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Posts by category: trail
Showing 1 to 20 of 29 posts in the category of trail
Bewl's full & Bewl 15 is filling too
19 Feb 2020
Bewl 15 mile trail race preview 2020
Bailey bounces back at Salisbury 54321
13 Aug 2019
Salisbury 54321 Trail race report 2019
Competitors fired up for Salisbury 54321
8 Aug 2019
Salisbury 54321 Wessex Trail event preview 2019
Hobbs' bounty at Bewl 15
9 Jul 2019
Bewl 15 trail race report 2019
Victory For Tom Carthey At Bude Pirate Run
12 Sep 2018
It was time to break out the rum to celebrate Tom Carthey's win at last Sunday's Bude Pirate Run.
Thirsting For Success At Beer Run
27 Jul 2018
British Beer Run Windsor launch 2018
Sevenoaks 7 Celebrates 25 Years
18 Jul 2018
Sevenoaks Seven report 2018
Bling It On At Farleigh Wallop
3 Jul 2018
Farleigh Wallop 10K Trail Race preview 2018
McDowell Sweats As Clements Shines
27 Jun 2018
North Downs Run 30k report 2018
Salomon Announces New Trail Workshops
28 Sep 2017
Learn how to hit the trails at Salomon's specialist workshops
30 Years Of Stomping Around The Stones
17 Jul 2017
Stonehenge Stomp celebrates 30 years of stomping in January 2018
Rollicking Good Run At Thornbury
19 Jan 2017
This muddy 9-miler featured an appearance by Mr Blobby and several other characters.
Snowdonia Climbs Even Higher For 2017
28 Sep 2016
With spectacular panoramic views of Snowdonia National Park, there's little surprise that the SCOTT Snowdonia Trail Marathon has proven to be highly popular among outdoor enthusiasts
Ups And Downs
28 Jun 2016
North Downs 30k Report 2016
Snowdonia Edging In On Sell Out
9 Apr 2016
Liminted number of places available for the acclaimed SCOTT Snowdonia Trail Marathon
Join Trail Running Giants At Keswick
1 Apr 2016
Trail running enthusiasts across the country will be delighted to hear of an opportunity to meet some of the top names in the field
Autumn Flyer In Surrey Hills
25 Aug 2015
The Hurtleberry Trail Run, a 10k through beautiful woodlands in the Surrey Hills, is set for Sunday, 13 September.
Way To Defend Salisbury Record
30 Jul 2015
Steve Way will return to the Salisbury Trail 54321 on 9 August to defend his record of 3:23 in the 50K Ultra.
Brutal Summer
15 Jul 2015
A chance of a summertime trail race has arisen thanks to the Minley Brutal event next month with 5k, 10k and 20k options.
Beach Chums
6 Feb 2015
Thanet Road Runners are set to host a beach race over some of Kent's most picturesque coastline in May.


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World Class
Data Centre Security
Data storage requires good stability and uptime on the basic infrastructure, as well as physical and operational data centre security. This is a service Green Mountain delivers and conserves so companies can focus on their core business.
Physical and operational data centre security
At Green Mountain, we understand that data centre security demands more than antivirus programs and firewalls to guard against cyber-crime, theft, vandalism, natural disasters, human-generated errors, accidental damage and other incidents. Our security system and our standard operational practices are in accordance with ISO 9001/14001/27001, and each of our data centres makes use of a full array of security tools to monitor and record access within our facilities.
Our data centre is among the safest in the world, not only because it is in the Norwegian mountains, but numerous barriers have been set up to prevent unauthorized access. The entire area is continuously monitored by an intelligent video analysis system; we carry out identity checks by all visitors, the centre is secured with biometric identification systems, and everyone has to go through mantraps before they can access the centre.
Green Mountain Security Standards
Security Sectioned Centre – The data centres are sectioned into different security zones for the different parts of the complex. The level for each zone may be adapted to customer requirements.
Biometric Access – All access to the centre is controlled by biometric identification system.
Man Trap Entrance – Each person entering the centre will gain access only through a man trap.
Intelligent Video Analysis – The outside and surroundings of the site are continuously monitored by an Intelligent video analysis system.
Security System – Our security system and our routines are in compliance with ISO/EN 27002.
Facility Security – All Green Mountain data centres have 24/7/365 security personnel present.
- Intelligent Video Analysis System
- Biometric Identification
- 24/7/365 Security Personnel
- Underground Data Centre in Norwegian Mountain
- Uptime Institute Tier III Certified
Due Diligence
Below are some important aspects that Green Mountain AS checked in our extensive due diligence of the sites:
- High stability of mountain rock
- Location is well protected against extreme weather conditions (does not pose a risk)
- The region has very little tidal movement (ca. 40 cm). Storm surge tides are registered since 1904. The site has very good clearance to any potential risk (does not pose a risk)
- Tsunami has never been registered in the region. The topography is such that there is no risk of a flood wave building up approaching the site (depth of 70 m on quayside)
- There is no heavy industry nor storage of flammable/explosive goods in the vicinity
- There is no hazardous chemical or oil refining in the vicinity
- Unprecedented geographical event in fjord system – does not pose risk
- Checked out risks related to global warming effects
- High stability of mountain rock foundation
- Location is well protected against extreme weather conditions (does not pose a risk)
- There is no heavy industry nor storage of flammable/explosive goods in the vicinity
- There is no hazardous chemical or oil refining in the vicinity
- River levels in the area confirmed as not being a threat to the site
Monitoring and Reporting
Our state-of-the-art award-winning DCIM (Data Centre Infrastructure Management) System provides a global holistic, common, real-time monitoring and management platform for all interdependent systems across IT and facility infrastructures. Giving our customers control by way of monitoring and configuring their Data Centre requirements without geographical constraints. Our monitoring solution also provides very detailed monthly reports on all aspects of the data centre environment.

subset: commoncrawl


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Grooming for men
Men's Biz
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Free shipping on all orders within Australia. Limited time only.
Taylor of Old Bond Street Mr Taylors Deodorant Stick is a paraben-free, aluminium-free deodorant lightly fragranced to neutralise natural body odour, keeping you fresh through the day. A perfect complement to the other products in the Mr Taylors range boasting notes of Tarragon, Lavender and Incense with hints of Green Galbanum, Citrus, Cedar, Sandal, Patchouli and Musk for an elegant manly scent. Made in England.
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Remove cap and apply to underarms as required. Allow to dry before dressing.
Propylene Glycol, PPG-3 Myristyl Ether, Stearic Acid, Alcohol Denat., Aqua (Water), Triethyl Citrate, Parfum (Fragrance), Sodium Hydroxide. Parfum contains: Linalool, Citronellol, Butylphenyl Methylpropional, Hexyl Cinnamal, Limonene, Alpha-Isomethyl Ionone, Eugenol, Coumarin, Geraniol, Cinnamal.
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Eton College Deodorant, 75ml
Mr Taylors Aftershave Balm, 75ml
Sandalwood Deodorant, 75ml
Mr Taylors Shaving Cream Tube, 75ml
Jermyn Street Deodorant, 75ml
Mr Taylors Bath Soap, 200g
St. James Deodorant, 75ml
Mr Taylors Shaving Cream Bowl, 150g
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Wednesday, January 27, 2010
this piece had nothing special at first i think . butdamn it came out awesome at the end )
i decided to use a french uniform for him hehe.
but question
why did u walk on him as he was changing ??????????
dont u see your embarrassing him ?
art and character belongs to Karmakat Silverwind
Tuesday, January 26, 2010
Fireman (wip)
been doing a vote on some of my friends today about what to draw
fireman got the priority.
since most of the time in usa movie they show a dalmatian to be their mascot i decided to make a firedog hehehe
and u just caught him as he was changing lol
soon for colors )
Posted by Karmakat at 2:49 PM No comments:
Sunday, January 24, 2010
out of the pool
i hope he likes it . thats to thanks him for all his work )
Rob belongs to Bluewolfr
art done and belongs to Karmakat Silverwind
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MYTILINEOS, one of Greece's leading industrial companies with a strong international presence in four Business Units, Metallurgy, EPC, Electric Power and Gas Trading, is currently seeking to hire Assistant Project Manager for Libya Project of METKA EPC Business Unit.
Assistant Project Manager (Arabic Speaking)
(Reference Code: APM 1218)
Key responsibilities:
- Keeps records with the correspondence between METKA and the Client (in English or Arabic if required).
- Arranges the preparation, submission and keeps record of the file with drawings, studies and equipment.
- Keeps, prepares and revises the budget of the Project in a monthly basis according to the requirements.
- Makes frequent site visits (Tobruk, Libya) or participates in meetings with Clients in Tripoli, Libya.
- University degree Electrical or Mechanical Engineering.
- 2-5 years of experience in a relevant position (preferably in the energy sector).
- Fluent use of Arabic and English language is essential.
- Strong knowledge of computer applications, especially Microsoft suite programs.
- Excellent communication skills.
- Strong Organizational skills.
- Critical thinking and problem-solving skills.
Interested parties are kindly requested to submit their curriculum vitae via the following URL, stating the position's reference code in the subject, no later than 28/2/2019.
URL: https://www.mytilineos.gr/en-us/cv/submission-form
All applications will be handled with confidentiality


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Thursday, August 17, 2017
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Getting There
On the day of admission
Please follow the instructions in your admission letter. If you are unwell or cannot attend, please call the relevant ward or department as soon as possible (on the telephone number listed in your letter), so that we can offer this appointment/admission to another patient.
Getting to hospital by bus
Ulsterbus service 338E (Newry City Centre - Camlough) serves the hospital from Newry Bus Centre. Click here for the timetable.
Getting to hospital by car
Daisy Hill Hospital is well signposted from the A1 bypass and Newry City Centre.


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- © 2015 American Society of Plant Biologists. All Rights Reserved.
Fluorescence-based protein-protein interaction techniques are vital tools for understanding in vivo cellular functions on a mechanistic level. However, only under the condition of highly efficient (co)transformation and accumulation can techniques such as Förster resonance energy transfer (FRET) realize their potential for providing highly accurate and quantitative interaction data. FRET as a fluorescence-based method unifies several advantages, such as measuring in an in vivo environment, real-time context, and the ability to include transient interactions as well as detecting the mere proximity of proteins. Here, we introduce a novel vector set that incorporates the benefit of the recombination-based 2in1 cloning system with the latest state-of-the-art fluorescent proteins for optimal coaccumulation and FRET output studies. We demonstrate its utility across a range of methods. Merging the 2in1 cloning system with new-generation FRET fluorophore pairs allows for enhanced detection, speeds up the preparation of clones, and enables colocalization studies and the identification of meaningful protein-protein interactions in vivo.
Two technological advances allowed the field of modern cell biology to emerge: the development of high-resolution microscopes and the groundbreaking work in the development of fluorescent proteins (FPs) that were originally isolated from jellyfish (for review, see Day and Davidson, 2009; Zimmer, 2009). Genetically encoded FPs can be fused directly to proteins of choice, offering insights into their functional properties by allowing researchers to monitor subcellular localization, chemical environment, interaction, movement, and/or turnover rate.
Research on the FPs themselves has exploded in the past two decades, and a range of technological improvements to and variations of the FPs were generated. These improvements include novel spectral properties from all areas of the color palette and from a diverse range of organisms. They also extend to a multitude of parameters, such as brightness, folding efficiency, chromophore oxidation rate, quantum yield, pH, and photostability (Day and Davidson, 2009). Taken together, these optimizations further the possibilities of available methods or prompt the development of new techniques.
In 1946, the German scientist Theodor Förster laid the theoretical foundation for resonance energy transfer, now known as Förster resonance energy transfer (FRET; Forster, 1946). According to this theory, a donor chromophore in an excited state can transfer the excitation energy to a nearby acceptor chromophore via dipole-dipole coupling without the emission of a photon (for review, see Clegg, 2009; Ishikawa-Ankerhold et al., 2012). FRET depends on the spectral overlap between the emission spectrum of the donor and the absorbance spectrum of the acceptor chromophores. It also depends on the fluorescence quantum yield and the excited state lifetime of the donor in the absence of an acceptor as well as the relative orientation of the two chromophores. Most importantly, FRET efficiency obeys an inverse sixth power law as a function of the distance between the donor and acceptor fluorophores. Thus, FRET is most effective for chromophores separated by distances of less than 10 nm, distances typical for proteins that are closely packed in multiprotein complexes. Accordingly, two interacting proteins in close proximity, which are labeled with an appropriate donor-acceptor FP pair, will generally give rise to a strong FRET interaction, whereas proteins that are located in the same compartment but that do not interact will exhibit little or no FRET activity (Peter et al., 2014). FRET has been used not only as a tool to study protein-protein interactions (PPIs) but also as a sensing principle of small molecule sensors for the determination of a number of cellular biochemical parameters such as calcium concentrations (Miyawaki et al., 1997), changes in membrane voltage (Tsutsui et al., 2008), and hormones (Jones et al., 2013).
Recently, we developed a Gateway-based 2in1 cloning technique that allows the simultaneous recombination of two genes of interest into two independent expression cassettes on the same vector backbone (transfer DNA [T-DNA]). Including an additionally expressed red fluorescent protein from this T-DNA, the system allows the application of a ratiometric bimolecular fluorescence complementation (rBiFC) assay, significantly enhancing the credibility of results compared with classical split-FP-based vector systems (Grefen and Blatt, 2012). This system has since been successfully employed to analyze interactions of a range of different proteins (Karnik et al., 2013b, 2015; Perrella et al., 2013; Hachez et al., 2014; Lipka et al., 2014, Zhang et al., 2015).
Here, we have merged the 2in1 cloning system with state-of-the art FPs for optimizing FRET and improving (co)expression. We demonstrate the technological advantages of using a 2in1 T-DNA over conventional two-plasmid transfection procedures. The new vector sets allow for transient as well as stable expression of FP-tagged proteins in plants. Colocalization of proteins as well as their potential interaction through FRET measurements, such as acceptor photobleaching (AB) and fluorescence lifetime imaging microscopy (FLIM), can reliably and easily be assessed.
The 2in1 System for FRET Applications
The 2in1 system makes use of the λ-phage recombination sites that are commercially exploited in the MultiSite Gateway system of Life Technologies (Grefen and Blatt, 2012). The rationale behind the 2in1 approach was to establish two independent, functional expression cassettes on the same vector backbone while maintaining the ease of recombination-based cloning with two different positive selection markers. This strategy thus allows for simultaneous and directional insertion of two genes into two expression cassettes flanked by individual promoters and tags, which then are expressed from the same vector backbone (Fig. 1A). As constructed, one of the cassettes is a modified version of the classical Gateway cassette. It consists of the chloramphenicol acetyl transferase and the Control of Cell Death B (ccdB) gene flanked by attR1 and attR4 sites (as opposed to attR1 and attR2 in the classical Gateway cassette). The ccdB gene is toxic for most Escherichia coli cloning strains, thereby precluding nonrecombinant clones (Bernard et al., 1994). The second recombination cassette contains the lacZ expression cassette delimited by attR3 and attR2 sites on either side. Both cassettes allow directional and site-specific integration of genes of interest via recombination from the Gateway entry vector constructs pDONR221-P3P2 and pDONR221-P1P4. The vectors designed for this work (Fig. 1, B–D) gave recombination efficiencies comparable to previously published 2in1 vectors of the rBiFC set (Grefen and Blatt, 2012). Vectors and constructs that are needed to perform 2in1 cloning are summarized in Table I.
Spectral Properties of the 2in1 FPs
Figure 2 shows the normalized absorbance and emission spectra, including the λ4-weighted spectral overlaps of mTRQ2-mVenus, monomeric enhanced green fluorescent protein (mEGFP)-mCherry, and mVenus-tagRFP pairs selected for the FRET 2in1 vectors. Exemplary pseudocolor images of the coexpression of nucleus-localized Basic Pentacysteine6 (BPC6), a GAGA motif-binding protein (Wanke et al., 2011), with itself were chosen for representation of the wavelength emission peaks of each FP (Fig. 2). Table II lists the crucial FRET-related spectral properties of the state-of-the-art FPs that were chosen. Particular attention is given to properties relevant for FRET-FLIM studies (see below), although the fluorophore pairs are also suitable for intensity-based FRET approaches and (co)localization studies. In terms of quantum yield and brightness, the donor chromophores mTRQ2 (Goedhart et al., 2012), mEGFP (Yang et al., 1996), and mVenus (Nagai et al., 2002) are the gold standards within their spectral ranges (Table II). These fluorophores also benefit from photostability, and their folding efficiency is among the highest of the latest generation of FPs (Kremers and Goedhart, 2009). Each donor can be excited with one of the three major argon laser wavelengths used in confocal microscopy, 458 nm (mTRQ2), 488 nm (mEGFP), and 514 nm (mVenus). Due to their excellent spectral overlap, mVenus, mCherry (Shaner et al., 2004), and tagRFP (Merzlyak et al., 2007) were chosen as FRET acceptors, respectively (Fig. 2). Each pair, namely mTRQ2-mVenus, mEGFP-mCherry, and mVenus-tagRFP, was used in generating four different vectors, thus allowing for the expression of all possible combinations of either N- or C-terminal FP fusions (Fig. 1, B–D). In addition, the chromophores chosen in this vector set are all monomeric, thereby avoiding dimerization or quenching due to fluorophore interaction (Zacharias et al., 2002; Zhang et al., 2002). While the FRET pairs mEGFP-mCherry (Tramier et al., 2006) and mTRQ2-mVenus (Goedhart et al., 2012) were demonstrated before (summarized by Müller et al., 2013), to our knowledge, the pair mVenus-tagRFP has not been used previously.
Dual Expression from One T-DNA
A prerequisite for PPI analysis is information on subcellular (co)localization of the proteins of choice. In spite of the reservations associated with heterologous (over)expression of genes depleted of their regulatory sequences (promoter, untranslated regions, and introns), transient or stable coexpression of two genes from a 2in1 vector is a feasible and fast way to determine subcellular (co)localization.
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins (SNAREs), which drive the final step in membrane fusion events, are often used as membrane markers due to their specific subcellular localization patterns. The plasma membrane (PM) SNARE Syntaxin of Plants121 (SYP121; Leyman et al., 1999), vesicle- and PM-localized Vesicle-Associated Membrane Protein721 (VAMP721; El Kasmi et al., 2013), and endoplasmic reticulum-localized SNARE VAMP723 (Uemura et al., 2004) were used here to assess the vector potential for (co)localization studies. Figure 3 and Supplemental Figure S1 demonstrate the use of pFRETgc 2in1 vectors to determine and distinguish the subcellular localization of the aforementioned proteins while using different transformation procedures and hosts. Figure 3 depicts the spatial differences in subcellular localization of the SNARE proteins VAMP723 and SYP121 in transient transfection of Nicotiana benthamiana leaves (Fig. 3A), Arabidopsis (Arabidopsis thaliana) leaf protoplasts (Fig. 3B), and stably transformed Arabidopsis Columbia-0 plants (Fig. 3C). In addition, Supplemental Figure S1 shows partial colocalization of SYP121 with VAMP721 in the PM while also highlighting vesicular structures that are solely tagged with GFP-VAMP721 in all three transformation procedures. Line histograms reveal the high spatial resolution providing information on each fusion protein's subcellular (co)localization or its distinction.
Despite heterologous overexpression, all gene products localize to their previously suggested, native subcellular compartments (Leyman et al., 1999; Uemura et al., 2004; El Kasmi et al., 2013). The vector set was primarily anticipated for transient studies in which the 35S promoter is still a powerful driver of expression despite its inherent silencing issues when used in stable transformants (Chalfun-Junior et al., 2003). Nevertheless, the T1 generation of stably transformed Arabidopsis seedlings was analyzed: out of 74 Basta-resistant seedlings that were screened for fluorescence signals, 10% expressed both mEGFP-VAMP723 and mCherry-SYP121 (Fig. 3C). None of the analyzed transformants expressed only one of the two FPs, confirming an expected coexpression and, thus, cotransformation efficiency of 100%. Taken together, the FRET 2in1 vectors prove their feasibility to easily assess subcellular (co)localization in both transient and stable transformation. In the latter case, the vectors avoid the time-consuming step of sequential insertion either by crossing two individually transformed lines or the simultaneous cotransformation (and T-DNA integration) of the two individual plasmids using floral dip transformation, which has been shown previously to yield only between 8% and 34% (Ghedira et al., 2013) cotransformants. Also, cosegregation excluded, the resulting heterozygous T1 lines will yield only 6.25% double homozygous T2 lines, as compared with 25% using one T-DNA (2in1 insertion).
Transient Transformation Efficiency of 2in1 Vectors Compared with Single Plasmid Cotransformation
For (semi)quantitative FRET studies, especially when they are intensity based, the accumulation of equal amounts of donor and acceptor FP fusion proteins is often ideal. Yet, no expression system can guarantee a 1:1 expression ratio of fusion proteins. Posttranscriptional or posttranslational regulation is likely to alter the stoichiometry even when the identical promoter is used.
Agroinfiltration has become a widely used technique for fast and reliable transient transformation of N. benthamiana leaves (Schöb et al., 1997). Many subcellular localization studies in transient N. benthamiana rely on coexpression of the protein of interest with a subcellular marker. While for colocalization, a 100% cotransformation efficiency is not crucial, for other methods, such as FRET, it is often best to have donor and acceptor fusion proteins in the same cell at nearly equal concentrations.
The issue of cotransformation ratios was raised in the paragraph above regarding stable transformation. Generally, literature values vary between 10% and 60% cotransformation probability for transient transformation of Arabidopsis root explants (Ghedira et al., 2013). In transiently transformed Arabidopsis seedlings using Agrobacterium tumefaciens, Marion et al. (2008) found a cotransformation efficiency of up to 70%. To our knowledge, there are no data on coexpression ratios of transient transformation of N. benthamiana leaves.
We have compared the number of cells that expressed both fusion proteins needed for FRET from either two individual plasmids or from the novel 2in1 vectors (Fig. 4). As the exemplary gene for our studies, we used nucleus-localized BPC6 (Wanke et al., 2011). To avoid the complication of comparing across different vectors, we chose to use the same 2in1 vector not only for the actual transformation of one plasmid with two expression cassettes but also for the two individually transformed plasmids that contain only one fusion protein (donor or acceptor). For vectors incorporating only one of the two fusion proteins in the 2in1 LR reaction, an E. coli resistance marker (dummy) was recombined in the unused of the two expression cassettes, thereby preventing translational read through of that expression cassette's C-terminal FP tag (Fig. 4). The resulting plasmid contained only one FP fusion protein (donor or acceptor), yet the same plasmid backbone was used for the infiltration.
We analyzed mean fluorescence intensities of nuclei from 30 different square sections (each roughly 0.084 mm2 in size) of four independently transformed N. benthamiana leaves that expressed either BPC6-mCherry, BPC6-mEGFP, or both (in pFRETgc-2in1-CC; for graphical description of the work flow, see Supplemental Fig. S2). Ratios of mCherry to mEGFP fluorescence were normalized by using the median value of all ratios as denominator for the absolute mCherry values. The box-plot diagram in Figure 4A shows ratios of 25% above or below the median in red boxes along with each value for mEGFP/mCherry signal, represented as black dots. Ratios 5 times higher (greater than 5) or lower (less than 0.2) than unity were designated as thresholds for coexpression. This analysis yielded an average cotransformation ratio for all four experiments of 77.6% (n = 1,221) for the individual and 99.3% (n = 1,099) for the 2in1 plasmid transformations. As the same promoter and backbone were used, the individual ratios also allow a rough estimate of coexpression efficiency. This suggests that, despite all potential posttranscriptional or posttranslational events that will be different for each pair of genes and fusion proteins to be analyzed, the 2in1 system allows for less variation in coexpression rates compared with individual plasmid transformation.
Values slightly below 100% coexpression in 2in1 constructs could have resulted from truncations of the T-DNA left border or from silencing of one of the two expression cassettes in individual cells. Nevertheless, the nearly complete cotransformation of both fusion proteins in the 2in1 system illustrates its advantage for assays that are based on transient expression.
Improving Intensity-Based FRET through Dual Expression
As demonstrated in Figure 4, the high variance of cotransformations makes finding cells with nearly equal expression levels cumbersome and time consuming. Naturally, the fluorescence of both fusion proteins would allow one to choose cells (in transient studies) that express both proteins to similar levels, but the temporal advantage of utilizing a dual expressing T-DNA is obvious.
In order to correlate the result of the cotransformation efficiency in Figure 4 with intensity-based FRET approaches, we performed AB experiments and measured the FRET efficiency of cells transfected with 2in1 constructs versus cells with two individual plasmids (Fig. 5). As a positive control, we again employed the nucleus-localized protein BPC6 fused to mEGFP and mCherry and its capacity to dimerize (Wanke et al., 2011). As a negative control, BPC6-mEGFP was coexpressed with nucleus-localized mCherry. Exactly as before, to avoid the complication of comparing across different vectors, the same 2in1 vector was used for individual and dual expression (see above).
For AB FRET experiments (Fig. 5), fluorescent nuclei were chosen that expressed mEGFP and mCherry at apparently equal levels (a routine that was more time consuming in the case of individual plasmids as compared with the 2in1 approach). Mean donor fluorescence intensity was measured in suitable nuclei (n = 30) before and after photobleaching of the mCherry acceptor fusion and used to calculate FRET efficiency (Fig. 5C). While the approach using two individual plasmids yielded FRET signals for the positive control sample that were significant when compared with the negative control, the values showed great variability. Contrary to that, the FRET efficiency values obtained using the 2in1 approach not only showed less variance but also resulted in higher FRET signals in general (FRET efficiency median value of 0.27 using 2in1 versus 0.11 using the conventional approach; Fig. 5C). Taken together, these results clearly show that a dual expressing T-DNA not only saves time in both preparing and performing the experiment but also allows for obtaining less variable, and hence more significant, results.
The Use of 2in1 Vectors for in Planta FRET-FLIM Studies
Quantitative as well as semiquantitative FRET-based interaction studies require for each FP a number of parameters to be optimal, such as quantum yield, oligomerization state, pH dependency, and photostability (Gordon et al., 1998; Kaminski et al., 2014). For the FRET pair, the spectral overlap, the acceptor's absorption coefficient, and the Förster distance (the distance between donor and acceptor where 50% FRET efficiency is reached) are among the most important parameters (Clegg, 2009; Ishikawa-Ankerhold et al., 2012). Intensity-based FRET techniques, such as donor dequenching through AB, are particularly sensitive to the relative concentrations of donor and acceptor, and their ratio should be kept as close to a constant value as possible (Fig. 5; Harter et al., 2012; Peter et al., 2014). Although this aspect can be tackled using the 2in1 approach, intensity-based FRET approaches are also subject to other difficulties, such as a cross talk caused by donor emission, which leaks into the acceptor channel, a direct excitation of the acceptor by the illumination source, or incomplete photobleaching of all acceptors in the microscopic detection volume (Gordon et al., 1998; Malkusch and Holloschi, 2004; Harter et al., 2012; Kaminski et al., 2014; Peter et al., 2014). These problems are circumvented by measuring FRET using FLIM. This technique takes advantage of the radiative rates of the transition from the excited to the ground state of the donor chromophore, which can be related directly to the distance between donor and acceptor fluorophores (Bücherl et al., 2014; Laptenok et al., 2014). If energy transfer to an acceptor occurs, an additional relaxation channel for the donor to lose its excitation energy opens. Accordingly, the radiative transition competes with this additional nonradiative pathway, causing the donor fluorescence lifetime to be shifted to shorter time values. This reduction in the fluorescence lifetime of the quenched donor is connected to the FRET efficiency and can be quantified, resulting in a unique value for the fusion protein pair (Harter et al., 2012; Peter et al., 2014). Although FRET-FLIM requires careful data analysis, this method is much less error prone than intensity-based FRET methods.
Figure 6 illustrates the use of the pFRETgc-2in1 and pFRETtv-2in1 vectors in measuring FRET-FLIM. The fluorescence lifetimes of donor BPC6-mEGFP with various mCherry acceptor fusions were visualized as bar charts (Fig. 6B) and as heat maps (Fig. 6C) that color code the fluorescence lifetime within the area of interest, here the nucleus. The results show that the donor lifetime of BPC6-mEGFP decreased significantly when coexpressed with BPC6-mCherry and, although slightly weaker, with an mCherry fusion protein encoding the nuclear localization signal-DNA-binding domain fusion of BPC1 (BPC1 NLS-DBD-mCherry). There is no lifetime decrease in the absence of an acceptor fusion or when BPC6-mEGFP is coexpressed with a gene coding for nucleus-targeted mCherry (Fig. 6B). These results indicate a specific homomerization of BPC6 and a heterotypic interaction of BPC6 with the DNA-binding domain of BPC1 inside the nucleus. This interaction has been detected before by yeast two-hybrid analysis (Wanke et al., 2011). The same set of proteins was also used to assess the feasibility of the mTRQ2/mVenus pair in FLIM analysis (Fig. 6, D–F), with results that virtually copy the mEGFP/mCherry study. Striking, though, is the difference in maximum donor lifetime of mTRQ2 (approximately 3.8 ns) as opposed to mEGFP (approximately 2.5 ns). This leads to higher absolute lifetime differences between positive and negative control samples, providing a broader range for quantitative and dynamic association measurements.
Comparing FLIM with rBiFC
Each PPI technique comes with a set of advantages and disadvantages when compared with alternative approaches. A comparison of FLIM with an alternative PPI technique, therefore, seems appropriate. To eliminate a bias against varying expression, we chose our previously published 2in1-based rBiFC (Grefen and Blatt, 2012) as an alternative method. An interaction between the soluble Qb+c SNARE SNAP33 and its corresponding Qa-SNAREs SYP111, SYP121, and/or SYP122 had been shown previously, mainly by using coimmunoprecipitation analysis (Heese et al., 2001; Kwon et al., 2008; Pajonk et al., 2008). To our knowledge, these interactions have not been demonstrated using either FLIM or rBiFC. As biological negative controls, we made use of the Qa-SNARE SYP21, which localizes to the prevacuolar compartment (Bassham and Raikhel, 1999) and does not cross interact functionally with SYP121 or SYP122 (Tyrrell et al., 2007). Additionally, and to use a control that localizes to the PM, we employed a truncated version of SYP121 that only contains its membrane-spanning C-terminal tail devoid of the known cytoplasmic interaction site (Qa motif; Lipka et al., 2007; Grefen and Blatt, 2008).
A direct comparison of the two methods using membrane-localized proteins revealed some differences. As expected, an interaction between SNAP33 and the three SYP1 family SNAREs was identified by both rBiFC (Fig. 7, B and C) and FLIM (Fig. 7, E and F). Both methods also reported a lack of interaction between the noncognate pair of SYP21 and SNAP33. However, SYP121, when truncated to its transmembrane domain only, yielded a significant rBiFC signal with SNAP33, whereas FLIM analysis showed no difference from the background. We suggest that this seemingly contradictory result highlights the potential for the split fluorophore to stabilize interacting proteins and thereby enhance weak or otherwise transient interactions. Qa-SNAREs are able to assemble in homomultimers as well as heterodimers with Qb+c-SNAREs, and this applies equally to SYP121 and SNAP33 (Karnik et al., 2013b). It is possible, therefore, that a weaker association with the SYP121 transmembrane domain, either with SNAP33 or with a SNAP33-SYP121 complex, could become locked through fluorophore annealing, a situation that would not arise when interactions were probed for protein complex dynamics using the FRET fluorophore pairs (Harter et al., 2012).
The vector set described here features a number of advantages compared with classical vector sets. Combining the ease of recombination-based cloning with the simultaneous insertion of two genes into two independent expression cassettes speeds up the process of preparing recombinant DNA. The resulting plasmids allow the reliable integration of both transgenes on one T-DNA into plants, transiently or stably, thereby increasing the percentage of cotransformation and enabling nearly balanced coexpression ratios.
In addition to the methodical advantages of cotransformation and coexpression efficiency, the choice of state-of-the-art FPs and their pairing for optimized physicochemical properties greatly enhance the feasibility for colocalization and challenging FRET studies. All three vector sets are equally suited for FRET-based experiments, so researchers can use the pair that fits their in-house microscope's specifications best. Nevertheless, the experiments performed in this study disclosed certain advantages of different FP pairs with respect to the applied FRET method. The mTRQ2/mVenus vector set benefits from a rather long lifetime of the FP mTRQ. A basal fluorescence lifetime of almost twice the GFP's makes it an ideal donor in FLIM experiments. The mEGFP/mCherry pair, on the other hand, is convenient in AB setups. Under our imaging conditions and in living plant cells, mCherry was very sensitive to photobleaching, an optimal prerequisite for an acceptor FP in AB experiments. This observation is contradictory to in vitro analysis of the fluorophore; however, it is known that the bleaching properties of mCherry depend on imaging modalities (Shaner et al., 2005). While the FRET pair mVenus/tagRFP does not stand out with such apparent optima, its benefit might be revealed in studying multimeric interactions. This vector set in combinatorial expression with the mTRQ2-mVenus FRET pair should enable triple FRET studies in the future (Haustein et al., 2003; Peter et al., 2014).
Creation of 2in1 FRET Vectors
Vectors were cloned using a combination of gene synthesis and classical cloning. Two different strategies were followed, one for vectors with both FPs at the C termini (CC) and one for the other tag orientations (CN, NC, and NN), as N-terminal FP fusions also carry a C-terminal epitope tag. A base vector (Supplemental Fig. S3A) was created through gene synthesis and subcloning of the synthesized cassette into the binary vector pBiFC-BB (Grefen and Blatt, 2012) via PmeI/SnaBI. This resulted in the binary vector pBBb that carries a codon-optimized (for Arabidopsis [Arabidopsis thaliana]) Basta expression cassette adjacent to the left border of the T-DNA and with four unique blunt-end restriction enzyme sites (RES; AfeI, PmlI, EcoICRI, and SnaBI) 3′ of the right border.
For the first strategy, a cloning helper vector (pUC35S-Tec; Supplemental Fig. S3B) was gene synthesized that contains the cauliflower mosaic virus 35S promoter and omega enhancer, unique XbaI-RES, 3xHA tag, unique MscI-RES, myc tag, unique NaeI-, SpeI-, PsiI-RES, and 35S terminator. This expression cassette is flanked by StuI-RES. pUC57-Tec was digested using XbaI/NaeI, and, using the 2in1 conversion vectors pUC-R3R2 and pUC-R1R4 (Grefen and Blatt, 2012), recombination cassettes were subcloned via SpeI (compatible to XbaI) and PsiI (blunt end, like NaeI). The resulting vectors pUC35S::R3R2 and pUC35S::R1R4 were used to introduce FPs at the C termini of the expression cassettes (via SpeI/PsiI). Venus and EGFP (Grefen et al., 2010) were mutated at residue Ala-206 to Lys via site-directed mutagenesis (Karnik et al., 2013a), rendering FP variants monomeric (Zacharias et al., 2002). Resulting mEGFP, mVenus, and mTRQ2 were introduced in pUC35S::R3R2, and mCherry, tagRFP (Evrogen), and mVenus were introduced in pUC35S::R1R4. C-terminally tagged R3R2 expression cassettes were excised via StuI and inserted into pBBb via SnaBI. The three resulting vectors are pFRETgc-intA, pFRETvr-intA, and pFRETtv-intA. With the exception of the mCherry expression cassette, which was PCR amplified and flanked by HpaI-RES, the R1R4 cassettes were excised with StuI and ligated into the intermediate vectors via AfeI, to yield pFRETgc-2in1-CC, pFRETvr-2in1-CC, and pFRETtv-2in1-CC.
The second strategy required another helper vector to be gene synthesized: pUC35S-Ctags (Supplemental Fig. S3C) contains the 35S promoter/omega enhancer followed by unique SpeI- and MscI-RES, a 3xHA tag, a unique NaeI-RES, a double myc tag, a unique PsiI-RES, and the 35S termination signal sequence. C-terminal 3xHA-tagged expression cassettes were created through the excision of 2xmyc tags via NaeI/PsiI and religation. The resulting vector was linearized using SpeI/MscI, and the R3R2 cassette of pUC-R3R2 was inserted (SpeI/PsiI). Via XbaI/HpaI, the PCR-amplified FPs mEGFP, mVenus, and mTRQ2 were introduced. Expression cassettes were excised via StuI and ligated into pBBb via SnaBI to create intermediate vectors pFRETgc-intB, pFRETvr-intB, and pFRETtv-intB. A C-terminally 2xmyc-tagged R1R4 cassette was generated through replacing the 3xHA tag in pUC35S-Ctags (SpeI/NaeI) with the R1R4 cassette of pUC-R1R4 (SpeI/PsiI). The FPs mCherry, tagRFP, and mVenus were PCR amplified and introduced via XbaI/HpaI 5′ of the R1R4 expression cassette. The resulting expression cassettes were excised via StuI (or PCR amplified in the case of mCherry) and introduced in intermediate vectors A and B. Also, the expression cassettes with C-terminally fused FPs were introduced into pFRET-2in1-intB vectors to generate another nine vectors: pFRETgc-2in1-CN, -NC, and -NN; pFRETvr-2in1-CN, -NC, and -NN; and pFRETtv-2in1-CN, -NC, and -NN. All vectors were verified via restriction digestion and/or Sanger sequencing. All maps, sequences, and/or DNAs are available upon request.
Gateway Cloning
Oligonucleotides for creating entry clones (Supplemental Table S1) were designed according to the guidelines in the Gateway manual (Life Technologies). PCR programs were based on a two-cycle routine using complementary DNA (cDNA) as a template: the first cycle comprises a five times repeated three-step program of denaturing at 95°C, annealing at 54°C, and synthesis at 72°C (20 s kb−1), followed by the second cycle, a 30 times repetition of a two-step routine, denaturing and synthesis, omitting the annealing step. After gel purification of PCR products, BP recombination reactions were performed as described previously (Grefen and Blatt, 2012). The 2in1 LR reaction was performed according to the supplemental data of Grefen and Blatt (2012). A molar ratio of vector to entry clones of 1:3:3 yielded optimal results as described previously (Grefen and Blatt, 2012). Colonies were selected from plates that contained 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside/isopropyl β-d-1-thiogalactopyranoside in addition to spectinomycin to distinguish partly recombined (blue) from fully recombined (white) clones. Plasmid DNA was verified via restriction digest and Sanger sequencing.
Nicotiana benthamiana Transformation
Agrobacterium tumefaciens strain GV3101 (Koncz and Schell, 1986) was used to transiently transform N. benthamiana with 2in1 FRET constructs through syringe-mediated infiltration (Schöb et al., 1997; Sparkes et al., 2006). Overnight cultures of agrobacteria were used to inoculate fresh Luria-Bertani medium (supplemented with 50 µg µL−1 rifampicin, 20 µg µL−1 gentamycin, and 100 µg µL−1 spectinomycin) to an optical density of 0.01 and grown for another 4 to 6 h up to an optical density at 600 nm of 0.5 to 0.8. Agrobacteria were harvested, washed in sterile water, and resuspended in ice-cold AS medium (10 mm MgCl2, 10 mm MES-KOH, pH 5.6, plus 150 μm acetosyringone; Grefen et al., 2008; Blatt and Grefen, 2014). After an incubation time of 30 to 60 min on ice, leaves of 3- to 4-week-old N. benthamiana plants were infiltrated as described previously (Schöb et al., 1997; Sparkes et al., 2006; Blatt and Grefen, 2014). Leaves were subjected to CLSM analysis 2 d post infiltration.
Arabidopsis Leaf Protoplast Preparation and Transfection
Leaves of 4-week-old Arabidopsis plants (Columbia-0), grown under standard greenhouse conditions, were perforated abaxially and incubated in TEX buffer (Gamborg's B5 salts, 500 mg L−1 MES, 750 mg L−1 CaCl2∙2H2O, 250 mg L−1 NH4NO3, and 0.4 m 13.7% [w/v] saccharose, pH 5.7), supplied with 1.5% (w/v) cellulase and 0.4% (w/v) macerozyme, overnight in the dark. After incubation, the cells were gently released, collected, and centrifuged at 100g. Intact cells, floating on top of the solution, were washed with W5 buffer (154 mm NaCl, 125 mm CaCl2, 5 mm KCl, and 5 mm Glc, pH 5.7).
Transfection of protoplasts was carried out using the small-scale transfection method described previously (Berendzen et al., 2012) with the following modifications. MMg (0.4 m Mannitol, 15 mm MgCl2, 4 mm MES, pH 6) was replaced by MM medium (0.4 m mannitol and 5 mm MES, pH 6), and the pH of the polyethylene glycol solution was decreased to pH 6. The concentration of cells was adjusted to 6.6 × 10−6 cells mL−1 with MM medium, and 2 × 10−5 cells were used per transfection subsequently. For the transfection, 4 µg of the respective plasmids were used according to Berendzen et al. (2012).
Arabidopsis Stable Transformation and Selection
A. tumefaciens strain GV3101 carrying the respective plasmids was precultured overnight at 28°C in 5 mL of Luria-Bertani medium supplied with antibiotics. A total of 200 mL of Luria-Bertani medium (including antibiotics) was inoculated with 200 µL of preculture and grown overnight again. Cells were pelleted and resuspended in 200 mL of infiltration medium (5% [w/v] Suc, 0.01 Silwett, and 10 mm MgSO4). Inflorescences of 5- to 6-week-old Arabidopsis plants were dipped into the suspension according to the floral dip method (Clough and Bent, 1998). The procedure was repeated after 1 week. T1 seeds were grown on soil and treated 7 d post germination with the herbicide Basta to select for successful transformants.
CLSM, AB, FLIM, and rBiFC Analyses
All CLSM, AB, and FLIM measurements were performed using a Leica TCS SP8 confocal microscope (Leica Microsystems) equipped with a FLIM unit (PicoQuant).
Images for Figures 2, 3, 5, 6, and 7 were acquired using a 63×/1.20 water-immersion objective. The rBiFC analysis was performed using a 40×/0.75 water-immersion objective. For excitation and emission of FPs, the following laser settings were used: mTRQ at excitation 458 nm and emission 465 to 505 nm; mVenus at excitation 514 nm and emission 520 to 560 nm; mEGFP at excitation 488 nm and emission 495 to 530 nm; mCherry at excitation 561 nm and emission 580 to 630 nm; tagRFP at excitation 561 nm and emission 580 to 630 nm.
AB was performed using a bleaching routine with the 561-nm laser (mCherry) line at 100% intensity and 10 frames. Data derive from two different biological replicates and measurements of up to 15 nuclei for each replicate. The mean FRET efficiency as well as the se values were calculated using Microsoft Excel.
FLIM data derive from four different biological replicates and measurements of up to 10 nuclei for each replicate. To excite BPC6-mEGFP for FLIM experiments, a 470-nm pulsed laser (LDH-P-C-470) was used, and the corresponding emission was detected with a FLIM-compatible photomultiplier tube from 495 to 530 nm by time-correlated single-photon counting using a Picoharp 300 module (PicoQuant). Each time-correlated single-photon counting histogram was reconvoluted with a corresponding instrument response function and fitted against a monoexponential decay function to unravel the GFP fluorescence lifetime of each nucleus. The average GFP fluorescence lifetimes as well as the se values were calculated using Microsoft Excel.
The measurements for rBiFC were performed as described previously (Grefen and Blatt, 2012). Data derive from four biological replicates with 20 different leaf sections each. Exemplary images define regions of interest for which mean fluorescence intensity was calculated and ratioed.
Vector Maps and Sequences
Vector sequences and maps can be downloaded at http://www.psrg.org.uk and http://www.zmbp.uni-tuebingen.de/dev-genetics/grefen.html and are available upon request through these Web sites.
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Colocalization analysis of mEGFP-VAMP721 with mCherry-SYP121 using different transformation approaches.
Supplemental Figure S2. Workflow to determine coexpression data of Figure 4.
Supplemental Figure S3. Maps of base vectors used to assemble the pFRET-2in1 vectors.
Supplemental Table S1. Oligonucleotides used for entry clone design and site-directed mutagenesis of FPs.
We thank Volker Lipka and Kenneth Berendzen for providing Venus and mCherry cDNAs; Dorus Gadella for providing the mTRQ2 cDNA and spectral data; Sabine Müller for valuable comments regarding figure preparation; and Lisa Asseck, Dietmar Mehlhorn, and Eva Schwörzer for technical support.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Christopher Grefen (christopher.grefen{at}uni-tuebingen.de).
A.H. and N.W. performed the experiments and analyzed the data; N.W. assisted C.G. with the cloning; S.P. and A.H. analyzed the spectra and helped with the data analysis; M.R.B. and K.H. helped in designing the experiments and assisted in writing the article; C.G. created the 2in1 vectors, designed and performed the experiments, assembled the figures, and wrote the article; all authors read and approved the final version of the article.
↵1 This work was supported by a Carl Zeiss Fellowship (to A.H.), by the Biotechnology and Biological Sciences Research Council (grant nos. BB/H0024867/1, BB/I024496/1, and BB/K015893/1 to M.R.B.), and by the Deutsche Forschungsgemeinschaft (grant no. SFB1101 to K.H. [project D02] and an Emmy Noether Fellowship to C.G. [grant no. GR 4251/1–1]).
↵2 These authors contributed equally to the article.
↵[OPEN] Articles can be viewed without a subscription.
- fluorescent protein
- Förster resonance energy transfer
- protein-protein interaction
- transfer DNA
- rBiFC
- ratiometric bimolecular fluorescence complementation
- acceptor photobleaching
- fluorescence lifetime imaging microscopy
- plasma membrane
- cDNA
- complementary DNA
- confocal laser scanning microscopy
- Received April 10, 2015.
- Accepted May 12, 2015.
- Published May 13, 2015.


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Actifio solves data explosion blues
Data virtualisation makes light of data backup, recovery complexities
If you have not heard of copy data virtualisation, you are not alone.
While there's a high degree of virtualisation in the IT industry today especially at the server and network level, the situation is less so in the backend -storage, backup or archiving. The reason for this, notes Grant Amos, general manager, MEA, for Actifio, is because data virtualisation is simply more complicated.
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Amend Your Ring
- 'At Commins & Co Jewellers we have over 600 rings set with certified diamonds.
- All of our rings are available in all precious metals
- We can change all centre diamonds in terms of size and quality to suit your budget
- All diamonds can be amended to any gemstone
- All rings can be made for your finger size
- We can redesign your ring with our CAD system and in house designer
Free Sonic Cleaning
- All our rings come with free sonic cleaning
- Cleaning takes 2 minutes
Free Independent Valuation
- All our engagement rings come with a free independent valuation valued at over €100
- It is provided by one of Irelands leading gemmologists
- Ideal for insurance purposes.
- We also provide a Commins & Co Jewellers internal valuation for insurance purposes which we can update annually at no charge
Categories: Antique Engagement Rings in Dublin by Commins & Co Jewellers, Engagement Rings in Dublin from Commins & Co Jewellers, Rings under €2,500, Side Stone Diamond Rings at Commins & Co Dublin.Tags: antique engagement ring, antique engagement ring dublin, engagement, engagement rings, engagement rings dublin, engagement rings ireland.


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Call for info and advice: 07781 409060. Shop open Saturday 12 – 4 pm
First club dive of 2019 on Zulu location TBC meet St Sampson marina ROPES OFF @ 15:00 , we have been working hard on her for the last couple of months for your enjoyment and installed a really nice new chart plotter to play with. There is still a lot I want to do yet but we have certainly made a great start with a complete service and replacement shaft seal.
We have had to increase the cost per dive to £15 as we have not had an increase for the last 10 years. click here if you need to hire kit.
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If you are an aspiring model, chances are, you want to look gorgeous in your photos every time. If you are scheduled for a photoshoot, or you simply want to look your best each time your photo is taken, there are some helpful tips and tricks you can try.
- If you want to avoid flashing a grin that's too wide, try putting your tongue behind your teeth to achieve that photo-worthy smile.
- A frizzy hair can have a great impact on the overall result of the photograph. You can apply a bit of shine spray or shine serum to make your hair look better.
- While the majority of professional photographers will most likely adjust to take your photograph at a better angle, it would be better if you know what your best angles at, yourself. You may practice taking your photographs at home to determine the perfect angle that will show all your beautiful features.
- Keep in mind that photographs taken from an elevated position would always look better than photos taken from a lower stance. So adjust your poses accordingly.
- Make sure to wear just the right makeup. A light makeup that would just enhance your natural beauty is best.
- Relax. A natural pose or posture would look better. In addition, physical tension and rigidness would be easily visible on the photograph.
- Choose the right pieces of clothing. You may want to steer clear or printed design that is too loud or distracting as they may have an overall effect on your photograph.
- While having your photos taken, look for a good source of light and turn your face slightly toward it to highlight your facial features.
- Remember that your eyes can play a significant role in the result of your pictures, so if your eyes look tired, use some eye drops to make your eyes look better.


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Download VIDEO: Wizkid ft Mut4y - Manya MP4 & 3GPFollow @Wapbaze_Com
VIDEO: Wizkid – Manya :Wizkid and MUT4Y of the Legendury Beatz drop the visuals to the fast rising smash 'MANYA" which is a first solo project for MUT4Y as a DJ/Producer.
The video was shot by 'Director Q" at the popular NEW AFRIKAN SHRINE with many special guest appearances. The song is Produced by Killertunes.
Download And Enjoy...
|Mon 20 Nov 2017
VIDEO: Wizkid ft Mut4y - Manya
VIDEO: MUT4Y x Wizkid - "Manya"
VIDEO & MP3: Wizkid ft Mut4y - Manya
Wizkid ft Mut4y - Manya
MP3: Wizkid & Mut4y - Manya
VIDEO: Wizkid - Manya
Wizkid - Manya


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Commercial Cloud Demo Center
Veeva CRM offers a seamless experience across all devices, enabling reps to get real-time insights and notifications to drive more meaningful HCP interactions. See how reps can leverage Veeva CRM for iPhone to be more productive on the go.
Multichannel Customer Engagement: Powered by Veeva Commercial Cloud
Delivering truly coordinated, multichannel engagement with healthcare providers is now a reality in life sciences. Veeva Commercial Cloud brings together customer data, compliant content and interactions across all channels to make multichannel communications seamless for you and your customers.
Veeva CRM Approved Email: Deliver Relevant and Timely Content
Veeva CRM Approved Email enables sales representatives to maintain a personalized, compelling dialogue with all customers, including no-see HCPs. It ensures that the latest, compliant information is presented and tracks every interaction for complete visibility. Reps are more effective and empowered to provide physicians with the content they want — when they most need it.
Key Account Management Teams. Empowered.
Take a look at how Key Account Managers (KAMs) and Sales Reps can work together on the same system while viewing only the data that's most relevant to them. Click the video below to begin. Please note the video may take a moment to load. Enjoy!
Veeva CRM Mobile: Improve Face-to-Face Interactions
Veeva CRM Mobile combines the power and capability of Veeva CRM with CLM in a single mobile application on the iPad. See how a primary care sales rep easily plans a call, delivers a CLM presentation, and automatically generates a call report.
Compliant Content & Digital Asset Management
See how Vault PromoMats accelerates your review and distribution of compliant content and enables global reuse with built-in digital asset management.
Vault PromoMats Auto Claims Linking
See how Vault PromoMats Auto Claims Linking enables you to automatically generate compliant reference links when reviewing promotional claims.
1:40 - Veeva Nitro: the Next-generation Commercial Data Warehouse Demo
Veeva Nitro delivers advanced analytics to the home office and embedded insights for field teams.


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Bainbridge Community Foundation
2017 Grants Report
Bainbridge Community Foundation awarded a record $269,887 to local nonprofit organizations through its 2017 Community Grants Cycle. Sixty-two organizations received funding in sectors including arts and culture, animal welfare, education, environment, health and human services, and recreation. This is the most dollars and the largest number of grantees ever awarded by BCF.
BCF has seen dramatic growth in its Community Grants Cycle, doubling annual grants by total dollars in just five years. In 2013, 49 local agencies received a total of $139,000 for an average grant of $2,800. 2017 saw an average award of $4,353, even as BCF funded a record number of organizations.
The Community Grants Cycle has made $1.84 million in local grants since the program began in 2005, providing support for specific programs, capital projects, as well as general operations of local nonprofit organizations.
After designing BCF's annual report for 2016, Mad Dog produced 2017's Grant Report using a similar visual style to establish baseline brand continuity. With a bright color palette and a focus on the surrounding community, MDG was able to showcase many of the grantees and their activities and contributions through the photos that were chosen for the report. "At-a-Glance" callouts help readers to quickly drill down to important facts and information.


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Horse Lover's RingLink
Ring homepage
25 sites out of 98
We're a high country ranch located west of Pikes Peak. We have American Bashkir Curly horses, American Quarter Horses, some grade horses and will be breeding Mini Australian Shephards.
my ponie & horse riding
Platinum Stables
Platinum Stables is a full service facility. A LARGE selection of horses & ponies always available for sale or lease. Registered welsh and half welsh foals are ready to go! Call today to see whats available! Make sure to check out our stallions!!!
Rocking G Riding Club
Site is for all Horse Lovers Anywhere. Meet new friends, start up your own riding group in your area. All Free, Free Classifieds, Free Games, advertise your horse site. Decorate your own web page. Come join in the Fun.
Buck Creek Fjords
Western themed products and gifts for the family that loves horses. Norwegian Fjords are the main horse of choice at our Buck Creek Horse Ranch. Come visit and see pictures of our wonderful Fjord horses.
My Florida Horse
All things horse in Florida including the best searchable horse event schedule.
WebRings for horse, cattle, ranch and farm websites. Your site must be a ag related ranch or farm homepage.
Ranch Links
Thousands of horse and ranch links!
A site for Horse Lover's!
Pep's Welsh Ponies
Breeders of Section A Welsh Ponies in East Texas. Top bloodlines in the U.S.
Lee's Arabians--Heritage Breeders for 50+ Yrs
Breeders of quality *Witez II and *Raffles Arabian horses. We stand three stallions, bringing the legendary bloodlines into today's Arabian horse. Breeding for substance and athleticism, and a willing, tractable temperament, are our top priorities!
Horse Pages
Thousands of horse related links and free classifieds!
Cream's Sketch Book
My website is about my love to drawing, horses, Arabians, Natural Horsemanship and more!
Horses, Graphics, music, Romancing time's son, Barrtime Romance! A great place for horse peope,
CTC Farm Quarter Horses
Our breeding program features performance bred quarter horses with the lines of Peppy San Badger, Doc O'Lena, Docs Lynx, Docs Prescription, Sonita's Last, and many more. Our stock is registered with AQHA, FQHR and NFQHA.
Ecovolunteer for horses
Join for an active holiday in hands-on participation in research and conservation on horses.
Shields Quarter Horse Farm
Located in NE Texas, specializing in foundation bred palominos, buckskins and duns. Offering stallions at stud, bred mares and other horses for sale. Pictures and info on all horses.
Equestrian Sport Photography-Photos by PD
Farm call equine photography, portraits, at liberty, conformation. Endurance ride photography. Horse shows in the Dayton, Ohio area.
Dockery Stables,Always Walkin' Horse Locators
All Breed Horse Locator Service, specializing in Gaited Horses.
Shilo Farms
The Joy Is In The Ride! We are totally dedicated to the equine profession, and look forward to helping you with all your horse needs.
Vineyard Equus's - The Stable Shop
Horse Tack at Wholesale Prices.. Horse Tack, Gifts Items, Bath and Body items, Horse related items. And more...
We are a family owned horse/cattle breeding ranch located in Blue, Grass, Ia. Heading our horse program is our stallion "Shinin Freckle Spark"
High Point Farm, Inc.
High Point Farm is a Full Care Equine Boarding Facility. We offer excellent care as well as activities for our clients. Nice selection of horses for sale and tack for sale.
Four Nickels Miniature Horses
AMHA/AMHR Top Quality Miniature Horses. Breeding, showing and selling since 1995.
Photo's and art work of the most beautiful horses, Arabian's Pinto's and there is always something new.
Previous 25 sites
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15.2.2019 – 27.4.2019 Exhibition: Plant+HumanAustralian Centre for Photography
Steven Cavanagh | Liss Fenwick | Phillip George | Hayley Millar-Baker | Polixeni Papapetrou | Pony Express | Merri Randell | Diana Scherer | Erica Seccombe
15 February – 27 April 2019
Tuesday – Friday 10 am to 5 pm
Saturday 11am to 4pm
Closed Public Holidays
ACP Project Space Gallery
21 Foley Street
Darlinghurst NSW 2010
Exhibition Open Event
Thursday 14 February 6-8 pm
For many the intimate connection between humans and the botanical has often been assumed, overlooked or not even considered. For others, plants are revered and appreciated through the filter of cultural beliefs and practices. The artists in Plant + Human approach their vegetal subjects with intensity, exploring both our historical and contemporary entanglements with ecology.
The performance-driven work of Pony Express assumes an equality in the plant–human relationship that importantly considers plants as sentient beings. While, Merri Randell's Touching the Mauri (2018) acknowledges heritage and wellbeing through old growth trees.
Our inter-species relationship is formalised in the aesthetics of Erica Seccombe's series Permutations (2018). Phillip George, Steven Cavanagh and Polixeni Papapetrou each consider flora as a cultural symbol of life and death, beauty and abundance. While the photography of Hayley Millar-Baker and Liss Fenwick explore the politics of place, plants and human habitation.
Through art and nurture, Diana Scherer has created delicate laces from root fibres, designed to wrap or clothe the human body. It seems nature has trained our understanding of existence more than we realise.
Image: Pony Express, Exosex pinup boy #1, 2016


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Tudor and Stuart Women Writers
Tudor and Stuart Women Writers
"... a nuanced, carefully argued work that reveals how women writers of the Renaissance, whether upper-class aristocrats close to court, daughters of successful merchants, Protestants, or Catholics, are inevitably affected by the gender biases that infuse all levels of Renaissance society and letters." -- Sixteenth Century Journal
"... quite effective at developing a critical vocabulary for analyzing the formal traits of early modern women's writing." -- Tulsa Studies in Women's Literature
From the perspectives of feminism, Marxism, sociology, and cultural semiotics, Louise Schleiner examines both familiar and obscure Tudor and Stuart women writers in a comprehensive study of those women who managed to go beyond translations or diaries and find a more individual voice in their public texts.


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This membership is designed for traders wanting to improve their results and consistency by learning how to be adaptive to the market through utilizing principles such as market analysis, technical analysis, and implied volatility analysis to make quality risk/reward/ probability decisions increasing probability and lowering risk while taking advantage of exciting opportunities to exponentially grow their returns.
Monthly Subscription $249.00*
Membership enrollment is open for a very limited time!
Enrollment Bonuses:
- Early access to the live Market Outlook sessions starting this Sunday, March 22nd at 9 PM ET.
- Early access to the live Options Trading for Income by the Guidelines sessions starting this Monday, March 23rd at 9:30 AM ET.
- Access to the live and recorded Trading Performance session on March 25th at 1 PM ET. This months topic will definitely be a session you will want to go back to help you propel your trading to the next level!
- Complimentary access to the soon to be released Enriching Session called "How To Best Utilize Calendar Spreads". You will be able to keep the recording of this session to refer back to as long as you remain a PRO member.
Our Pro level membership is all-inclusive. You'll experience views from multiple coaches and other top traders as they go through their quality decision making processes during live sessions. All different types of options trading strategies are welcome as we discuss how to flow trade strategies and improve their probabilities in the current state of the market. This is for traders who take their trading seriously. They are Pros! So, we will give them every possible resource that we have at our disposal towards their trading success.
Nothing is off limits. It is the best of the best. All of our monthly membership offerings are included.
It is all here:
|Access to live biweekly hour Trading with the Pros sessions hosted by our professional trading coaches with recordings of those sessions ($200 value)
|Access to live weekly Market Outlook sessions with recordings of those sessions($49 value)
|Access to live monthly Trading Performance sessions with recordings of those sessions ($99 value)
|Annual Trader Check-Up with John Locke for those who have been a Pro member for at least 12 consecutive months ( $250 value)
|Private PRO Trading Skype group for subjective traders to discuss market movements and adapting trades
|Access to the private PRO forum to post questions for prepared, detailed answers by a professional trader.
|Guidelines Options Trading – GO Membership ($99 value)
|Learn THREE different trading strategies: The Bull, Quick and Dirty RUT Broken Wing Butterfly, and The Super Bull
|Access to the entire Fundamental In Trading (FIT) library with over 25 videos, podcasts, templates, and bonus content
|Access to the Fundamentals In Trading (FIT) dictionary to help clarify trading terms, jargon, and acronyms
|Access to GO Ask A Trader sessions once a month with a professional trader who will be there to provide detailed, specific answers to your trading questions.
|Access to the private GO forum to post questions for prepared, detailed answers by a professional trader.
|Access to live the Options Trading for Income Weekly Webinar
|Immediate access to over 720 recorded episodes since 2011 of the Options Trading for Income Weekly Webinar (180 months of webinars at $40 = $7,200 value)
|Access to trade logs of the trades that are reviewed during the Options Trading for Income Weekly Webinar since 2015 (44 months at $20 = $880 value)
|Immediate access to over 40 Successful Options Trader of the Month videos
|Access to bonus, live and recorded webinars by exceptional, experienced professionals
|Automatic registration to public webinars hosted by Locke In Your Success, LLC
|Member only resources to include sample trading plans, tips & tricks for your analytical software
|Private GO Trading Skype group for guideline options traders to keep the discussions solely focused on guidelines
|Email notifications with a new Winning Trade episode when it is released with a bonus video
|Email notification every time a new Trading Performance Podcast is released
|20% discount on one-on-one coaching sessions (A savings of up to $500 off per purchase)
|60% discount on Enriching Sessions (Regular cost $125 for Pro members each session is just $49)
|Trade journal subscription discounts with templates specific to our trades but can also be customized.
Monthly Subscription $249.00*
Membership enrollment is open for a very limited time!
*All subscriptions will begin on the 1st of the month. Subscriptions can be canceled by email at any time. However, cancellations must be received via email by the 20th of the month for the cancellation to occur by the 1st of the following month.


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Us And Our BrandsFully Authorised Lelli Kelly StockistSuperfit And Gore-Tex, Guaranteed Waterproof FootwearBrand New & Exclusive Lelli Kelly Shoes - 'California Lights'Bobux Pre-walkers and First Walkers - Supporting What Nature IntendedFrom Toddlers to Teenagers - Designer Shoes from Hop, Skip & JumpBaby Shoes - For those First Precious StepsThe Latest School Shoe Designs from Lelli KellyP.E. Pumps - Fitted for SafetySchool Shoes & Summer Sandals - Shipping Sorted!
Ricosta Diddi Beige Sandal
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This page lists additional sources of information on
lowland grassland issues, partnerships and initiatives.
See also management of UK lowland grassland habitats and
JNCC Grassland publications.
Please email any recommended additional links to:
email address: comment_[AT SYMBOL]_jncc_DOT_gov_DOT_uk (replace _DOT_ with full stop/period and _[AT SYMBOL]_ with the usual @ symbol)


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Status: Closed
Know more about this business than we do? Cool! Please submit any corrections or missing details you may have.Help us make it right
Be the first SILive.com user to add photos or videos of B & W Automotive
Now here's the good old fashioned service shop where you actually speak with the same person, they know your car and they remind you of services when needed! They are reliable,honest,clean workers and will try hard to accomodate your schedule. Best of all they are really good mechanics. I have gone here for the past 15 years with several family vehicles and no complaints. Because they have...
B & W Automotive is located at 21 Amboy Ave, Metuchen, NJ. This business specializes in Auto Repair & Service.


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Bell of Fortune, developed by Play N' Go is a five reel slot with only one row and only the one payline; the game is designed to look like a classic arcade-style slot machine with tubular reels for that sense of authenticity, and there's the pay table on the right-hand side where all the pay out and wins information is stored. Located above the pay table is the current progressive jackpot total which players will need to keep close attention on.
Bell of Fortune delivers players a classic treat with a delicious and tasty selection of fruity icons, including juicy oranges and plump plums, plus some strawberries. Also, Bell of Fortune has a golden bell (as the game's name suggests) that acts as the game's most lucrative prize paying symbol. Players should not expect significant explosive action and mind-blowingly realistic 3D graphics when playing this game. This is a classic retro-style slot game.
Players are able to take advantage of three coin levels betting their stakes; however, they should note that to obtain the highly lucrative progressive jackpot they will need to be playing at the maximum coin level to stand any opportunity of winning the valuable progressive jackpot. To achieve the cash prize, players will need to land at least three golden bells on the one payline.
Bell of Fortune is indeed a slot for those who appreciate something a little more simplistic, but it does also have the gold-plated progressive jackpot that keeps ticking over and thus players engaged. However as explained, to maintain this level of excitement – players must not forget to bet at the maximum coin level available.
There are tonnes of classic slot machines on the internet, the plethora of choice is gargantuan and deciding which one to play can be a headache itself. However, if players want a retro three-reel game that is perfectly pleasant on the graphics front and that has the ability to pay some substantial progressive wins, then this slot machine is indeed worth a punt.
Words: David Bailey-Lauring
Images: Bitcasino
Login here and have fun with our top games!
Don't have an account yet? Get one now!


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Explore Basic Concepts with these fun Dinosaur themed activities!
60 Pages of Preschool Fun!
In this pack they will count to 12 with counting clip cards, build 4 and 5 piece puzzles, sort shapes and types, and much more!
What is inside:
- Colors
- Prewriting Skill Builders
- Puzzles
- Counting
- Hunt and Find
- Size Sorting
- and much more!
My son helped me create this clip art and I have been so busy that I hadn't had a chance to use it. I decided it was time. He did such a great job, I needed to share it! I hope you all enjoy this pack as much as we did making it!
Suggestions, requests, questions, corrections, or comments? Use our worksheets or activities?
Send a picture to be featured in a future post! Email to: email@example.com
This download is mostly color and free to use for any daycare, church, school, personal, co op, or home school use. We all make mistakes sometimes, if you happen to find one of mine feel free to email me and let me know so I can correct it!
TERMS OF USE: All activity images, printables, and games are free to download and/or use as you choose, with the exception of claiming as your own, modifying, or redistributing as your own. Please feel free to use in any classroom setting! Feel free to link to this page but not to the download or image itself, if you would like to share downloads or ideas. All shares are appreciated. Permission to use 1 image of your choice is given. Thank you for respecting these guidelines.


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Critical Care Unit Practice Areas
Not all critical care units are the same, nor do they all treat the same kinds of patients with the same conditions. There are several practice areas within the critical care world that a nurse can choose to pursue. Each practice area unit has its own unique patient base and specialties, which is an important consideration for nurses wishing to pursue a career in critical care. A nurse can choose to work in the traditional ICU, or work in the specialized PICU and NICU. Learn more about each practice area below.
The infographic below explains what critical care units are, as well as the different types of practice areas within critical care units. Critical care units are different areas in a hospital that focus on trauma patients in critical condition. Patients in these areas often need full attention because of the intensity of their complications. These care units are divided into smaller units known as the ICU, PICU, and NICU. Patients in the ICU are usually adults over the age of 18, while the PICU cares for children up to age 17. The NICU treats newborns with health complications, usually as a result of premature birth.


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For those of you who don't know us, the Fair Custodian team consists of four people who share a passion for data. We are tired of how personal data is processed and shared without the person's explicit consent. We possess the skills and experience to be able to redefine how personal data is handled during everyday interactions online. So we created Fair Custodian and we've been fortunate enough to be accepted on the world renowned Ignite pre-accelerator programme, based in our home city of Newcastle.
Finding out what people think about Privacy
We recently published a survey about data privacy (this one to be precise). We've laid out the results below for you to explore and we've now also created another one, which we hope you'll fill out too (there's a link at the bottom of this post)! Complete it and you can help us deplete our bank account for some good causes.
What people have told us
The first stage of our work was to understand how people feel about their data, how companies use it and what they actually do to change things. The scariest thing we came across from the survey was the lack of password managers, and that people were quite willing to actually share how they built their password (e.g. mother's maiden name + date of birth), despite us not asking! The most interesting thing we found was the extreme lengths people would go to create new emails when signing up to services rather than use their existing email addresses.
Our first survey was the initial step towards creating an ecosystem that will allow everyone to safely manage access to and the sharing of their personal data. The next step we're taking is to launch a beta of our first product. We support the work of a large range of privacy advocacy organisations (Open Rights Group & EFF to name two) but we also understand that the wheels of industry must keep turning. People will always be making cool, new stuff and other people will always want to buy it. Connecting those two groups of people requires the transfer of personal information and we want to make sure that happens in the most fair, transparent, and secure way possible. We've planned a long term roadmap of technologies and services; which include machine learning backed intelligent email management for individuals, single sign-on authentication for e-commerce platforms, and the creation of a marketplace where people can share data and directly benefit when it's sold. That said, we do appreciate the need to start small and we'd like you to help us shape our first product.
Launching the beta
This product, enables you to register for 3rd party sites and services using a disposable, private email address instead of sharing your personal one. You can create as many of these disposable email addresses as you wish. Our platform will receive any messages sent to the disposable accounts and you can access them via our website without them clogging up your personal inbox.
You can sign up for the beta here.
Jake, Ben, Saul, and Jay
Fair Custodian is building a platform to create a new type of relationship between consumers and businesses. A relationship based on trust, transparency, and personal empowerment. To find out more about us, check us out at www.faircustodian.com.


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Discover a Career - What the future holds for positions in underbalanced drilling.
Despite the drilling industry's shift toward managed pressure drilling (MPD) over the past half-decade, underbalanced drilling (UBD) continues to be a desirable option for many operations today, especially when drilling into pressure-depleted formations and/or attempting to reduce skin damage for better productivity. Because of this sustained demand, largely by operators willing to pay top dollar for skilled UBD personnel, a fundamental knowledge of the technique can benefit young petroleum engineers looking to excel in their field.
UBD Overview
The philosophy of UBD is diametrically opposed to traditional/conventional drilling methods and MPD. Conventional drilling uses an equivalent mudweight (EMW) that results in a bottomhole pressure (BHP) greater than that of the pore pressure exposed to the open hole to prevent formation fluids (oil, gas, or water) from entering the wellbore. MPD uses equivalent circulating density—a combination of hydrostatic pressure, circulating friction pressure, and applied surface pressure—to create a BHP equal to or greater than formation pore pressure.
Looking for more?
Some of the OnePetro partner societies have developed subject- specific wikis that may help.


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Casio, the company you may best remember for supplying calculator watches to contestants on a 90's game show, has announced that it's adding 4G LTE to its 4-inch G'zOne Commando rugged smartphone. Priced a $99.99 on Verizon's network, Casio says the Commando has been military tested to withstand everything from drops to drowning.
Powered by Google's now outdated Android Ice Cream Sandwich OS, the G'zOne Commando packs a 1.5-GHz dual-core processor, 1GB of RAM and 16GB of onboard storage. A 4-inch 800 x 480 TFT LCD display sits up front, above which is a 1.2-megapixel camera, that Verizon, unfortunately, says is great for take "selfies." Around back is the handset's 8-megapixel shooter. The internal specs are an improvement over the previous model of the same phone, released 2 years ago, at which time it earned a star rating of 3.5 on our G'zOne Commando review.
MORE: Top 10 Smartphones
Casio says the G'zOne Commando meets MIL-STD-810G standards for drops from up to 5 feet, dust, vibration, humidity, salt fog, solar radiation, blowing rain, lower pressure, high temperature and low temperature and water resistance. Of course, that kind of ruggedness adds some bulk to a smartphone. Measuring 5.1 x 2.7 x 0.54 inches and weighing 6.17 ounces, the Commando is a rather hefty handset.
The electronics maker has filled the Commando with a variety of apps that should keep outdoor aficionados happy including a Barometer that can be used to to forecast weather trends, compass, walking counter, tides, thermometer, sun/moon and star gazer. The G'zOne Commando goes on sale on June 27 and we'll be sure to bring your our full impressions when we get our hands on a review unit.