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Jan Wright, Doune Macdonald
Published May 22, 2012
Reference - 220 Pages
Series: Routledge Studies in Physical Education and Youth Sport
Published May 24, 2010
Reference - 220 Pages
Series: Routledge Studies in Physical Education and Youth Sport
Published June 10, 2010
Reference - 220 Pages
Series: Routledge Studies in Physical Education and Youth Sport
June 10, 2010
by Routledge
Reference - 220 Pages
Series: Routledge Studies in Physical Education and Youth Sport
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Despite society's current preoccupation with interrelated issues such as obesity, increasingly sedentary lifestyles and children's health, there has until now been little published research that directly addresses the place and meaning of physical activity in young people's lives. In this important new collection, leading international scholars address that deficit by exploring the differences in young people's experiences and meanings of physical activity as these are related to their social, cultural and geographical locations, to their abilities and their social and personal biographies.
The book places young people's everyday lives at the centre of the study, arguing that it this 'everydayness' (school, work, friendships, ethnicity, family routines, interests, finances, location) that is key to shaping the engagement of young people in physical activity. By allowing the voices of young people to be heard through these pages, the book helps the reader to make sense of how young people see physical activity in their lives.
Drawing on a breadth of theoretical frameworks, and challenging the orthodox assumptions that underpin contemporary physical activity policy, interventions and curricula, this book powerfully refutes the argument that young people are 'the problem' and instead demonstrates the complex social constructions of physical activity in the lives of young people. Young People, Physical Activity and the Everyday is essential reading for both students and researchers with a particular interest physical activity, physical education, health, youth work and social policy.
Introduction: Theorising Youth and Physical Activity Part 1: Physical Activity and Geographical Locations 1. The Place of Physical Activity in the Lives of Rural Young People: Stories From the 'Outback' 2.'The Police be Comin' so That's Why I Didn't go Over There': Young People's use of Neighborhood Spaces in a US City 3. Young People, Cities and Physical Activity Part 2: Social and Cultural Location and Physical Activity 4. Social Class, Schooling and Young People's Meanings of Physical Activity and Health 5. Physical Activity and Indigenous Young People 6. Physical Activity and Confucianism: the Relations Between Hong Kong Children and Their Parents 7. 'Our Prophet Said We Should Play Sport': Young Muslim Women Negotiating Islam, Popular Culture and Physical Activity Part 3: Physical Activity and Constitution of Selves: Health, Fitness and Bodies 8: Eating Well, Being Active and the Production of the "Good Citizen" 9: Young People, Transitions and Physical Activity 10. 'Pump Weights, Eat Right': Young People's Engagements with Health Discourses 11. 'Making Castles in the Sand': Community Contexts and Physical Culture 12. Diversity, Subjectivity and Constructions of Fitness and Health by Young Canadians Part 4: Methodological Issues and Future Directions 13. Reflections on Methodological Issues and Lessons Learned From the Life Activity Project
We provide complimentary e-inspection copies of primary textbooks to instructors considering our books for course adoption.

subset: commoncrawl


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Hendrik | Introduction
Hendrik is a Netherlands based artist. A reserved family man, he uses art as an escape to a engage with with his geeky fantasies. He talks to Graff.io Art's about his latest collab in Augmented Reality (AR), "Hunk of Junk"
I guess bringing art to life is an interesting topic to explore. By animating it, you can do so much more - it can be a vehicle for discovery - for example, the viewer could find out more about a cause. it could be nothing more than an exploration of the artform itself.
More & more artists are actually using the physical piece as the starting point and the AR piece as the artwork itself.
I love showing it to kids, as it seems to actively encourage the mystery of life, all those things that can get crushed out of you the longer you are part of this world...


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If there's anything that every business owner can agree on, it's that sales are the driving force of their business. Without customers, a business won't survive; this is true for both product and service-based businesses. Below are some of the best ways to improve your business's sales so that your business can stay in business longer.
1) Simple Surveys
Is there anything more difficult than forecasting sales in any business? It can be tricky to know what customers want in a business– especially if business owners forget to ask their customers what they want. One of the best ways to know what customers are looking for is to provide them with a survey.
With a survey, business owners can ask customers exactly the questions they need the answers too. This is much faster than simply waiting around for customers to give their opinions, unprompted. Once you have the answers to these questions, you can implement any changes necessary to get more customers interested in your business.
2) Customer Loyalty Programs
One way to keep customers coming back for more is to give them a reward for doing so. Implementing a customer loyalty program is a great way to incentivize customers. There are a variety of ways to set up customer loyalty programs– giving out membership cards, signing up online, or assigning them a customer number.
Once the loyalty program is in place, set up a reward for their patronage. Providing a discount or free items after the customer spends a certain amount of money is a great way to encourage them to shop more often. Punch cards, in which a customer earns a punch after each sale, is a great way for customers to keep track of their progress towards a reward without the need of a computer.
3) Regular Sales
Having sales on a regular basis is a great way to get new customers into your shop. However, it is important for customers to know when there is a sale. After all, if a sale is not advertised, then only regular customers who go into the shop anyway will notice it.
Advertising sales is simple and can be relatively cheap. If a business has a social media presence, a social media manager can easily make a post so customers know about the sale. If you are less tech-savvy, then try printing out posters to put around a mall or the town so people can learn about the sale that way.
Try these tips to get new customers into your shop and to keep old customers coming back for more. By doing this, you are sure to increase your overall sales, which is great news for any business!
Additional Resources:
Why You Should Analyze Customer Churn
Why Surveys Are Important For Your Business
4 Creative Ideas to Use Instagram Polls for Your Business
Why SEO & Online Marketing is Key for Business Growth
Your Marketing Automation Strategies to Optimize Marketing and Sales Funnels


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Cutaneous Oncology
Chen ST, et al. J Am Acad Dermatol. 2019;doi:10.1016/j.jaad.2019.09.026.
Dermatology consultation may improve the long-term implications of hospitalization for suspected dermatologic immune-related adverse events, according to researchers in Journal of the American Academy of Dermatology.
"Current practice guidelines recommend consideration of systemic corticosteroids for the treatment of even mild dermatologic [immune-related adverse events]," Steven T. Chen, MD, MPH, of Harvard Medical School and departments of dermatology and medicine, Massachusetts General Hospital, and colleagues wrote. "This may be detrimental to oncologic outcomes, at least at high doses."
The aim of the study was to determine the impact of dermatology consultation among a cohort of hospitalized patients with suspected dermatologic immune-related adverse events. Patients had received immune checkpoint inhibitors, and the analysis included only those who experienced a disruption to their oncologic management as a result of dermatologic immune-related adverse events.
Among 47 patients admitted to the hospital with a suspected dermatologic immune-related adverse event, 33 cases were confirmed after consult by a subspecialist, of which 15 patients were hospitalized primarily or exclusively for dermatologic immune-related adverse events. Six of the other 18 patients developed dermatologic immune-related adverse events during hospitalization.
Dermatology consultation was requested in 22 of the confirmed cases and all 14 cases that were deemed improbable. The confirmed patients that were evaluated were less likely to receive systemic immunosuppression when compared with patients who did not undergo dermatology consultation (18% vs. 55%, P = .049). Additionally, 0% of patients who underwent dermatologic consultation discontinued immune checkpoint inhibitors compared with 36% who did not undergo dermatology consultation (P = .014).
"Our finding that all improbable cases received dermatology consultation indicates that dermatologists may help rule out dermatologic [immune-related adverse events], thereby allowing providers to explore other etiologies and continue cancer treatments uninterrupted," Chen and colleagues wrote.
Disclosures: The authors report no relevant financial disclosures.
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Dear Parents and Guardians,
Below you will find a list of interactive read aloud books we have read in our classroom. Please ask your child to tell you about the book and our discussions. Through the interactive read aloud and discussions students have the opportunity to learn to think within the text, about the text and beyond the text.
During this time we are getting to know and learn about authors, explore different genres, expand vocabulary, character traits, main idea, recognize themes, style and language. These skills are then transferred to their own reading and writing.
If you should have any questions, please feel free to contact me.
Miss Nelson is Missing - Fiction
Camping Spree with Me and McGee - Fiction
Fine, Fine School - Fiction
Stand Tall, Molly Lou Melon - Kindness
My Mouth is a Volcano - Behavior
Fire Boat- 9/11 Tribute
September 12th, I Knew Everything Would Be Alright
The Dot - Growth Mindset
We the People - Constitution Day
Edward the Emu - Courage to be Yourself
Commotion in the Ocean - Poetry
The Bad Seed - Character Traits
Johnny Appleseed - Tall Tales
Chester the Raccoon and the Big Bad Bully - Bullying
Sarah, Plain and Tall - Reading Strategies
Blackberry Mouse - Friendship/Sharing
Picture Book of Christopher Columbus - Explorers
The Most Magnificient Thing - Mindset/Writing
Do Unto Otters - Behavior
Creepy Carrots - Fiction
Who Wants Arthur? - Kindness/Respect
The Day the Crayon Came Home - Theme
The Best Witch Poems - Poetry
How Martha Saved Her Parents from Green Beans
More Than Anything - Character Traits
Lighthouses - Nonfiction Text Features
Cactus Hotel - Nonfiction
Tigers - Nonfiction Text Features
The Good Egg - Behavior
Turk and Runt - Character Traits
Crazy Like a Fox - Similes
Dear Santa Claus - Letter Writing
Colonial Times - Pilgrims
I'm Thankful for Each Day
Chanukah Lights Everywhere
Olive, the Other Reindeer
Those Darn Squirrels - Story Elements
Aesop Fables
Town and Country Mouse - Fable
Lousy, Rotten, Stinkin' Grapes - Fractured Fable
Princess Kim and Too Much Truth - Honesty
Two Bad Ants - Point of View
True Story of the Three Little Pigs - Point of View
Martin Luther King - Biography/Timelines
Rosa Parks - Biography/Timelines
Voices in the Park - Point of View
Three Hens and a Peacock -Fiction
Be Kind - Kindness
Unbeatable Bread - Fiction
Ming Lo - Folktale
Babushka, Yaga Baga - Folktale
Martina the Beautiful Cockroach - Folktale
100 Day Worries - Fiction
Chocolate - Nonfiction - How To
Myths - Various Titles
Kindness is Cooler with Mrs. Ruler - Kindness
Alexander and the Terrible, Horrible, No Good Day - Cause and Effect
Dr. Seuss Books - Various Titles
The Invisible Boy - Empathy


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UKI Canada Contacts
Sarah Mairs
UKI Canada Liaison – direct contact with Laura Derrett
Paula Collins, WAO Team Canada Manager
UKI Canada Liaison – direct contact with Laura Derrett
Rebecca McKay, Prairie and West UKI Canadian Development Coordinator
Sandy McDavid, East UKI Canadian Development Coordinator
Leslie Lord – Atlantic UKI Canadian Development Coordinator
Shona Thirsk, WAO Team Sponsorship and Fundraising Coordinator
Shona Thirsk, Website Administrator
The above mentioned VOLUNTEERS are your KEY contacts for answering information about hosting a UKI Trial or adding UKI events to your current trial of another organization.
They are contacts for any questions you may have with regards to earning byes for the Canadian Open.
They are contacts for any questions you may have about earning points towards your titles and how you move up the ladder.
They are strong WAO supporters who will encourage WAO fundraising at local trials as best they can.


text: Undergraduate Admission Publications Drexel University provides students with a wide variety of programs and opportunities that allow ambitious students to explore and pursue their academic goals. Below is a sampling of some of our admission publications, each developed to provide helpful insights and answer questions you may have. Be sure to request more information to receive these and other useful materials. Undergraduate Viewbook [PDF] Transferring to Drexel [PDF] Drexel Welcomes the World — International Brochure [PDF] Gateway to Drexel — The International Gateway Program Brochure [PDF] The Veteran Student Experience at Drexel University [PDF]


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Here, we report on a novel PCR targeting-based strategy called 'PCR duplication' that enables targeted duplications of genomic regions in the yeast genome using a simple PCR-based approach. To demonstrate its application we first duplicated the promoter of the FAR1 gene in yeast and simultaneously inserted a GFP downstream of it. This created a reporter for promoter activity while leaving the FAR1 gene fully intact. In another experiment, we used PCR duplication to increase the dosage of a gene in a discrete manner, from 1× to 2x. Using TUB4, the gene encoding for the yeast γ-tubulin, we validated that this led to corresponding increases in the levels of mRNA and protein. PCR duplication is an easy one-step procedure that can be adapted in different ways to permit rapid, disturbance-free investigation of various genomic regulatory elements without the need for ex vivo cloning.
Citation: Huber F, Meurer M, Bunina D, Kats I, Maeder CI, Štefl M, et al. (2014) PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast. PLoS ONE 9(12): e114590. https://doi.org/10.1371/journal.pone.0114590
Editor: Joseph Schacherer, University of Strasbourg, France
Received: September 4, 2014; Accepted: November 10, 2014; Published: December 10, 2014
Copyright: © 2014 Huber et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All data are available as Supporting Information files, except microscopy images, which are available from the Dryad Digital Repository: http://doi.org/10.5061/dryad.gj51n.
Funding: FH was supported by the SFB1036 of the DFG (Deutsche Forschungsgemeinschaft, www.dfg.de/en), MM and MS by the CellNetworks Cluster of Excellence (www.cellnetworks.uni-hd.de), MS in addition by the Alexander von Humboldt Foundation (www.humboldt-foundation.de/web/home.html), IK by the HBIGS graduate school (Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology, www.hbigs.uni-heidelberg.de) and DB received funding from the Boehringer Ingelheim Stiftung für medizinische Grundlagenforschung (www.bifonds.de). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Yeast possesses efficient homologous recombination systems that permit the targeted integration of foreign DNA into the genome [1]–[3]. This enables rapid genomic manipulations, such as gene deletions, plasmid integration or even the induction of complex chromosomal rearrangements [4]. Homologous recombination is highly efficient and works well using short identical sequences of 40–50 bp for targeting the foreign DNA to a specific genomic locus [5]. In combination with marker genes that permit two-way selection - for their presence or their absence - genomic DNA can be modified in many different ways simply by using homologous sequences provided by PCR oligonucleotides. Over the years, a variety of methods and strategies have been developed to achieve virtually any desired genomic modification, from the introduction or removal of sequences to the editing of single nucleotides [6]–[12]. Today, most of these manipulations are based on a plethora of available PCR templates that provide functionality, such as selection markers, protein tags etc. Since the DNA fragments for transformation are made by PCR, this method has been called PCR targeting: a one-step procedure, involving PCR amplification of a generic cassette, the subsequent transformation of the fragment followed by selection of the marker and validation of the correctness of the manipulation. Typically, >80% of the obtained clones are correct. This value depends on the quality of the oligos and the uniqueness of the sequences chosen for homologous recombination.
One-step PCR targeting strategies enable the following genomic manipulations: gene and sequence deletion, C-terminal protein truncation, C-terminal protein tagging, fusion of a heterologous promoter to a gene of interest and N-terminal tagging using heterologous promoters. Large numbers of suitable cassettes are available, see e.g. refs [10], . Recovery of markers for selection can be achieved through a second step and tailored features of the used cassettes. The presence of tandem repeats, for example, enables the spontaneous loop-out of the marker [7], [20], [21], a process which can be further stimulated by endonucleases [17]. Alternatively, Cre- or other recombinases can be used to remove markers, thereby leaving only short sequences behind [16]. Perfect removal of the introduced sequences can also be achieved by a second round of transformation in which a larger fragment of DNA is introduced that contains the desired manipulation (e.g. a single nucleotide exchange) and sequences flanking the marker. Using counter-selectable markers this method was optimized to yield a low rate of false positives, with typically >80% of correct clones [8].
Yet, no simple cloning-free PCR targeting methods exist that enable the generation of gene duplications or promoter fusions to reporter genes. For locus duplication, Scherer and Davis presented a pioneering method, which also enables seamless genome modification, referred to as 'Pop-in/Pop-out' replacement (1979) [6]. There, a two-step procedure is applied. First, the modified allele of the target locus is cloned into a plasmid containing a two-way selectable marker such as URA3. The plasmid is linearized by restriction enzyme cleaveage of the plasmid in a sequence homologous to the target locus, followed by transformation into yeast, yielding a duplication of the targeted region (Fig. 1a). However, this method requires ex vivo cloning of the initial plasmid.
(a) The 'Pop-in' strategy. First, the desired genomic region is cloned into a plasmid containing a marker selectable in yeast. Homologous recombination with the target locus can be stimulated by linearization of the plasmid containing the cloned DNA using a unique restriction site within the cloned fragment. (b) The PCR targeting–based duplication strategy (PCR duplication). A PCR product containing a selection marker and short sequences of homology (45–55 bp) to a target locus is transformed into yeast cells. The homologous sequences are provided by the oligonucleotides used for PCR. This leads to the formation of two copies of the target locus, separated by the selection marker. Depending on the template used, PCR duplication can be used for simple duplication of a target locus such as a gene, or it can be used to generate reporter fusions to a specific sequence, e.g. a regulatory element such as a promoter, while a functional copy of the element is retained in its native context. (c) Primer design guidelines for PCR duplication. For designing the primers the following rules apply: Duplication primer A = 55 bp from 3′-end of target region in sense orientation + annealing site I. Duplication primer B = 55 bp from 5′-end of target region in antisense orientation + annealing site II.
Expression reporters to monitor the activity of a specific promoter constitute another experimental strategy that may involve the duplication of genomic elements. Here, a regulatory sequence is cloned into a plasmid harbouring a reporter, followed by chromosomal integration. Alternatively, PCR targeting is used to integrate a reporter gene downstream of a regulatory sequence, such as a promoter, thereby disrupting the function of the original gene.
The strategy we present here consists of a single step and avoids cloning of fragments into plasmids or the disruption of gene function. Instead of a cloned full-length DNA fragment for integration, as it is used for 'Pop-in' (Fig. 1a), only the outmost 45–55 bp on each side of the target locus are used for targeting the transformed DNA fragment to its genomic locus. These short sequences can be generated by PCR and when targeted to the genome they faithfully produce a duplication of the target locus with the duplicates flanking the amplified selection marker (Fig. 1b). Presumably, this process is mediated by endogenous DNA recombination and repair systems.
If the target locus encompasses the ORF of a gene along with its 3′- and 5′-regulatory sequences, this leads to gene duplication. If the upstream intergenic region of a gene, which contains its promoter, is targeted and a reporter gene is included in the transformed PCR product, transformation will lead to a duplication of the upstream intergenic region, one of them now controlling the reporter construct and the other one controlling the original downstream sequence. Hence, a promoter reporter is generated while preserving an endogenous version of the promoter and the gene it controls (Fig. 2a). In this work we demonstrate this method using selected examples and two different applications, and we suggest further applications of the method.
(a) Generation of genomic promoter-reporter fusions. The PCR duplication strategy was used to obtain from a wild type strain (i) a strain containing GFP fused to the FAR1 upstream intergenic region, including its promoter (FAR1prom), plus a copy of the full wild type FAR1 upstream intergenic region and the FAR1 ORF (ii). Subsequently, PCR targeting was used to delete the FAR1 ORF copy left in that strain using a second selection marker (iii). (b) Cells from the three different strains (i to iii) shown in (a) were grown to logarithmic phase. After the addition of α-factor (10 µg/ml) samples were removed at the indicated time points and used for microscopy to visualize shmoo formation and quantify GFP fluorescence. DNA content was measured using flow cytometry. Scale bar: 3 µm.
Materials and Methods
Yeast culturing conditions and strain constructions
Yeast strains used in this study are listed in S1 Table. Cells were cultured according to standard methods [22] at 30°C in either synthetic complete (SC) or YPD medium with antibiotics as required. Strain manipulation procedures were based on PCR-targeting methods as previously described [11], [13]. Cassettes were amplified using a high fidelity DNA polymerase. Plasmids used are listed in S2 Table and oligos used are listed in S3 Table. Strains were validated by colony PCR using Taq DNA polymerase.
Primer design for duplication of genomic loci
PCR-based duplication of genomic loci was performed by transformation of the PCR product of a pair of primers, which we term "duplication primer A" and "duplication primer B" (Fig. 1c). Conveniently, these primers can be designed in a way that they contain generic annealing sites for PCR amplification of resistance or reporter/tagging cassettes in plasmids with the appropriate binding sites [11], [13], [15]. Upon transformation, the amplified region is inserted in reverse orientation between the duplicated loci. Duplication primer A has a 55 bp overhang that is homologous to the sense strand of the 3′ end of the locus that is to be duplicated. Duplication primer B has a 55 bp overhang that is homologous to the reverse complement of the 5′-end of the target locus. For an illustration of the scheme see Fig. 1c and for detailed design instructions see S1 Methods.
FACS analysis after pheromone induction
Cells from an overnight culture grown in SC medium were diluted to an OD600 of 0.025 (∼2.5×105 cells/ml) and grown to an OD600 of 0.4, followed bystimulation with 10 µg/ml of α-factor for 2.5 h. FACS analysis of the DNA content using propidium iodide staining was done as follows: Cells were fixed in 70% ethanol, centrifuged and resuspended in 1 ml of 50 mM sodium citrate containing 0.25 mg/ml DNAse free RNase A (Roche, 10109169001) and incubated for 1 hour at 50°C. 50 µl of 20 mg/ml proteinase K (Merck Millipore, 1.24568.0100) were added followed by another hour of incubation at 50°C. Next, 1 ml of 50 mM sodium citrate containing 16 µg/ml propidium iodide was added. Cells were kept at 4°C overnight prior to FACS analysis using a BD FACS Canto II machine and the FACS Diva v6.1.3 software. 10,000 cells were measured per sample.
Time course and quantitative light microscopy after pheromone induction
Cells from an SC overnight culture were diluted to an OD600 of 0.025 and after reaching an OD600 of 0.2 stimulated with 10 µg/ml of α-factor. 20 min before imaging 200 µl of cells were immobilised in 96-well glass bottom plates (Matrical BioScience, MGB096-1-2-LG-L). Time points denote the time after stimulation and were as follows: before stimulation as well as 45, 90, 135 and 180 min after stimulation. Imaging plates were treated with 1∶1000 triethoxysilane aldehyde (UCT Specialties, PSX1055-KG) in 100% ethanol for 15 minutes, washed once with ethanol and water each, treated with 0.1 mg/ml concanavalin A (Sigma, C7555) for 15 minutes and washed twice with water.
Imaging was done with an Applied Precision DeltaVision microscope system consisting of an inverted epifluorescence microscope (IX71, Olympus), a Lumencor Spectra ×LED light source, a 60×NA 1.42 oil immersion objective (Olympus), an sCMOS camera (pco.edge 4.2, PCO) and a temperature-controlled chamber. Eight images were acquired per well from focal plane corresponding to the center of the cells. In addition, imaging of a well containing mCherry-GFP fusions in 1×PBS 5% BSA were taken and served as reference images to correct for uneven illumination. Images without illumination were used to subtract the camera offset from the images. Images were segmented and quantified using ImageJ [23] and custom MATLAB (MathWorks) scripts. For whole-cell segmentation, the Canny edge detection algorithm [24] was used, exploiting the autofluorescence of yeast cells using 390/510 nm (excitation/emission) filter sets.
RT-qPCR and qPCR
Extraction of genomic DNA (gDNA): 107 cells of a log phase YPD culture were centrifuged for 5 min at 500 g and resuspended in 0.25 ml of S buffer (10 mM K2HPO4, 10 mM EDTA, 25 µg/ml zymolyase (AMSBIO), pH 7.2), incubated for 30 min at 37°C and then 155 µl of lysis solution (25 mM Tris-HCl, 25 mM EDTA, 2.5% SDS, pH 7.5) were added. 90 µl of 3 M KOAc were added, samples were incubated on ice for 10 min and after centrifugation for 10 min at 16,000 g DNA in the supernatant was recovered by ethanol precipitation. 100 ng of gDNA were used per qPCR reaction. Isolation of RNA: RNA was extracted from 107 cells of a log phase culture in YPD using the EURx GeneMatrix Universal RNA Purification Kit (E3598) according to the manufacturer's instructions. cDNA was synthesised from 2.5 µg of input RNA per sample using M-MLV reverse transcriptase (RT) RNase H- mutant (Promega, M368A) and random hexamers (Thermo Scientific, SO142) according to the manufacturer's instructions. Samples without RT served as negative controls. qPCR runs and quantification: qPCR reactions were performed using a LightCycler 480 II (Roche) with 7.5 µl of LightCycler 480 SYBR Green I Master mix (Roche, 04707516001) and 2.5 µl of either the gDNA solutions or a 1∶5 dilution of the cDNA reaction samples. Primer efficiencies for gDNA and cDNA were determined for all primer pairs by measuring dilution series. Actin was used as a reference gene and relative copy numbers were calculated using the method by Pfaffl [25]. Samples were measured using 5 technical replicates and dilution series using 3 technical replicates. The correctness of the obtained amplification products was verified by melting curve analysis and agarose gel electrophoresis.
Trichloroacetic acid (TCA) precipitation and Western blotting
Cell lysates for Western blotting were prepared from log phase YPD cultures using the NaOH/β-mercaptoethanol/TCA method as described [13]. For detection of specific proteins, the following antibodies were used: 1∶500 rabbit α-Tub4 (polyclonal, antibody raised against a C-terminal fragment fused to GST, for details see [26]), 1∶5000 mouse α-Pgk1 (monoclonal, Molecular Probes, catalog no.: 459250, clone 22C5D8), 1∶5000 goat α-mouse HRP (polyclonal, Dianova, catalog no.: 115-035-003) and 1∶5000 goat α-rabbit (Jackson Immunoresearch Laboratories, catalog no.: 111035003, lot: 86831). Bands were detected using the Immobilon Western Chemiluminescent HRP Substrate kit (Merck Millipore, WBKLS0500) and an LAS-4000 (Fujifilm) imaging system. Quantification: Western blot images were quantified using ImageJ. Bands were segmented manually and intensities were corrected by subtracting a local background. To correct for unequal loading of lanes intensities were normalized to the Pgk1 signal.
A PCR-based 'Pop-in' strategy to generate duplicated chromosomal loci
The 'Pop-in' strategy of integration of a cloned fragment at the locus homologous to the fragment leads to a duplication of the locus (Fig. 1a). This strategy involves the linearization of the cloned fragment followed by transformation of the fragment into yeast cells. Recombination between the two fragments on either side of the cleavage site then leads to an integration of the fragment and duplication of the locus. In order to avoid cloning of the fragment, we tested if it might be possible to replace the cloning step by a PCR step. Instead of cloning the whole fragment, oligonucleotides that provide short sequences of homology flanking the target locus would be used. This would then result in a duplication of the locus with the duplicates flanking a selection marker. If desired, a tag or any other sequence of choice can be introduced downstream of one of the duplicated loci (Fig. 1b and 1c, for a detailed protocol see S1 Methods).
Construction of a transcriptional reporter for the FAR1 gene
In a first experiment to demonstrate this idea, we investigated the possibility to use it to construct a transcriptional reporter. We chose the upstream intergenic region of the FAR1 gene, which contains the promoter of this gene. FAR1 is classically used to monitor the activity of the pheromone signalling mitogen-activated protein (MAP) kinase pathway. FAR1 is induced upon treatment with α-factor and the produced Far1 protein is critically important for cell cycle arrest prior to mating of haploid yeast cells [27], [28]. FAR1 is not essential for signalling per se, but deletion of this gene leads to a failure in cell polarization and shmoo formation and cells are no longer able to arrest the cell cycle in G1.
We applied PCR targeting using a cassette containing GFP as a reporter gene and kanMX6 encoding for geneticin resistance as a selection marker. The original purpose of this cassette was to use it for C-terminal GFP fusions using PCR targeting (S1 Table)[11]. The primers prFAR1-dupA and prFAR1-dupB were designed in a way that the resulting PCR product contained 55 bp homologies to the entire FAR1 upstream intergenic region. This would then yield a fusion of that region with the GFP ORF in the cassette followed by the selection marker (kanMX6) and a second copy of the FAR1 upstream intergenic region and the FAR1 ORF (Fig. 1b, Fig. 2a and S1 Figure). After PCR targeting, several clones were selected and PCR was used to probe for the presence of the new junctions (S1 Figure). In our experience, correct clones were retrieved at a frequency similar to that of other standard yeast genetic manipulations based on PCR targeting. Pheromone treatment of these new strains led to the expected G1 arrest and shmoo formation, similar to control cells, as shown by microscopy and flow cytometry (Fig. 2b), indicating the presence of a functional FAR1 gene. Quantification of GFP signals showed a strong increase in GFP intensities (Fig. 2b), indicating the presence of a functional FAR1 promoter in front of the GFP ORF. As a control we next deleted the FAR1 ORF in this strain using a second step of PCR targeting and a natNT2 marker encoding for nourseothricin resistance (S1 Figure) [13]. As expected, this yielded a strain that retained GFP induction upon pheromone treatment, but without showing shmoo formation or G1 arrest, indicative of a failure to arrest the cell cycle [27]. GFP expression was weaker in this strain as cells only respond to α-factor in G1 (Fig. 2b) [29]. This experimentally confirmed that PCR targeting can be used to generate chromosomally localized and disturbance-free expression reporters based upon a local duplication of regulatory sequences.
Generation of a duplication of the TUB4 gene
In a second application, we used our PCR-targeting strategy to generate gene duplications. As an example we chose the TUB4 gene. This gene encodes the essential γ-tubulin required for microtubule nucleation. Duplication was validated by testing for the newly generated junctions with colony PCR (S2 Figure). Using specific antibodies to detect the protein, we observed a ∼2.5-fold increase in Tub4 protein levels, consistent with the presence of two functional copies of this gene. Likewise, RT-qPCR and qPCR confirmed a corresponding increase in the amount of TUB4 mRNA and a 2-fold increase in TUB4 DNA levels (Fig. 3).
(a) PCR duplication of the TUB4 gene including the associated intergenic regions 5′- and 3′- to the TUB4 ORF. (b) Western blot and detection of Tub4 using 4 biological replicates of the strain containing the duplication, as well as 4 clones of the wild type strain. Tub4 and, as a reference, Pgk1 were detected using specific antibodies. Quantifications are shown in c. A GFP-tagged version of Tub4 shows the specificity of the antibody. (c) Western blot quantification, RT-qPCR and qPCR quantification of TUB4 protein, mRNA and the chromosomal gene copy number, respectively, in the different strains. qPCRs were performed on 1 biological replicate done in 5 technical replicates each. Error bars denote S.D. In all cases, differences between wild type and duplications are statistically significant (two sample t-test, unequal variance, p<0.05).
Our results show that the 'PCR duplication' method presented here is an easy, cloning-free way to generate duplications of genomic loci in yeast. We demonstrate that this method can be used to selectively increase gene dosages and that it enables the simple generation transcriptional reporters in a disturbance-free manner.
For many applications of this method there is no need for the construction of new PCR templates. Instead, published cassettes can be used. For example, any cassette originally constructed for C-terminal gene fusions can also be used to generate an expression reporter. Since a plethora of such cassettes exists (see e.g. refs: [10], [13]–[16], [30], [31]), many different possibilities with respect to reporter genes and selection markers are available. For planning a duplication experiment, care has to be taken to choose the appropriate region for duplication so that, for example, accidental truncation of functional elements is avoided. For this reason the construction of the FAR1-expression reporter involved the duplication of the entire SSY5-FAR1 intergenic region, and not only the region spanning the presumed promoter sequence of the FAR1 gene. Similarly, the duplication of the TUB4 gene involved the full intergenic regions on the 3′- and the 5′-side of the TUB4 ORF, as it is possible that regulatory ncRNAs originate outside of the presumed promoter or terminator sequences of an ORF [32], [33].
A critical step in the construction of such genomic duplications is the validation of the correctness of the genomic manipulation. We observed that >80% of the picked clones contained the desired modification as judged by chromosomal PCR on junctions formed upon duplication (S1 and S2 Figures). In principle, this form of genotyping does not discern clones with multiple insertions. Such cases can only be identified by methods such as Southern blotting or qPCR. We investigated the possibility that multiple integration events occur by using a fluorescent protein reporter linked to the marker gene of the PCR cassette. We found that insertion of more than one copy appears to be a rare event: less than 5% of all colonies showed increased levels of fluorescence as compared to the other colonies, which exhibited uniform fluorescence (Matthias Meurer and Michael Knop, unpublished data). Nevertheless, if the exact copy number is critical additional tests to exclude clones with multiple insertions are recommended.
Depending on the experiment, sequencing of the insertions should also be considered due to the possibility of PCR errors or faulty oligos. These may lead to the duplication being associated with a mutation in the sequence used for generating the transformed construct. Experimenters also need to be aware of the fact that recombination between the duplicated loci can lead in rare cases to a spontaneous loss of the duplicated region and the associated cassette. Therefore, we recommend to always maintain selection pressure for the inserted marker.
Finally, we want to note that PCR duplication can be easily adapted for a wider variety of functional studies. In principle, any tag or reporter can be inserted downstream of one of the duplicated loci while preserving the endogenous structure of the other locus. To name just one more example, one could duplicate a gene while simultaneously replacing its C-terminal tag or terminator sequence with another sequence of interest in one of the copies in order to study the effect of that sequence on the expression of the gene. Moreover, we are optimistic that our approach is not limited to yeast but can also be adapted for functional studies in other eukaryotes.
Supporting Information
S1 Figure.
Genomic duplication of gene regulatory regions and genomic reporter gene construction. (a) Construction of strains: a GFP-kanMX6 cassette [11] is used to generate the duplication of the SSY5-FAR1 intergenic region and to construct simultaneously a FAR1-promoter fusion with the GFP ORF. In a next step the wild type copy of FAR1 is deleted using a natNT2 cassette [13]. (b) Validation of the strains using PCR and chromosomal DNA (see Materials and Methods) to validate specific new junctions and to confirm the disappearance of other junctions. Strains: FAR1 → ESM356-1; prFAR1::GFP::FAR1 → FHY144-1, prFAR1::GFP::Δfar1 → FHY151-2 Exact genotypes are listed in S1 Table. PCR-A: Far1_j1f & Far1_ur (5′-Junction), Far1_ORF_f & Far1_termrev (3′-Junction); PCR-B: Far1_j1f & Far1_j1r (5′-Junction), Far1_j2f & Far1_j2r (3′-Junction); PCR-C: Far1_j1f & His_and_Kan_Tag2 (5′-Junction), Nat-sense & Far1_termrev (3′-Junction). Oligo sequences are listed in S3 Table.
S2 Figure.
Validation of clones carrying TUB4 duplications. Colony PCR was used to validate the new junctions, as indicated in the top panel, using 4 independently obtained clones. Primers for the 5′ and 3′ junctions were Tub4_j1f, Tub4_j1r and Tub4_j2f and Tub4_j2r, respectively. wt = wild type, ESM356-1. Sizes in bp are indicated on either side of the gel.
S1 Table.
Yeast strains.
S2 Table.
S3 Table.
S4 Table.
Per well averages of microscopy quantifications of this study. The underlying images are available from the Dryad Digital Repository: http://doi.org/10.5061/dryad.gj51n.
S1 Data.
FACS raw data.
S2 Data.
qPCR raw data.
S3 Data.
Western blot quantifications.
S1 Methods.
A step-by-step protocol for PCR duplication.
We gratefully acknowledge our colleagues who have tested the method of PCR duplication; Monika Langlotz from the ZMBH FACS facility; and Anton Khmelinskii and Brian Luke for comments on the manuscript.
Author Contributions
Conceived and designed the experiments: FH MK. Performed the experiments: FH. Analyzed the data: FH MK. Contributed reagents/materials/analysis tools: C. Mongis. Wrote the paper: FH MK. Performed validations of different applications of the method: MM DB IK C. Maeder MS.
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Rim-to-Rim-to-Rim (R2R2R)
ElevationAscent: 10,919' 3,328 m
Descent: -10,919' -3,328 m
High: 8,178' 2,493 m
Low: 2,454' 748 m
GradeAvg Grade: 9% (5°)
Max Grade: 61% (31°)
Current trail conditionsUpdate Conditions
Popular runs nearby
Rim Trail
13.1 mi 21.0 km • Point to Point • 469 ft Ascent 142.87 m Ascent
Bright Angel Point
0.5 mi 0.8 km • Loop • 70 ft Ascent 21.3 m Ascent
Cape Royal Trail
0.9 mi 1.5 km • Loop • 32 ft Ascent 9.71 m Ascent
Uncle Jim Complete Loop
4.8 mi 7.8 km • Loop • 494 ft Ascent 150.7 m Ascent
Cape Final Trail
4.3 mi 6.9 km • Out and Back • 425 ft Ascent 129.56 m Ascent
Tusayan Ruins Loop
0.2 mi 0.4 km • Loop • 30 ft Ascent 9.06 m Ascent
Navigate on-trail with our free app
"Run across one of the Seven Wonders of the World... TWICE!"— Cyndi Wyatt
Features River/Creek · Views · Waterfall
The unique aspect of crossing the Grand Canyon is that you first descend 9 miles, then run across the floor for 7 miles, before hitting the steepest part of the North Kaibab Trail where you'll climb 7 miles up a 15% - 20% grade. Pacing yourself is the key to finishing R2R2R.
The views in the canyon as the sun kisses the walls change with every hour. Take your time to enjoy all that the Grand Canyon has to offer.
Make time to visit Ribbon Falls just off the North Kaibab Trail. Take a dip but don't stay too long as you have a long day ahead of you. The falls are about a mile off the trail. Take the bridge exit off of the trail to get there.
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Need to Know
Runner Notes
Descend the Bright Angel Trail to the Colorado River, through Bright Angel Campground, and then head up the North Kaibab Trail.
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Now you are at the half way mark! Go back and cross the same way that you came.
Flora & Fauna
History & Background
Run this trail?
Is something wrong? Let us know. Have photos to share? Help fellow runners know what's here.
Land Manager: National Park Service - Grand Canyon National Park


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Whether you're looking for the perfect St. Patty's Day Recipe, or just an awesome hearty dinner, this Irish Shepherd's Pie recipe gives you an awesome bite from the Emerald Isle. An awesome recipe that's the perfect bite of meat, potatoes and yummy veggies! So, confession time. I hadn't tried shepherd's pie until a few years'…
Sous Vide Steak Recipe with Whiskey Pan Sauce
What's better than a great sous vide steak recipe? How about a sous vide steak recipe finished with a simple, delicious whisky pan sauce? This simple recipe will deliver some of the best steak you've ever had! The first time you pull of a sous vide steak recipe, you'll wonder what took you so long…
How To Smoke A Brisket
Want to know how to smoke a brisket, but don't know where to start? Learn how to smoke a brisket to get the payoff of a perfectly smoky, buttery soft cut of meat to make those long, patience trying hours worth every bit of sleep you just lost.


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Posh for a Purpose
October 2017
Although Kemery is only nine-years old, she has already participated in five Susan G. Komen Races for the Cure. As we have participated in each race, we become aware of more ladies who are warriors, angels, and survivors, including Kemery's Grandma.
Kemery Kreates chose Posh for a Purpose for the first organization to donate 5% of the month's online revenue. "Posh for a Purpose" (PAP) is a creative organization, designed to produce moments of stylish and artistic ambience, for the purpose of raising money for various causes that effect our society. It prides itself in the ability to promote change through creativity and education." <~~~Excerpt from the Posh for a Purpose website


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The 19th Bladesman by S.J. Hartland
Nomination badges earned:
His duty is to die young, but fate has something more lethal in mind…
Kill. Die. That's what a swordsman bonded to the ancient gods does. If Kaell breaks, the kingdom breaks with him. And prophecy says the 19th Bladesman will break…
As a vengeful god escapes his ancient prison and draws Kaell into his web of deception, even Kaell's lord can't protect him from the dark prophecy awaiting him—unless he's prepared to do the unthinkable
S.J. Hartland is an emerging fantasy writer from the Darling Downs, Australia. She is a journalist and fencer who has spent too many holidays roaming around obscure castles. The 19th Bladesman is her first novel. The second in the series, The Last Seer King, will be released in May.


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Sale ends soon!
Main features and functions of this machine: The frame can rotate arbitrarily in 360 degree The platen can print four color Equipped with four handles on both sides of the chuck adjust the screen printing frame After trimming complet of the adjustment the handle is fixed on the front of the chuck The screen frame support arm is equipped with fourhandles on both sides adjust the screen printing. Adjustment the two screws before the screws fixed after trimming fix up two screws. The frame clamp can achieve 2cm height adjustment according to the substrates height .the platen can move 10cm front and back All the frame clamps can achieve the best up-down tension by adjusting the spring tension according to the frames weight There are metal positioning wheel,positioning groove and positioning steel sheet.To make sure the overprinting accurate All the machines adopt advanced electrostatic spraying .the parts adopt stainless steel aluminium ,nickel platingand galvanized process,it is corrosion resistant and no rust. All the packing adopt the export standard five layer corrugated paper ,there are hard and soft foam and three-ply board in the box. All the metal parts are separated by the soft foam. Each machine is equipped with the complete set of installation tools. The main application of this machine: This machine is four color screen printing machine,suitable for two operators. The operator just need to stand in one station to work,the platen and frame can rotate in 360 degree,the operator donât need to move. Mainly used for T-shirt printing,also printed cloth and other products. Parameter: Platen size:45*55cm(18*22inch) Machine size: 185*91*37cm/72*35*14inch Net weight:35KG/78lb Packing size:86*50*25cm/34*17*10inch Gross weight:42KG/96lb


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Sean Nwankwo – the son of Kanu Nwankwo and his wife Amara Kanu – clocks 15years old today 14, February 2020 and his parents have taken turns to celebrate him.
The proud father also used the opportunity to showed love to his lookalike son as he also used the opportunity to celebrate Valentine's day.
Kanu wrote, "Happy birthday my Son Daddy loves you very much. I wish you Gods protection,blessings and happiness and May all your heart desires come true Amen thanks. #happyvalentinesday #happy birthday."


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Recently it has been reported that PlayFusion will team up with Crytek to bring enhanced augmented reality (AR) tech to CryEngine. Additionally, this deal will bring multiplayer AR capabilities to CryEngine, too.
According to gamesindustry.biz it has been reported that in a deal between the two companies, folks will see the industry's "most feature-rich and performant AR technology" come through this Enhanced Reality Engine.
Moreover, the multiplayer augmented reality capabilities set to hit one of the later versions of CryEngine was announced by Cambridge-based enhanced reality developer: PlayFusion.
According to the publication site, the collaboration will see PlayFusion license its Enhanced Reality Engine development platform to Crytek, adding said tech to "CryEngine's capabilities."
Crytek founder and joint CEO, Avni Yerli, made mention that the platform is "feature-rich" and that it's pretty much noteworthy tech. In addition to Crytek thoughts on the whole thing, a newly published press release reads that…
"Together, our proprietary technologies comprise the world's first comprehensive mixed reality platform-as-a-service spanning IoT hardware, computer vision, audio recognition, machine learning and deep data," said Crytek in a statement.
"PlayFusion enables entirely new and compelling experiences that are seamlessly connected, telemeterized, and dynamic on the fastest, most efficient, feature-rich platform on the mobile market today."
If you don't know, PlayFusion is founded by many industry veterans, and has worked on "leading" artificial intelligence (AI) and robotic technologies in order to pursue mixed reality development and this very collaboration.
There's no telling how this partnership will pan out in the future, and most importantly if gamers and fans alike will take to this tech at launch, however time will tell what's to come of this move.
Lastly, Crytek announced not too long ago CryEngine 5.5. This new build includes "260 improvements" and full access to the editor source code.
You can read more about the aforesaid by visiting gamesindustry.biz or press.cryengine.com.


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One day after the official announcement of Long Island City as the future second headquarters of online retail giant Amazon, Councilmember Ben Kallos participated in a protest at the planned site of HQ2, on Wednesday. Kallos was among a chorus of voices expressing concerns about the impact of Amazon settling across the river from us on four million square feet of space. Many Long Island City community members attending the protest brandished Amazon prime boxes with the logo upside down, making a frown.
Amazon officially announced a plan to bring its second headquarters to Long Island City on November 13, following a 14-month long contest between hundreds of cities across the country. The company will also open a second new headquarters in Arlington, Virginia, with each location expected to house 25,000 new employees; Nashville will become home to Amazon's "Operations Center of Excellence."
Council member Kallos at Rally to Protest Amazon Deal
Living on Roosevelt Island for Amazon Employees
It is likely that families moving to New York City to work at Amazon would settle on Roosevelt Island where the living is easier; there are better options here for both school and transportation, and the commute to work would be one stop away on the ferry. Long waitlists for entrance into Long Island City schools have already brought some students here. To handle the overflow, busing to Woodside has been a community conversation until recently. The school's zone size has increased by 124 percent since the 2011-12 school year. The neighborhood is also transportation poor; commuters to Manhattan rely solely on the 7 train.
Roosevelt Island Operating Corporation President Susan Rosenthal agrees, "We think the move will also increase awareness and interest in Roosevelt Island as an attractive place to settle and raise a family, as well as a convenient location where retailers can prosper."
Trump Countdown Clock is Part of HQ2
According to the agreement with the City, HQ2 will consist of 4,000,000 square feet of commercial space and the creation of 25,000 new jobs with an average annual wage of over $150,000 within ten years. HQ2 will be constructed on a site bordered by Vernon Boulevard, 44th Road, 46th Avenue, and the East River. The site will be just four blocks north of the Long Island City ferry stop. The land is a mix of city-owned and private property owned by the plastics company Plaxall. The building with the Trump countdown clock, visible from the east side of the Island is on that property.
"At a moment in time when our City is facing infrastructure, housing, and transit deficits, we should not be subsidizing one of the largest multi-billion dollar corporations in the United States to the tune of $3 billion," said Council Member Ben Kallos. "Any deal with Amazon must go through the established community zoning-change process and ensure that Amazon hires locally and ethically, meeting our City's standards and prevailing wage requirements. As chair of the committee on Planning Dispositions and Concessions, residents on Roosevelt Island need to know that I will do my best to monitor developments of this deal closely, advocating for transparency and accountability."
With Roosevelt Island and Manhattan to the West, HQ2 will be on one side of Anable Basin, used by LIC Boathouse for kayaking, with the ferry and residential LIC on the other side.
Local Council Members Oppose Deal
Kallos' chief concern is that the ULURP (Uniform Land Use Review Procedure) process, a standardized procedure where applications affecting the land use of the city are publicly reviewed, wasn't followed. Kallos also listed rising rents and infrastructure being unable to keep up, as other problems with the deal. Councilmember Jimmy Van Bramer, whose district will include HQ2, tweeted from the protest, "Our community has been fighting for better infrastructure & more open space in #LIC FOR YEARS. I'm sick of hearing there isn't enough money for new schools, parks, transportation & affordable housing. $3 Billion should be invested in longtime residents, not #AmazonHQ2."
Corey Johnson, speaker of the New York City Council, issued a statement the morning of November 13 echoing both council members' concerns. "Amazon is one of the richest companies in the world, but you can't put a price on community input, which has been missing throughout this entire process." He added, "I find that lack of engagement and the fact that negotiations excluded the City Council – which is elected by New Yorkers to guide land use projects with communities in mind – extremely troubling. I also don't understand why a company as rich as Amazon would need nearly $2 billion in public money for its expansion at a time when New York desperately needs money for affordable housing, transportation, infrastructure, and education."
Amazon's potential move into the waterfront properties would typically be contingent on a city rezoning, which includes a City Council vote. The state, however, is reportedly set on executing a controversial "General Project Plan" or GPP, that would bypass this procedure. This has been used in several other recent large-scale projects, Queens West and Atlantic Yards, for example, and is open to non-binding feedback from the city.
In a press conference held on November 13, attended by Congresswoman Carolyn Maloney, Mayor Bill De Blasio, Governor Andrew Cuomo and others, De Blasio said, "It's a great day for NYC; it's an extraordinary day for Queens." Noting that Queensbridge is the largest public housing development in the country with 3,142 apartments, he said "[One of the] biggest companies on earth will be next to the biggest housing project in the country," and explained that baked into the deal will be "a lot of things to help a lot of people, including job trainings."
De Blasio did address the pink elephant in the room, transportation. He said, "Ferry service is benefitting LIC and western Queens right now. We have to look at every conceivable way to improve," and said "interesting options" could include a new Long Island Rail Road stop and adding a shuttle bus from the ferry stop to the larger community. He said, "We will look at every kind of option. Amazon or no Amazon; this is something we have to address."
On the subject of transportation, Roosevelt Island Residents Association President, Lynne Shinozaki, agrees that transportation to and from Long Island City needs to improve, saying "I can't imagine a scenario where the ferry picks up the slack. Who do I see benefitting from this? Real estate interests."
Silver Lining for Cornell Tech Students
Not everyone believes this is all bad for us. Roosevelt Island Operating Corporation Susan Rosenthal said, "RIOC applauds the announcement that Amazon will be establishing a headquarters in nearby Long Island City. We hope Roosevelt Island residents, including those at Cornell Tech, will be among the pool of prospective new employees."
Hudson Related's David Kramer also thinks the benefits for Roosevelt Island will be long-term, saying "I think Roosevelt Island, like the rest of NYC, will be an indirect beneficiary to what'll happen over the long run in LIC."


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Need a fun day with colleagues or to nurture customer relations?
Looking for an unusual activity?
We will help you!
We offer custom-made events with fantastic animals and environments.
We have many alternative activities for you e.g
Yoga in the Alpaca pasture
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*När du trycker på "Skicka" samtycker du att Dalslands Alpacka får behandla dina uppgifter.


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Roganic To Host Singapore's Jaan In British-Themed Four-Hands Collaboration This September
With numerous exciting chef collaborations this summer, we must add just one more, as new restaurant Roganic is hosting a four-hands collaboration with Singapore's Jaan this September.
On the evenings of September 4 and 5, Rogan will join forces with Jaan's Kirk Westaway to present a British-themed menu highlighting seasonal produce and foraged items for a 10-course dinner. This collaboration marks Westaway's return since his last chef collaboration at Mandarin Oriental's Krug Room. The 10-course collaboration menu will feature signature dishes from both restaurants, including Jaan's roasted potato and black truffle soup and summer bean salad with burrata and quinoa, as well as Roganic's stuffed hen of the woods with kohlrabi, roasted yeast and elderflower. The exclusive two-night collaboration menu is priced at HK$1,980 per person, with an optional wine pairing package designed by Sean Oakford, available at HK$980 per person.
RoganicRestaurant, $$$$, British, Causeway Bay
Book Online


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Project Overview: Pan card (NSDL & UTI) Physical Verification Project in PAN India
Project Description/Type of Work: – Life Insurance risk management survey.Monthly data provided by the L1 Company ,Verification needs to be done in given TAT.
Rework only if there is error in report.
90 days payment cycle.
Survey involve the following stages:
1.Objective-setting and planning (design),
2.Orientation (information gathering about the sampling environment)
3.Survey sampling and follow-up.
4.Data presentation and analysis.
Duration: – 11 Months
For more enquiry call at : 9319368445 or Email Id : email@example.com


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talk of the town.
How to use it
Add 1/3 teaspoon of Edible Flavoured Cocktail Glitter Dust to any drink of your choice. Watch it dissolve and instantly change the colour & flavour, with a beautiful iridescent shimmer.
Nutritional info
Coming soon...
- 4 calories per serving.
- The perfect party starter.
- Delivers a delicious taste as well as looking incredible.
Galaxy-like cocktails, whenever, wherever 🎉
Why not make your party cocktails the talk of the town? Our Edible Cocktail Glitter Dust promises to create beautiful, iridescent. galaxy-like cocktails that are bound to wow people.
Ingredients 🍸
Raspberry Rose Gold
Ingredients: Sugar, Acidity Regulators (Citric Acid, Bicarbonate of Soda, Malic Acid), Natural Raspberry Flavouring, Emulsifier (Gum Arabic), Maltodextrin, Colours (E171, E129), Sweetener (Sucralose).
Black Cherry
Ingredients: Sugar, Acidity Regulators (Citric Acid, Bicarbonate of Soda, Malic Acid, Tartaric Acid), Natural Cherry Flavouring, Emulsifier (Gum Arabic), Colours (E171, E151, E172), Maltodextrin, Sweetener (Sucralose).


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School Closure - Year 1
Year 1- Home Learning Resource Pack
Year 1 - Practical Ideas
Year 1- Home Learning Pack
Year 1 - Home Learning Pack Guidance and Answers
RWI Phonics lessons at home from Monday 23rd March
We're scheduling free Read Write Inc. Phonics lessons on Facebook and YouTube for children to watch at home; three short Speed Sound lessons with one of our trainers every day for the next two weeks. Films will only be available at the times below:
Set 1 Speed Sounds at 9.30 am and again at 12.30 pm
Set 2 Speed Sounds at 10.00 am and again at 1.00 pm
Set 3 Speed Sounds at 10.30 am and again at 1.30 pm
Note: films are streamed live and won't be available at other times.
Please share with RWI schools and parents.


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Working Girl
Custom Designed Leggings by ELB Designs. Full length, Yoga waistband. 92% Polyester 8 % Spandex buttery soft material. One Size (OS) fits most sizes 2-10, Plus Size (PS) fits most sizes 12-18.
25 pairs of OS
25 pairs of PS
4-6 weeks from order to deliver.
To add any other sizing please contact us at
Disclaimer- we can not control customs and factory time frames. Most orders will be delivered within 4 weeks however sometimes can take closer to 6 weeks or more for customs problems.
Colors may slightly vary depend on screen resolution and lighting


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A - Papers appearing in refereed journals
Kanyuka, K., Mcgrann, G. R. D., Alhudaib, K., Hariri, D. and Adams, M. J. 2004. Biological and sequence analysis of a novel European isolate of Barley mild mosaic virus that overcomes the barley rym5 resistance gene. Archives of Virology. 149, pp. 1469-1480.
|Kanyuka, K., Mcgrann, G. R. D., Alhudaib, K., Hariri, D. and Adams, M. J.
|Year of Publication
|Archives of Virology
|Journal citation
|149, pp. 1469-1480
|Open access
|Published as non-open access
|Funder project or code
|Characterisation of plant -virus- vector relationships, with particular respect to transmission by plasmodiophorids
|Project: 4425
Permalink - https://repository.rothamsted.ac.uk/item/8935z/biological-and-sequence-analysis-of-a-novel-european-isolate-of-barley-mild-mosaic-virus-that-overcomes-the-barley-rym5-resistance-gene


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You've just finished reading q.e.d. Iff - Shoumei Shuuryou - chapter 12: Sudden Change by Katou Motohiro
If you like the manga, please click the Bookmark button (Heart icon) at the bottom left corner to add it to your favorite list. If you find any errors, contact us so we can fix it as soon as possible!


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|13th September 2020
Edinburgh Playhouse
18 - 22 Greenside Place, Edinburgh New Town EH1 3AA
From £16
Book Tickets
|Visit the event website here
The smash hit film as you have never experienced it before.
Our live host will warm up your voices in true Freddie Mercury style, teach you some iconic dance moves and show you how to use your interactive prop bags.
Then sit back and sing along with the on screen lyrics to "Bohemian Rhapsody", "Under Pressure" and "Somebody to Love" not forgetting YOU the audience becoming part of their famous Live Aid concert.
We guarantee to have you walking out feeling like the "Champions of the World!"
Please note: this is a screening of the movie not a live stage show.
While we take every opportunity to ensure the details for Sing-a-Long-a Bohemian Rhapsody are accurate, we always advise that you contact the event organiser before setting out for the event to avoid disapointment. All information (whether in text or photographs) is given in good faith but should not be relied upon as being a statement of representation or fact.
Looking to reach new customers? We offer a variety of advertising options with prices starting from just £30 per month!


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Other Vacations
We have had the privilege of planning vacations to several European countries, such as Iceland, Ireland, England, Italy, Norway, France, Spain, Germany, Denmark and Portugal. If you'd like to travel to Europe, you're in good hands!
Whether your travels include Europe, Asia, Africa, French Polynesia or South America, Dreams are Forever Travel can help create just the vacation experience you are looking for!
"We wander for distraction, but we travel for fulfillment."
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Customized travel and vacation plans tailored to your specific vacation interests. We want to create a unique vacation experience, not just a trip!


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Ned V. - Top Academic Tutor with Years of Experience in Most Subjects
$75 / hr
Roswell, GA
20 miles Travel Radius
In-person + Online
Dear student or parent,
I am a professional academic tutor with 20+ years of experience tutoring most subjects at the high school, college and graduate school levels. These include most
* Math courses including algebra, geometry and pre-calc, calculus, linear and coordinate math, statistics differential equations and computer modeling and more.
* Science courses including biology, chemistry, physics, organic chemistry, biochemistry, physics, astronomy, genetics and more.
* Business courses ...
I am a professional academic tutor with 20+ years of experience tutoring most subjects at the high school, college and graduate school levels. These include most
* Math courses including algebra, geometry and pre-calc, calculus, linear and coordinate math, statistics differential equations and computer modeling and more.
* Science courses including biology, chemistry, physics, organic chemistry, biochemistry, physics, astronomy, genetics and more.
* Business courses ...
ReviewsSearch More Tutors in the UCLA Area
Ned, has been tutoring me on HTML5 and CSS3, he has been of great help. Ned is really fantastic in breaking it down, and not only that but he makes it fun- It can certainly feel pretty overwhelming learning what feels like a foreign language. I can say I no longer feel like I am lost in a foreign land after Ned taking me through Web Design, and now I am enjoying it all the more. Last, but not least, I also have to attribute Ned's ability as a professional and tutor to achieving an A in my Web Design class...On another note, I will be embarking into deeper realms into Web Design with JQuery and Javascript. There's no question I plan on calling on Ned for his guidance on this next phase in Web Design. He's the best!
- Caroline B. from Cape Elizabeth, ME
Ned taught me the iterative process of creating a website or app with the minimal amount of elements. I was failing miserably in my class - but with Ned's help I scored a 91.20.
- Stephen P. from Suwanee, GA
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The time spent with Ned has been very productive. Ned is good tutor and easy to work with, he appears very knowledgeable about WordPress in general, implementing WP Themes, working with WP Plugins, CSS, HTML, and general web design concepts and marketing best practices, including social media.
- Rene S. from Atlanta, GA
Ned has been very helpful in setting me up and providing training on Full-Stack Web development, including HTML, CSS, JavaScript, PHP and MySQL. He knows his stuff both in the academic sense and also from a practical, real-world perspective, and has been amazing in helping me to jump start a planned career change. He is amazing and I recommend him highly.
- Patrick J. from Norcross, GA
Ned quickly established a trusting rapport with my 11th grade son who does not want to ask anyone for help. I was concerned that he would go once and not want to go back, but after the first session he was engaged and trying to figure out a good weekly schedule. Ned makes my son feel at ease, treats him like an adult and helps him break down his Calculus problems into terms he can understand. He also spends additional time when needed to help cement that understanding. It's been a very good experience so far!
- Rita H. from Acworth, GA
Ned helped me with coursework and projects for my MS Office 365 Intermediate course. We worked together both in person and online, and he helped me with my questions on MS Word, PowerPoint, Excel, and Access. Ned is patient and explains the material well and made lessons interesting, fun, and very helpful in the real world. I recommend him strongly.
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Ned helped me with my physics 2 course . We did numerous lessons remotely online. He was very conversant and very knowledgeable about the different topics. I went from a C in the class at midterm to having a final grade of A-. I have no regrets and would definitely use him again in the near future.
- Debbie O. from Atlanta, GA
Ned reviewed derivatives with my son and went over notes with him until he felt more comfortable about the subject. He was very patient and answered many questions.
- Jane J. from Marietta, GA
Ned really knows his stuff, he helped me catch up in Physics 2 when I was struggling significantly. He explains things in a way that is easy to understand and is a very likable guy. Ned also provided me with a TON of notes from our sessions that were invaluable in studying for the final exam.
- Davis O. from Kennesaw, GA
Need I say more? The subject was college level physics. My son was really lost and thought he would flunk the exam. Ned really rates 10 stars - but that's not an option.
- Ethan B. from Kennesaw, GA
Patient, intuitive and experienced tutor. Doesn't oversell his services......seems to focus on what is needed to enable the success of the student.
- Douglas W. from Acworth, GA
Ned was incredibly helpful in helping me with last-minute preparation for my taking the MCAT. I had previously taken the Kaplan Course, but Ned was far more helpful in explaining concepts from biochemistry, general chemistry, physics, genetics, enzyme regulation, metabolism, titrations, thermodynamics and electricity. He was very helpful in analyzing CARS passages and in particular he helped me focus on the MCAT test questions themselves, and understanding the special style and thought process involved in understanding these questions and strategies for arriving at the correct solution. He helped me organize what for me was an overwhelming amount of material and to develop connections between various topics that helped me plug holes in my understanding. He also works with students remotely online, and is just as skilled presenting and explaining material via video chat as in person. Mostly, he helped me feel confident with the material and organize my time and study plan. And he helped me by finding supplemental videos and diagrams that helped me understand and see relationships more clearly! I definitely recommend Ned and one of the best tutors and MCAT coaches available.
- Sue G. from Alpharetta, GA
Ned is outstanding at helping to grasp crucial concepts and working through problems. Ned helped teach me derivatives and infinite limits for calculus 1. He also taught me boiling point elevation/freezing point depression, osmotic pressure, molarity, and molality for a Chem 1212 exam.
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Ned was fantastic. VERY knowledgable of the issues I needed help with HTML, CSS with DIV tags and Dreamweaver. He understood from the start what my needs were and was able to provide expert, friendly, and thorough instruction with concrete guidance and explanations. He's extraordinarily conscientious, patient, polite, and knowledgable. He truly cares about your success and is competent in every way. He gets 5 stars and my highest recommendation.
- David V. from Dublin, OH
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- Nancy B. from Roswell, GA
We appreciated Ned's concise explanations and the way he directs the student to the most important aspect of a concept.
- Priya J. from Kennesaw, GA
Such a wonderful tutor! He went over my Physics quiz and broke it down to where I could understand my mistakes and made sure I wouldn't make those same mistakes again. Super patient and friendly. Highly recommended! :)
- Trinh P. from Kennesaw, GA
If you are looking for a tutor for Calculus, do yourself a favor and contact Ned. You'll thank me later. He is extremely organized when it comes to making appointments, and is very easy to reach. Not only was I understanding the material and making good grades, but I found that I was even enjoying working problems that I would have otherwise avoided before I began tutoring with Ned. He is extremely kind, patient, and makes you feel comfortable right away. If I had any complaints, I would write them here. If you are in the area, Ned should be your next tutor.
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- Christian A. from Marietta, GA
Posted ByNed V.


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Code: RG 10/1101.
Description: Unknown or not given.
Registration district: Petworth.
Address: Shorts Cottage.
Type: Inhabited.
Show all the households at this property
Show everyone in this property (Shorts Cottage)
Schedule: 33.
Head of household: Ayling, Edward.
Show everyone in this household (schedule)
Folio: 60.
Page: 10.
Name: Ayling, Harriott.
Occupation: Unknown or not given.
Age: 24.
Gender: Female.
Marital status: Married.
Relationship to head: Wife.
Born In County?: Yes (based on birth parish).
Birth parish: Duncton.
Birth county: West Sussex.
Birth country: England.
Transcribed by: John Morrison.
Batch: CEN_Kirdford_1871 Kirdford.csv.
Source: 1871 Census Kirdford Transcription.


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Four Quick Ways to Have A Whiter, Healthier Smile
If you're just like the rest of the population, then you take your oral health for granted. You skip dental check-ups at Kim Norman, DDS Dentistry for the Individual in Maryville, TN and you cannot be bothered to floss your teeth. … Continued


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Barbara Jergensen
Professional Hairstylist
Barbara has educated herself intensively to suit her clientele. In 2004, she became Susan Alan's 1st Deva Inspired Stylist. She has mastered the revolutionary method for cutting, coloring and styling curly hair. Barbara will teach you to love and care for your curly hair.
Her on-going education includes: Advanced training in Loreal professional hair color, Toni and Guy Academy, Aquage, Sebastian International, Framesi hair color, and is also certified in Bio Ionic straightening, Brazilian Blowout, and Keratin smoothing systems.
If you are looking to donate your hair, Barbara also works with Beautiful Lengths and the American Cancer Society.
Experience: 30 Years