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Now through February 5th, 2013, head on over to the WomanFreebies.com Facebook page, "like" them, and click on the "Maybelline app" tab to possibly win one of 100 FREE bottles of Maybelline Color Show nail polish. Good luck! :)
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December 1, 2015
PALISADE, Colo - The Bulldogs played host to the 2009 Palisade Invitational wrestling tournament on Saturday. The Central Warriors took first place with 168.5 points, followed by Palisade (128.5), Cortez (114) and Fruita Monument (109).
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At first,I was just like everyone else aboard this ship
Who still had the desire to take this outer space trip
And see what see what the vast cosmos has to offer
Like a new world that has life under the water.
But that was before I had bitten my bottom lip
And stared out at one of the windows of the worlds and
Realized that leaving Earth may not be such a grand
Idea after all because there still is life back on our
Homeworld and the sorrow alone waits for the one hour
On which it would force me to abandon our hearty little band
Of deep space explorers and make the return trip to Earth.
As a matter of fact,I still want to return to the planet of my birth
Each and every time I stare at my own reflection on a computer
Screen and wish that our ship's commanding officer
Would let us make the trip back to where we belong...Earth.
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- Eats
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Everyone has a unique sense of style, so there can really be no "mistakes" when it comes to your home's interior design, right? Wrong. Whether it's ignoring classic proportions or failing to select cohesive colors, design blunders are a common...


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Part 1 of 5.
In which a werewolf, a vampire, and an invisible man come together and then split off in the pursuit of happiness.
Produced by Minty Pineapple Entertainments and Tasteful Tuna.
Starring: Josh Spurgin as Seth, Joe Heath as Bob and the Announcers, Vincent E.L. as Nick, Jillanna Babb-Cheshul as Bethany, Brad Heath as Death and the Zombie, Ray Friesen as Gary, Jez Layman as Random Person, Daniel McCulloch as Kid #3, Lizzie McCulloch as Kid #1, Samuel McCulloch as Kid #2, Rachel Erin Scott as Kid #4, Jamie Spurgin as Deadpan Actor, Lewis Lovhaug ( http://atopfourthwall.blogspot.com/ ) as The Host, Dennie Eugene Thompson III as Roger, Hope Heath as Mall Woman, Helen Hamilton as Opening Credits Victim, and Joshua Wooten as Additional Voice in Crowd.
Death Shemp: Joe Heath.
Peeing into toilet by Twitterdj. http://www.freesound.org/
Mon Anon, Cool Science and Lizzy Winters logo by Ray Friesen. http://www.DontEatAnyBugs.com
Camera Operators: Joe Heath, Brad Heath, Hope Heath, Josh Spurgin, and Bruce Scott.
Mic Operators: Joe Heath, Brad Heath, Hope Heath, and Josh Spurgin.
Executive Producer: Joe Heath.
Producer: Brad Heath.
Opening Theme by Vincent E.L.
Closing Theme by Joe Heath and Brad Heath.
Additional music by Vincent E.L.
Written and Edited by Joe Heath and Vincent E.L.
Directed by Joe Heath.
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Macklemore - Thrift Shop
Hey, Macklemore! Can we go thrift shopping?
What, what, what, what... [x7]
Bada, badada, badada, bada... [x9]
I'm gonna pop some tags
Only got twenty dollars in my pocket
I - I - I'm hunting, looking for a come-up
The rest of this lyric is blocked due to country restrictions.
Wat andere gebruikers ook bekeken
|Macklemore - Same Love
|Macklemore - can't hold us
|Lorde - Royals
|Miley Cyrus - Wrecking ball
|Macklemore - White walls


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Does anyone know the approximate cab fare from Orange County airport to the hotel? With or without tip? I'm thinking it might be the same (or cheaper ?) for the 3 of us and more convenient than taking one of the recommended shuttles.
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Exploitation of DNA-based analyses of microbial pathogens, and especially simultaneous typing of several virulence-related genes in bacteria is becoming an important objective of public health these days.
A procedure for sample processing for a confirmative analysis of enterohemorrhagic Escherichia coli (EHEC) on a single colony with DNA chip array was developed and is reported here. The protocol includes application of fragmented genomic DNA from ultrasonicated colonies. The sample processing comprises first 2.5 min of ultrasonic treatment, DNA extraction (2×), and afterwards additional 5 min ultrasonication. Thus, the total sample preparation time for a confirmative analysis of EHEC is nearly 10 min. Additionally, bioinformatic revisions were performed in order to design PCR primers and array probes specific to most conservative regions of the EHEC-associated genes. Six strains with distinct pathogenic properties were selected for this study. At last, the EHEC chip array for a parallel and simultaneous detection of genes etpC-stx1-stx2-eae was designed and examined. This should permit to sense all currently accessible variants of the selected sequences in EHEC types and subtypes.
In order to implement the DNA chip array-based analysis for direct EHEC detection the sample processing was established in course of this work. However, this sample preparation mode may also be applied to other types of EHEC DNA-based sensing systems.
Enterohemorrhagic Escherichia coli (EHEC) strains comprise a subset of Shiga toxin (Verocytotoxin) – producing E. coli associated with serious endemic outbreaks [1-3]. They cause food-borne infections and severe, potentially fatal illnesses in humans especially among children, such as haemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS) [4-6]. The infections with EHEC are often sporadic but they can also give rise to epidemics of great extent. EHEC strains that cause human infections belong to a large number of O:H serotypes. Actually, a total of 472 serotypes recovered from human infections are listed in http://www.lugo.usc.es/ecoli/index.html webcite, including more than 100 serotypes from patients with HUS [7]. Certain EHEC strains belonging to serotypes O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 have been more frequently isolated from humans with severe illnesses [8,9]. Among them, most outbreaks of HC and HUS have been attributed to strains of the enterohemorrhagic serotype O157:H7 [7]. EHEC strains of the O157:H7 serotype are the most important EHEC pathogens in North America, the United Kingdom and Japan but several other serotypes can also cause disease and are more prominent than O157:H7 in many regions in the world such as Europe, Australia, Canada, South America [10,11]. The infection source is difficult to trace because the EHEC cells are hidden among the ubiquitous non-pathogenic E. coli. A standard method (ISO 16654:2001) for EHEC determination is based on a confirmative analysis of the presence of the O157 antigen after a primary enrichment culture [12]. The whole procedure takes about 4 days. However, there is a low degree of correlation between the O157 presence and pathogenicity [13,14]. It was reported in the literature that many other serogroups than O157 are associated with the diseases [9,13,15,16]. There are at least two genes coding for two Shiga-toxins in E. coli (stx1 and stx2) [3,4,17]. Furthermore, the intimin protein, encoded by the gene eae, is assumed to be essential for the virulence since it accounts for the attachment of the cell to epithelial cells [18-20]. In general, the use of DNA-based analyses for identification of EHEC, rather than traditional classification in species or serological strains, offers a great advantage in the assessment of health hazards [14,21]. Here, we report on development of a method for sample processing for alternative confirmative analysis of EHEC colonies from primary enrichment cultures with the use of electric DNA chip array. The EHEC chip array for a parallel and simultaneous detection of genes etpC-stx1-stx2-eae was designed and examined. It is believed that for the assessment of E. coli pathogenicity, a DNA chip array with the capacity to detect the presence of the etpC gene, the two stx genes and the eae gene should be more efficient and rapid than the ISO method.
Cell number count of colony
The E. coli strains, EDL933, CB571, 86–24, and DH5α were cultured on agar plates at 37°C for colony formation. The average diameter of the colonies was 2 ± 0.5 mm. The cell numbers in these colonies were determined by flow cytometry and evaluated against data of viable cell counting on agar plates (cfu). Both methods showed comparable values of 5 × 107 - 1 × 108 cells per colony.
EHEC DNA preparation for chip array analysis
To evaluate the cell disruption during ultrasonication, samples containing 1 × 108 cells (corresponding to one agar colony) were subjected to ultrasonic disintegration followed by flow cytometry analysis (Fig. 1). The forward scatter profiles obtained for each sample are shown. At first, one broad peak with a strong signal representing non-disrupted cells was visible. With increasing ultrasonication time, this signal progressively became weaker and most of the main peak corresponding to the undisrupted cells disappeared after 150 sec sonication. Thus, the 2.5-minute sonicated sample was selected for further handling.
Figure 1. Kinetics of cell disruption by ultrasonication as shown by a histogram of forward scatter values from E. coli EDL933 cells subjected to 0 – 300 sec ultrasonication.
In the next test, the samples sonicated previously for 2.5-min were subjected to an extraction step after heat treatment and centrifugation (see Methods). DNA released was extracted two times with a phenol:chloroform:isoamyl alcohol mix, and afterwards subjected to second sonication for 0 – 15 min. Agarose gel electrophoresis was used to determine the size distribution of DNA subjected to post-extraction ultrasonic fragmentation (Fig. 2). Highly fragmented DNA was evident from the presence of a DNA smear rather than high-molecular weight bands that were eliminated from samples sonicated for 2.5 min or longer. Longer sonication gradually reduced fragment lengths to approximately 150 – 600 bp, and sonication for 15 min further degraded these fragments, as can be seen mostly by the upper part of the smear. Thus, the average DNA fragment size gradually declined with ultrasonication time and the 5 min treatment allowed to obtain the sizes of DNA fragments most suitable for chip array assays [as proved previously by 22,23]. At last, the DNA analyte preparation procedure comprising first 2 min of ultrasonic treatment, DNA extraction (2×), and subsequent 5 min sonication, was established.
Figure 2. Electrophoretic analyses of distribution of genomic DNA of E. coli EDL933 subjected to 0 – 15 min ultrasonication. L indicates the DNA Ladder.
Bioinformatics study
The bioinformatics-based design was managed with all sequences of target genes (stx1: 74, stx2: 178, eae: 18 and etpC: 7) available in GenBank. The cases were subjected to a BLAST analysis against the totality of the sequences composing the database in GenBank (revised September 2007), in order to check their specificity for target genes with a threshold of e-value of 0.004. Data from literature were used as indicators for the conserved regions and were evaluated during the complete study. The PCR primers and array probes were determined by the multiple alignments of whole sequences of each virulence genes. All selected oligonucleotides are presented in details in Table 1.
Table 1. Oligonucleotide primers and probes used in this study.
Identification of virulence-related gene sequences by PCR
Genomic DNAs from all E. coli strains included in this study, EHEC and non-EHEC, were applied for PCR amplification of four virulence-related sequences. Fragments with expected sizes being amplicons of the etpC, stx1, stx2 and eae genes were obtained from the EDL933 and CB571 (Fig. 3A). When the E. coli 86–24 DNA was used as a template in the PCR, predicted fragments of etpC and eae genes were amplified (Fig. 3A). Additionally, the DH5α was found to be positive for stx2. However, some parasitic bands for the amplification of etpC and stx2 sequences were observed. In order to improve the hybridization of primers and the work of the Taq polymerase, a longer time for the annealing at slightly higher temperature, and prolonged elongation was experienced. In a consequence, 2-min at 57°C, instead of 1-min at 56°C primer annealing, as well as 3- instead of 2-min elongation allowed to appreciate the optimization's effects on the PCR program with the vision of clean bands (Fig. 3B).
Figure 3. Electrophoretic analyses of PCR amplicons: etpC (219 pb); stx1 (302 bp); stx2 (519 bp); and eae (346 bp). A. Genomic DNAs from E. coli strains: (1) EDL933, (2) CB571, (3) 86–24, (4) DH5α, (5) MG1655, and (6) W3110, were applied as the PCR templates, respectively. Reaction conditions: 1-minute primer annealing at 57°C, and 2-minute elongation step. B. Genomic DNAs from E. coli strains: (1) EDL933, (2) CB571, (3) 86–24, (4) DH5α, were applied as the PCR templates, respectively. Reaction conditions: 2-minute primer annealing at 56°C, and 3-minute elongation step. NC is a blank PCR assay. L stands for DNA Ladder.
In addition, amplification reaction conditions were determined to elaborate a multiplex PCR test allowing a quick typing of EHEC (Fig. 4). An annealing temperature gradient from 55 to 65°C was realized (Fig. 4A). No parasitic bands were seen with the increase of the annealing temperature. This analysis revealed that the 56°C as annealing temperature in the PCR assay was appropriate, so in consequence used to test all strains with the multiplex mix (Fig. 4B).
Figure 4. Electrophoretic analyses of multiplex PCR amplicons: etpC (219 pb); stx1 (302 bp); stx2 (519 bp); and eae (346 bp). A. Temperature gradient of annealing step in the multiplex PCR using E. coli EDL933 as DNA template. Line 1 presents the anealing temperature of 55°C; line 2: 55.3°C; line 3: 55.8°C; line 4: 56.7°C; line 5: 57.8°C; line 6: 59.3°C; line 7: 61°C; line 8: 62.4°C; line 9: 63.5°C; line 10: 64.3°C; and line 11: 65°C. For all and 3-minute elongation step was performed.B. Genomic DNAs from E. coli strains: (1) EDL933, (2) CB571, (3) 86–24, (4) DH5α, (5) MG1655, and (6) W3110, were applied as the PCR templates, respectively. Reaction conditions: 2-minute primer annealing at 56°C, and 3-minute elongation step. NC is a blank PCR assay. L stands for DNA Ladder.
EHEC chip array design and examination
Capture oligonucleotides, i.e. O157 cp, Stx1 cp, Stx2 cp, and Eae cp (see Table 1) were designed and afterwards immobilized on randomly chosen positions of the chip array. Detection probes, i.e. O157 dp, Stx1 dp, Stx2 dp, and Eae dp (see Table 1) labeled with a biotin at the 3' end were selected to bind adjacent to the capture region of the target. Additionally, four array positions with negative control probe relevant for target sequence of B. subtilis were used for validation of the probes' specificity and assay performance. The EHEC chip arrays were used for hybridization assays, A1, A2, A3 and A4, with corresponding amplicons of etpC, stx1, stx2 and eae, products of simplex PCR. 0.4 nM of each purified PCR amplicon was sequentially applied to the chip test (Fig. 5). Basically, in each particular assay only specific signals were generated from the target corresponding positions. None of the spots resulted in a signal after exposure to the non-relevant PCR amplicon, indicating that no significant unspecific binding occurred. Also, no cross-reactions were observed for the negative control positions.
Figure 5. EHEC DNA chip array assessment. Four individual assays, A1, A2, A3 and A4, were serially conducted with four various products of simplex PCR, etpC, stx1, stx2 and eae, respectively, used as targets, and with corresponding detection probes (10 nM). 0.4 nM of each purified PCR amplicon was sequentially applied onto the chip array functionalized with capture probes. Each column is an average of three independent determinations. NC is non-biotinylated negative control capture probe of acoC of B. subtilis.
Most cases of so-called food poisoning are caused by microorganisms, among them by enterohemorrhagic E. coli. Routine bacteriological techniques take at least several days for detection of these bacteria or even more for their complete characterization. Since there is no correlation between serotype and pathotype, a genotypic determination is therefore necessary for the identification of these pathogenic strains. Thus, the development of EHEC detection and identification methods that cover the pathogenicity pattern is an important issue in the field of clinical diagnosis and food safety. The developments in bioinformatics of EHEC have widened the basis for its identification to include also nucleic acid analysis. As a result, new analytical techniques, monitoring devices and rapid tests have been created [21,24]. Among them, conventional and real-time PCR-based amplification methods itself or chip-based detection systems combined with bench-top PCR prior the analysis, are playing an increasingly important role [21,25,26]. Although, very sensitive, these methods remain limited for in field applications because of their inherent complexity. They are also time-consuming and offen give results problematical for data interpretation. In this respect, the most promising breakthrough in the area of rapid detection turned into platforms for real samples, is endogenous nucleic acid analysis without previous PCR. To date, only a handful of scientists worldwide have reported such analytical procedures, including optical or electrical sensing of nucleic acids isolated from cells [22,27,28]http://www.alderonbiosciences.com webcite. This has a great impact on the development of rapid assays, as first prototypes of fully integrated genetic assays for sample-to-answer nucleic acid analysis already appeared, and were reported for example by Liu and co-workers [29]. The biochip device consisting of microfluidic mixers, pumps, valves, tubes and microarray sensors, allows performance of all functions including sample preparation, mixing steps, chemical reaction and electrical detection. With the use of such fully integrated biochip device the researchers reported functional detection of pathogenic bacteria from blood samples [29]. Moreover, Rudi et al. [30] developed methods of nucleic acid-based microbial sensors that cover both the sample preparation and detection approaches.
A new generation of an automatic electric chip measuring systems for the detection of pathogenic Shiga-toxin producing E. coli was reported lately [21]. In the most recent instrumental version added microfluidics, i.e. a complex network of valves and tubes, permits users to move liquids on and off the chip allowing for performance of multi-step assays. These miniaturized amperometric devices, based on electrical biochips made in silicon-technology, have been constructed for field applications and point of care diagnoses. An advantage of the developed system is the use of a semiconductor technology that avoids any mechanical adjustments of sensing elements, as it is necessary for optical devices. However, in this case artificial analogues such as PCR products were used as targets in the assays for biochip measurements. In the meantime, however, considerable work on the development of a procedure for bacterial sample processing for a confirmative analysis of EHEC, via simultaneous screening of virulence genes, on a single colony with DNA chip array was made. It was understood that this major technical challenge needs to be established in order to implement the technology for direct EHEC detection, and in consequence to reduce the risk of EHEC outbreaks in a rapid manner. The protocol for sample preparation includes application of fragmented genomic DNA from ultrasonicated colonies. Thus, the method requires the bacteria first to be disrupted to make the endogenous DNA available for further processing. As the extent of exposure to ultrasound increased, the formation of fragmented DNA molecules amplified. It is believed that this gives an improved diffusion-driven target movement and results in a sufficient hybridization reaction [22]. Among different methods, physical disruption is preferred, as most chemical agents inhibit the following processes, requiring removal in subsequent additional steps [31]. The use of ultrasound itself to extract DNA from cells is not a new issue, however, in this work, the procedure comprising ultrasonication implemented for the DNA fragmentation was developed and optimized with the intention to prepare samples for DNA hybridization on a chip array.
Currently, a DNA extraction step is also included in the procedure. In total, the sample preparation time for a confirmative analysis of EHEC on a single colony is app. 10 min. At the same time, the EHEC chip arrays for a parallel and simultaneous detection of genes etpC-stx1-stx2-eae were designed and examined. For this, four capture probes, each complementary to the selected target sequence, were immobilized on a 16-position chip array. Detection was realized by a biotin label placed at the 3' end of a detection probe and with the PCR products as targets. The chip arrays responded correctly with significant positive signals of the present PCR amplicons without any false positive signals.
In conclusion, the sample processing for the performance of DNA chip array-based confirmative analysis of enterohemorrhagic Escherichia coli (EHEC) on a single colony was demonstrated. The results obtained in the course of this work will be exploited in further study. The sample processing method will be implemented and tested in utilization of EHEC DNA chip array-based analysis performed directly on bacterial cells. It is also intended to combine the developed sample processing procedure with the chip array-based detection system in order to obtain a fully integrated unit. Less than 30 minutes will be required to perform the whole analysis. To our knowledge, this may offer the most rapid procedure for the genetic identification of EHEC described to date (commercially available GenoType EHEC enables the investigator to obtain the results of the analysis in 3 h). At last, it is worth mentioning that this sample preparation mode might also be applied to other types of EHEC DNA-based sensing systems.
4-Aminophenyl β-D-galactopyranoside (pAPG), bovine serum albumin 30% solution (BSA, protease-free), ethylene-diamine-tetra-acetic acid (EDTA), ethidium bromide solution (10 mg/ml), polyoxyethylensorbitan monolaurate (Tween 20), deoxynucleotide mix (each dNTP 10 mM), Taq DNA polymerase (5 units/μl) and PCR buffers were purchased from Sigma-Aldrich (Steinheim, Germany). Streptavidin-β-Galactosidase conjugate (Str-β-Gal) was obtained from Roche Diagnostics Corporation (Bromma, Sweden). Agarose was bought from Boehriger (Manheim, Germany), while 6× Loading dye solution and GeneRuler™ 100 bp DNA Ladder Plus (14 bands, 100 – 3 000 bp) from Fermentas (St Leon-Rot, Germany).
All salts used for buffers were obtained from Merck KGaA (Darmstadt, Germany). Water used in all experiments was ultra-pure Milli-Q water (Millipore purification system). Phosphate-buffered saline (PBS) was prepared by dissolving 150 mM NaCl and 10 mM Na2HPO4 in water and adjusting to pH 7.4. TPBS buffer (pH 7.4) was completed by adding 0.01% (v/v) Tween 20 to PBS. DNA hybridization buffer contained 4 mM EDTA in 4× PBS solution. Working buffer was prepared by dissolving 120 mM NaCl, 30 mM K2HPO4 and 1 mM MgCl2 in water and adjusting to pH 7.2. Flushing buffer was prepared by adding 0.01% (v/v) Tween 20 to working buffer. Enzyme-conjugate dilution buffer was prepared by mixing 1% (v/v) BSA to flushing buffer. Tris-borate (TBE), a 0.5× solution was prepared by dissolving 0.045 M Tris(hydroxymethyl)-aminoethan-borate and 0.001 M EDTA in water and adjusting to pH 8.0. Dulbecco's buffered saline (DBS) was prepared by dissolving 160 mM sodium chloride, 3 mM potassium chloride, 8 mM disodium hydrogen phosphate dihydrate, and 1 mM potassium hydrogen phosphate dihydrate in water and adjusting to pH 7.3. All buffers were sterilized by autoclaving before use.
Bacterial strains and cultivation
The E. coli strains used in this work, MG1655 (ATCC 47076), W3110 (ATCC 27325), DH5α (ATCC 53868), EDL933 (ATCC 700927), CB571 [32], and 86–24 (from Dr Gail Christie, Virginia Commonwealth University, USA) were grown aerobically in nutrient broth (LB or LA media, Merck KGaA, Darmstadt, Germany) at 37°C. Microorganisms with ATCC numbers were purchased from the American Type Culture Collection, Manassas, USA.
Flow cytometry
Flow cytometry was realized in order to determine precisely cell counting. It was used to analyze the number of cells in isolated colonies from agar plates, and to study cell disruption by applying ultrasound to bacterial samples. All measurements were done on a Partec PAS flow cytometer (Partec, Münster, Germany) with 488 nm excitation from an argon-ion laser at 20 mW. Interferences from system noise and non-microbial particles were minimized by appropriate instrument setup, careful calibration, and filtration (0.2 μm) of all solutions prior to use. The suspended colony was further diluted 10× with DBS buffer, resulting in 1 to 2 × 106 cells/ml, which is the recommended cell density for the flow cytometry measurements. The suspension was analyzed at a flow rate of 1500–2500 counts/s. Partec Flo-Max software (version 2.4b) and MATLAB (The MathWorks, Inc) were used for data analysis and for collecting histograms of forward scatter (FSC) as a function of time. The forward scatter is considered to represent the size of cells and other measured particles [33,34].
Arrangement of cell lysates and extracted DNAs
Bacterial pellets suspended in PBS to the desired final concentration were treated with ultrasound disruptor UP100H (Dr Hielscher GmbH, Stuttgard, Germany) equipped with a microtip 1 mm in diameter. The operating frequency was 30 kHz and effective output power was 100 W. During the operation, samples were cooled in an ice-water bath, mixed and centrifuged. The samples were utilized for flow cytometry studies, while for later handling, samples were subjected to a heat treatment (95°C, 5 min). The crude cell lysates were processed with a mixture of phenol:chloroform:isoamyl alcohol (25:24:1). An equal volume of this mix was added to the lysate sample, the solution was vortexed vigorously for 15 s and centrifuged at 15,000 xg for 2 min at room temperature (RT) around 22°C. The top aqueous phase containing the genomic DNA was carefully separated and collected in a new sterile Eppendorf tube.
Subsequently, samples were sonicated to fragment the DNA. The sonication step was realized in the same conditions as described above. To evaluate the fragmentation effects on the genomic DNA, samples were analyzed by using agarose gel electrophoresis.
Design of PCR primers and probes
After selection of target genes as specific genetic markers of EHEC, primers and probes design was realized on the common areas of the sequences available in GenBank after bioinformatics analyses. The corresponding sequence accession numbers are GenBank:AF401292 (etpC), GenBank:M17358 (stx1), GenBank:AF500187 (stx2), and GenBank:AF022236 (eae). The OLIGO Primer Analysis Software, Verssion 6.88 (MedProbe, Oslo, Norway) was utilized. The upper and lower primers were designed to be single-stranded oligonucleotides of 25 nucleotides complementary to the selected region of each virulence factor representative sequence. Capture and detection probes were designed to be complementary to antisense DNA strand of each toxin-encoding sequence. An extra four nucleotides spacer (non-complementary to selected targeting sequences) at the 5' end of capture probe was added and labeled with a thiol group via a C6 chain linker, and at the 3' end of detection probe this spacer was labeled with a biotin. One negative control probe was used and described as non-biotinylated and non-relevant to any of selected four target sequences. It was designed in Bacillus subtilis genome (accession number: Z99108).
All oligonucleotide sequences were purchased from Thermo Electron GmbH (Ulm, Germany), and are listed in Table 1.
PCR assays and product purification
Two types of PCRs, i.e. simplex and multiplex assays were performed in this work. Genomic DNAs used as templates for amplification of selected sequences were obtained from single colonies transferred to microtubes with a sterile loop and suspended in 50 μl of the PCR mixture. The reaction volume contained also 200 μM each of four kinds of dNTPs and 2.5 units of Taq polymerse in reaction buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, pH 8.3). 0.5 μM or 0.125 μM primers were used in simplex or multiplex PCR, respectively. The amplification program consisted of 4 min of initial denaturation at 95°C, 35 cycles of 45 s of denaturation at 95°C, 1- or 2-min primer annealing at 57°C, and 2- or 3-min elongation at 72°C. The program ended with 10-min final extension step at 72°C. All PCR products were analyzed with agarose gel electrophoresis.
The purifications of the PCR amplicons were performed with Montage™ PCR Cleanup Kit from Millipore (Billerica, USA). Afterwards, 1 μl of each purified solution was analyzed by UV spectrum using a spectrophotometer ND-1000 (Nanodrop, USA). The purified PCR products' solutions were diluted to a concentration of 20 nM. Aliquots of 30 μl were stored at -20°C until their utilization for chip analyses. Purified products were additionally analyzed with agarose gel electrophoresis.
Chip array analysis
Chip arrays with 16 electrode positions, contact pads and their connections, and the corresponding instrument 'eMicroLISA' were obtained from Fraunhofer Institute for Silicon Technology and AJ eBiochip GmbH (Itzehoe, Germany), respectively. Details of the chip array elements and instrument were described previously [35].
All electrode positions were functionalized by DNA probes via thiol-gold interaction by using a piezo electric nanodispenser NP 2.0 (GeSiM, Groβerkmannsdorf, Germany). The comprehensive protocol was provided by Elsholz et al. [35]. Each chosen capture probe was spotted on random triplicate electrode positions of the chip array. Furthermore, four negative control probe position (i.e. non- biotinylated and non-relevant to any of selected eight toxin representative sequences) were used, in order to validate detection specificity and assay performance. After functionalization, chip arrays were stored in the dry condition under the protection of nitrogen gas at RT. Before assay, single chip array was applied to the reaction chamber and flushed with 4× PBS at 50°C for 5 min.
Assay program and detection
Purified PCR amplicons were used as target DNA analytes and mixed with detection probes (1 μM working conc. for each) in hybridization buffer. The mix (200 μl total volume) was incubated at 95°C for 5 min, then immediately put on ice for 1 min, and directly transferred in the chip reaction chamber for assay performance. The assay program was designed with steps in a sequence (Table 2). An internal iridium oxide reference electrode (+100 mV anode/-400 mV cathode) was used for all measurements.
Table 2. Assay program at 'eMicroLISA'
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
PB performed the experiments and helped to draft the manuscript. GW edited the manuscript. SOE coordinated the study. MGC supervised the work and wrote the manuscript.
We thank Dr. Bruno Elsholz from Fraunhofer Institute for Silicon Technology (Itzehoe, Germany) for performance of the chip array functionalization. This work was supported by the European Commission (EC project LSHB-CT-2004-512009 eBIOSENSE) and the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS).
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Blanco M, Blanco JE, Dahbi G, Alonso MP, Mora A, Coira MA, Madrid C, Juárez A, Bernárdez MI, González EA, Blanco J: Identification of two new intimin types in atypical enteropathogenic Escherichia coli.
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Call DR, Brockman FJ, Chandler DP: Detecting and genotyping Escherichia coli O157:H7 using multiplex PCR and nucleic acid microarrays.
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Gerrish RS, Lee JE, Reed J, Williams J, Farrell LD, Spiegel KM, Sheridan PP, Shields MS: PCR versus hybridization for detecting virulence genes of enterohemorrhagic Escherichia coli.
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Gabig-Ciminska M, Andresen H, Albers J, Hintsche R, Enfors S-O: Identification of pathogenic microbial cells and spores by electrochemical detection on a chip.
Microb Cell Fact 2004, 3(1):2. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Metfies K, Huljic S, Lange M, Medlin LK: Electrochemical detection of the toxic dinoflagellate Alexandrium ostenfeldii with a DNA-biosensor.
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Liu RH, Yang J, Lenigk R, Bonanno J, Grodzinski P: Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection.
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Rudi K, Nogva HK, Moen B, Nissen H, Bredholt S, Moretro T, Naterstad K, Holck A: Development and application of new nucleic acid-based technologies for microbial community analyses in foods.
Int J Food Microbiol 2002, 78:171-180. PubMed Abstract
Belgrader P, Okuzumi M, Pourahmadi F, Borkholder DA, Northrup A: A microfluidic cartridge to prepare spores for PCR analysis.
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Beutin L, Montenegro MA, Orskov I: Close association of verotoxin (Shiga-like toxin) production with enterohemolysin production in strains of Escherichia coli.
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Reynolds Yacht Sales Receives 2014 Best of Rock Hall Award
ROCK HALL August 9, 2014 -- Reynolds Yacht Sales has been selected for the 2014 Best of Rock Hall Award in the Yacht Brokers category by the Rock Hall Award Program.
Each year, the Rock Hall Award Program identifies companies that we believe have achieved exceptional marketing success in their local community and business category. These are local companies that enhance the positive image of small business through service to their customers and our community. These exceptional companies help make the Rock Hall area a great place to live, work and play.
The 2014 Rock Hall Award Program focuses on quality, not quantity.
The Rock Hall Award Program is an annual awards program honoring the achievements and accomplishments of local businesses throughout the Rock Hall area. Recognition is given to those companies that have shown the ability to use their best practices and implemented programs to generate competitive advantages and long-term value.
41' 2000 Sea Ray 410 Sundancer in North Carolina
Turn Key!


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No surface to be left untouched in update of The Woodland Hills Academy
Crews will paint walls, rebuild stairs and replace doors as part of a multimillion-dollar renovation of The Woodland Hills Academy.
Officials estimate it will cost $12 million to $15 million to update the building, the former Turtle Creek High School on Monroeville Avenue.
Woodland Hills school directors approved the project in a split vote in May after disagreeing over whether to put money into the school, which was built in 1917, or spend it elsewhere.
"Money will always be a debate," said Kia Williams of Turtle Creek, a mother of four children who attend Woodland Hills schools. "However, the importance of education and our children should always be put first. Investing money into education is a wonderful thing."
As part of the renovation, crews will replace front steps, paint lockers and install an elevator. They will fix drinking fountains, remodel restrooms and improve accessibility for people with disabilities.
The Academy houses classes for children in kindergarten through eighth grade.
Mechanical work and installation of some light fixtures has started. The renovations are scheduled to be finished next year.
"I know that change can provoke both excitement and anxiety," Academy Principal Kelly Berthold said.
Paige Mitchell is a contributing writer for Trib Total Media.
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Thank this author by sharing:
By Rob Farley,
How many times do you have a bunch of code that you want to comment out in Query Analyzer, so you put a /* and later, put a */. Then you run it, and you find that there was a 'go' in there somewhere, giving you not only an error saying that it couldn't find the pairs of the comment brackets, but also running anything in between that you really didn't want to have run! Well, Management Studio is nicer than that. A 'go' statement surrounded by comment brackets is also ignored. So there's no need to highlight it all and do Ctrl-Shift-C (or Ctrl-K, Ctrl-C in Management Studio). Actually, there's another thing - commenting is now consistent with Visual Studio. This is a tremendous thing for anyone in the developer space. Probably not so great if you're a DBA, but I find it good. I do wish that Ctrl-Shift-C still worked, but only because there are times I hit it and find that nothing happened. And you can now undo comments! In Query Analyzer, I used to get really frustrated that I couldn't hit Ctrl-Z and undo my bulk-comment. But now, Ctrl-Z will undo a Ctrl-K, Ctrl-C. And I'm a much less frustrated coder.
use pubs
select * from authors
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Cannot execute queries from SQL Server 2008 Management Studio
Microsoft SQL Server Management Studio Question
"Linked Server" Tables "SQL Server Management Studio"
I made the decision today to start using SQL Server 2008 Management Studio at work to manage my 2000...
A new article from Steve Moore shows us how to use SQL Server Management Studio with your SQL Azure ...
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- Filed Under
Officers of the Lancaster Police Department now are wearing video cameras attached to their uniforms, a measure that - if used properly - should provide extra protection for police and residents alike.
Of course, even the best of intentions can lead to problems and abuses, so we urge Lancaster police to carefully monitor how the cameras are being used on a daily basis. However, the potential benefits of the cameras are too numerous to ignore.
Officers will have the ability to record any interaction they have with members of the general public, which should eliminate at least some of the ...


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John Sheridan declines to get too excited
Posted: March 21, 2013|
Manager John Sheridan is refusing to get carried away by Plymouth Argyle's recent improved results.
The Pilgrims have won three and drawn two of their past six games to move out of the npower League Two relegation zone and up to 20th position.
The Devon side's latest victory came when they beat Southend United 2-0 at Roots Hall on Tuesday. Argyle will now set their sights on a third win in a row when they travel to one of Sheridan's former clubs, mid-table Chesterfield, on Saturday.
Sheridan said: "I don't get carried away. I like my players to be consistently good. If they do silly little things, I will still be on their case whether we are winning 2-0, 3-0, or whatever. I want them to improve as players.
"I have been a manager for quite a while now and I don't get carried away, but I'm pleased for the players and the supporters. We had brilliant support for a Tuesday night in Southend.
"We have just got to carry on doing what we are doing. If we keep picking points up, we will be all right," the Pilgrims' boss added.
Argyle's victory over Southend came on the same night as relegation rivals Accrington Stanley, Barnet and Torquay United all won. York City and Aldershot Town, who were not in action, now occupy the bottom two positions.
Sheridan said: "I think the performance [against Southend] was very good. To get a clean-sheet and win the game 2-0 was a great effort from the players."
Argyle took the lead in the 57th minute at Roots Hall with a penalty from substitute Paul Wotton. Yet had they squandered the chance to open the scoring in the first half when Jason Banton's goalbound shot was touched into the net by Paris Cowan-Hall.
As soon as he did so, Cowan-Hall was flagged offside and Southend were reprieved.
Sheridan said: "I don't really understand what Paris was trying to do. It was a great 'goal' from Jason and, unfortunately, Paris made the wrong decision. But I thought our performance in the second half was excellent. We defended when we had to, as a team, and looked really lively on the counter attack."
Wotton's spot-kick came after on-loan Bristol City midfielder Joe Bryan raced at full speed into the Southend penalty area and was felled by Bilel Mohsni. Sheridan said: "It was a brilliant run. When he's direct, he's a big threat.
"I think he ran from near enough the halfway line and showed what he can do. It was pleasing when we saw the referee had given the penalty.
"I don't watch penalties. I'm always happy when I hear the players and the staff celebrating. I just usually pretend to tie my shoe laces and, touch wood, it works more often than it doesn't."
Wotton started the game against Southend on the bench after Anthony Charles returned to the team. Charles had been kept out by a groin injury for the previous two matches, and Wotton had filled in for him at centre-back.
However, Charles suffered a recurrence of his problem in the first half and Wotton replaced him in the 38th minute. Sheridan said: "Charlesy is an experienced player and told me he was fit, so I threw him back in. Unfortunately, he slipped and got a reaction."
Charles for Wotton was one of two team changes made by Sheridan, with Cowan-Hall taking over from Andres Gurrieri. Sheridan said: "I think the players understand how we are going to play no matter who I pick.
"They all know how I want them to play their roles. I left Andres out, who has been one of my best players over the last two or three games," he added. "I was thinking of making three or four changes but, luckily for me, I made the right decision."
Argyle went 2-0 up in the 75th minute when striker Reuben Reid scored his first goal in his 11th game on loan from Yeovil Town.
Sheridan said: "I'm pleased for Reuben. I thought he did really well again. He worked his socks off and, hopefully, that will give him a boost now and a bit of confidence."
Sheridan also allayed any injury fears over Banton. The 21-year-old was replaced by substitute Ronan Murray in the 79th minute. Sheridan said: "Jason had a bit of cramp. They [the players] had all had a long journey up here."


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Ocular Sarcoidosis: Learn More About Its Dangers and Prevention
Ocular sarcoidosis is a manifestation of sarcoidosis, which is an inflammation of the habit tissues that affects lungs, lymph nodes and offbeat organs.
Ocular sarcoidosis develops money around 30% of the sarcoidosis patients, although in Japan the percentage is much considerable. This inflammation culpability be chronic, and if solitary untreated it leads to a permanent impairment of the visual system and matched to a complete vision loss.
Sarcoidosis granulomas formed pressure the eyes, are not unlike granulomas that form prominence pursue of the areas affected by sarcoidosis. They form when the immune system tries to wall bump off an infection or a exterior substance, and they attend as solitary, yellow nodules.
Ocular sarcoidosis manifests mainly considering uveitis (swelling and irritation of the middle layer of the persuasion called uvea) but indubitable incumbency also interest parts of eyes. First off or posterior granulomatous uveitis is incalculably common, besides may be accompanied by conjunctival and scleral lesions.
Anterior uveitis may cause pain also extreme sensitivity to light, but some patients don't experience bite symptoms. Chronic anterior uveitis estranged untreated may be the set up of glaucoma, sash keratopathy (corneal disease) and cataract.
Intermediate uveitis is another type of uveitis indicative of sarcoidosis. It is characterized by inflammation in the vitreous (thick, conspicuous substance that fills the bull's eye of the eye), pars plana (layer of the uvea), or peripheral retina.
Posterior uveitis affects the back of the uvea called the choroid. This causes an inflammation called choroiditis, which may progress further affect inconsistent areas of the eye.
undocked types of uveitis may introduce to a disease by the propose of cystoid macular edema (growth of the macula, which is a small, highly sensitive central area of the retina).
Ocular sarcoidosis may and manifest now other eye conditions like: optic neuropathy, which is a rare pickle of sarcoidosis that leads to vision loss; and orbital disease, which mainly affects lacrimal glands, further may drive to speculation entrapment further diplopia. Lacrimal gland illness is a humdrum symptom besides may loom before all the other signs of sarcoidosis.
When sarcoidosis is suspected, the compassionate should receive a voluminous examination of the eyes, especially if the eye problems are noted.
Ocular sarcoidosis may be treated spell peculiar ways including topical therapy, immunosuppressive therapy and methotrexate (a commonly used steroid-sparing agent). Topical corticosteroids are used if the inflammation is mild, while money additional intense cases steroids may be injected, considering a rapid up. After the use of corticosteroids, reoccurrence of the disease is possible, stretch immunosuppressive therapy has more long term effects.
The pump with sarcoidosis is the monotonous decrease of knowledge in latter medicine on the triggers of the ailment. But, lately, some zestful tidings be credulous been reported on the conclusions about underlying chemical unbalances and causes drag a holistic treatment picture protocol known as the Aden covenant. You can learn supplementary about ocular sarcoidosis and the groundbreaking treatment options.
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Sheh Bertram: Giving Others a Hand Up
Utah Innovation Awards
Measured Progress
Sheh Bertram: Giving Others a Hand Up
Eric Olson: Reinforcing the Backbone of Utah's Economy
Outstanding Directors
Around Utah
Around Utah
Money Talk
Every Little Bit Helps
Legal Briefs
Tax Free Asset Protection
Economic Insight
Inflationary Fears
Lessons Learned
Mall Makeover
Body of Work
Smooth Operator
Business Trends
Hard Hat Zone
Living Well
Charming and Rustic
Editor's Note
Celebrating Utah's Past, Present and Future Innovators
Well Traveled
Industry Outlook
Advertising and Marketing
Reader's Choice
Better Biking
By the Books
To Sell Is Human
Utah has a long and storied legacy of innovation, forged by industry leaders such as Alan Ashton and Bruce Bastian, co-founders of WordPerfect; Ray Noorda, co-founder of Novell; Edwin Catmull, president of Pixar Animation Studios; and Nolan Bushnell, founder of Atari. And in the life sciences, the state leads the way in genetics, cancer research and even next-gen prosthetics stemming back to Willem Kolff's ground-breaking work with artificial organs up to today's healthcare innovators like Edwards Lifesciences, Merit Medical and Myriad Genetics, among others.
Utah's innovative and entrepreneurial spirit remains strong today despite the increasingly complex business and political environment, as evidenced by the state's many accolades and accomplishments. For example, the University of Utah has been a leading producer of startup companies, even ranking as the No. 1 startup launcher for two years in a row (the U came in second place this year, just below the Massachusetts Institute of Technology). Utah's record of entrepreneurialism is further solidified by the number of patents filed each year; according to the U.S. Patent and Trademark Office, Utah consistently ranks as one of the top patent-producing states per capita.
In this issue, we're excited to celebrate the latest and greatest innovations coming out of the Beehive State. As the 2013 Utah Innovation Awards program demonstrates, Utah has no shortage of innovation and entrepreneurialism. This annual awards program, which is presented by Stoel Rives and the Utah Technology Council, celebrates the state's top innovations in categories ranging from biotechnology to clean energy to consumer software, among others. Their inventions are revolutionizing the way the world works, and we applaud their accomplishments and contributions. Read their stories beginning on page 58.
Utah is home to many innovative, inspirational and devoted individuals who work diligently to improve our world. Perhaps no Utahn is better known for his innovative and entrepreneurial spirit, as well as his work improving the health of many throughout the world, than Jon M. Huntsman. A true pioneer in both the business world and in his devotion to cancer research, Huntsman has changed the world as we know it.
In this issue, we honor Huntsman's legacy as the founder and director of the Huntsman Cancer Institute in our 2013 Outstanding Directors Awards program. We admire his vision and devotion to this cause that touches so many lives.
We also pay tribute to two other giants in our community: Jerry Atkin, CEO of SkyWest, Inc. and decades-long board member of Zions Bancorporation; as well as community builder Dave Winder for his service to Alsco, Inc. These three leaders are not just anchors in the companies they serve—they are pillars in our state. Beyond their extensive business-building backgrounds, they each are actively involved in helping other businesses, and the community, reach greater heights. Read their stories beginning on page 76. (One fun tidbit you won't learn about Jon Huntsman in the article—he's a major Elvis fan.)
Whether it's the storied works of giants like Jon Huntsman, Dave Winder or Jerry Atkin, or the up-and-coming innovators who are advancing healthcare, clean energy and aerospace exploration, Utah is a place where a solid idea combined with hard work and perseverance can take off.
From the Editor
Sarah Ryther Francom
firstname.lastname@example.com | 90 South 400 West, Ste 650 Salt Lake City, Utah 84101 | (801) 568-0114
Advertise with Utah Business


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|Back to Home > Events > Media Events > Mobile Marketing Strategies
Mobile Marketing Strategies
Nov 20, 2012, 8:00AM to 4:40PM
|4-18 Harrington Gardens
London, UK
|Waitrose, Clarks, Savills, Amnesty International, Visa UK and many more discuss how to leverage the mobile channel to engage your audience.
Global brands and marketing moguls will join together to:
- Hear how globally successful brands including Waitrose, Clarks, Savills, Amnesty International and Visa UK are leveraging this unique and multi-faceted channel to drive customer engagement and optimise revenue
- Find out about new and emerging technologies and the impact that these will have on the strategies that you have in place including new generation handset devices and the availability of 4G
- Gain the know-how needed to develop an effective business plan around mobile marketing including the tricky topic of measuring ROI
- Hear cutting-edge case studies from experts who have savoured the success of using this marketing channel effectively including how Vouchercodes.co.uk developed and launched their iPhone app which grew to 1.6 million downloads in under a year
- Discover how to bring in real money via the mobile channel; keep up with the trends in mobile payments; encourage repeat usage and multi-purchases via mobile; integrate mobile into your overall marketing strategy and track and evaluate the impact of your mobile marketing.
One Day Rate £340 + VAT
Standard Rate £795 + VAT
Early Bird Rate £595 + VAT
Twitter https://twitter.com/MarketTheMobile
Booking https://www.eventsforce.net/cmpi/frontend/reg/tSelectBookingMode.csp?pageID=331570&eventID=793&tempPersonID=673771&eventID=793
Website http://www.mms-conference.com/
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|Millenium Gloucester


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Dear readers:
Every few months some harried freelancer charged with coming up with a novel spin on something sexual or other contacts me for a pithy quote. And since I am all about the pith, I will oblige if at all possible. Most recently, the writer was a staffer at Details, which I used to read when it was sort of sceney and kinda gay back in the '80s but which sunk beneath my radar when it morphed into some sort of younger, more metro GQ. What did I think, the writer wanted to know, about the demise of the hand job? Had the rise of more exotic pursuits among American teenagers sealed its fate, or was good old manual release doomed to fade into obscurity by dint of its own lack of pizzazz? What was the hand job's appeal, if any? And by the way, did I know any really good horror stories, Indian burns, that sort of thing?
It got me thinking, first about horror stories. As a collector of (other people's) horrible sex accident stories, I know that hand jobs hardly figure. Skin-to-skin virus transmission is possible, sure, but nobody ever seems to accidentally yank anybody's equipment clean off or anything. Not even close. Even CBT, cock and ball torture, is rarely as grizzly as it sounds. I did once demonstrate my most successful technique, a two-fisted opposite swivel, for a friendgirl who'd had only girlfriends but was considering branching out. Damned if my little pantomime didn't look very much like I was administering an Indian (sorry, Native American is it?) burn, something I'd never noticed when doing it for real. Of course, hand jobs are best administered with a generous shot of lube or, at the very least, a palmful of spit. It's really hard to hurt somebody with a palmful of spit.
So, hand jobs are safe, I concluded, but are they sexy? Is nobody doing them anymore because there's so much hotter stuff to do, or is it simply that they're not worth doing? These I couldn't answer because I'm not sure I buy the premise. There's no question that there has been a steady trickle (ew) of articles and TV scare pieces about the oral sex "epidemic" among young people, going back at least 10 years. But not only do these fail to convince me that more young people (well, women - these articles are never about a cunnilingus epidemic) are going down, they never say a thing about them eschewing hand jobs in favor of blow jobs. If you compare The National Survey of Family Growth, the best recent research on Americans' sex habits, published in 2005, with Sex in America, the last decent survey, done in the early '90s, there isn't much increase in the incidence of oral sex. Period. There is, intriguingly, an increase in the incidence of anal sex, potentially a much greater health risk. But it doesn't say a thing about hand jobs, which are, presumably, relegated to the catchall category "any" sex. So no matter how many articles are published insisting that life for the typical American teen these days is one big blow job party (the parts that aren't taken up with pornographically violent video games and being obese, anyway), I haven't seen anything supporting it. And before people start freaking out about all those teenagers having anal sex, the increase there was among people in their 20s.
My own take is that hand jobs aren't dead, they're just boring. Or at least, boringish. Boringish to receive, depending upon the recipient's level of desperation and the donor's skill, of course; boringish to perform (at least compared to the raunchier, more dramatic blow job), and above all boringish to write TV magazine scare pieces about. Nobody dies from them, so nobody cares. Also, while the hand job may figure prominently in some gay male scenes, most straight people kind of forget about them as they leave their teenage groping days behind.
Also from this author
alt.sex.column says so long -- and thanks for all the fish


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Werner Elementary's Trimathlon Team front row, left to right: Mitchell Knipp, Ethan Kramer, Espen Marston, Cameron Carcasson. Second row: Veronica Knipp, Elijah Froiland, Isaac Chong and Sebastian Mahal. Third row: Mr. Reggie Kellum, Center Director of Mathnasium Fort Collins, Mr. Hayden Camp, Werner Elementary Principal, and Mrs. Tracy Bean, Werner Elementary Gifted and Talented teacher. Not pictured: Price Schaeffer.
- Filed Under
Math-minded Werner Elementary students take first in regional Trimathalon
Nine students from Werner Elementary bested more than 40 Northern Colorado competitors to win first place at the second annual Timathalon.
Held last month in Fort Collins, the Trimathalon pitted students from seven elementary schools against one another as they used math skills to take a three-part numeric exam. Werner's team took the lead and was awarded $275.
Mathnasium, The Math Learning Center, at 2733 Council Tree Ave., hosted the event at its offices. The Trimathalon is designed to increase excitement and engagement in mathematics among grade-level students, parent Wanda Molinary Knipp said in an email to the Coloradoan.
Poudre High students to quiz candidates
American Government students at Poudre High School will tonight ask questions of candidates running for seats in State House Districts 52 and 53.
The forum starts at 7 p.m. in the board room at the Johannsen Support Services Center, 2407 LaPorte Ave.
Students on Monday night moderated another forum with political hopefuls from Colorado's Senate District 14.
Classified, adjunct employees honored
This week is Classified School Employees Week in Colorado, which, in Poudre School District, will celebrate 1,508 classified employees.
Those among that employee group are paraprofessionals, who work one-on-one with students, and office personnel who call parents when their child is sick, maintain student records and take care of emergencies. But there are many more employed in departments such as information technology, school services, payroll and more.
Classified employees work in PSD schools and in central office positions but aren't required to have teaching certificates, according to PSD's website. They will be recognized in various ways at individual sites, departments and schools.
At the same time, CSU is celebrating Campus Equity Week, focused on the role of adjunct faculty at the university.
"As universities have come to rely on faculty who work off the tenure-track to teach undergraduate courses, disparities in working conditions and compensation have moved adjunct or contingent faculty to advocate for reforms," a news release said.
Various Campus Equity Week events "encourage those efforts," the release said.
President of New Faculty Majory Maria Maisto will deliver the keynote address at 7 p.m. tonight in the main auditorium of the Institute for Teaching and learning building on the Oval.
She and member of the College of Liberal Arts Adjunct Faculty Committee are also scheduled to host a public forum from 9:30 to 11:30 a.m. Wednesday in the new Main Floor Event Hall at Morgan Library.
These events are free and open to the public.
Global environmental experts to speak
Chris Holmes, U.S. Agency for International Development global water coordinator and senior executive, will speak about water, health and food security at 4 p.m. Thursday in the Lory Student Center's Grey Rock Room. This event is free and open to the public.
Sponsored by the University Water Strategy for Global Issues Research Team, Holmes will highlight the water-food nexus and address the importance of linkages across disciplines, agencies and countries, according to a news release on CSU's website.
As a part of the USAID, Holmes has investigated the impact the global community has had on meeting water needs around the world.
Additionally on Thursday, the Graduate School and the Global Women Scholars Network will host an expert in international science policy.
Ian Simon, of the U.S. Department of State and American Association for the Advancement of Science fellow in Science and Diplomacy, will present "World Experiences in Treaties, Trade & Sustainable Development" at 11:30 am in the Francis Clark conference room, 215 Natural Environmental Sciences Building.
For more information on these events contact Melinda Laituri at firstname.lastname@example.com or visit www.sustainability. colostate.edu.
Coloradoan staff


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Vejle Windmill
Vejle Windmill
The landmark of Vejle is situated on a hill in the southern end of the city. The original mill from 1847 burned down, but the current mill from 1890 soon followed. The furniture of the mill is from the 1930s. Vejle Windmill has a grinding loft, rollers, belt drive, etc. It functioned as a mill up to 1960.
Today, the mill houses an exhibition about the history of the mill and the running of mills in the Vejle area.
The Vejle Museums, VejleMuseerne, host an annual mill market in June. See the event calendar at www.vejlemuseerne.dk for dates and details.
Enjoy a spectacular view of the city, the river valley and the bay from the circular corridor of the mill.
|Opening hours
|02/04/2015 - 18/10/2015
|Tuesday - Sunday
|11:00 - 16:00
Last updated byVisitVejle firstname.lastname@example.com
Søndermarksvej 1
7100 Vejle
+45 76813100
City / Area
- Vejle
- free entrance
General facilities
- coach/truck parking
Museums, Galleries and crafts
- cultural history
- History
- industry
- local history
- mill
- museum
- packed lunch allowed
CoordinatesLongitude : 9.5305419999
Latitude : 55.699693


text:
Posted February 2, 1999
젨젨젨젨젨HOLLAND -- Bruce Himebaugh, CFP, director of human
젨resources at Hope College, has been authorized by the
젨Certified Financial Planner Board of Standards (CFP Board)
젨to use the CFP certification mark and the marks CFP and
젨Certified Financial Planner in accordance with CFP
젨certification and license renewal requirements.
젨젨젨젨젨Himebaugh is responsible for all personnel and
젨benefits programs at Hope, including retirement planning.
젨He has worked at the college since 1970.
젨젨젨젨젨The marks identify those individuals who have met
젨the experience and ethical requirements of the CFP Board,
젨have successfully completed financial planning coursework
젨and have passed a certification examination covering the
젨following areas:젨the financial planning process, risk
젨management, investments, tax planning and management,
젨retirement and employee benefits, and estate planning.젨CFP
젨licensees must also agree to meet ongoing continuing
젨education requirements and to uphold the CFP Board Code of
젨Ethics and Professional Responsibility.
젨젨젨젨젨Himebaugh completed his coursework and the two-day
젨examination in November.
젨젨젨젨젨The CFP Board is a nonprofit professional
젨regulatory organization established in 1985 to benefit and
젨protect the public by maintaining and enforcing high
젨standards of ethical professional conduct among Certified
젨Financial Planner licensees.젨An independent certifying
젨body, the CFP Board owns the CFP certification mark and the
젨marks CFP and Certified Financial Planner, the most widely
젨recognized marks in the financial planning profession.젨More
젨than 31,000 financial planners are presently licensed to use
젨the marks.
젨젨젨젨젨젨젨젨젨젨젨젨젨젨젨젨 -30-


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Riyadh, March 2014: flynas, Saudi Arabia's National Carrier, is participating as a gold sponsor at the Saudi Travel and Tourism Investment Market 2014 which opened yesterday in Riyadh under the patronage of HRH Prince Khaled bin Bandar bin Abdulaziz Al Saud, Governor of Riyadh and Chairman of the Tourism Development Council. The opening ceremony was attended by HRH Prince Sultan bin Salman, President of the Supreme Commission for Tourism and Antiquities at the Riyadh International Convention and Exhibition and the event will continue until the 4th of April, bringing togetherspecialized private and public sectors companies from the tourism and travel industry in the Kingdom. During the opening ceremony,, flynas was recognized for supporting and participating in the weeklong event.
The theme of the event, which is in its 7th year, is "A partnership towards sustainable development" featuring 5 key scientific sessions and a series of workshops run by local and international experts which will address key topics related to the tourism industry.
HRH the Governor of Riyadh and HRH the President of the Supreme Commission for Tourism and Antiquities visited the flynas pavilion following the official ceremony where flynas was recognized as a gold sponsor during the Tourism Excellence Awards Ceremony which announced the leading companies and organizations working in the Saudi tourism industry.
flynas Director of Marketing & Communications, Mr. Wael Al Sarhan expressed his delight in sponsoring this important event this year stating, "This forum allows us to exchange information and experience with local and global experts in the travel and tourism industry. It also gives us an opportunity to meet with all visitors, sharing our new services and key values which revolve around availing smart choices to our Customers wanting to travel from and to the Kingdom." Al Sarhan also underscored the airline's continuous participation in key conferences and forums across the Kingdom.
Wael Al Sarhan confirmed flynas commitment to supporting and developing local tourism, working with the various organizations of relevance to develop this important economic sector in line with the strategic vision for national tourism. flynas participation as a gold sponsor of STTIM 2014 is one of the airline's many initiatives to encourage tourism in the Kingdom.
STTIM 2014 is a unique opportunity for exhibitors to gain direct and quick access to the decision-makers in the Saudi tourism sector, to strengthen partnerships with ministries and agencies, departments and governmental and private committees as well as provincial tourism development councils, paving the way for all tourism development firms, tourist products and companies, hotel management and operation companies, as well as manufacturers and suppliers of tourism industry needs in general, such as design, services and luxury hotel, restaurants and café operating companies; to benefit from the opportunities offered by the growing movement of tourism projects in the Kingdom.
STTIM 2014 comprises an exhibition to promote domestic destinations to engage investors and operators of various facilities and tourism services and other developed destinations.
About flynas:
flynas is a leading low-cost carrier with added value (Low Cost Carrier Plus) from Saudi Arabia with a young fleet of 27 aircraft, operating nearly 950 weekly flights on 26 destinations within and outside of Saudi Arabia. Launched in 2007, flynas has successfully operated over 140,000 flights carrying more than 15 million passengers, a number that has steadily grown over the years.
flynas unveiled a new modern identity in 2013 and made significant investments in enhancing its aircraft fleet and introducing a dedicated Business Class Cabin.
In 2014, flynas has launched and pioneered the Low Cost Carrier Plus (LCC+) concept that allows the carrier to offer its passengers value-added, services-driven, yet affordable international air travel on both Business Class and Economy Class, as part of its Global Flight Routes program. This program will drive the future growth of flynas, as it expands its presence on three continents – Asia, Africa and Europe.
In 2013, flynas carried 3.3 million passengers; its highest ever since launch.
For more information, please visit www.flynas.com.


text:
ibis budget Sheffield Arena2
298 Attercliffe Common, SHEFFIELD, S9 2AG
Price FromView Rooms
- Very Good
- 88 guest reviews
"Good room, spacious. Very basic, but had everything you need! Receptionists were helpful, however weren't very inviting upon arrival..."
"Basic, clean accommodation. Friendly, helpful staff...."
"Basic but fine for a quick stay...."
"Good but basic..."
"This really is a unique and innovative type of hotel room. Short of the standard carpet, desk and usual 'creature comforts' you may think yo..."
About the hotel
Ibis budget Hotel Sheffield Arena is a low-cost hotel situated close to the city center. Book this hotel to be close to Sheffield Arena and Meadowhall Shopping Center with easy access to city center nightlife. The hotel has 122 guestrooms and is 100% non-smoking.
Each room has en-suite shower, air cooling and flat-screen TV. A continental breakfast buffet is served every morning. There is a chargeable limited amount of outdoor parking and free WIFI throughout.
Read MoreLess
Room Availability
Showing Tuesday 1 December 2015 to Wednesday 2 December 2015Change Search
Facilities Show/hide
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- Disabled Facilities
- Hotel services
- 24 Hour Reception
- Family friendly
- Pets Allowed
- Business
- Wi-Fi Available In Public Areas
Useful Info Show/hide
- From 14:00 hours
- Until 12:00 hours
- The price shown is based on the occupancy and dates you have searched for. Prices and availability are subject to change based on the arrival date, number of nights and occupancy searched for. All prices are inclusive of all local taxes.
- Pets are allowed
Customer Reviews
Our reviews are from genuine LateRooms.com customers. We verify that the customer has stayed before leaving a review.
Very Good
78% would recommend it to a friend
Customer service
Food quality
Room quality
Good room, spacious.
Very basic, but had everything you need. Receptionists were helpful, however weren't very inviting upon arrival
Read More Less


text:
Join Now
Join ePHOTOzine, the friendliest photography community.
Upload photos, chat with photographers, win prizes and much more for free!
My partner is a music teacher and she wants some pictures of her instruments (Clarinet, Sax, Flute and Piano) to hang in her teaching room in her new house. I'm going to get some printed on to canvas for her when they're all done.
This is the first one (possibly!)
Please feel free to leave comments. Thanks
|Nikon D60 Check out Nikon Nation!
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|22 Mar 2010 - 5:52 PM
|Black & white, Bobthomas, Clarinet, Close-up / macro, D60, Nikon, Still life
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Nominating Constructive Critique
This is a cracking image, well done.
Nominating Constructive Critique
Thanks a lot
Phil: I think it might just need more depth of field? Also, if you look at the top of the sheet music, you can see the page is slightly turned up which I totally missed when I was taking the photo! I guess I need to look a bit more carefully before I take the picture! I only got the camera a couple of weeks ago (it's my first DSLR), so I'm still learning that too!!
At least I have a few more weeks before I need to get anything made up, so I can have another crack at it!
Nominating Constructive Critique
great shot, i think it needs to be a little sharper on the middle keys, my pov. I'm trying to do the same with my flute, how did you light this shot?
Nominating Constructive Critique
Hi Kevin,
That's one of the reasons I was thinking of re-taking it with more depth of field, so that I can get the whole thing in focus.
It was just natural light coming through a window. It was a pretty sunny day, but I took this quite early and the sun was coming in through the window at the time. There were actually two windows, in an L shaped wall, with a window on each wall which probably helped.
I'm still thinking about how to do the flute!! It's going to be a lot more reflective than a clarinet, so I'm still debating that one. I was considering trying to get reflections of the other instruments, but I think that might be a bit of a mare to achieve and it wouldn't even notice unless you looked REALLY carefully! I guess it's the little things that make the difference though!
I'm also struggling to get a shot of the piano I like. I tried down the keyboard from one end, but that didn't work out as I'd hoped. I think it needs something else in it. I'll post it when I've worked out what to do!
Nominating Constructive Critique
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- This event has passed.
Annual Picnic
August 24, 2014 @ 2:00 pm - 3:00 pm
Event Navigation
H UBBARDTON The Castleton Historical Society will hold its annual potluck picnic at 2 p.m. on Sunday, Aug. 24 at the Hubbardton Battlefield. Tom Hughes, manager of the Crown Point historic site, will give a presentation "The Battle of Hubbardton: 1777 and 1977." Bring a chair, place setting and a dish to share; beverages are provided. Free and open to anyone interested in local history. Information: Holly Hitchcock, 802-468-5105.


text:
- Filed Under
Goggles and swimsuits aren't normally associated with January in Cincinnati, but the third weekend of 2013 will bring plenty of both to Southwest Ohio.
The Mutual of Omaha Breakout! Swim Clinic will be held at Keating Natatorium on Jan. 21 from 9 a.m. to 1 p.m. Swimmers ages 11 to 18 will have the opportunity to improve their stroke techniques under the instruction of former and current swimming champions.
Among the presenters will be North Bend native Josh Schneider. Schneider graduated from Taylor High School in 2006 before attending the University of Cincinnati on a swimming ...


text:
Consumer Survey Finds Big Upswing In Favorability Toward Clean Energy
NAW Staff, Thursday 02 January 2014 - 13:58:10
Public attitudes toward clean and renewable energy concepts have tended to fluctuate and, in several cases, decline. According to a new
consumer survey
from Navigant Research, however, favorable attitudes for a number of these concepts - particularly wind energy, solar power, hybrid vehicles and electric cars - have rebounded significantly from their 2012 levels.
The survey, which polled 1,084 U.S. adults in fall 2013, found that wind energy was viewed as either favorable or very favorable by 72% of respondents - up from 66% in 2012. Wind was only slightly less popular than solar energy, which held the top spot at 79% favorability - an increase from 69% in 2012.
Notably, Navigant found that just 7% of respondents held unfavorable opinions of wind power, and approximately one in five respondents was neutral or had no opinion on the energy resource.
From a demographic perspective, Navigant says there was a 9% difference in favorability toward wind energy between men and women, although this difference was close to the margin of error in each direction. Consumers with incomes of $75,000 or higher, respondents 45 years of age and older, and those with four-year college degrees and graduate school held more favorable views of wind as well, the research firm adds.
Navigant says the similarly high levels of favorable views toward solar and wind energy indicate that consumers are generally supportive of the more established renewable energies that harness naturally occurring power sources. Since these two concepts have retained their most favored status year after year, the firm asserts that consumers consider these renewable energies to be important pieces in the power generation portfolio of the future.
Overall, the average favorability rating for the 10 concepts that fall under the categories of clean energy, clean transportation, smart grid and building efficiency rose to the highest level seen in Navigant Research's annual survey since 2010. The rating rose from 44% in 2012 to 51%.
"Between 2009 and 2012, there were steady declines in favorability for some clean energy concepts, particularly the most favorable concepts, such as solar energy, wind energy, and hybrid and electric vehicles," says Clint Wheelock, managing director at Navigant Research. "[2013] saw statistically significant increases in favorability for seven of the 10 concepts, and a decline for only one - nuclear power."
Navigant Research's white paper regarding the consumer survey can be found
this content item is from North American Windpower
( http://www.nawindpower.com/e107_plugins/content/content.php?content.12452 )


text:
When a patient's life expectancy is limited, comfort is just a phone call away.
Wayne Memorial's Hospice Team, part of the hospital's Home Health Department, provides a full range of compassionate care for the dying so they may live their lives to the fullest.
Our Hospice Team Leader can be reached at the Honesdale number below.
Wayne County
Stourbridge Mall, Honesdale
(570) 253-8431
Pike County
Pike County Medical Center, Rte 739, Lords Valley
(570) 775-4266
Toll free
(800) 675-3003
Brochure: HospiceBrochure.pdf


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Join RSC and save on every purchase
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Photos: Tornado moves through southside of OKC
Published On: May 20 2013 04:21:34 PM CDT Updated On: May 21 2013 03:39:46 PM CDT
At least one tornado touched down near Oklahoma City Monday afternoon.
The views expressed are not those of this company or its affiliated companies. Please note by clicking on "Post" you acknowledge that you have read the Terms Of Service and the comment you are posting is in compliance with such terms.blog comments powered by Disqus


text:
Roundup: Wabtec to acquire U.K.-based manufacturer for $215M; MSA 4Q profit rose 30% on higher sales; more
Wabtec to acquire U.K.-based manufacturer for $215M
Wabtec Corp. agreed to acquire Fandstan Electric Group Ltd., a rail and industrial equipment manufacturer based in London, for about $215 million in cash. Fandstan has annual sales of about $235 million. Wabtec, based in Wilmerding, expects the transaction to be completed by March 31. The company plans to issue fourth quarter 2013 results on Feb. 19, and a 2014 forecast that will include Fandstan. Fandstan Electric makes electrical and data collection products for rail and tram transportation, industrial and energy markets. It has about 1,000 employees and operations in the United Kingdom, Europe, China, Australia, and the United States. "Fandstan Electric will expand our high-technology content on transit vehicles and provides another entry into the infrastructure segment of the market," said Wabtec CEO Albert J. Neupaver.
MSA 4Q profit rose 30% on higher sales
Mine Safety Appliances Co. said on Wednesday that profit rose 30 percent in the final three months of 2013 as sales increased. The Cranberry-based safety equipment maker said fourth-quarter net income was $25.4 million, or 67 cents a share. Revenue rose to $291.4 million from $282.3 million in the same period of 2012. The company's stock price rose to $51.63 as the market opened, up from $49.25 at Tuesday's close. "Consistent growth in our five core product groups, combined with a strong fourth-quarter performance in emerging markets, certainly provides momentum as we start 2014," said CEO William M. Lambert.
Lexus tops 2014 dependability list
Lexus, Mercedes-Benz and Cadillac had the vehicles with the fewest reported problems, according to J.D. Power and Associates' annual survey of vehicle dependability. Lexus had just 68 problems per 100 vehicles, the only brand with fewer than 100 problems. Owners of three-year-old vehicles are reporting more problems than they did a year ago — an average of 133 problems per 100 vehicles, up from 126 a year ago. It's the first time since 1998 that the average number of problems per vehicle has increased. J.D. Power, a California-based ratings and consulting company, said engine issues accounted for most of the increase in problems reported by the original owners of cars and trucks from the 2011 model year. David Sargent, J.D. Power's vice president of global automotive, said the company recorded an increase in complaints about engine hesitation, rough transmission shifts and lack of power.
Rice Energy to buy new pipeline system
Rice Energy Inc. announced on Wednesday that it will buy a new pipeline system in eastern Washington and Greene counties. A subsidiary of the Cecil drilling company is paying $110 million in cash for the M3 Appalachia Gathering LLC network. The assets include 28 miles of pipe from 6 to 16 inches wide that gathers gas from the wells, and permits and rights of way to build an 18-mile track of 30-inch pipe to connect the local system to the interstate system, it said. Rice, which recently went public, expects to have a total capacity of more than 1 billion cubic feet of natural gas per day.
— Staff and wire reports
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You are deciding to take a small risk, but, I guess, a very dangerous one, in putting your thumb out. There's no better way to feel the beating pulse of a place though. You can wind up anywhere. Eating bee larvae in a cliff-top restaurant, watching girls audition to study opera in a musikschule in Cologne - who knows. Of course, you could also end up chopped into pieces. Here are some of my guidelines to successful hitch-hiking.
When you get on the side of that road, and raise your thumb, you are getting in contact with the world again. When you wake up that morning, you have no idea where you will be when the sun sets that night. It usually ends up being somewhere pretty sweet.


text:
Thailand forum
Horseback Riding
Joined Travelfish
25th August, 2008
Posts: 8
So we're hoping to some elephant trekking, but I also read somewhere that several islands have horseback riding on the beach... I'm pretty sensitive to things about animal care, and I'd be sad to show up at a place with skinny unkept horses... Can anyone recommend anywhere to ride in Ko Samui or Ko Lanta? (or even Krabi or somewhere close that we could just do a day trip for)
#1 Posted: 16/9/2008 - 18:09
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Joined Travelfish
30th December, 2007
Posts: 295
Pattaya is close to Bangkok (2 hrs. by bus.) There are some horse riding facilities in that area.
More info shows up with Google check if bottom link doesn't open.
Cheaper hotels in Pattaya than your other places.
Good luck.
#2 Posted: 19/9/2008 - 02:26
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Joined Travelfish
27th January, 2007
Posts: 560
Try Googling "Horseshoe Point" - it is a "HI_SO" equestrian centre outside Pattaya....they have accommodation too.
#3 Posted: 29/9/2008 - 10:14
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