench and Patton has been good for us since he joined the rotation. He is just a big body and doesn't know any better than to be huge and that is what he does," Worthington said.
"Owen has been getting better game for game as well.
"Against this team there is no more important game for us to get a contribution from the bench."
Melbourne United won't get much rest time after the Perth game as they host Adelaide 36ers at Hisense Arena on Thursday night at 7pm.


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PR Newswire
LOVELAND, Colo., Oct. 19, 2016
LOVELAND, Colo., Oct. 19, 2016 /PRNewswire/ -- Heska Corporation (NASDAQ: HSKA - News; "Heska" or the "Company"), a provider of advanced veterinary diagnostics and products, plans to announce its third quarter 2016 financial results in a press release to be issued on Tuesday, November 1, 2016 before the market opens. The Company plans to host a conference call to discuss the results at 9 a.m. MT / 11 a.m. ET on the same day, Tuesday, November 1, 2016.
To access the conference call:
The conference call will be webcast live from the Company's website on www.heska.com.
A telephonic replay of the conference call will be available beginning two hours after the end of the call and continue through 11:59 p.m. ET on Tuesday, November 8, 2016.
To access the replay:
The webcast will also be archived on www.heska.com until December 31, 2016.
About Heska
Heska Corporation (NASDAQ: HSKA - News) sells advanced veterinary diagnostics and specialty products. Heska's state-of-the-art offerings include blood testing instruments and supplies, digital imaging products, software, and single use offerings such as in-clinic heartworm diagnostic tests, heartworm preventives, allergy immunotherapy and allergy testing. The Company's core focus is on supporting veterinarians in the canine and feline healthcare space. For further information on Heska and its products, visit the company's website at www.heska.com.
At Heska Corporation:
At Hayden IR:
John McMahon, Chief Financial Officer
Brett Maas, Managing Partner
To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/heska-schedules-third-quarter-2016-earnings-release-and-conference-call-for-november-1-2016-300347335.html
SOURCE Heska CorporationPR Newswire
Last updated on: 19/10/2016
PharmiWeb.com is Europe's leading industry-sponsored portal for the Pharmaceutical sector, providing the latest jobs, news, features and events listings.
The information provided on PharmiWeb.com is designed to support, not replace, the relationship that exists between a patient/site visitor and his/her physician.

subset: commoncrawl


text:
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Thursday, 28 February 2013
It's been a while...
I guess it's been a while since I posted but as I've working extra hours as well as rehearsing for a play I've had no time! As I have a spare half hour thought I'd post a quick card or two
I have promised myself to post every day in March so we will see how I get on. We these cards were made with the docrafts goody bag for Feb, enjoy...
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John Lockwood
Thursday, February 28, 2013
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Microbes commonly exist in milieus of varying complexity and diversity. Although cultivation-based techniques have been unable to accurately capture the true diversity within microbial communities, these deficiencies have been overcome by applying molecular approaches that target the universally conserved 16S ribosomal RNA gene. The recent application of 454 pyrosequencing to simultaneously sequence thousands of 16S rDNA sequences (pyrotags) has revolutionized the characterization of complex microbial communities. To date, studies based on 454 pyrotags have dominated the field, but sequencing platforms that generate many more sequence reads at much lower costs have been developed. Here, we use the Illumina sequencing platform to design a strategy for 16S amplicon analysis (iTags), and assess its generality, practicality and potential complications. We fabricated and sequenced paired-end libraries of amplified hyper-variable 16S rDNA fragments from sets of samples that varied in their contents, ranging from a single bacterium to highly complex communities. We adopted an approach that allowed us to evaluate several potential sources of errors, including sequencing artifacts, amplification biases, non-corresponding paired-end reads and mistakes in taxonomic classification. By considering each source of error, we delineate ways to make biologically relevant and robust conclusions from the millions of sequencing reads that can be readily generated by this technology.
Microbes in nature typically exist in complex communities, which can be highly variable in both the composition and the abundance of their constituent species. Initial surveys of microbial community diversity applied cultivation-based methods, but these were often biased and of limited value owing to the inability to cultivate many (more likely, the majority of) naturally occurring species. Such methods were supplanted by molecular approaches that did not rely on cultivation but were based rather on the PCR amplification, cloning and Sanger sequencing of universally conserved molecules, usually the 16S ribosomal RNA gene. Current sequence databases contain over a million full-length 16S rRNA sequences spanning a broad phylogenetic spectrum than can serve as a benchmark for assessing the bacterial taxa (also referred to as 'phylotypes' or 'ribotypes') present in environments worldwide (Cole et al., 2009).
Although the traditional 16S-cloning-and-sequencing approach has the potential to provide an in-depth view of the richness and evenness of bacterial species within a community, its application is somewhat laborious and costly, with the result that most applications have assayed on the order of only 100 sequences per sample, which may not be sufficient to fully characterize all but the simplest communities. This has led to the development of alternative techniques for assessing rRNA variation (for example, ARISA, DGGE, tRFLPs) (Muyzer et al., 1993; Liu et al., 1997; Fisher and Triplett, 1999); however, the most revolutionary innovation has been the application of 454 pyrosequencing to survey hundreds of thousands of 16S rRNA gene amplicons (termed 'pyrotags') in a single sequencing run (Sogin et al., 2006). Since its initial introduction, pyrotag analysis has been widely implemented (for example, Dethlefsen et al., 2008; Bowers et al., 2009; Cardenas et al., 2010; McLellan et al., 2010) and further optimized in order to obtain thousands of sequences from multiple samples in parallel (Parameswaran et al., 2007; Hamady et al., 2008; Costello et al., 2009; Engelbrektson et al., 2010).
Advancements in sequencing technologies, by offering greater numbers of sequencing reads at much lower costs, might eventually supplant pyrotag analysis for the characterization of microbial communities. Recent interest has focused on the applicability of other sequencing methodologies, most notably Solexa/Illumina (Lazarevic et al., 2009; Claesson et al., 2010; Gloor et al., 2010; Caporaso et al., 2011; Zhou et al., 2011), which is currently less than 1/100 the cost per read than 454 pyrosequencing. Although realizing much shorter read lengths, the Illumina technology can be tailored to yield sequences of increased lengths, as can be obtained by merging the paired-end reads generated from the same amplicon (Gloor et al., 2010; Rodrigue et al., 2010; Zhou et al., 2011). By integrating sample-identifying barcodes into the amplification primers, the Illumina platform, like 454 pyrosequencing, is amenable to a high level of multiplexing, which further increases its utility for examining large and complex sets of samples (Gloor et al., 2010).
Here, we use the Illumina sequencing platform for 16S rRNA amplicon analysis (called 'iTags') and assess its generality, practicality and potential complications. We first fabricate and sequence a paired-end library of hyper-variable 16S rDNA fragments amplified from samples that varied in contents from a single bacterium, an artificial community and a highly complex natural community. The inclusion of identifying barcodes and multiple primer pairs, combined with the selected amplicon lengths, allowed us to evaluate the level of sequencing error, amplification biases and association between paired-end reads, among other variables. Despite recovering over 30 million reads, our imposition of stringent quality filters resulted in up to an 85% reduction in the number of potentially informative reads. Nevertheless, tens to hundreds of thousands of informative reads were returned for each of the multiplexed samples. These results uncovered both the major advantages and obstacles in the application of these methods, and provide insights into the ways that such impediments can be ameliorated prior to the application of these procedures.
During the development of this project, the Illumina GAIIx sequencer could produce reads of up to 100 nucleotides (nt) in length from both ends of single DNA fragments or amplicons (that is, paired-end reads). We exploited this technology to assemble reads spanning a >100-nt region of the 16S rDNA. To accomplish this, we identified candidate universal primer pairs spanning the 16S rRNA variable loops (Neefs et al., 1991) that would maximize sequence information and be short enough to allow adequate read overlap.
The phylogenetic distribution of each primer sequence was analyzed using the RDP Probe Match (Cole et al., 2009). The goal was to obtain broadly distributed primers that amplify regions of approximately 100–150bp in length such that the paired-end reads from an amplicon would show some overlap. Of primer sets meeting these criteria, two flanking the variable loop-6 (V6) were chosen (XXX denotes sample-specific barcodes): 967F 5′-XXX CAACGCGAAGAACCTTACC-3′ and 1046R 5′-XXX CGACAGCCATGCANCACCT-3′ (Short 1 V6 (S1V6); 98bp in Escherichia coli) (Sogin et al., 2006), and 917F 5′-XXX GAATTGACGGGGRCCCGC-3′ and 1061R 5′-XXX CACGRCACGAGCTGACGAC-3′ (Long V6 (LV6); 163bp in E. coli) (Keijser et al., 2008). We further modified the S1V6 primer pair to increase both the extent of overlap between paired-reads and the number of potentially informative sites as follows: 970F 5′-XXX CGCGAAGAACCTTACC-3′ and 1050R 5′-XXX ACGACAGCCATGCANC-3′ (Short 2 V6 (S2V6); 96bp in E. coli).
Sample-identifying barcode sequences were included at the 5′ ends on both the forward and the reverse primers. The barcodes were designed (1) to be short, 3 or 4bp, to minimize loss of sequence information; (2) to have two or more differences from one another (such that a single sequencing error could not convert one into another); and (3) to have the 3′ nucleotide of the barcode be a low-frequency match of nucleotide upstream from the 5′ end of the priming site. The barcodes for the primer pairs were as follows: S1V6–ATG, CAG, TCG, ACT, CGT, GCA, GAT, and TGA; S2V6–AGC, ATT, GTC, TCC, CCAT, CGGC, GCGT and TTAT; and LV6–GTC, AAC, CCA, TAA, AGGA, GCGC, TTGC and ATAC.
We tested 16S primers on sets of samples representing a range of microbial diversity. First, we used two well-defined samples containing either DNA extracted from a single sequenced strain of E. coli K-12 (MG1655) (Blattner et al., 1997) or from a mixture of genomic DNAs of known concentrations from 19 cultivated strains of bacteria (Table 3). Next, we analyzed a sample of unknown microbial diversity: a fecal sample from a laboratory mouse. The QIAamp DNA stool mini kit (Qiagen, Valencia, CA, USA) was used following the manufacturer's protocol to extract total DNA from the feces of an individual lab-reared mouse, Mus domesticus strain WSB.
PCR amplifications were performed in triplicate 30-μl reactions using 30–50ng of template DNA and one of two DNA polymerases. Samples amplified with the barcoded S1V6 primer pairs were performed using 1.2U of Taq DNA polymerase (5 PRIME) and final concentrations of 1X polymerization buffer, 1m dNTPs and 0.4μ of each primer. The reaction mixtures were subjected to an initial denaturation cycle of 95°C for 2min, followed by 25 cycles at 95°C for 30s, 55°C for 45s and 72°C for 1min, and a final extension step at 72°C for 10min. The samples amplified with the barcoded S2V6 or LV6 primer pairs used the high-fidelity Advantage 2 polymerase mix and the Advantage 2 SA PCR buffer (Clontech, Mountain View, CA, USA) following the manufacturer's instructions. Amplifications with the S2V6 and LV6 primer pairs were performed in a laminar-flow hood using screw cap tubes to minimize contamination. The thermal cycling conditions were identical to those detailed above except that the annealing temperature was 58°C.
Triplicate reactions for each sample were pooled, visualized on 1% agarose gels, purified with the MinElute PCR purification kit (Qiagen) and eluted in 11μl of EB buffer. The concentrations of the amplification products were quantified on a Bioanalyzer using the DNA 1000 LabChip kit (Agilent Technologies, Santa Clara, CA, USA). The barcoded products of the S1V6 and S2V6 primer sets were combined in equimolar amounts, re-purified with the MinElute kit, eluted in 30μl of EB buffer and submitted for 2 × 75-nt paired-end sequencing at the Yale Center for Genomic Analysis. Similarly, the LV6 products were mixed in equimolar amounts, but these longer amplicons were subjected to 2 × 100-nt paired-end Illumina sequencing.
Initial base-calling using Bustard (v 1.7) and quality filtering of paired-end reads were performed by the Yale Center for Genomic Analysis. Upon receipt, these reads were evaluated for quality (mean quality scores; presence of sites denoted as 'N'), and identifiable primers and barcode sequences. Read pairs with perfectly matching primer and barcodes were extracted and binned by barcode. The resulting Illumina fastq files, containing quality values equivalent to Phred scores, were converted to Sanger fastq files using Maq ('maq ill2sanger', v 0.7.1) (Li et al., 2008). Individual read pairs were converted to fasta and qual files, and then assembled using Phrap (v 1.080812) using the following parameters: '-vector_bound 0 -trim_start 0 -forcelevel 3 -preassemble -bandwidth 10 -repeat_stringency 0.98 -ace -minmatch 5 -maxmatch 20 -minscore 10'.
To investigate the effect of quality scores on estimates of diversity, the assembled reads for each barcoded sample were further filtered by consensus quality scores. Barcodes were pruned from sequences and the data sets were aligned in the RDP pyrosequence aligner (Cole et al., 2009), which implements the Infernal algorithm (Nawrocki and Eddy, 2007). The resulting alignments were edited to remove primer sequences and clustered to generate operational taxonomic units (OTUs) using the RDP clustering algorithm. The taxonomic assignments of OTUs were derived from the RDP Classifier using a 50% bootstrap cutoff, as recommended for reads less than 100bp. Custom PERL scripts were written to generate a pipeline to analyze, organize and format these data. The PERL scripts and raw sequence data presented and analyzed here are available at www.yale.edu/ochman/data.
Over 30 million paired-reads were collected from each of the two multiplexed Illumina sequencing runs. Initial filtering removed 85% of the reads from run1 and 54% from run2 owing to errors stemming from several sources (Table 1), which is comparable to other studies that report filtering 40–70% of their initial reads (Gloor et al., 2010; Caporaso et al., 2011). We secured a total of 4.5 and 16.0 million paired-end reads for the two runs in spite of the high number of reads that were discarded because they did not meet our thresholds for accuracy; this, in turn yielded tens of thousands to millions of raw read-pairs per multiplexed sample (run1 =380253±176319; run2 =3936966±2299439) (Table 2). Because the runs were founded with equimolar concentrations of amplicons, some of the variation in the number of reads returned per sample is most likely attributable to amplification biases that occurred during the synthesis of the Illumina sequencing library. Future experiments might avoid this bias by implementing an amplification-free library approach (Kozarewa et al., 2009).
We used a strict set of quality filters to minimize the impact of erroneous reads on measures of microbial diversity (Kunin et al., 2010). Errors in the barcode/primer sequence of either of the paired-end reads necessitates the removal of both reads in the pair; and in the majority of read-pairs that were culled, either only one of the reads perfectly matched the primer sequence or the paired reads lacked identical barcodes. The primer and barcodes are not informative for sequence classification and together constitute over 20% of the sequencing read, but the inclusion of both sequences provide a useful filter for removing erroneous reads and estimating sequencing error rates.
Error frequencies were estimated for the barcode/primer regions of all reads with high sequence quality (q30) for the first 25nt and with single-nucleotide differences from the expected sequence. This included reads initially filtered out for not containing a perfect primer or barcode sequence (see above). Across the 14 barcode/primer combinations analyzed from run1, the error rates per nucleotide varied over three-fold (1.6 × 10−4 to 5.6 × 10−4), and rates varied only two-fold (4.1 × 10−4 to 8.4 × 10−4) among the four barcode/primer combinations from run2 (Supplementary Table 1). For two reasons, we ascribe these variants to errors generated during sequencing rather than to artifacts that arose during PCR amplification: (1) Oligonucleotide synthesis is typically very accurate, such that errors in primer regions are more likely generated during the sequencing step; (2) we performed independent experiments using two different Taq polymerases each having unique error rates, but obtained approximately the same rate (analysis of covariance; F1,15=1.07, P=0.317) and spectrum of mutations with each. The fact that error rates were independent of the source of Taq polymerase further suggests that errors are introduced during DNA sequencing.
There were large numbers of paired-end reads with mismatched barcodes, but owing to our use of barcodes that require at least two mutations to convert one barcode into another, not many of these mismatched read-pairs were attributable to sequencing errors. In fact, many read-pairs contained recognizable barcodes that differed between the corresponding reads, which could result from (1) over-clustering during the sequencing run; (2) the production of chimeric amplicons during the amplification step of the Illumina library or (3) primer contamination, the likelihood of each of which is discussed below.
Over-clustering can result from low nucleotide diversity in the first four sequenced nucleotides and high cluster density on the flow cell. Although the first four bases of our barcodes and primers did not contain equal frequencies of each of the four nucleotides, over-clustering is an unlikely cause of the mismatched barcodes detected. An analysis of the run showed that read signal intensities and the percentage of clusters passing filter were both above normal expectations (intensity >200 for first cycle, >80% clusters passing filter). Alternatively, chimeric amplicons are a common by-product of 16S rDNA amplifications (Qiu et al., 2001); however, our protocols used a low number of amplification cycles (10–12) and the high-fidelity Taq polymerase for library construction, both of which reduce the frequency of chimeric amplicons. This leaves primer contamination during the initial PCR amplifications as the principal contributor of mismatched barcodes. Although we implemented precautionary measures intended to minimize contamination (using screw cap tubes; removing primers and templates individually; setting up the reactions in a laminar flow hood), contamination occurred in these samples. With current interest in multiplexing scores (or even hundreds) of samples, we let these results serve to forewarn investigators that highly stringent procedures are necessary to prevent cross-contamination.
We first evaluated the efficacy and accuracy of the iTag approach by sequencing a single template, E. coli K-12 (MG1655). The E. coli genome contains seven rRNA operons, six of which have identical alleles at V6 and the seventh encoding a V6 allele with nine nucleotide differences (Figure 1a). At both 100% and 97% identity thresholds only two OTUs are expected.
The V6 loop of E. coli was amplified using three primer pairs (S1V6, S2V6 and LV6), and we performed a technical replicate of S2V6 (Table 2). Nearly all reads assembled with well-supported overlaps of their distal 3′ regions (S1V6 and S2V6, ≈50bp; LV6, ≈30bp). Alignment and clustering of the assembled iTag sequences showed that each of the E. coli samples contained much more than the two predicted phylotypes (n=4090–159224; Table 2).
Previous work has shown that quality trimming of pyrotags is essential for accurate prediction of community composition (Kunin et al., 2010); therefore, we filtered the assembled reads according to consensus quality scores estimated by Phrap. We compared two quality filters, requiring that all sites had error probabilities of either 10−2 or 10−3 (corresponding to Phred scores of q20 or q30). These measures reduced the number of unique phylotypes (aka 100% OTUs), but failed to remove all of the spurious OTUs (Table 2).
Among these confounding OTUs, we found that 3–7% of high-quality reads (q30) differed by only 1nt from the expected E. coli alleles (Figures 1b–e). Variants occurred at every nucleotide position regardless of the primer pair or the source of Taq polymerase. Given the numbers of variants, it is not likely that each represents a new mutation that occurred during growth in culture. Therefore, these phylotypes are attributable to errors that occurred during sequencing.
The very large number of sequence variants made them impossible to cluster into the two expected OTUs, even when applying very relaxed thresholds (for example, 90% OTUs). Most of the remaining OTUs (Figure 1) represent phylotypes that were detected in other samples and are best explained by primer contamination (discussed above). In addition, some of the contaminants in the LV6 samples were because of nonspecific amplification of other chromosomal loci; for example, we recovered a 123-bp fragment of uridylyltransferase (glnD) from E. coli and a 78-bp fragment of an ATP transport permease from Streptococcus sp.
To rid samples of artifactual phylotypes generated by sequencing errors and PCR contamination, we used a sequence abundance threshold in a manner similar to that of Caporaso et al. (2011). We based this threshold on the representation of a unique phylotype (100% OTU) given the number of q30 reads for that barcode. Through stepwise increments in the abundance threshold, we followed the degree to which spurious reads were eliminated (Figures 1 and and2).2). Eventually, by applying a 1.0% abundance threshold (that is, removing all OTUs occurring at frequencies less than 1.0%) we were able to recapitulate the two phylotypes expected to occur in the E. coli genome.
Having addressed the sequence variation detected within a single species by the iTag approach, we analyzed the data generated for a synthetic community of 19 strains of bacteria (Table 3). This sample was amplified using each of the three primer sets, which should yield 19 100% OTUs, and 16 (S1V6, S2V6) or 15 (LV6) 97% OTUs. A similar pattern showing an excess of erroneous and contaminating high-quality OTUs emerged even after stringent quality filtering (Table 3). Although the application of a 1% abundance threshold removed artifactual reads, it also removed several of the expected OTUs that occurred below this threshold (Figure 2). The initial template DNA concentrations of 12 of the 19 strains represented less than 1% of the total DNA mass, so this result was anticipated (Table 3); however, we also found that the actual read abundances for the 19 100% OTUs departed from the expected values (Figure 3). The likely cause of the deviation between the relative amount of input DNA and the relative number of reads could be primer specificity. For example, the Streptococcus cristatus OTU was similarly abundant in the CAG–S1V6 (1.7%=2754/162125) and ATT–S2V6 (1.8%=10207/556184) data sets, but nearly absent from the AAC–LV6 data set (0.0022%=9/407785).
Short 16S tags can be difficult to assign taxonomically and alone provide limited phylogenetic resolution (Liu et al., 2007). We found that the RDP Classifier accurately identified all of the 19 expected OTUs to taxonomic Class, but its success rate progressively decreased for lower taxonomic divisions, with only about 60–90% assigned correctly. Alternative BLAST-based classification approaches can increase accuracy provided related full-length 16S rRNA sequences are available (Huse et al., 2008). Of course, neither of these approaches are able to distinguish among species that have identical V6 loop sequences (for example, E. coli, Escherichia fergusonii and Salmonella enterica; Figure 3).
By way of analyzing a complex microbial community whose composition and constituents are uncharacterized, we assessed the V6 sequence diversity within a fecal sample from the house mouse M. domesticus strain WSB/EiJ. This sample was amplified with GCA–S1V6 and TCC–S2V6, generating, respectively, 384480 and 223708 binned reads. As before, even with rigorous quality filtering, a large number of low-frequency 100% OTUs remained (Table 2). We applied similar abundance thresholds (10−5 to 10−2), which resulted in a dramatic reduction in phylotypes (Figure 4). The utilization of the same threshold as that applied to the E. coli samples (that is, 10−2) leaves only 12 OTUs, which represent 67% (GCA–S1V6) and 74% (TCC–S2V6) of the high-quality reads. Thus, it appears that application of a 1.0% threshold is too stringent for actual biological samples and probably removes many tags that are likely to represent the actual microbial constituents of these communities. The two amplified samples show an extensive overlap in 100% OTUs (Sørensen distance, 0.010; Jaccard distance, 0.020), which results in a highly similar taxonomic distribution regardless of the abundance threshold applied (Figure 4).
Massively parallel sequencing platforms supply an efficient and sensitive means of characterizing microbial communities, but the sequencing depths offer by these methods are accompanied by artifacts that can affect the interpretation of community diversity. We have identified and quantified several sources of errors inherent to 16S amplicon sequencing (iTags) on the Illumina platform (contamination, amplification and sequencing errors), thereby enhancing the utility of these data for assessing community diversity. Most recent efforts to interrogate microbial communities have relied upon 16S rRNA gene sequences generated by 454 pyrosequencing (for example, Dethlefsen et al., 2008; Bowers et al., 2009; Costello et al., 2009; Cardenas et al., 2010; McLellan et al., 2010); however, Illumina sequencing technologies currently produce much larger numbers of much shorter reads for a fraction of the cost and will likely supplant the use of 454 pyrotags.
Our experimental iTag design used barcoded primers flanking the V6 segment of 16S rRNA, a region commonly amplified in pyrotag experiments (Sogin et al., 2006; Keijser et al., 2008; Galand et al., 2009). Individual samples were amplified, mixed and then used as templates to construct and sequence two standard Illumina paired-end libraries. Although Illumina sequence quality decays along the length of reads, our motivation for assembling paired-end reads was to increase the quality and confidence of the overlapping region (Gloor et al., 2010; Rodrigue et al., 2010; Zhou et al., 2011). Therefore the amplified V6 region was restricted to 100–160bp to ensure an adequate overlap of the forward and reverse paired-end reads. We subsequently filtered out low-quality tags, leaving only assembled reads that contained barcode and primer sequences at each end, which bounded a phylogenetically informative amplified region. This design allowed the simultaneous estimation of sequencing error rates and microbial diversity.
Over half of >30 million paired-end reads from each of Illumina runs were subsequently discarded owing to either (1) sequencing errors in one or both of the primer regions or (2) paired-end reads that contained mismatched barcodes (Table 1). Other Illumina-based 16S rRNA studies have encountered similarly high sequencing error rates, making such extreme read filtering necessary, but these studies did not report a high incidence of mismatched barcodes (Gloor et al., 2010; Caporaso et al., 2011). After ruling out over-clustering and sequence chimeras, primer contamination during the initial sample amplifications was viewed as the most likely cause of the mismatched barcodes (see above). For example, >60% of the contaminants detected in the E. coli libraries were present in one or more of the other samples, and frequently these contaminants represented the more abundant phylotypes in the other samples (data not shown). The sequencing depth afforded by the Illumina platform greatly increases the likelihood of detecting contaminants; however, we stress the utility of barcoding and sequencing both ends of multiplexed amplicons in order to detect this source of errors. In the absence of paired barcodes, removing reads that lack a significant match to a reference database can be implemented. Although this latter method is of limited utility when examining novel or poorly characterized communities.
To establish baseline levels of sensitivity of the iTag approach, we performed several controls, including the amplicons generated from a single bacterial genome and from a defined mixture of DNA from 19 strains. In both cases, we found an excess of high-quality, 100% phylotypes regardless of the primer pair, Taq polymerase or sequencing run. However, sequencing reads bearing a single mutation relative to the two expected E. coli phylotypes, or to the 19 phylotypes from the mixture of strains, represented 2.2–8.2% of the reads. Based on these reads we estimate a sequencing error rate of 1.3 × 10−3 to 4.1 × 10−4 per nucleotide (Supplementary Table 2), which is slightly greater that the rate estimated from errors confined to the primer regions (run1 =3.8 × 10−4; run2 =6.4 × 10−4). This difference is in line with expectations as, in the Illumina platform, error rates are known to increase with distance from the priming site (Rodrigue et al., 2010; Zhou et al., 2011). These errors impact taxonomic identification using the RDP Classifier, particularly reducing the ability to assign Order, Family and Genus identities to the shorter S1V6 and S2V6 tags.
To account for the large number of spurious reads in these samples, we applied strict abundance thresholds to our samples. We came the closest to recapitulating our expected diversity from these samples when we applied an abundance threshold at which all phylotypes at frequencies <1% were removed. This cutoff is based on the total number of high-quality reads for a given sample and more-or-less corresponds to the 0.01% threshold applied by Caporaso et al. (2011), whose threshold is based on the total number of read-pairs recovered for their entire experiment (10000 out of 87507177 reads). Application of such stringent thresholds is not particularly satisfying, but will remain a necessary measure until sequencing error rates are improved.
Mammalian guts house a diverse array of resident microbes and are numerically dominated by the Firmicutes and Bacteriodetes (Ley et al., 2008). A previous pyrosequencing effort identified 1000 bacterial phylotypes (97% OTUs) in healthy Mus musculus individuals using the same S1V6 primers (Antonopoulos et al., 2009); however, our sample from a lab-reared, wild-derived inbred strain of M. domesticus WSB yielded only 12 phylotypes (97% OTUs) after application of the equivalent quality and abundance filters derived from the contrived samples (q30 and 10−2). Reducing the stringency of the abundance threshold increased the number of recovered iTag phylotypes, but did not alter the taxonomic distribution of the iTags (Figure 4). (Lowering the abundance threshold from 10−2 to 10−4 yielded about 20% more iTags containing only a single-nucleotide difference relative to the 12 high abundance OTUs.) The source of the difference in the number of phylotypes detected in the Mus fecal samples is unknown, and direct comparisons between iTags and pyrotags can only be assessed from the corresponding analyses of 16S diversity within E. coli (Kunin et al., 2010; this study). Therefore, we suggest the amplification and sequencing of a control sample (for example, E. coli) during each multiplex experiment to empirically determine appropriate abundance thresholds and to account for differences in sequencing error rates between runs.
Interest in leveraging the Illumina sequencing platform to examine 16S rRNA gene diversity has led to the recent development of several strategies and observations about its implementation (Claesson et al., 2010; Gloor et al., 2010; Caporaso et al., 2011; Zhou et al., 2011). Until recently, the Illumina sequence read length was limited to ≈100nt. As a result, paired-end sequencing of single, short variable loops (V6) has been assembled (Gloor et al., 2010; Zhou et al., 2011; this study), and longer variable region(s) (255–465bp) were artificially merged or analyzed separately (Claesson et al., 2010; Caporaso et al., 2011). Assembly of overlapping, paired-end reads results in phylotypes of considerably better quality because it identifies and rectifies problems that arise from the deterioration of read quality as a function of read length (Claesson et al., 2010; Gloor et al., 2010; Zhou et al., 2011). But because PCR and sequencing errors persist, application of abundance thresholds are still necessary to avoid inflated estimates of species richness (Gloor et al., 2010; Zhou et al., 2011). Moreover, conservative estimates of error rates using contrived DNA samples vary six-fold, from 2.4 × 10−3 to 4.1 × 10−4 per nucleotide (Zhou et al., 2011; this study).
The iTag approach holds enormous promise as means to investigate microbial diversity, but in its current applications there are three major obstacles: (1) contamination, (2) the utility and classification of short read lengths, and (3) sequencing error rates. Contamination can likely be mitigated by changes in laboratory procedures, including use of liquid-handling robots and sterile work environments. Although not considered here, contamination derived from reagents or consumables is also possible and would require alternative strategies for its detection and elimination. Despite the prevalence of studies using the V6 region and evidence that short reads perform adequately for community analyses (Liu et al., 2007), short V6 tags appear to systematically overestimate species richness (Youssef et al., 2009). Current Illumina read lengths (≈150nt) will allow the recovery and assembly of larger (V4) or more (V6+V7) 16S variable regions that better reflect the microbial diversity obtained when analyzing the entire 16S rRNA molecule. Additionally, longer read lengths will facilitate the use of additional barcodes that will allow multiplexing hundreds of samples in a single run. Although the observed error rates are of little consequence to most genomic studies, as errors are resolved through sequencing depth, amplicon tagging approaches treat each unique sequence read as a novel OTU. Until there are improvements to the Illumina sequencing chemistry and analysis programs, erroneous reads will need to be filtered through the application of abundance thresholds (Gloor et al., 2010; Caporaso et al., 2011). The cost and capacity of the Illumina sequencing make it the preeminent platform for assessing microbial community diversity, and we have called attention to the errors that need to be resolved in its application and in the interpretation of results.
We thank Michael Nachman, Nancy Moran and Zakee Sabree for contributing the template material used for library construction, and Zakee Sabree for coining the term 'iTag'. Funding for this project was provided by the National Institutes of Health to HO.
Supplementary Information accompanies the paper on The ISME Journal website (http://www.nature.com/ismej)
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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I'm one of those weird hybrid punk / uber geek. Actually, it's so trendy and acceptable to be a geek today that I prefer the made up term nerdpunk.
Random interests: Technology, roller derby, rock'n'roll, video games, dungeons and dragons (yeah, i know).
More recent punk rock (Boris The Sprinkler, The Queers, Screeching Weasel, The Riverdales)
Some anarcho-punk (Crass)
Recent smaller punker stuff (Unbelievers, Le Volume Était Au Maximum, Le Forfait de Base)
'Various other punk' stuff (Dead Kennedys, Vulgaires Machins)
Digital Hardcore (Ambassador 21, Atari Teenage Riot)
Industrial / Noise (Memmaker, scrap:edx)
.. and some blues, old rock'n'roll, drum and bass, breakcore.
Classic 30's horror / sci-films
B-movies (robots, giant apes and giant lizards)
Musicals (guilty pleasure)
The 5 Kevin Smith Jersey Trilogy movies
80's classics (John Hugues among others)
Repertoire (Citizen Kane)
And lots lots lots more. I love cinema.
Don't watch much of it but loved: Angel, Buffy, Firefly, all Star Treks, BattleStar Galactica (old and new) Lost, Heroes.
Books: Read a lot but not a lot of novels. I have a few fantasy books and recently listened to some audiobooks (Hitchhiker's Guide to the Galaxy, The Areas of my Expertise and Millenium).


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Thank you to all who nominated me for the "Rockin' Girl Blogger Award"! What fun! I'd like to tag these 5 chicks because their blogs are awesome and ones that I visit at least once a day!
1. Nancy's Creative Mess
2. Orange Moose
3. Quilt Nut
4. Stampinsars
5. Card Creme


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CASES of child rape are on the increase in Cheshire, latest figures have revealed.
According to Her Majesty′s Inspectorate of Constabularies, there has been an increase in rape offences against adults and children.
The report uses statistics released by the Rape Monitoring Group, covering a 12 month period, up to the end of March 2013.
It shows there were 98 children reported to have been raped.
That was up from 87 in 2012, and 62 in 2011.
Police say the increase is due to victims feeling more able to come forward.
During the same period, there were 142 rapes reported, compared to 129 in 2012, and 108 in 2011.
Ass Chf Con Ruth Purdie said: "Cheshire Police welcomes the publication of this data.
"We are committed to working with our partner agencies to support victims of rape and bring offenders to justice.
"We encourage anyone who has been a victim of rape to come forward with the assurance that we will take their report seriously and investigate it rigorously."
Det Chf Insp Nigel Wenham, of Cheshire Police's Strategic Public Protection Unit, said: "Cheshire Police has a dedicated rape unit which is made up of trained detectives who respond to and deal with all reported rapes.
"The unit works alongside a team of co-located specialist Crown Prosecution Service lawyers to develop strategies around cases and secure prosecutions.
"Cheshire Police works closely with St Mary′s Sexual Assault Referral Centre and the Rape and Serious Sexual Assault Support Service to ensure victims of rape receive the support they need during this traumatic time."
The report also showed the detection rate - when a suspect is charged - was 14 per cent for adult victims, and 22 per cent for children.


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IELTS Writing Task 2 Sample 292 - Teaching children at home is best for a child's development
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- Last Updated: Saturday, 03 September 2016 17:01
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IELTS Writing Task 2/ IELTS Essay:
You should spend about 40 minutes on this task.
Some people believe that teaching children at home is best for a child's development while others think that it is important for children to go to school.
Discuss the advantages of both methods and give your own opinion.
You should write at least 250 words.
Sample Answer 1:
Education is the fundamental building block for children's development. However, the growth of students is very much dependent on the way of coaching. Some prefer educating students at home while others think it is necessary for children to attend schools. In this essay, I will analyse both viewpoints before reaching a reasonable conclusion.
(by - Aqueela Mansuri)
Sample Answer 2:
Today's children become tomorrow's citizens. So allowing them to interact with the community is very important. More than that ever human is a social being. Schools play an important role in forming children's social behaviours. So, in my opinion teaching children at school is better than teaching at home.
The main reason why kids need to be sent to school is to get awareness about the society he/she will be a part in the future. Till 4-5 years, children normally with their family. Only after sending to school they will get a chance to mingle with the same age group. This will help them to improve communication skills, social awareness, skills to handle difficult situations etc. They will be able to do at least their own things themselves. Also, I think this is required to reduce their selfishness- they will learn how to share in schools.
The other important fact is discipline. At home, kids always feel easiness. It will be always a tension free atmosphere for them since they are used to it from the childhood. Also, parents and other family members may not be as strict as a teacher in many aspects. So it is hard to get discipline in life for kids those who miss schooling. Main reason for this is the freedom they get at home.
Apart from all these disadvantages, learning at home has some merits too. The main advantage is kids will be more comfortable. Parents can decide or prioritise the topics to teach, unlike in some schools give over work-load to kids. The time for study can be flexible so that children will get more free time. And parents may be comparatively tension free too.
In a nutshell, even though both methods have its own merits and demerits, parents should make important decisions about their children according to their circumstances and convenience.
Sample Answer 3:
One school of thought is that the best approach for child's development is that if parents teach their children themselves at home. On the other hand, the second school of thought is that children should attend school for their better development. There is no general consensus over this and both approaches have advantages.
Let's see the advantages of the first approach. It is understood that no one can better understand their child except his/her parents. A child is with the parents since birth and parents know each and every aspect of the child. While teacher has to teach the whole class and take every student along with including slow learners and fast learners. Whereas parents have one child to teach and they can give him/her their hundred percent attentions. They can schedule the teaching as per their child's pace. Moreover, parents can better look after the child.
Another approach is attending school. This method has its own advantages. In school, a child has to spend his time in the class with other children of the same age. He/she starts making new friends. They not only talk about studies but also discuss other happenings around them. They share knowledge and information with each other. All the children take part in group activities including extracurricular activities. As a result, their confidence increases many folds.
As we have seen, both methods have their own advantages. However, I strongly feel that it is very vital for children to attend school. This way, school and parents share the responsibility of development of the child and it is the best approach.
Homeschooling has been a practice throughout history, as the mother serves as the first teacher. At the present, homeschooling has been a choice for parents who commonly not satisfied with the school system. It provides learning freedom, giving children enough time to focus on one topic at the time as well as giving them enough rest periods. Commonly it is facilitated by the mother, giving them a chance to spend much time with their children creating a great bond. Moreover, making them aware of the children's aptitude, attitude and abilities, therefore making it easier to come with individualised lesson and facilitate one on one tutorial.
Another benefit of it is that it helps in reducing emotional pressure brought by bullies, peer pressure and boredom. In the contrary, this requires a full attention by the parents, limiting their time to work. Thus, requires parents to be creative in facilitating the lesson in order to gain interest and children's attention in each lesson. In the financial note, although you are not obliged to pay for tuition fees, being homeschool is expensive as you need to buy certain educational resources that are easily available in school settings.
On the other side, attending school provides a lot of positive aspects; one of this is that the school provides a wide range of up to date educational resources. Moreover, teaching is facilitated by a well-trained teacher that can provide effective teaching strategies. Although bullies and peer pressure are present in the school settings, this will help children to be socially exposed to these factors, thus, giving them a chance to cope with it and further gain their confidence and high self-esteem.
To sum it up, both choices has its positive and negative side, but in my opinion attending school provide a wide range of necessary information as well as skills that children will help them develop well. Even though attending school is beneficial, education supplementation from home will help students to attain a high level of education. School and home teaching will greatly help students understand more and help them mould their character and attitudes that will help them in the near future.
Add comment
Some people consider that studying at home is the best for children's development. There are several reasons why this method seems to be helpful. Firstly, children learning at home provide them comfort in the knowledge that they are with their parents. Moreover, they can concentrate more on their studies because the ambience of being at home provides them ease while learning. Secondly, children are more flexible with the time they spent studying. For example, they may have their breaks anytime when they feel the need to. Lastly, parents can attend to focus more on their child's weaknesses where it can lead to more productive and better academics.
However, other individuals insist that attending schools is still essential. Educational institutions, for example, offers students shared knowledge and activities. Moreover, schools may improve children's interpersonal skills. For instance, children are given responsibilities and tasks where they should attend and deliver the activities together with other pupils. As a result, children are more active in their social activities. Having this said, it can also be a factor to reduce the number of depression cases around the globe.
In conclusion, it is true that education plays a vital role in our society. In my own honest opinion, both methods have their own merits and can actually coexist in the way that parents should be encouraged to have their children to attend schools and studying at home would be a supplement if parents need the feel of having their children to develop more and lead them to good and better academics.


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Billings Montana Police Patch
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- Item Category: Fraternal, Political, Organizations
- Source: eBay
- Sold Date: Feb 20, 2011
- Channel: Auction House
Billings Montana Police PatchThis patch is in used condition; size is 3 ¾ inches long by 2 ¾ inches wide, year unknown; Cheese cloth backing. Shipping Information
Shipping: $1.00 by first class mail Combined Shipping Discount: Shipping discount given on multiple items purchased! If more than one item is won PLEASE wait for seller to update your invoice for all shipping discounts. All items must be paid for within 10 days Feedback: Feedback is left ONLY after it has been left for me. This is only done because some buyers frequently forget to leave it after they receive their item(s). All patches are sold as collectibles or for display purposes only! Please feel free to send an ebay Message with any questions &


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Know the market value of your used two-wheeler
Find out how much your bike or scooter is worth for.
KMs Run
Bikes ›› Ducati ›› Diavel ›› XDiavel S
XDiavel S from Ducati is said to be the union of two worlds; that of a cruiser for long journeys and short distance relaxing rides. It is said to be one of the most sophisticated motorcycles of new generation, which comprises of Italian style, high performance and technical intelligence. The S version of the new XDiavel uses top level technology to bring out most unique and unmatched design to life.
The XDiavel S is built around the Ducati's iconic Testastretta DVT 1262 engine. DVT stands for Desmodromic Variable Timing. This system helps in supply of incredibly full and at the same time fluid torque at low running speeds and guarantees sports oriented performance, when throttle is fully opened. This engine is Euro-4 approved and it is completely new. This twin cylinder liquid cooled engine is capable of producing 156bhp power @ 9500 rpm and a maximum torque of 128.9Nm @ 5000 rpm. XDiavel features three different riding modes such as, Ride-by-Wire (RbW), Ducati Traction Control (DTC) and ABS Cornering Bosch Systems.
Xdiavel may be the first cruiser bike to use a belt driven system for transmission. It is the most reliable and convenient way of power transmission. Low noise level, low maintenance and easy cleaning are the advantage of belt driven motorcycles from chain driven. The braking force of XDiavel relies on the twin 320mm semi floating discs with radial Brembo monobloc 4-piston M50 calipers at the front and 265mm disc and 2-piston floating calipers at the rear. A 50mm adjustable USD fork at the front adjustable preload single shock absorber at the rear comprises suspension department.
Kerb weight of this bike is 247 kg and wheelbase is 1615 mm. Mileage of the vehicle is 18.3 kmpl and fuel tank capacity is 18 litres. Triumph Thruxton R and Harley Davidson 1200 custom are the alternatives for this beast.
|Engine Displacement
|1262.00 cc
|Engine Type
|Ducati Testastretta DVT (Desmodromic Variable Timing), L-Twin, 4 Desmodromically actuated valves per cylinder, Dual spark, Liquid cooled
|Bore x Stroke
|106mm x 71.5mm
|Compression Ratio
|Slipper and self-servo wet multiplate clutch with hydraulic control
|Maximum Power
|156 hp @ 9500 rpm
|Maximum Torque
|128.9 Nm @ 5000 rpm
|6 speed
|Front Brake
|2 x 320 mm (12,60 in) semifloating discs, Radial Brembo monobloc 4-piston M50 callipers and radial m
|Rear Brake
|265 mm (10.43 in) disc, 2-piston floating calliper, Bosch cornering ABS as standard equipment
|Chassis and Suspension
|Tubular steel Trellis frame
|Front Suspension
|Adjustable 50 mm (1.97 in) usd fork with DLC treatment
|Rear Suspension
|Single shock absorber, Adjustable preload and rebound, Remote reservoir, Single sided cast/trellis frame swingarm, natural anodized and brushed swingarm
|Wheel Type
|Front Tyre
|Pirelli Diablo Rosso II, 120/70 ZR17
|Rear Tyre
|Pirelli Diablo Rosso II 240/45 ZR17
|Length x Width x Height
|2310 x 1010 x 1133 mm
|1615 mm
|220 kg
|Petrol Tank Capacity
|18 litres
Know the market value of your used two-wheeler
Find out how much your bike or scooter is worth for.


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Jimmy Martin
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James Henry Martin, 10 August 1927, on a farm near Sneedville, Tennessee, USA, d. 14 May 2005, Nashville, Tennessee, USA. Martin learned to play the guitar as a boy and first appeared on radio in Morristown in 1948. He joined Bill Monroe in 1949 and remained with him (except for a short break) until 1954. Many rate Martin to be the finest lead singer and guitarist ever to work with Monroe. He played on some of Monroes best recordings and sang notable duets with him, including Memories Of Mother And Dad. In the mid-50s, he worked with the Osborne Brothers, with whom he recorded 20-20 Vision, before forming his own Sunny Mountain Boys. Martin went on to become a legend of bluegrass music, playing the WJR Detroit Barn Dance, The Louisiana Hayride and all major venues. His band contained some great bluegrass musicians, including J.D. Crowe, Doyle Lawson and Alan Munde. He recorded for Decca Records from the late 50s to the mid-70s and...
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text: Remote Control Interior Car Heater November 13, 2012 By Outblush What would we love to have in our cars now that temperatures are plummeting? A remote starter. What's really expensive and difficult to install? A remote starter. This Remote Control Interior Car Heater ($150), on the other hand, is a breeze to set up, and costs a fraction of the alternative. We can even set its timer to start exactly when we want it to, so our morning-blurred brains won't even have to remember to press the button.


text: 12 February 2001 Ra, ha, ha Television - Andrew Billen enjoys Love in a Cold Climate's anthem for doomed glamour Sign up for our weekly email * On Omnibus (31 January, BBC1), Deborah Moggach pointed out that two things count against Nancy Mitford: first, she is posh and, second, she is funny, "and we have an equivocal attitude to humour". I think she is right - certainly about our resistance to what another contributor called the "poshocracy" - and it is a tribute to her adaptation of Mitford's Love in a Cold Climate and The Pursuit of Love that even the fiercest class warrior will be moved as well as amused by this story of overindulged lives lived in the vernacular of flippancy. To the chorus of three actresses identified simply as "the Twitterers", babies miscarry, marriages are ruined, sons go off to war and hearts shatter. Mind you, Love in a Cold Climate (Sunday, BBC1) is still very funny. The head of the Alconleigh family, the xenophobic Uncle Matthew, has the best lines, simply because he does not know he is being funny. On Omnibus, John Julius Norwich almost wept with laughter as he read out Uncle M's denunciation of the nurse in Romeo and Juliet ("Bet she was Roman Catholic, dismal old bitch") and in the dramatisation, Alan Bates gave this speech welly. But his children themselves get superb throwaways of the "Nanny, where do you keep that powder you used to dust our bottoms with?" variety. As they grow into women, the humour becomes more sulphurous. Our central heroine, Linda, is not pleased by her infant daughter. "Isn't it horrid? Like a howling orange in a black wig," she tells her visitors. One of Nancy Mitford's two surviving sisters, the Duchess of Devonshire, mourned on Omnibus that Nancy is now known more for her silly essay on U and non-U speech than for her fiction, which is taken to be an extension of the journalistic noblesse oblige. But the documentary made it clear that her novels were actually a form of jumbled autobiography, with Linda standing in for Nancy and Uncle Matthew for her father, Lord Redesdale. The dilemma of how unsophisticated, undereducated but intelligent young women could find purpose in Thirties England was, in fact, played out on an even more bizarre scale in real life. Nancy's sister Unity fell in love with Hitler; Diana married Oswald Mosley; and the baby of the family, Deborah, nabbed the Duke of Devonshire and inherited Chatsworth. Make it up, you could not. Perhaps it was because the story was so underpinned in biography and because the production was shot at the Mitfords' home, Batsford Park in the Cotswolds, that Moggach and her producer, Kate Harwood, opted against telling the story through high camp. Although their adaptation begins with an exaggeration - Lord Redesdale actually pursued his daughters through his grounds with a single hound rather than a pack - they achieve a remarkable degree of credibility, helped by the unflashy camerawork of the director, Tom Hooper, who only begins to let his hair down visually in the concluding instalment. The affecting young actresses who play the leads, Linda, Fanny and Polly (Elisabeth Dermot Walsh, Rosamund Pike and Megan Dodds), look older than their years and, if beautiful, then only in the most unfashionable way. At one point, Polly's ghastly mother, Lady Montdore, complains about this new quality of "sex appeal" that girls are required to have. It is a problem. In comparison with, say, The Camomile Lawn or the recent Channel 4 adaptation of Sword of Honour, which also featured Dodds, this is almost dangerously unsexy television. But libido's loss is credibility's gain and, with similar good taste, the accents are U but not Drones Club U. It is all frightfully well judged. I mean, compare Alan Bates's Uncle Matthew to Peter O'Toole's appallingly over-the-top Lord Emsworth for the BBC a few years ago. O'Toole was all affectation: Bates provides an almost interior rendering. He is a faulty boiler house of trapped emotion. Listening to an aria, his face twitches with a kind of spiritual indigestion. He is superb, but his co-stars are almost his equals: Celia Imrie as his wife, a portrait of agonised passivity; Sheila Gish, as Lady Montdore, showing all the vulnerability of a rusting battleaxe; John Wood, as Linda's worldly confidant Lord Merlin, proof that an overdeveloped intellect can lead you as far into eccentricity as anything else. My only reservation, having watched both parts, is that they fool you into thinking you are watching something more substantial than it actually is. As far as a moral can be drawn, it is that love rarely presents itself appropriately, that it cannot be guaranteed, even between parent and child, and, as such, that there is no fool like a fool in love. As Diana Mosley said of Nancy: "The only thing that was wrong in her life were the men in it. They were hopeless." Towards the end, Moggach's script emphasises that this is also a story of a doomed and forgotten class - a cliche that, unlike the "storm clouds gathering over Europe", she leaves unridiculed. But English writers have never forgotten the stately Hons of England, who, even today, enjoy a lingering and undeserved half-life in English literary productions. Off with their heads, I say. But this production was still utter, upper bliss. Andrew Billen is a staff writer on the London Evening Standard Andrew Billen has worked as a celebrity interviewer for, successively, The Observer, the Evening Standard and, currently The Times. For his columns, he was awarded reviewer of the year in 2006 Press Gazette Magazine Awards. Subscribe from just £1 per issue This article first appeared in the 12 February 2001 issue of the New Statesman, Exclusive: how Labour could lose More


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The Great Recession made us poorer.
It may also have made us smarter. And more likely to live longer.
According to the U.S. Census Bureau, the number of people in the U.S. with doctoral degrees jumped 40 percent between 2008 and 2013, while the number with master's degrees rose 18 percent and the number with associates degrees rose 33 percent – rates of growth faster than in the prior four year period.
A press release accompanying the latest data on educational attainment also noted that the number of people with some graduate education had increased 24 percent.
Among economists and social scientists who study recessions, that result is expected. One rational response to a poor job market or a layoff is to stay in school (or go back to it) and use the time to develop skills that are in demand.
Recent research by Andrew Barr and Sarah E. Turner at the University of Virginia, published in an edition of the Annals of the American Academy of Political and Social Science, noted that a quick upturn in college enrollment coincided with the financial crisis and economic downturn.
"Even as demand for many goods and services tends to decline during a recession, demand for postsecondary education tends to increase," the two wrote.
Judging from the Census data, that appears to be paying off in degrees awarded.
One other possible upside of the downturn is what other researchers expect will show up as a decline in overall mortality during the recession years. Data haven't confirmed it yet, wrote the University of Michigan's Sarah A. Burgard and two others.
But past recessions have coincided with a decline in overall death rates, a trend broadly attributed to things like fewer traffic accidents (not as many people are rushing to work; and if they drink they are more likely to stay home rather than go to a bar and drive home intoxicated). There is more time for exercise, and improved nutrition as families economize on food.
That, of course, is offset by the strains a recession puts on individuals and families – suicide rates tend to climb, the stress of long-term unemployment is unhealthy, and that giant Cheesecake Factory plate of pasta may actually be swapped for McDonalds rather than a balanced home-cooked meal.
But Burgard said that the expectation is that, as the data are more closely analyzed, "we expect some reduction in overall mortality" during the recession.


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We are 4 partners from the U.S. and Mexico who founded a production company in Buenos Aires, Argentina. What started out as a hobby has turned into a (fairly) serious business. For the past 2 years we've been photographing models from Argentina and around South America. We're now producing videos for DVDs, the iPod, and the Internet. We've recently launched a new project called 'mariposaHD' which is the world's first original HDTV show for the Internet.
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Hi there! My name is Zoe Allamby and I am a 23 year-old Model based in Miami.
I left my beloved island of Barbados in 2007 to further my education and pursue a career in the fashion industry. While enrolled at the Miami International University of Art and Design, pursuing my Bachelor of Arts Degree in Fashion Merchandising,I juggled modeling, being a full-time student and a part-time sales associate. Yes, I was always tired.
Along the way there was something I learnt about hard work- It paid off. I was blessed with amazing some amazing modeling opportunities and able to book shows for Rachel Roy, Diane Von Furstenberg, Nanette Lepore, Missoni, Alberta Ferreti and Saks Fifth Avenue.
Everything I had learnt from make up artists or even my own personal research, I wished to share with young women… and that's where CaribBeauty came into play. CaribBeauty is my beauty and fashion channel on Youtube that I use to connect with girls around the world. Whether it was after class or a show, I found unpretentious joy in coming home and filming make up tutorials (even though I made so many mistakes!). To know that I inspired at least one girl, was the best feeling.
I've graduated, interned worked in corporate and now it's back to full-time modeling.
My mission is to teach and encourage young women to look and feel their best by methods of beauty and style, whilst encompassing the essence of my Barbadian culture.
Carib Chic is my new hub to document my journey, hope you enjoy the ride.
Love Always,


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Posted on 12 November 2004
- 12:00
Robbie Keane is calling for a repeat of what he described as the most 'electrifying atmosphere' he has experienced when Arsenal return to the Lane for the Derby on Saturday.
Their last visit at the back end of April saw the team claw back a two-goal deficit, the leveller coming through Robbie's dramatic last-minute penalty. The afternoon was just as memorable for the sizzling atmosphere in the stadium before, during and after a game the striker declared was one of the most memorable of his career.
"On a professional level for me, for the fans and everyone associated with the club, the end of that game will be up there due to the importance of the game - even though they won the league that day.
"The performance from the lads was excellent and to score the equaliser in the last minute with a penalty was very special.
"I was asked recently what the most electrifying atmosphere I have known in a stadium was and the Arsenal one came straight into my head.
"It was electric and the crowd was absolutely fantastic. When you are out there it really helps when the crowd are cheering and singing and it does become a 12 man. Last year we definitely had that and we hope Saturday will be the same."
Meanwhile, Robbie offered his seal of approval for the appointment of Martin Jol as Head Coach.
"He is good to work with, he is a straightforward guy who is honest and will tell you how he feels. For a player that is good, you want to hear things and will want to know if you are left out or whatever - he will come and speak to you.
"He is someone who is respected by all the lads, not only now he's the manager but from when he first came. His training sessions have been good and he always goes out of his way to speak to you."
15 May 2017
All the stats, facts and figures you need to know about our competitive matches during our 118-year stay at White Hart Lane.
23 March 2017
Spurs Ladies will play at White Hart Lane for the first time on Wednesday, April 19.
27 February 2017
Take an in-depth look into the Club's history and ethos and go behind the scenes of our new stadium with NBC's Men In Blazers' host Roger Bennett as he interviews the likes of Chairman Daniel Levy, Manager Mauricio Pochettino, Head of Coaching and Player Development John McDermott and Captain Hugo Lloris.
View mobile site
© Tottenham Hotspur Ltd 2017. All rights reserved.
All photography copyright Getty Images (unless otherwise stated)


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Rosie O'Donnell will never wed again
Rosie O'Donnell will never get married again.
The 54-year-old comedienne, who has walked down the aisle twice before, is adamant she'll never tie the knot again after her divorce from Michelle Rounds was finalised at the Manhattan Supreme Court in New York yesterday (31.03.16).
Speaking to reporters as she left the matrimonial court, according to the New York Post newspaper, she declared: "She'll never marry again. You can print that!"
Rosie married Michelle, 44, in 2012 but their romance was short-lived as they split just two years later in 2014.
The brunette beauty filed for divorce from the headhunter shortly after they announced their split, but they have spent the last 15 months fighting over money and how they would raise their three-year-old adopted daughter, Dakota.
And this wasn't the first split Rosie has been through as she also went through a similar ordeal when she called time on her relationship with Kelli Carpenter-O'Donnell in 2007.
The couple, who have children Chelsea, Blake and Vivienne together, got married in 2004 after just a short time of dating, but their union was annulled later that year when the California courts ruled gay marriages were illegal.
The pair stayed together but separated three years later.
Rosie also has now-grown-up son Parker, who she took in as an infant in 1995.


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Bay a wedding hot spot
The economy may be flat-lining but the bridal business is booming in the Far North.
Shane and Pip Lloydd's Copthorne Hotel and Resort Hokianga has hosted 20 weddings since the start of September and more are booked until July.
"The market is definitely increasing and demand has grown," Mrs Lloydd says.
Most couples holding their ceremonies and receptions at the resort are from Auckland or overseas with 70 to 140 guests.
"They love the location because it's beachfront and has a tropical feel to it. They often liken it to Fiji."
Revenue from weddings is a big part of the Copthorne's income.
"They basically book up the resort."
Tour operators and motels in Omapere also benefit when the resort hosts weddings because it only has 33 rooms and guests usually stay for three days.
"A lot of guests hire fishing charters or take forest or boat tours. It becomes a community thing."
The Duke of Marlborough Hotel in Russell will have hosted 16 weddings by the end of May – compared with two last season – thanks to extensive renovations and targeted marketing.
Bridget and Anton Haagh and Jayne Shirley and Riki Kinnaird have refurbished the hotel's function room and kitchen and upgraded waterfront rooms to five star quality since they bought it last year.
Hosting nuptials at the historic hotel has boosted business and brought visitors to the Bay of Islands, Mrs Haagh says.
"Weddings range from 60 to 180 people and most have been out-of-towners."
Marketing the Duke as a wedding venue is a big part of the hotel's business strategy.
"We're going to the Grand Wedding Show and Bride and Groom Show in Auckland in April and May," Mrs Haagh says.
Kerikeri marriage celebrant Heather Townsend has helped more than 20 couples, including four of the same sex, tie the knot since the start of September.
She says the last spring and summer has been an extremely busy wedding season.
"I'm usually quite busy but these are more than I would normally do," she says. "I still have four weddings to go."
Beaches have been the most popular locations for non-religious wedding ceremonies.
Other popular venues include the Waitangi Treaty Grounds, Eagles Nest in Russell and The Pear Tree, Wharepuke and Kingston House in Kerikeri.
Pakaraka wedding planner Debra Shand says a growing number of upmarket but relatively affordable venues are capitalising on the Bay's closeness to Auckland.
''Some venues have realised this is a good market and they're making efforts to refurbish and make their premises more appealing,'' she says.
Two weddings she is organising for 75 and 85 Australians reflect the bay's popularity as a venue across the Tasman.
''Some in Sydney or Melbourne are exceptionally expensive and they have to book years in advance, so in some ways it's cheaper for them to come here.''
Ms Shand would like tourism agencies to promote the Bay of Islands as a wedding destination, especially in Asia.
''There are some beautiful places to marry here and it's relatively unspoilt.
''I think it's just a matter of marketing that.''
Northern News


text:
The budget deficit doubled in June and reached 0.5 percent of the GDP at the end of the first half, as revenues went up by 2.9 percent in nominal terms on a year-on-year basis and expenses were cut by 0.3 percent, according to the Ministry of Finance data.
After five months, the budget revenues went up by 3.7 per cent whereas the expenses were 2.4 percent smaller. At the end of June, the revenues totaled RON 100.4 billion (15.2 percent of the GDP) and the budget expenses reached RON 103.9 billion (15.7 percent of the GDP), leading to a deficit of RON 3.5 billion.
Romania committed within the agreements with the international financial institutions that its budget deficit of this year should not exceed 2.2 percent of the GDP.
In terms of revenues there were higher figures in the first half at the social security contributions revenues (RON 1.47 billion), taxes and fees for property (RON 957.8 billion), excise taxes (RON 1.08 billion), tax on profit (RON 689.5 million) and VAT (RON 459.3 million).
There were higher revenues at the local administrations for property taxes and fees (5.2 percent), taxes on usage of goods (3.6 percent) and non-fiscal revenues (2.2 percent).
The amounts reimbursed by the EU in the account of payments made decreased by 10.8 percent on a year-on-year basis. The general consolidated budget reported only the reimbursed amounts for the projects where the beneficiaries are public institutions, without including the private sector.
The amount spent from the budget for goods and services was 5.7 percent higher, as the major increase was reported at the national fund of healthcare, 11.3 percent, following the payment of arrears by the pharma drugs suppliers.
The investments expenses, including the capital expenses as well as the development programs funded from internal and external sources, amounted to RON 9.6 billion, which represents 1.5 percent of the GDP, compared to RON 12.2 billion last year during the same period. The Finance Ministry notes that over the first half of last year the arrears of the National Company of National Roads and Motorways were paid, in amount of roughly RON 2 billion, as well as arrears of the local administrations from loans from the treasury in amount of RON 800 million.
Budget deficit doubled in June
The budget deficit doubled in June and reached 0.5 percent of the GDP at the end of the first half, as revenues went up by 2.9 percent in nominal terms on a year-on-year basis and expenses were cut by 0.3 percent, according to the Ministry of Finance data.


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Mountain Views Villa
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- About Hotel
- Reviews
About Mountain Views Villa
Mountain Views Villa is ideally located in most famous locale of the city of Srinagar, near the Nishat Mughal Gardens across the famous Dal Lake. Food served on order with special Kashmiri dishes available. Laundry service facility available with hair dryers in each room. Enjoy breakfast in the garden or go out for a stroll along the stream running a few miles away from the Villa. Peaceful accommodation in Kashmir with luxurious bedrooms covered with beautiful wooden ceiling and filled with antiques. Each room equipped with heating facility, LCD, WiFi internet, direct telephone line, attached bathrooms, 24/7 room service. The Mountain View Villa is conveniently located near all major tourist attractions of the city like the famous Mughal gardens, Handicrafts centers, Shankaracharya temple and the Dal Lake. While staying at the Villa experience the boat ride in the Dal Lake which is just a short stroll away!
No of floors: 0 | No of rooms: 23
Checkin Time: 1200 pm
Checkout Time: 1200 pm
Hotel Amenities
- Room Service
- Medical Services
- Luggage Storage
- Laundry Facilities
- Front Desk
- H/C Running Water


text: Well now that Omega is out and been played twice over by me, I find myself looking to the future and wondering what other DLC will probably be coming out for ME3. I am assuming another costume skin pack will likely be on the way and possibly another weapons pack as well, but what I'm more interested in is what other playable content will be coming out. Some people mentioned that inside the code for either leviathan or omega there was some kind of reference to the citadel, has anyone else heard this? I would like to ask what people have heard about the future of ME3 dlc, and what, regardless of how remote, is blowing around as a possibility.


text: Viewing Speakers Corner TV - 34 of 62 You Arrogant Bastard - Clodagh | Speakers Corner Clodagh reveals a weird and life changing event that occurred in her life. Some... more »Clodagh reveals a weird and life changing event that occurred in her life. Some people have horrible bosses. Clodagh's had a horrible boss who forced her to work ANOTHER weekend. Well, until..... « less Speakers Corner May 14, 2013 66 Views More Info Full Credits Speakers Corner Uploader Stats & Data 4Funny2Die 66 Views May 14, 2013 Published Category Web Series Real Life Rants Tags Clodagh Boss Bastard Fired Arrogance Family Angry Death Rant Rants Speakers Corner Simplynew Marc Scarpa Up Next: The Sensuality Of Feathers - Jena | Speakers Corner AUTOPLAY Up Next In: 10 The Sensuality Of Feathers - Jena | Speakers Corner Mother's Day 2013 - Yvonne | Speakers Corner Freedom Of The Press - Esther | Speakers Corner America Is Not Ready For A Woman President - Charlie | Speakers Corner Follow Browse Related Comments Speakers Corner TV 60+ more View All ▶ 90+ more View All ▶ Speakers Corner Vaguely Related Videos Vaguely Related Pics & Words Advertisement Tags: Clodagh Boss Bastard Fired Arrogance Advertisement


text:
login with
Haha I was just telling my husband that the grass was starting to look green!
It made me stop in my tracks! I was walking by the back window and it's like I ran into a wall! lol
I see it! I'd forgotten what that looks like...
I know, ours is slowly turning green. The poultry pasture is the greenest besides the new pasture we planted last spring. All that chicken manure!
About Alissa
Current: Chinook, Montana
Birth: September 17
On Moms.com since: Jan 30, 2014
*Site Hostess* I am a first time mom living in North Central Montana. I have a fiance, the most gorgeous baby boy, and a goofball dog named Lexi to fill up my plate :)


text:
Copy A Prevent Copying Memo...
This Article Covers
Administration Tools
Download: IT certifications that stand out
Are you looking to advance your career? Expand your knowledge? Boost your credibility? Our editors put together this complimentary 19-page guide on everything you need to know about obtaining an IT certification—with special attention given to cloud and desktop certifications.
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sender has selected "Prevent copying" in the Delivery options. Just make it
run on selected documents.
Dim session As New NotesSession
Dim db As NotesDatabase
Dim collection As NotesDocumentCollection
Dim doc As NotesDocument
Set db = session.CurrentDatabase
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text:
The European Aviation Safety Agency (EASA) has issued an emergency airworthiness directive (AD) for ATR cockpit windows. AD Number 2009-0159-E requires inspection and, if necessary, replacement of cockpit forward-side windows on ATR models 42-200, 42-300, 42-320, 42-400 and 42-500, and 72-101, 72-102, 72-201, 72-202, 72-211, 72-212 and 72-212A equipped with PPG Aerospace glass windows. The inspection must be completed within 10 days after accumulation of 2,000 total flight cycles on a given cockpit forward-side window, or within 10 days after July 20, 2009, the effective date of the AD, for windows that have accumulated 2,000 or more total flight cycles . The reason for the AD is a recent event in which a left hand, forward side glass window of an ATR 72-212 blew out while the airplane was performing a ground pressure test. The investigation revealed some anomalies on the forward side window at the level of the z-bar on the window's external side and at the level of the inner retainer on the window's internal side considered precursors of the failure. Neither ATR nor PPG Aerospace authorizes repairs.
EASA Issues ATR 42/72 Airworthiness Directive
- July 22, 2009, 10:37 AM


text:
A fantastic two bedroom, end of terrace home located in a quiet cul-de-sac in the highly sought after Quelm Park estate.
The ground floor of the property comprises of entrance hallway with cloakroom and storage cupboard, recently refitted kitchen, spacious lounge/diner and rear conservatory leading to a partially decked rear garden with hot tub, lighting and side access. At the front of the property is a garage with electricity supply and driveway parking.
The first floor of the property comprises of two evenly sized bedrooms, with recently refurbished en-suite to the master bedroom, family bathroom and loft access.
Within convenient distance of local schools, shops and with excellent road and public transport links.
Please see the many photographs that accompany this listing and the indicative floor plan for further details. The best way to appreciate this property is to arrange a viewing appointment which you can do 7 days a week by calling the number above or by clicking on the contact link provided.
Approximate Floor Area 140 sq. ft.(13.0 sq. m)
Garage 16'10" x 8'3"(5.13m x 2.52m)
Ground Floor
Approximate Floor Area 441 sq. ft.(41.0 sq. m)
Living Room/Diner 17'4" x 12'7"(5.28m x 3.84m)
Kitchen 9'10"x 6'7"(3.00m x 2.00m)
Conservatory 10'0" x 9'10"(3.05m x 3.00m)
First Floor
Approximate Floor Area 344 sq. ft.(32.0 sq. m)
Bedroom 12'7" x 8'4"(3.84m x 2.55m)
Bedroom 12'7" x 9'4"(3.84m x 2.84m)
En-suite 6'2" x 6'1"(1.88m x 1.85m)
Bathroom 6'1" x 6'2"(1.85m x 1.87m)
Book a viewing online in minutes. Just click below to choose the days and times
you're able to view the property and you'll receive an email and SMS as soon as the owner confirms one of your options.
Get started by requesting your free valuation from today. With our fees at just £795, you can save thousands selling with the UK's best rated online estate agent.Get Your Free Valuation


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Royal Doulton 'Heroes of the Sky' plates. All boxed with certificates. Eleven in total. Will separate for £10 each. Bromley area.
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text:
My guy mentioned that we girls criticize each other more than guys about appearance. Most of the time, this is true. Why do we do this and more importantly, why does our culture judge us so much harsher than guys when it comes to appearance? Is it tradition? Expectations? Is it learned? Is it biological? Any opinions will be appreciated. Thanks!
Most Helpful Girl
Girls are very competitive, generally.00|00|0
Yes. We are. In some ways more than men over the "long run."


text:
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text:
So lol there's this "hot" girl I've been attracted to for a year and now she was showing heavy interist but I was too shy. She came back and found out we live in the same building and wants to catch up. I don't trust her but may learn too. Then there is a close friend of mine who I have known for 6 years and she has been through a lot with me and we are close. She broke with her bf and I was closer to her than him and I am not as physically attracted to her but I love her personality and truly trust her. There's a better shot with the girl I don't trust cause she is giving me hints.
Most Helpful Girl
Just go for the hot girl sounds like your kinda shallow and would be uninterested in the nice girl after a short while00|00|0
Well I just started thinking about the nice girl and considering it cause I dont want to be hurt.
- Show AllShow Less
Ok, so I read what you wrote in the other comments and I wanted to say that's really nice that you were there for your friend of 6 years when she had relationship problems I didn't see those details before. That's really kind of you, maybe more will come of it, either way good luck!
Ya I don't trust people easily and Ill admit I this other girls sketchy cause she magically came back into my life and even brought a basketball from home. Like I don't even remember talking to her about how I played basketball in HS. I don't know I asked her to hangout and she said she was busy. Then stopped texting me back when I asked her when she was free. Then she asks questions and tried to get to know me lol.


text:
Iron Man Officially Joins Marvel's Avengers Line-Up!
by Alex Billington
October 28, 2008
It's now official. Tony Stark aka Iron Man has officially joined The Avengers. Marvel has announced today (the full press release can be found on SuperHeroHype) that Robert Downey Jr. will appear in both Iron Man 2 and The Avengers as Tony Stark, with Jon Favreau set to direct Iron Man 2 and executive produce The Avengers. Additionally, Marvel confirms the announcement made a few weeks ago surrounding Don Cheadle replacing Terrence Howard as Col. James "Rhodey" Rhodes. Cheadle is officially signed on to appear in Iron Man 2 and other Iron Man sequels (and presumably spin-offs) as well as The Avengers. Additionally, Marvel confirms that the full line-up will include: Iron Man, Captain America, Thor, the Hulk and more. Sounds like every comic book geek's wildest dreams are finally coming true!
It's happening. It has begun. After years of waiting, we're finally going to get the ultimate crossover movie that we've all been dreaming of! Unfortunately it doesn't look like Jon Favreau will be directing this, as initially rumored. Nor will Hulk be one of the villains, as also initially rumored. Those confirmations aside, I don't think I'm the only one who is already incredibly excited to see this come together. This is only the first of many exciting announcements to come. And to give us a taste of things to come, Marvel leaves us with this. "In a movie event, The Avengers will bring together the super hero team of Marvel Comics characters for the first time ever, including Iron Man, Captain America, Thor, the Hulk and more, as they are forced to band together to battle the biggest foe they've ever faced." Who could that be?
Follow Alex's main profile on :


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pygmy squirrel
Definition of pygmy squirrel
: any of numerous minute African and Asiatic tree squirrels (as of the genus Myosciurus)
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