text:
Made in Chelsea's Francis Boulle backs Young Enterprise
Diamond trader and financier makes inspirational speech to 800 at Titanic Belfast
Mending broken surfboards and selling them on to tourists gave the star of Channel 4's Made in Chelsea Francis Boulle his start in business.
The heart-throb revealed the beachside enterprise as he addressed more than 800 students at a Gala Celebration to mark the success of all the Young Enterprise companies formed and traded over the 2012-2013 academic year at Belfast's newest tourist attraction, Titanic Belfast.
He said: "My first business, I was about 11 or 12, and it was repairing broken surfboards and selling the on to tourists. I think it is absolutely integral to have a foundation of understanding key business concepts moving forward, not just for the workplace but for creating your own job, creating your own company and potential legacy.
"It is self evident that the children are the future and we should be investing in them as we should be investing in the small and medium sized enterprises out there today."
He added: " I am Francis Boulle founder and CEO of fund mine .com and I support Young Enterprise Northern Ireland."
Francis Maximilien Yvan Christophe Boulle, born 20 October 1988, is a British entrepreneur, best-selling author and cast member of Made in Chelsea.
Of French, Norwegian and Cherokee descent, Francis was born in London but spent most of his childhood living between France and the United States. He completed his secondary school education at The Oratory School and later went on to study Philosophy at The University of Edinburgh and an MA in International Business at Regent's College London.
Francis began sourcing diamonds for a private client base whilst at boarding school and went on to set up an independent commodity brokerage in his second year of study at the University of Edinburgh. In November 2011, Boulle announced that in response to the lack of current financing options available to SMEs (small and medium sized enterprises), he would be launching fundmine.com, a new type of intelligent angel investment platform.
Fundmine.com provides start-ups and SMEs with a solution to securing seed and venture capital online. Through a system developed with the protection of the entrepreneurs' IP at its core, Fundmine.com provides an environment for entrepreneurs and private companies operating across all sectors to dynamically present their visions to a network of investors.


text:
Still working on my Captain Mothstorm illustration.
Added some colour:
I've updated the character profiles for my digital comic project again. Basically, I've given the moths some funky mind powers!
First name: Sylen
Middle names:
Family name: Benevolen
Height: 184cm
Weight: 80kg
Continue reading Project ADC-1 Character Profile Sheet V4
This guy is one of the baddies from my comic project. A big impassive henchman type.
OK so this script has been through TPG twice and I have just gotten round to re-working page 1 after the last round of critiques.
My main concern is the dialogue now. The setting is on another world so I originally tried medieval type speech but I think I over did it so I have toned that down.
I have also tried to ease up on the info dumping .
After some good ideas from Ryan Kroboth on the panel layouts, I've altered that as well.
Page 1 (7 panels in 3 rows)
Panel 1 (full width of top row):
Establishing shot. It is dusk. The planetary rings are visible in the purple cloud-streaked sky. We are looking out at a misty mountain scene with the Dark Temple of Mymosule visible in the distance on the right of the panel. In the foreground on the left of the panel we see a full length side view of Captain Sylen Benevolen gazing at the temple with his helmet in his right arm. He is actually stood inside a wide rectangular hangar type space. He is in a derelict skyscraper that is leaning to the right of the panel so the walls, floor and ceiling are angled accordingly. At the edges of the panel we can see vines growing on the walls and ceiling. The walls and ceiling are covered with green moths. At the top left of the panel, there is a green moth in extreme close up on a vine.
Benevolen: We used to race sky-panthers together as children.
Benevolen: Have you found her?
Green moth (telepathically): Not yet, but we have counted 107 individual acolytes so far inside the temple, Sylen. They have guns and blades.
Panel 2 (middle row left):
Half body shot of Benevolen from the waist up. He has just put on his helmet (his hands are still on his head). Some of the moths have taken wing.
Green moth (telepathically): Lady Umia is in a metal cage. They are carrying her to the sacrificial chamber! We need to help her quickly!
Benevolen: Their rituals are horrific. I thought the Acolytes Of Mymosule had ceased to exist long ago, during the Breaking Of The World.
Panel 3 (middle row centre):
Rear, full length shot of Benevolen who is throwing himself out of the hangar. The moths are streaming after him.
Benevolen: Once Umia is safe, I will return with a battalion of the Suborean Guard and eradicate them.
Panel 4 (middle row right):
Reverse the camera and move it back so we are now facing the derelict skyscraper. Front full shot of Benevolen who is hurtling down the side of the derelict skyscraper towards the swirling mist below. The moths are swarming down after him. We can see the mist at the bottom of the panel.
Panel 5 (bottom row left):
Benevolen has plummeted into the mist sending it billowing up. The moths are swarming into the mist after him.
Panel 6 (bottom row centre):
Shot of the dark temple in the background with the moths swarming down into the mist in the foreground. No sign of Benevolen.
Panel 7 (bottom row right):
Shot of the dark temple in the background with the moths swarming up out of the mist in a batwing formation in the foreground. Benevolen is suspended beneath them. The mist is swirling in their wake.
I've updated my character profiles for my digital comic project and also included some pictures.
First name: Sylen
Middle names:
Family name: Benevolen
Height: 184cm
Weight: 80kg
Continue reading Project ADC-1 Character Profile Sheet V3
In an attempt to flesh out my comic book characters a bit more I'm going to write some quick micro fiction about them.
This piece is supposed to be an audio journal entry from Umia as she is out riding on Torina, her winged panther.
The Audio Journal Of Lady Umia Yulon
Date: Wutanday 17th Ortumum 7117
Time: 06:13 hours
"The sun rise is breath taking this morning. I'm taking Torina out for our morning flight. "
"It's silly but I've often dreamed of going all the way up through the atmosphere and touching those planetary rings above us. Beautiful."
"Anyway enough nonsense, back to the interval training."
"We'll warm up for another 10 minutes and then do the first sprint."
"Wait … what was that?"
"Someone's shooting!"
"They're coming for us Torina! Fly like the wind girl!"
"No no no no!"

subset: commoncrawl


text:
In June, 1863, the Sixth Corps made its third successful crossing of the Rappahannock, storming the works that defended the passage, and making prisoners of the garrison. Battery D at once took position, with other artillery of the Sixth Corps, out in the fields, near the ruins of the Mansfield House, where Gen. Bayard was killed, at the time of the first crossing by General Burnside in 1862. This picture was made as the guns were engaging the enemy, the gunners who had just received the order, "cannoniers to your posts," calling to the photographer to hurry his wagon out of the way, unless he was anxious to figure in the list of casualties. In line to the rear of the Battery, is the Veteran Vermont Brigade, acting as a support. Further still, is the bank of the river, skirted by the trees; those to the right being a grove of white poplars around the Mansfield House. With characteristic coolness some of the troops had already pitched their little shelter tents, and were sleeping beneath their frail cover. Better protection was soon afforded by the fine line of earthworks which soon sprung into existence, embrasured for the artillery, and impregnable to attack when lined by the heroes of the Sixth Corps, nobly commanded, as it has always been, by such Generals as Franklin—who organized it—Sedgwick, and Wright. This Battery was present at the first battle of Bull Run, where the enemy got a taste of its mettle, while attempting to turn our extreme left, and fall upon the rear of the army. On the organization of Franklin's Division, its commander, Captain—now Colonel—Platt, was made chief of a brigade of artillery in that organization, and Lieutenant, since General, Upton succeeded to the command. Under the latter officer the Battery gained much commendation, and on his promotion to the command of a New York regiment of volunteers, continued its glorious career under Lieut. Williston, being finally transformed into a horse-battery, and ordered to the cavalry, where it remained to the close of the war, without ever losing a gun, although the list of its actions was so long that its battle-flag had no space to transcribe them upon.
taken from original text, Plate 31, Vol. I,
Note: Though the book plate is labeled as Battery D, 5th U.S. Artillery, it is actually Battery D 2nd U.S. Artillery that is pictured.
Battery D, 5th U.S. Artillery, in Action. Fredericksburg, Virginia. June, 1863 Albumen print photograph by Timothy H. O'Sullivan.
© 2002 Division of Rare & Manuscript
reference questions, send mail to:


text:
Vote for Your Favourite Project in the National Lottery Awards from Today
Last Updated: Wednesday 24th June 2015, 10:12 am
Voting is now open for the National Lottery Awards, in which the public chooses their favourite projects that have been made possible by the National Lottery Good Causes fund. A panel of judges narrows down each year's entries until they are agreed on the seven most worthy programmes in seven different sectors - arts, education, environment, health, heritage, sport and voluntary.
Each cause nominated has benefitted from lottery funding, with 28p of every pound played on Lotto, EuroMillions and instant win games being placed into the Good Causes pot. In addition, any prizes that remain unclaimed 180 days after the draw date will also be used to fund community projects across the UK. Find out more about where your money goes at the National Lottery Good Causes Fund page.
You have until Wednesday 29th July to make up your mind about your favourite entries, with the winners in each category receiving £2,000 to boost their coffers.
Projects up for the vote in the arts section include a scheme to promote music concerts in libraries, an educational project that commemorated the First World War through music and dance and the £15 million renovation of the Whitworth Gallery in Manchester. The education category is honouring programmes such as the High Street Experiment, which helps young people who have missed out on schooling learn new skills and earn qualifications.
The restoration of Lincoln Castle, which provided an improved area in which to display its copy of Magna Carta on its 800th anniversary, is nominated in the heritage category. It is up against projects such as the St Columba Heritage Centre in Derry/Londonderry. A charity called ORCA that educates children about whales is represented in the environment section, along with a scheme to plant hedges in Ballymoney, Northern Ireland, along an avenue that has featured in hit TV show Game of Thrones.
Music Matters, which provides live musical performances for people in care homes, and an alcohol-free bar and cafe in Liverpool are amongst the hopefuls in the health category. In the sport section there is a wheelchair rugby league team competing against the This Girl Can programme to encourage women to celebrate being active, and the voluntary category sees a helpline for lonely older people, a West Midlands foodbank and five others hoping to claim the prize.
Every time you play National Lottery games, you are ensuring these and thousands more fantastic schemes across the country can carry out great work. Buying tickets for tonight's £5.6 million Lotto draw will help those in need and could win you a life-changing amount of money. Use the Lotto Number Generator to help you pick a truly random selection of numbers that could well bag you the big prize this evening!


text:
Figuring out a way to keep in touch with your family and friends? Well, I have stumbled upon one, nothing intricate. Keeping in touch made easy in a budgeted weekend project and here is a few items required.
- Droprint – An app that manages your pictures and have them printed at an affordable price. Available for Android and iOS.
- Envelopes
- Sharpee / Markers
- Stamps
Droprint delivers your desired printed pictures and have them delivered to your door step.
Here is what I did, I got the pictures printed, put them into individual envelopes and have them snail-mailed to my friends and families (without informing them). Surprises are meant to be surprises 🙂
The app is easy to use.
More info/update, find/follow:
Website: www.droprint.my
Facebook: Droprint


text:
Routes : North America : Mexico : Nuevo León : El Potrero Chico : Culo De Gato : 10. The Sick Dimension
10. The Sick Dimension - 5.14bAverage Rating : 0.00 out of 5
Route sequence (left to right): 1
Route Summary | Ascent Notes (0)
Premier Sponsor:
16 quick draws
Description:the Hardest climb in Mexico, originally graded 5.14b by First Ascentionist Rodman (Rodney Blakemore), more than 10 key holds broke since he climbed it upping the grade considerably. This route will never be downgraded, it is SOLID 14B now for sure. This is the route directly to the right of the Crawling King Snake. It starts at the 3rd bolt (it's 5.6 up to there) then some 5.13 climbing brings you to the crux V10/11 (where a key hold broke making one single move from V2 to V9), after the crux enjoy the only rest on the route before punching it through the 45 degree overhanging sustained 5.13c to the anchors (don't worry, if you can do the crux, you won't fall here because compared to the crux, this 5.13 section feels more like 5.8). you can fix a rope to the anchor and go for the ride of your life (MASSIVE SWING) from the 1st bolt, but don't let go or you'll be picking cactus spines outta your butt for weeks!
Submitted by: manboy on 2001-08-14
Views: 898
Route ID: 2786


text:
Are capacity bottlenecks in the brain's information processing pathways, at the heart of many mental conditions and disorders?
Differences between monkey and human mirror-neuron systems.
Ageing affects people's ability to correctly interpret proverbs.
Comparing the mental health of children and teenagers across 12 European countries.
You have read this article Extras with the title Extras. You can bookmark this page URL http://psychiatryfun.blogspot.com/2008/07/extras_9.html. Thanks!


text:
BioMed Research International
Volume 2013, Article ID 526069, 14 pages
Molecular and Immunogenic Properties of Apyrase SP01B and D7-Related SP04 Recombinant Salivary Proteins of Phlebotomus perniciosus from Madrid, Spain
Unidad de Entomología Médica, Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera Majadahonda-Pozuelo s/n, Majadahonda, 28220 Madrid, Spain
Received 12 April 2013; Revised 26 July 2013; Accepted 16 August 2013
Academic Editor: Hirotaka Sakamoto
Copyright © 2013 Inés Martín-Martín et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Sand fly salivary proteins are on the spotlight to become vaccine candidates against leishmaniasis and to markers of exposure to sand fly bites due to the host immune responses they elicit. Working with the whole salivary homogenate entails serious drawbacks such as the need for maintaining sand fly colonies and the laborious task of glands dissection. In order to overcome these difficulties, producing recombinant proteins of different vectors has become a major task. In this study, a cDNA library was constructed with the salivary glands of Phlebotomus perniciosus from Madrid, Spain, the most widespread vector of Leishmania infantum in the Mediterranean basin. Analysis of the cDNA sequences showed several polymorphisms among the previously described salivary transcripts. The apyrase SP01B and the D7-related protein SP04 were successfully cloned, expressed in Escherichia coli, and purified. Besides, recombinant proteins were recognized by sera of hamsters and mice previously immunized with saliva through the exposure to uninfected sand fly bites. These results suggest that these two recombinant proteins conserved their immunogenic properties after expression in a prokaryote system. Therefore, this work contributes to expand the knowledge of P. perniciosus saliva that would be eventually used for the development of tools for vector control programs.
1. Introduction
Leishmaniasis is still one of the most important vector-borne diseases in terms of incidence as two million people per year are affected worldwide [1]. The causative agent of the aforementioned disease is Leishmania spp., a flagellate parasite which is transmitted by infected phlebotomine sand flies (Diptera: Psychodidae) during blood feeding. Arthropod saliva is actively involved in the transmission of pathogens to its host as it contains a complex cocktail of antihaemostatic and immunomodulatory molecules that are inoculated into the host skin during blood feeding of both infected and noninfected sand flies [2]. Concretely, sand fly salivary components are known to play an important role in the establishment of Leishmania spp. infection [3]. In the last years, research on sand fly salivary proteins has greatly increased, suggesting that salivary proteins could be successfully assayed both as anti-Leishmania vaccine candidates and as markers of exposure to sand flies [4, 5]. As hosts are bitten, they develop both humoral and cellular responses against sand fly saliva [6]. Moreover, a positive correlation has been observed between the number of bites and antibody levels [4, 7, 8]. Therefore, host exposure to sand flies can be measured by evaluating humoral responses against salivary antigens. This methodology is being applied by mainly using salivary gland extracts [4, 9–12]. Recombinant salivary proteins have already been produced for sand fly species such as Lutzomyia longipalpis and Phlebotomus papatasi [4, 8, 10, 11, 13, 14]. Some of these proteins have been already proved as good markers of exposure [11, 14]. However, since salivary proteins display high specificity, it is necessary to produce immunogenic salivary proteins for other sand fly species.
In the western Mediterranean basin, Leishmania infantum is mainly transmitted by Phlebotomus perniciosus in an anthropozoonotic cycle where dogs have been traditionally considered the main reservoir [1]. However, other potential reservoirs such as hares (Lepus granatensis) have been recently involved in L. infantum transmission at a human leishmaniasis outbreak in Madrid [15, 16]. As a part of a management plan to control the disease in this environment, measuring exposure of reservoirs to the main vector involved, P. perniciosus, through the detection of anti-saliva antibodies, would be useful to evaluate whether actions taken to reduce leishmaniasis have been effective as previously done in India and Nepal [9].
In previous studies, our group described immunogenic salivary proteins of P. perniciosus and P. argentipes. These proteins were identified through the combination of two-dimensional electrophoresis and Western blot with sera of mice and hamsters experimentally exposed to uninfected sand flies [17, 18]. Therefore, in this study, our aim was to obtain some of the P. perniciosus salivary immunogenic molecules as recombinant proteins, through a cDNA library from salivary glands, from P. perniciosus and subsequent studies of the immunogenicity of the purified salivary proteins. Moreover, several polymorphisms between transcripts of the P. perniciosus cDNA from Madrid were compared to the previously annotated ones that belonged to specimens from Italy [19].
2. Material and Methods
2.1. Sand Flies and Salivary Glands Collection
P. perniciosus sand flies were maintained at 27°C and 17 : 7 light-darkness photoperiod at the Medical Entomology Unit of the Instituto de Salud Carlos III (ISCIII), Madrid, Spain. This colony was established in 1987 from sand flies captured at a leishmaniasis endemic area of Madrid [20]. Salivary glands from recently emerged up to 1-day-old sand flies were dissected and stored in RNAlater (Invitrogen, San Diego, CA).
2.2. Salivary Gland cDNA Library Construction
A cDNA library was constructed with mRNA isolated from 165 salivary glands using the Micro-FastTrack mRNA isolation kit (Invitrogen, San Diego, CA). After isolation, mRNA was reverse transcrbted to cDNA and subsequently amplified by PCR following the instructions of the SMART cDNA library construction kit (Clontech). cDNAs were then fractionated by column chromatography before cloning. Directional cloning into λTriplEx2 vector (Clontech) was achieved through SfiIAB flanking sites incorporated by PCR during the cDNA amplification. The pool of cDNAs cloned into λTriplEx2 vector was packaged into phage particles following the manufacturer's instructions (Gigapack III Gold Packaging Extract, Agilent). The resulting cDNA library was amplified following general molecular biology protocols [21].
2.3. cDNA Library Screening, Sequencing, and Bioinformatics
After plating the library by infecting log phase XL1-blue cells (Clontech) in the presence of β-D-thiogalactoside (IPTG) and 5-bromo-4-chloro-indolyl-β-D-galactopyranoside (X-Gal), white plaques were randomly picked from the agar plates and analyzed by PCR using λTriplEx2 primers (forward: 5′-TCCGAGATCTGGACGAGC-3′ and reverse: 5′-TAATACGACTCACTATAGGGC-3′). Recombinant phages which presented the greatest insert sizes were converted into pTriplEx2 plasmid through Cre recombinase-mediated site-specific recombination. Plasmid DNA was then isolated and sequenced using the primer 5′λTriplEx LD: 5′-CTCGGGAAGCGCGCCATTGTGTTGGT-3′ and the BigDye Terminator v3.1 Cycle Sequencing kit (PE Biosystems, Foster City, CA) in an ABI PRISM 3730XL DNA Analyzer (Applied Biosystems). Both strains of clones of interest were subsequently sequenced with reverse primer 3′λTriplEx LD: 5′-ATACGACTCACTATAGGGCGAATTGGCC-3′. DNA electropherograms were manually inspected and corrected using Chromas program (McCarthy, Queensland, Australia). Sequence identities were determined by BLAST (http://blast.ncbi.nlm.nih.gov/). DNASTAR software (Lasergene, Madison, WI) was used for nucleotidic alignments (SeqMan program), and protein features were assigned by Protean tools. Amino acid sequences were aligned with ClustalW (http://www.ch.embnet.org/software/ClustalW.html) and refined using Boxshade server (http://www.ch.embnet.org/software/BOX_form.html). Glycosylation sites were predicted using NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) and NetOGlyc 3.1 Server (http://www.cbs.dtu.dk/services/NetOGlyc/) for N- and O-glycosylation sites, respectively [22, 23]. Phosphorylation sites prediction was done using NetPhos 2.0 Server (http://www.cbs.dtu.dk/services/NetPhos/) [24].
2.4. Cloning of Salivary Gland cDNAs
Salivary coding sequences of the apyrase SP01B and the D7-related protein SP04 were amplified by PCR from the corresponding pTriplEx2 plasmids using specific primers. cDNAs were cloned into PCR4-TOPO plasmid and transformed into TOP10 competent cells (Invitrogen, San Diego, CA). Restriction sites were incorporated by PCR from the recombinant PCR4-TOPO vector. SalI (forward and reverse) was used as a restriction site (represented as bold letters): SP01B: 5′-GTCGACATGATATTGTTGAAATTG-3′ and 5′-GTCGACTTACTTAATTCCTTTGGG-3′ and SP04: 5′-GAGAGAGTCGACATGAATACCTTATTG-3′ and 5′-GAGAGAGTCGACCTAATAATTTGTTAATG-3′. The primers were manually designed according to the sequences obtained. All primers were synthesized at the Genomic Unit of ISCIII. The high-fidelity polymerase Pfu Turbo Hotstart (Stratagene, La Jolla, CA) was used in order to avoid point mutations. Coding sequences were excised from the plasmid by enzymatic digestion and ligated into pqE31 vector (Qiagen, Hilden, Germany). The recombinant expression vectors pqE31 were transformed into competent M15 (Qiagen, Hilden, Germany). All construction steps were verified by sequencing.
2.5. Expression and Purification of Recombinant Salivary Proteins
Different expression and purification conditions were tested in order to optimize the processes for these specific proteins. Cultures of M15 cells that contained the pqE31 recombinant plasmid for SP01B and SP04 were grown in Luria-Bertani medium containing ampicillin (100 μg/mL). Protein expression was induced by the addition of IPTG to a final concentration of 1 mM, and cultures were grown at 37°C for 3 hours. Bacterial cells were collected by centrifugation and lysed in 6 M guanidine hydrochloride, 100 mM NaH2PO4, 10 mM Tris-HCl, and pH 8. Cell debris was then separated by centrifugation, and the resultant supernatant was submitted to affinity chromatography by using Ni-NTA Superflow resin in prepacked 5 mL columns. Proteins were purified under denaturing conditions following the manufacturer's instructions (Qiagen, Hilden, Germany). Protein refolding was done by removing urea through dialysis against PBS (SnakeSkin Dialysis Tubing 10 kDa MWCO, Thermo Scientific, Goettingen, Germany). Proteins were then concentrated using the stirred ultrafiltration cell with 10 kDa MWCO membranes (Millipore, Bedford, MA) and quantified by gel in comparison with BSA standards.
2.6. Western Blotting
Western blots were performed following standard protocols. Briefly, 1 μg of recombinant protein was separated by SDS-PAGE and electroblotted onto a PVDF membrane which was incubated overnight in blocking buffer (3% BSA, Sigma, St. Louis, CA; 2% ECL Blocking reagent, Amersham, Piscataway, NJ). After washing, membranes were incubated for 2 hours with pooled sera (1 : 25) of either hamsters that were immunized against P. perniciosus saliva by the bite of uninfected sand flies through the exposure to 100 sand flies on a weekly schedule over 10 weeks [17] or mice exposed 13 times to 150 P. perniciosus (unpublished). Goat anti-hamster IgG and goat anti-mouse IgG peroxidase-conjugated antibodies (1 : 3500, Southern Biotech, Birmingham, AL and 1 : 500, AbD Serotec, resp.) were used, and immunogenic proteins were visualized by CN/DAB reagent following the manufacturer's instructions (Thermo Scientific, Goettingen, Germany). Experiments with sera of immunized and nonimmunized animals were carried out in parallel. For His-tag detection, mouse anti-RGS-His antibodies (1 : 3000, Qiagen, Hilden, Germany) were used in combination with the goat anti-mouse IgG peroxidase-conjugated antibodies (AbD Serotec).
3. Results and Discussion
3.1. cDNA Library Analysis
The expression library showed a titer of 3.6 × 106 and 17.6% of nonrecombinant clones. The amplified library displayed a larger number of independent clones (1.2 × 1012) and a low number of nonrecombinant clones (1.92%). Therefore, this amplified library was used for subsequent analysis and cloning processes. 201 white plaques were randomly picked from IPTG/X-Gal plates and analyzed by PCR. 36 out of 201 plaques showing the greatest insert size were selected, converted into pTriplEx2, and further sequenced. Among the 25 complete transcripts, they were identified mostly as apyrases (SP01, SP01B), D7-related proteins (SP04, SP04B), yellow proteins (SP03B), ParSP25-like proteins (SP08), lufaxin-like proteins (SP06), and PpSP15-like proteins (SP02, SP09, and SP11). Complete sequences were annotated at EMBL nucleotide database, and NCBI accession numbers are shown in Table 1.
Overall, high degree of conservancy was found among the salivary transcripts we sequenced, and their best matches available in nonredundant databases were obtained from a cDNA library from P. perniciosus from Italy [19]. This finding is in contrast to the high level of divergence found for the salivary protein maxadilan of L. longipalpis from distinct locations [25].
In the case of maxadilan, the high level of polymorphisms elicits variant-specific antibodies with little cross-reactivity, and it has been suggested that sand flies may have evolved diversity in maxadilan as a strategy to evade the host immune response against this essential vasodilator peptide that facilitates blood feeding of L. longipalpis. Balancing selection might be maintaining many maxadilan alleles with equivalent vasodilatory potencies [25, 26]. On the other hand, our results match the description of a high degree of conservancy among salivary transcripts in two geographically distant Phlebotomus duboscqi sand fly populations in Africa, and it was suggested that sand flies belonging to the genus Phlebotomus show greater degree of conservancy than Lutzomyia spp. [27]. Following this trend, in previous experiments of our group, we did not find qualitative differences in the salivary protein profile among three colonies of P. perniciosus collected from different areas of Spain and reared under identical laboratory conditions [17]. Moreover, a recent comparison between transcripts from cDNA libraries constructed with P. papatasi strains from Israel and Tunisia showed a high level of conservancy [28]. Indeed, the high degree of conservancy found between the salivary transcripts of P. perniciosus from Spain and Italy may be a reflection of little evolutionary pressure from the host immune response on the analyzed salivary proteins in contrast with previous observations of maxadilan. Moreover, studies on the evolution of apyrase from several Phlebotomus species show high degree of conservancy, and the geographical pattern of genetic variation was consistent with neutral demographic processes mainly regional isolation and isolation by distance resulting from the changes that occurred during the late Pleistocene [29]. In the case of secretary salivary proteins, it is possible that the presence of multiple copies reflects an adaptation to increase the production of these important proteins for sand flies [30]. Salivary peptides often occur within and between gene families, for which there is evidence for overlapping functions [19]. Redundancy is a common property among salivary proteins of sand flies and other arthropods [17, 31], and it has been suggested that it might play a role in ensuring blood-feeding success [32].
Therefore, several polymorphisms were found when comparing the sequenced salivary transcripts in this work with their corresponding annotated transcripts. Although these polymorphisms are expected among populations, we aimed to in silico study in detail whether these polymorphisms could lead to changes in phosphorylation and glycosylation processes, structure, immunogenic properties, and functionality through bioinformatics-predictive programs. In order to confirm these single nucleotide polymorphisms (SNPs) both 5′ and 3′ strands were sequenced and aligned. Most of the SNPs were located among the 5′ and 3′ untranslated regions (UTRs) which are known for the high variability [33]. On the other hand, several SNPs were found on the translated region, and some of them resulted in amino acid changes. Substitutions in coding regions may influence protein structure, and therefore electrostatic forces and ligand affinity may involve changes in protein function [34]. Prediction of locations of alpha and beta regions, as well as the antigenic index and phosphorylation sites for the sequenced proteins, confirmed the presence of several changes from their best matches in databases as shown in Table 1. Polymorphisms found in SP01B and SP04 resulted in changes of the antigenic index from positive to negative values and vice versa when compared to the Italian strain (located at T166 for SP01B and R51 for SP04). In the case of phosphorylation residues, most of the analyzed transcripts presented changes regarding to the P. perniciosus Italian strain. For instance, in the apyrase sequence SP01B, the amino acids T163 and Y148 were predicted to be phosphorylated in the Italian strain but not in the Spanish one (Figure 1). Although point mutations can change the ability of the protein to bind carbohydrates, the SNPs were not located at the N- and O-glycosylation sites as in silico predicted through bioinformatic programs (Table 1).
A description of the salivary proteins from P. perniciosus Spanish strain is listed below.
3.1.1. Apyrases
SP01 and SP01B belong to the protein family of Cimex apyrases which are typically found in sand flies and bed bugs. These enzymes hydrolyze nucleotide di- and triphosphates to orthophosphates and mononucleotides and act as potent antihaemostatic factors [13]. Transcripts coding for SP01 and SP01B in our library are conserved with the respective genes from P. perniciosus from Italy (Figure 1).
3.1.2. Odorant-Binding-Related Proteins
In sand flies two salivary protein families are included among the odorant-binding protein superfamily; the D7-related proteins and the PpSP15-like proteins [35, 36].
The SP04 and SP04B are classified as D7-related proteins. D7-related proteins are widely distributed in the saliva of haematophagous Diptera. One of the first cloned proteins from the saliva of Aedes aegypti was arbitrarily called D7 [37], and since then similar proteins have been identified in the saliva of other Nematocera such as mosquitoes, black flies, Culicoides, and sand flies [36]. Further studies showed that the odorant-binding-protein superfamily had a multigene organization and due to gene duplication processes produced two distinct forms of proteins called long and short forms [38]. Sand fly D7-related proteins are related to the long form [39].
In mosquitoes, D7 proteins are able to bind biogenic amines and leukotrienes, in addition to various components of the coagulation cascade, thus interfering with the haemostatic and host immune responses [40]. The role they play in sand flies has not yet been clarified however, their great representation at both transcriptomic and proteomic levels [17–19, 28, 41] and the conservation of the cysteinyl leukotrienes-binding motifs [42] suggest an involvement in counteracting the coagulation cascade [41]. Concretely, the binding site for thromboxane A2 (TXA2) analogs in A. stephensi D7 protein (GenBank ID: 315583502) lies in a hydrophobic pocket in the N-terminal domain which accommodates a large portion of the fatty acid chain. Tyrosine at position 52 in A. stephensi D7 protein is known to stabilize the TXA2 analogs through hydrogen bonds [42]. In A. aegypti D7 protein (GenBank ID: PDB:3DXL_A), a mutation of this position from tyrosine to phenylalanine leads to a failure to bind TXA2 analogs [43]. In sand flies, most of the D7-related proteins conserved a residue of tyrosine in this position (Y65); however, both SP04 from P. perniciosus Spanish and Italian strains contain a phenylalanine in that position (Figure 2). Moreover, in the D7-related protein SP04 from the P. perniciosus Spanish strain (GenBank ID: CCK73754), one of the cysteinyl leukotriene-binding motifs located at R51 showed a polymorphism (Figure 2). However, without experimental confirmation of the functionality of D7-related proteins in sand flies, these changes in the binding motif remain to be elucidated. The D7-related protein SP04B from the Spanish strain (GenBank ID: HE980443) showed two deletions between T546 and A547 that lead to a switch in the reading frame of the gene and an earlier stop codon (546TAG548, data not shown). The predicted protein should appear in the polyacrylamide gels at a theoretical molecular weight (Mw) of 20.7 kDa, however, it had not been found at that Mw [17, 19]. In order to check whether all SP04B clones present in this library included this mutation, we amplified the SP04B gene with primers flanking the CDS, the cDNA library as a template, and further sequenced the extracted DNA. We found the complete sequence for SP04B without that mutation, confirming that in our library both clones are present.
SP02, SP09, and SP11 belong to the PpSP15-like family which have been found only in sand flies and represent the most abundant family among sand fly salivary proteins [19, 41, 44]. PpSP15-like proteins were described as highly polymorphic and derived from a multicopy gene family [19, 30]. The sequences of these proteins show a high degree of variability in addition to certain conserved cysteine residues [28]. In P. papatasi, large numbers of variants of the PpSP15 gene were observed, and differences between alleles were small and resulted in only few amino acid substitutions [30]. Therefore, polymorphisms within transcripts are expected, as shown in other species. Some transcripts, such as SP02, were found to be highly representative within the cDNA library (Figure 3). One of the sequenced clones of SP02 (GenBank ID: HE985075) presented a single nucleotide deletion between A340 and T341 that leads to a switch in the reading frame of the gene. As a consequence, the putative-translated protein (GenBank ID: CCM43815) would differ in sequence and length since it contains an earlier stop codon (417TGA419, Figure 3). Although these changes do not seem to affect its immunogenicity, as the polymorphisms were found within an area of low antigenicity according to the Jameson-Wolf index (data not shown), the function of this protein family remains unknown. Interestingly, immunization with P. papatasi SP15 protected mice from cutaneous leishmaniasis caused by Leishmania major [45].
3.1.3. Yellow Proteins
The yellow proteins are found in the saliva of insects, and they were named after observations in Drosophila, where mutation of a given gene gave a yellow phenotype, indicating that they are involved in melanization processes and pigmentation [46]. In sand flies, yellow proteins are widely represented at both transcriptomic and proteomic levels, showing molecular weights around 41–45 kDa and a wide range of isoelectric points [17, 19, 35, 41, 44]. Recently, it has been demonstrated that yellow proteins from L. longipalpis are able to bind and therefore inhibit the effects of several biogenic amines such as serotonin, norepinephrine, epinephrine, and histamine. Therefore, they act as vasodilators and as inhibitors of platelet activation, itch, and pain [47]. Yellow proteins are conserved among sand fly species as shown in Figure 4.
3.1.4. Par25-Like Proteins
This protein family displays a molecular weight around 25 kDa and an isoelectric point of 4.4–5 due to the great proportion of acidic residues (Figure 5). There are conserved regions rich in certain amino acid residues, and this protein family has been identified in Adlerius (P. arabicus), where it represents the second most abundant protein family in the salivary glands cDNA, and Larroussius (P. ariasi, P. perniciosus, and P. tobbi) [19, 41, 44, 48]. Although its function is still unknown, several members of the sand fly ParSP25-like family stand to be highly immunogenic. Concretely, P. perniciosus SP08 is highly recognized by the sera of mice, hamsters, and dogs exposed to the bites of this vector [7, 17]. Moreover, a plasmid encoding P. ariasi SP25 strongly elicits both humoral and delayed-type hypersensitivity responses in mice [48].
3.1.5. Lufaxin-Like Proteins
SP06 from P. perniciosus Spanish strain (GenBank ID: CCK18305), a component of the 33 kDa family protein, shares 44% sequence identity with lufaxin from L. longipalpis (GenBank ID: AAS05319, Figure 6) whose antithrombotic and anti-inflammatory effects have been recently described [49]. These alkaline proteins of molecular weight around 32–36 kDa seem to be specific for sand flies, and they have been identified in species of both genera Phlebotomus and Lutzomyia [19, 28, 35, 41, 49–51].
The transcript coding for a hypothetical protein P119 (GenBank ID: HE985078) was found among the sequenced cDNAs. This predicted protein shares homology with several hypothetical proteins of different insects, being the best match from A. aegypti (GenBank ID: XM_001663068). However, since it lacks signal peptide, it will not probably be secreted into the salivary gland lumen and would possibly be found in the cells of the gland walls.
3.2. Cloning, Expression, and Purification of Salivary Proteins
cDNA sequences of the salivary proteins SP01B and SP04 were successfully cloned in frame into expression vectors as confirmed by sequencing. Different expression conditions of the apyrase SP01B (37.3 kDa) and the D7-related protein SP04 (28.9 kDa) were assayed, and the best results were obtained when we induced cultures with 1 mM IPTG during 3 hours at 37°C. Batch purification of both 6xHis-tagged proteins was achieved by His-tag affinity chromatography using Super flow Ni-NTA resin under denaturing conditions. Adequate expression pattern was attained for these salivary proteins as the corresponding Coomassie blue-stained bands matched the theoretical Mw, and Western blot revealed the presence of His-tagged protein (Figure 7 and Figure 8(b), resp.).
The apyrase SP01B and the D7-related protein SP04 were chosen for expression and purification since they had previously described as antigens for mice, hamsters, and dogs [7, 17], and they are highly represented in the saliva as seen in P. perniciosus salivary proteome [17, 19]. Apyrases have been pointed as good vaccine candidates since they display putative MHC epitopes, and a recombinant apyrase of P. ariasi produced protective cellular DTH responses in mice [27, 29, 48]. Besides, apyrases are conserved proteins among sand flies and therefore good candidates for a wider vaccine [29].
3.3. Immunogenicity of Recombinant SP01B and SP04
Both recombinant proteins SP01B and SP04 were recognized by the sera of hamsters and mice exposed to sand fly bites in Western blot (Figure 8(a)).
Therefore, immunogenicity of the recombinant proteins was conserved for at least these animals after expression in a prokaryote system. Thus, we hypothesize that part of their immunogenicity is due either to lineal epitopes or conformational ones after an appropriate refolding. Posttranslational modifications that may occur in nature might contribute to their immunogenicity but do not seem to be essential for the recognition of mice and hamster sera as the recombinant proteins lack them. Previously, we had found that SP01B was recognized by the sera of both hamsters and mice while SP04 was highlighted only by hamster sera [17]. However, in the current experiments, both recombinant proteins were recognized by hamster and mice sera. In the case of SP04, the discrepancy observed with previous results may be due to the differences in the schedule followed to immunize the mice (100 flies weekly over 10 weeks versus 13 times exposure to 150 flies). Moreover, the anti-saliva IgG levels from P. perniciosus mice immunized 13 times to 150 times were much higher than those sera from the other set of mice (data not shown).
4. Conclusions
Several polymorphisms were found between transcripts from the cDNA library of salivary glands of P. perniciosus and the cDNA library previously annotated [19], and further studies should be done in order to determine the biological meaning of all these polymorphisms. On the other hand, high level of conservancy was found between salivary transcripts of P. perniciosus from Spain and Italy thus, little antigenic variation is expected suggesting that recombinant salivary proteins could be used in different geographic areas where this sand fly species is present. Moreover, we successfully cloned, expressed, and purified SP01B and SP04 salivary proteins of P. perniciosus. Further characterization of these recombinant proteins will give additional information about their function, especially for SP04, as the functionality of the D7-related proteins has not been experimentally confirmed in sand flies. In addition, we preliminary tested the immunogenicity of these proteins with hyperimmune sera of mice and hamsters experimentally exposed to sand fly bites. Yet, these proteins should be further tested with sera of other reservoirs such as dogs, cats, hares, rabbits, and also with human sera in order to assess if they preserve immunogenicity for these species or not. In addition, further characterization of cellular immune responses of these recombinant proteins should be carried out to determine whether they could be selected as vaccine candidates against leishmaniasis.
Furthermore, to ensure that the best epidemiological markers are selected, it should be necessary to evaluate several recombinant proteins with sera of different hosts to select the most widely recognized proteins and test them alone or in combinations to cover a wide range of host immune responses. In this sense, additional works are in progress to obtain other recombinant proteins.
Conflict of Interests
The authors declare no conflict of interests.
This study was partially funded by the Spanish Ministry of Science & Innovation (Project AGL2008-01592) and by EU grant FP7-2011-261504 EDENext and is catalogued by the EDENext Steering Committee as EDENext 082 (http://www.edenext.eu/). The contents of this paper are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. Inés Martín-Martín is a Ph. D. recipient of a fellowship from the Spanish Ministry of Science & Innovation (FPI-MICINN).
- WHO, Control of the Leishmaniases, Technical Report Series no. 949, World Health Organization, Geneva, Switzerland, 2010.
- J. M. C. Ribeiro and I. M. B. Francischetti, "Role of arthropod saliva in blood feeding: sialome and post-sialome perspectives," Annual Review of Entomology, vol. 48, pp. 73–88, 2003. View at Publisher · View at Google Scholar · View at Scopus
- S. Kamhawi, "The biological and immunomodulatory properties of sand fly saliva and its role in the establishment of Leishmania infections," Microbes and Infection, vol. 2, no. 14, pp. 1765–1773, 2000. View at Publisher · View at Google Scholar · View at Scopus
- A. Barral, E. Honda, A. Caldas et al., "Human immune response to sand fly salivary gland antigens: a useful epidemiological marker?" American Journal of Tropical Medicine and Hygiene, vol. 62, no. 6, pp. 740–745, 2000. View at Google Scholar · View at Scopus
- R. Gomes and F. Oliveira, "The immune response to sand fly salivary proteins and its influence on Leishmania immunity," Frontiers in Immunology, vol. 3, article 110, 2012. View at Google Scholar
- I. Rohoušová and P. Volf, "Sand fly saliva: effects on host immune response and Leishmania transmission," Folia Parasitologica, vol. 53, no. 3, pp. 161–171, 2006. View at Google Scholar · View at Scopus
- M. Vlková, I. Rohoušová, J. Drahota et al., "Canine antibody response to Phlebotomus perniciosus bites negatively correlates with the risk of Leishmania infantum transmission," PLoS Neglected Tropical Diseases, vol. 5, no. 10, article e1344, 2011. View at Publisher · View at Google Scholar · View at Scopus
- M. Vlková, I. Rohoušová, J. Hostomská et al., "Kinetics of antibody response in BALB/c and C57BL/6 mice bitten by Phlebotomus papatasi," PLoS Neglected Tropical Diseases, vol. 6, no. 7, article e1719, 2012. View at Google Scholar
- K. Gidwani, A. Picado, S. Rijal et al., "Serological markers of sand fly exposure to evaluate insecticidal nets against visceral leishmaniasis in India and Nepal: a cluster-randomized trial," PLoS Neglected Tropical Diseases, vol. 5, no. 9, article e1296, 2011. View at Publisher · View at Google Scholar · View at Scopus
- C. Teixeira, R. Gomes, N. Collin et al., "Discovery of markers of exposure specific to bites of Lutzomyia longipalpis, the vector of Leishmania infantum chagasiin Latin America," PLoS Neglected Tropical Diseases, vol. 4, no. 3, article e638, 2010. View at Publisher · View at Google Scholar · View at Scopus
- A. P. Souza, B. B. Andrade, D. Aquino et al., "Using recombinant proteins from Lutzomyia longipalpis saliva to estimate human vector exposure in visceral leishmaniasis endemic areas," PLoS Neglected Tropical Diseases, vol. 4, no. 3, article e649, 2010. View at Publisher · View at Google Scholar · View at Scopus
- M. F. Clements, K. Gidwani, R. Kumar et al., "Measurement of recent exposure to Phlebotomus argentipes, the vector of indian visceral leishmaniasis, by using human antibody responses to sand fly saliva," American Journal of Tropical Medicine and Hygiene, vol. 82, no. 5, pp. 801–807, 2010. View at Publisher · View at Google Scholar · View at Scopus
- J. G. Valenzuela, Y. Belkaid, E. Rowton, and J. M. C. Ribeiro, "The salivary apyrase of the blood-sucking sand fly Phlebotomus papatasi belongs to the novel Cimex family of apyrases," Journal of Experimental Biology, vol. 204, no. 2, pp. 229–237, 2001. View at Google Scholar · View at Scopus
- S. Marzouki, M. Abdeladhim, C. B. Abdessalem et al., "Salivary antigen SP32 is the immunodominant target of the antibody response to Phlebotomus papatasi bites in humans," PLoS Neglected Tropical Diseases, vol. 6, no. 11, article e1911, 2012. View at Google Scholar
- R. Molina, M. I. Jiménez, I. Cruz et al., "The hare (Lepus granatensis) as potential sylvatic reservoir of Leishmania infantum in Spain," Veterinary Parasitology, vol. 190, no. 1-2, pp. 268–271, 2012. View at Google Scholar
- M. Jiménez, E. González, A. Iriso et al., "Detection of Leishmania infantum and identification of blood meals in Phlebotomus perniciosus from a focus of human leishmaniasis in Madrid, Spain," Parasitology Research, vol. 112, no. 7, pp. 2453–2459, 2013. View at Google Scholar
- I. Martín-Martín, R. Molina, and M. Jiménez, "An insight into the Phlebotomus perniciosus saliva by a proteomic approach," Acta Tropica, vol. 123, no. 1, pp. 22–30, 2012. View at Google Scholar
- I. Martín-Martín, R. Molina, and M. Jiménez, "Identifying salivary antigens of Phlebotomus argentipes by a 2DE approach," Acta Tropica, vol. 126, no. 1, pp. 229–239, 2013. View at Google Scholar
- J. M. Anderson, F. Oliveira, S. Kamhawi et al., "Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis," BMC Genomics, vol. 7, article 52, 2006. View at Publisher · View at Google Scholar · View at Scopus
- R. Molina, "Laboratory adaptation of an autochthonous colony of Phlebotomus perniciosus Newstead, 1911 (Diptera: Psychodidae)," Research and Reviews in Parasitology, vol. 51, no. 1–4, pp. 87–89, 1991. View at Google Scholar
- J. Sambrook and D. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, NY, USA, 2001.
- R. Gupta, E. Jung, and S. Brunak, "Prediction of N-glycosylation sites in human proteins," in preparation, 2004, http://www.cbs.dtu.dk/services/NetNGlyc/.
- K. Julenius, A. Mølgaard, R. Gupta, and S. Brunak, "Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites," Glycobiology, vol. 15, no. 2, pp. 153–164, 2005. View at Publisher · View at Google Scholar · View at Scopus
- N. Blom, S. Gammeltoft, and S. Brunak, "Sequence and structure-based prediction of eukaryotic protein phosphorylation sites," Journal of Molecular Biology, vol. 294, no. 5, pp. 1351–1362, 1999. View at Publisher · View at Google Scholar · View at Scopus
- G. C. Lanzaro, A. H. C. S. Lopes, J. M. C. Ribeiro et al., "Variation in the salivary peptide, maxadilan, from species in the Lutzomyia longipalpis complex," Insect Molecular Biology, vol. 8, no. 2, pp. 267–275, 1999. View at Google Scholar · View at Scopus
- R. S. Milleron, J.-P. Mutebi, S. Valle et al., "Antigenic diversity in maxadilan, a salivary protein from the sand fly vector of American visceral leishmaniasis," American Journal of Tropical Medicine and Hygiene, vol. 70, no. 3, pp. 286–293, 2004. View at Google Scholar · View at Scopus
- H. Kato, J. M. Anderson, S. Kamhawi et al., "High degree of conservancy among secreted salivary gland proteins from two geographically distant Phlebotomus duboscqi sandflies populations (Mali and Kenya)," BMC Genomics, vol. 7, article 226, 2006. View at Publisher · View at Google Scholar · View at Scopus
- M. Abdeladhim, R. C. Jochim, M. Ben Ahmed et al., "Updating the salivary gland transcriptome of Phlebotomus papatasi (Tunisian Strain): the search for sand fly-secreted immunogenic proteins for humans," PLoS One, vol. 7, no. 11, Article ID e47347, 2012. View at Google Scholar
- S. S. Mahamdallie and P. D. Ready, "No recent adaptive selection on the apyrase of Mediterranean Phlebotomus: implications for using salivary peptides to vaccinate against canine leishmaniasis," Evolutionary Applications, vol. 5, no. 3, pp. 293–305, 2012. View at Publisher · View at Google Scholar · View at Scopus
- D.-E. A. Elnalem, C. Meneses, M. Slotman, and G. C. Lanzaro, "Genetic variation in the sand fly salivary protein, SP-15, a potential vaccine candidate against Leishmania major," Insect Molecular Biology, vol. 14, no. 2, pp. 145–150, 2005. View at Publisher · View at Google Scholar · View at Scopus
- A. Oleaga, A. Escudero-Población, E. Camafeita, and R. Pérez-Sánchez, "A proteomic approach to the identification of salivary proteins from the argasid ticks Ornithodoros moubata and Ornithodoros erraticus," Insect Biochemistry and Molecular Biology, vol. 37, no. 11, pp. 1149–1159, 2007. View at Publisher · View at Google Scholar · View at Scopus
- I. M. B. Francischetti, V. M. Pham, B. J. Mans et al., "The transcriptome of the salivary glands of the female western black-legged tick Ixodes pacificus (Acari: Ixodidae)," Insect Biochemistry and Molecular Biology, vol. 35, no. 10, pp. 1142–1161, 2005. View at Publisher · View at Google Scholar · View at Scopus
- L. Patthy, "Alternative splicing: evolution," eLS, 2008. View at Publisher · View at Google Scholar
- C. Walsh, M. Gangloff, T. Monie et al., "Elucidation of the MD-2/TLR4 interface required for signaling by lipid IVa," Journal of Immunology, vol. 181, no. 2, pp. 1245–1254, 2008. View at Google Scholar · View at Scopus
- J. M. C. Ribeiro, B. J. Mans, and B. Arcà, "An insight into the sialome of blood-feeding Nematocera," Insect Biochemistry and Molecular Biology, vol. 40, no. 11, pp. 767–784, 2010. View at Publisher · View at Google Scholar · View at Scopus
- J. G. Valenzuela, R. Charlab, E. C. Gonzalez et al., "The D7 family of salivary proteins in blood sucking diptera," Insect Molecular Biology, vol. 11, no. 2, pp. 149–155, 2002. View at Publisher · View at Google Scholar · View at Scopus
- A. A. James, K. Blackmer, O. Marinotti, C. R. Ghosn, and J. V. Racioppi, "Isolation and characterization of the gene expressing the major salivary gland protein of the female mosquito, Aedes aegypti," Molecular and Biochemical Parasitology, vol. 44, no. 2, pp. 245–254, 1991. View at Publisher · View at Google Scholar · View at Scopus
- B. Arcà, F. Lombardo, A. Lanfrancotti et al., "A cluster of four D7-related genes is expressed in the salivary glands of the African malaria vector Anopheles gambiae," Insect Molecular Biology, vol. 11, no. 1, pp. 47–55, 2002. View at Publisher · View at Google Scholar · View at Scopus
- R. Charlab, J. G. Valenzuela, E. D. Rowton, and J. M. C. Ribeiro, "Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly Lutzomyia longipalpis," Proceedings of the National Academy of Sciences of the United States of America, vol. 96, no. 26, pp. 15155–15160, 1999. View at Publisher · View at Google Scholar · View at Scopus
- E. Calvo, B. J. Mans, J. F. Andersen, and J. M. C. Ribeiro, "Function and evolution of a mosquito salivary protein family," Journal of Biological Chemistry, vol. 281, no. 4, pp. 1935–1942, 2006. View at Publisher · View at Google Scholar · View at Scopus
- I. Rohoušová, S. Subrahmanyam, V. Volfová et al., "Salivary gland transcriptomes and proteomes of Phlebotomus tobbi and Phlebotomus sergenti, vectors of leishmaniasis," PLoS Neglected Tropical Diseases, vol. 6, no. 5, article e1660, 2012. View at Google Scholar
- P. H. Alvarenga, I. M. B. Francischetti, E. Calvo, A. Sá-Nunes, J. M. C. Ribeiro, and J. F. Andersen, "The function and three-dimensional structure of a thromboxane A2/cysteinyl leukotriene-binding protein from the saliva of a mosquito vector of the malaria parasite," PLoS Biology, vol. 8, no. 11, Article ID e1000547, 2010. View at Publisher · View at Google Scholar · View at Scopus
- E. Calvo, B. J. Mans, J. M. C. Ribeiro, and J. F. Andersen, "Multifunctionality and mechanism of ligand binding in a mosquito antiinflammatory protein," Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 10, pp. 3728–3733, 2009. View at Publisher · View at Google Scholar · View at Scopus
- J. Hostomská, V. Volfová, J. Mu et al., "Analysis of salivary transcripts and antigens of the sand fly Phlebotomus arabicus," BMC Genomics, vol. 10, article 282, 2009. View at Publisher · View at Google Scholar · View at Scopus
- J. G. Valenzuela, Y. Belkaid, M. K. Garfield et al., "Toward a defined anti-Leishmania vaccine targeting vector antigens: characterization of a protective salivary protein," Journal of Experimental Medicine, vol. 194, no. 3, pp. 331–342, 2001. View at Publisher · View at Google Scholar · View at Scopus
- P. K. Geyer, C. Spana, and V. G. Corces, "On the molecular mechanism of gypsy-induced mutations at the yellow locus of Drosophila melanogaster," EMBO Journal, vol. 5, no. 10, pp. 2657–2662, 1986. View at Google Scholar · View at Scopus
- X. Xu, F. Oliveira, B. W. Chang et al., "Structure and function of a "yellow" protein from saliva of the sand fly Lutzomyia longipalpis that confers protective immunity against Leishmania major infection," Journal of Biological Chemistry, vol. 286, no. 37, pp. 32383–32393, 2011. View at Publisher · View at Google Scholar · View at Scopus
- F. Oliveira, S. Kamhawi, A. E. Seitz et al., "From transcriptome to immunome: identification of DTH inducing proteins from a Phlebotomus ariasi salivary gland cDNA library," Vaccine, vol. 24, no. 3, pp. 374–390, 2006. View at Publisher · View at Google Scholar · View at Scopus
- N. Collin, T. C. Assumpcao, D. M. Mizurini et al., "Lufaxin, a novel factor Xa inhibitor from the salivary gland of the sand fly Lutzomyia longipalpis blocks protease-activated receptor 2 activation and inhibits inflammation and thrombosis in vivo," Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 32, no. 9, pp. 2185–2198, 2012. View at Google Scholar
- T. R. de Moura, F. Oliveira, M. W. Carneiro et al., "Functional transcriptomics of wild-caught Lutzomyia intermedia salivary glands: identification of a protective salivary protein against Leishmania braziliensis infection," PLoS Neglected Tropical Diseases, vol. 7, no. 5, article e2242, 2013. View at Publisher · View at Google Scholar
- H. Kato, R. C. Jochim, E. A. Gomez et al., "Analysis of salivary gland transcripts of the sand fly Lutzomyia ayacuchensis, a vector of Andean-type cutaneous leishmaniasis," Infection, Genetics and Evolution, vol. 13, pp. 56–66, 2012. View at Publisher · View at Google Scholar
- J. Dai, J. Liu, Y. Deng, T. M. Smith, and M. Lu, "Structure and protein design of a human platelet function inhibitor," Cell, vol. 116, no. 5, pp. 649–659, 2004. View at Publisher · View at Google Scholar · View at Scopus


text:
Noisey's YouTube series, Guitar Moves, features host Matt Sweeney interviewing and playing with some big-name guitarists. The latest episode's guest was ZZ Top guitarist and Master of Beards, Billy Gibbons. The two jam out to some old blues, talk about using the guitar to make people dance, and Billy's choice in strings. It's an exceptionally laid back interview that takes place in Kid Rock's barn, and if you ever wanted to see Billy Gibbons improvise a song about how much he likes Kid Rock, then this is really your only chance to do that.
Also, if you want to watch Kid Rock pour wine into a solo cup for Billy Gibbons, you can kill two birds with one stone here. Stick around till the end to see the three of them jam out with Tina Turner's "Nutbush City Limits."


text:
Cruise Quot Movie Premiere Sees Anti Scientology Protests
Uk. The superstar, however, looked unperturbed as he made his way through Time Warner Building, where he joined the British co-stars Bill Nighy and Eddie Izzard. This is not the first time anti-Scientology groups have protested against the cruise, a staunch supporter of the cult, and his family. Co. Anonymous, an anti-Scientology group, held at the protests before Tom Cruise Valkyrie last film in New York. Anonymous made their presence felt with signs and singing, reports thesun.


text:
Russian university confers Honorary Degree on the Dalai Lama
The Russian Federal State Education Institute 'Tuva State University' awarded an Honorary Doctorate degree to Tibetan spiritual leader the Dalai Lama yesterday, here in town.
"Russian Academic Council of the Tuva State University has decided to confer the Honorary Degree to His Holiness the XIVth Dalai Lama of Tibet for his outstanding contribution to the development of science, and his personal contributions towards solving pressing issues such as protection of human rights, promotion of religious harmony, conservation of environment, strengthening of moral and ethical principals in the society," said Telo Rinpoche, the spiritual leader of Kalmykia.
Telo Rinpoche along with officials of the university awarded the Honorary Degree to the Dalai Lama at the end of his three-day teachings at the main temple Tsug-la Khang.
"For the past many years, we made every possible effort to prepare His Holiness the Dalai Lama's visit to Russia but unfortunately we have faced many difficulties," said the spiritual leader of the Kalmyk people.
After the collapse of the Soviet Union, the Dalai Lama paid his first and only visit to Kalmykia and Tuva in 1992, regions whose once-rich Buddhist heritage was destroyed in the 1930s by the dual Soviet policies of collectivization and atheism.
"I have visited Tuva once and learnt of the Tuva people's great interest in Buddhism. Tuva has a long-standing strong cultural ties linked to Tibet for the past hundreds of years," said the 76-year old Tibetan leader.
"However, in recent years due to political reasons, I couldn't visit Tuva," added the Dalai Lama.
Thousands of Buddhists from Russia have regularly visited Dharamshala, the exile seat of the Dalai Lama to receive teachings and listen to him.
From December 19 – 21, at the request of the Russian devotees, the Dalai Lama gave a three-day teaching on Tsongkhapa's "Destiny Fulfilled" (Tokjo Dunlekma) and Geshe Langri Thangpa's "Eight Verses of Training the Mind (Lojong Tsikgyema).
Thousands of devotees, including more than 1300 Russians, over one thousand Tibetans from Tibet, and hundreds of devotees from across the world and India attended the teachings.


text:
Howdy Leaky Shed Studio Fans! Juliana here with a simple, yet fun, chipboard technique to share with you. While horsing around with my Leaky Shed Studio chipboard, I came up with an idea for embellishing it with stamps.
Plain stamping wasn't enough though. I repeatedly stamped the same image (a small star) across the Stampede Border using Versamark Watermark Ink. While the ink was still wet, I sprinkled the images with white embossing powder and then heat set it using a heat gun. I only stamped one or two stars at a time before heat setting the embossing powder. Once I had finished stamping and embossing, I colored the chipboard with Distress Ink in Walnut Stain using a foam applicator.
I colored two chipboard corners with the Distress Ink in Walnut Stain to coordinate with the border and placed one on each of the top outer corners of my two photos. The placement of the border and coordinating corners helps frame the photos and draw your attention to them.
For a complete list of other supplies used on this layout, please visit my blog - 17turtles.
Leaky Shed Studio Chipboard:


text:
This is video meant to show CATIA's capability in the composites workbench. It's not a training video but it just shows what V5 can accomplish in this growing sector in the industry.
Wednesday, July 11, 2012
CATIA V5 - Composites
Posted by m1turbo at 11:49 PM
Subscribe to: Post Comments (Atom)


text:
At the next Ferry Sloops monthly meeting on Wednesday, April 2, 2014 at 7:30PM, Riverkeeper Patrol Boat Captain John Lipscomb will a talk about the Hudson River, water quality, and insights from his 13 years piloting the R. Ian Fletcher on monthly patrols of the Hudson River estuary.
FERRY SLOOPS NEW LOCATION. Join us in Ossining at the Shattemuc Yacht Club, host for the Ferry Sloops "Music & Lecture" series.
Admission is FREE and open to the public. Come with a dish to share at 6:30PM and join us for a potluck supper or arrive at 7:30PM for the presentation.
Ferry Sloops' monthly meetings are held at the Shattemuc Yacht Club in Ossining, NY, on the first Wednesday of each month. Our potluck supper begins at 6:30PM, followed by our meeting at 7:00PM and guest (speaker or musician) at 7:30PM.
For more information, please visit the Ferry Sloops website at http://www.ferrysloops.org


text:
In 2008 we got to go to Madame Zingara's Theatre of Dreams in Durban, South Africa. A unique supper theater spectacular that is housed in one of the last remaining Mirror Tents in the world; a magnificent tent named "Victoria" that has a 25 metre velvet top, mirrored columns, velvet drapes and beveled booths; and has travelled around the globe for the last 80 years! We went to see this show as we knew it would be an amazing experience (and it was, especially since the group of us that went all dressed up accordingly), so if you're in South Africa and haven't yet been to see Madame Zinagara, make a plan to get tickets when they're in your city next! (They're currently in Johannesburg).
And then there's Cirque du Soleil …
For years we'd been mesmerized by reruns of Cirque du Soleil shows broadcast on TV and always said that one day we would love to go and actually see a live show. That day finally arrived and last night we got to watch Cirque du Soleil live in action. Their production of Totem is still on in Auckland and runs from 22 August to 28 September, before they move across to Australia from October 2014 to July 2015 (showing in various cities).
According to Cirque du Soleil's website "TOTEM traces the fascinating journey of the human species from its original amphibian state to its ultimate desire to fly. The characters evolve on a stage evoking a giant turtle, the symbol of origin for many ancient civilizations.
Inspired by many founding myths, TOTEM illustrates, through a visual and acrobatic language, the evolutionary progress of species.
Somewhere between science and legend TOTEM explores the ties that bind Man to other species, his dreams and his infinite potential."
Words can't really describe how amazing it was, but I'll try as best as I can to give you an idea.
Upon arrival at Alexandra Park in Greenlane, Auckland, we parked our vehicle and made our way through the main entrance to see the enormous yellow and blue striped tents looming before us!
Apparently these tents are home to about 170 people who travel with the Totem production and once inside, you're suddenly in a magical world; a world where people can somersault, leap, spin and fly, all while making it look so effortless and easy. Tasks and tricks that seem impossible to us in our day-to-day world, suddenly have us completely captivated and our mind's are transported and amazed by what the human body can actually achieve!
Humans were miraculously transformed into various animals, with a bright array of colorful costumes and skillfully applied make-up and I really do admire these performers who apply their own make-up during the shows, as they do a superb job! Yes, each performer applies their own make-up by following step-by-step instructions given to them by the Make-up Designer who creates the looks for the show, but there is a make-up supervisor on staff who oversees them to make sure it's all done right.
The show began with us all waiting in anticipation and watching this giant turtle-shaped mound in the centre of the stage. Suddenly the cloth cover of the turtle's "shell" was whisked away, revealing the carapace underneath – a jungle gym-like frame complete with parallel bars. The shell represented the origins of life on earth and inside the frame colourfully dressed amphibians and fish came alive to playfully leap, jump and soar during a parallel bar sequence.
An Amerindian dancer was up next, with hoops that she tossed, rolled and jumped through while creating magical shapes using the hoops and her body, to evoke images of various animals, symbolising the endless circle of life. She ended her act by creating a completely circular ball made entirely out the hoops she'd used during her performance!
There were so many great acts and amazing performances, that I could be here all day telling you about it: monkeys, neanderthals and modern man all sauntering on stage to create an 'accidental' line representing our evolution; a Crystal Man that dangled and spiralled down from the ceiling; Crystal Ladies spinning and swapping squares of sparkling fabric on their hands and feet, in a high-speed co-ordination; an agile performer contorting his body on an hourglass-shaped frame; a Scientist in his 'laboratory', spinning sparkling balls in a giant glass beaker (representing atoms or planets?), while his colleagues beat out music on brightly coloured test tubes filled with mysterious plants and fluids; Men leaping, jumping and flying on Russian bars, weightlessly defying gravity!
But there were two acts that really stood out for us … 5 ladies on unicycles; and an Amerindian couple on roller-skates with their act symbolising a wedding ceremony.
The five ladies on unicycles represented the abundance of Fall (Autumn), as they juggled metal bowls while displaying amazing agility, balance, synchronisation and grace. The bowls were tossed using one foot, while controlling the unicycle with the other! The bowls were then caught on their own heads or tossed across (or backwards over their shoulders, depending on which way they were facing) onto the heads of the other unicyclists!
Once again, the stage lighting changed and the floor became a river; a beautiful Amerindian woman arrived in a canoe, followed shortly by a chief in a separate canoe. Both were wearing white and on their feet were knee-length, laced up roller-skates. What happened next was absolutely astounding … they both climbed up onto a 1.8 metre diameter, drum-shaped platform and holding onto each other, spun around in a tight circle, whirling and twirling at heart-stopping speeds! One slight mistake and it all could have gone horribly wrong. At one point, she was attached to him by a harness around his neck which went fitted across the base of her skull and head, nothing else was holding them together. He spun them around in this small circle, while she "barrel-rolled" in a blur of tassels and wheels!
As I said before, words cannot come near to describing this magnificent show, so watch the official Totem trailer for a sneak peek of it really looked like … Cirque du Soleil – Totem.
If you ever get a chance to see Cirque du Soleil live, don't even think about it, just buy those tickets and go! It was an awesome night out and a truly amazing experience. Definitely worth every dollar spent!
Note: Some of the information above and all photos are from Cirque du Soleil's website, as photographs are not allowed to be taken in the tent.


text:
Trust and Deference between Legal Authorities in Europe
A workshop organised by the Amsterdam Centre for European Law and Governance (ACELG) and the Paul Scholten Centre (PSC). Financed by Law and Justice Across Borders.
This workshop, financed by Law and Justice Across Borders, seeks to explore the conceptual and empirical assumptions and justifications underlying the mechanism of trust and deference between legal authorities in Europe.
Trust and deference are crucial mechanisms of interaction and cooperation between legal authorities in Europe. Deference means that an authority does not make its own assessment or decision in a particular matter but relies on the decision of another authority. The basis for this deference is trust in the second authority. It ultimately means that the deferring authority puts its trust on the authority it defers to. It has trust that the second authority takes a correct decision in matters of fact and law, especially the rule of law. The workshop seeks to explore the conceptual and empirical assumptions and justifications underlying the mechanism of trust and deference between legal authorities in Europe.
Scope and background
The mechanism of trust and deference operates between various authorities at different levels in Europe. The scope of the workshop is thus deliberately vast in terms of areas of law to be covered.
- ECtHR deferring to Parties to the Convention, cf. the margin of appreciation.
- ECtHR deferring to the EU, cf. the Bosphorus doctrine.
- Police and judicial authorities of one Member State deferring to a judicial authority of another Member State cf. mutual trust and recognition in the Area of Freedom, Security and Justice.
- Civil enforcement authorities of a Member State deferring to a judicial authority of another Member State, cf. mutual recognition of European Enforcement Orders.
- Administrative agencies of a Member state deferring to an administrative agency of another Member State, cf. Common European Asylum System, esp. Dublin Regulation; European passport for financial services (MIFID).
Domestic law
- Criminal courts deferring to administrative tribunals, cf. criminalization of migration offences.
- Administrative tribunals deferring to the administration, cf. marginal review of the exercise of administrative discretionary powers; judicial review of factual assessment done by the administration, instead of a de novo examination of the fact.
- Legislative and judicial authorities deferring to administrative authorities in the context of emergency regimes.
Both in case law and legal scholarship, we find justifications and explanations for this mechanism of trust and deference. A deferring authority may put its trust in other authorities because of their expertise. Deference may express a particular constitutional and political arrangement whereby some authorities are deemed more legitimate to make certain judgments, e.g. policy choices and weighting of interests. Trust and deference may also operate as a compromise between full harmonization and integration of public functions and retaining national or institutional autonomy, eg European Arrest Warrant.
Confirmed Speakers
- Evelien Brouwer (Vrije Universiteit Amsterdam)
- Anne Meuwese (Tilburg University)
- Valsamis Mitsilegas (Queen Mary University of London)
- Marc de Wilde (University of Amsterdam)
- Lauréline Fontaine (Université Sorbonne Nouvelle - Paris 3)
- Ronald Janse (University of Amsterdam)
- Marie Linton (Uppsala University Sweden)
- Eva Storskrubb (Uppsala University Sweden)
Practical matters
Attendance is free of charge, registration per email to Yvonne ter Horst is appreciated.
Seminar room A3.01
Roeterseilandcampus - building A
Nieuwe Achtergracht 166 | 1018 WV AmsterdamGo to detailpage
+31 (0)20 525 5340
This workshop will be followed by a follow-up event on 17 September taking place at the Asser Institute in The Hague and focusing on the operation of trust and deference between legal authorities in the world.


text:
Your Savannah Technology Experts
We are headquartered in Savannah and have been helping businesses in the area with their technology needs since 1984.
Susan Speros began her career in the telecommunications industry in the 70's, and started Speros in 1984 in order to better help businesses with their technology.
We can take care of your technology needs from phone systems, managed IT services, surveillance systems, or web design. Fill out this form to schedule a meeting with us today.
We look forward to working with you.
2141 Rowland Ave.
Savannah GA 31404
(912) 354-8900
(912) 352-9191 fax


text:
Keven Carlton – Registered Dental Hygienist
"Hi, my name is Keven Carlton and I am one of the registered dental hygienists who will be working with you routinely, helping you maintain your teeth for a lifetime. I feel so privileged to be able to work with so many wonderful patients everyday and help them with my dental skills and my sense of humor! I have been practicing with Dr. Westermann for over 25 years and cherish the relationships I have made with my patients. I love seeing them every few months and sharing what has gone on in their life since our last appointment. It gives me great pleasure to be able to educate them about their oral health, and uplift their spirits. A personal caring attitude is a must in our profession, in my opinion!"
Keven graduated from the Univ. of Louisville School of Dentistry in 1985, and received her certification in laser treatment of gum disease in 2011. She enjoys spending time with her nieces and nephews, and is also a polymer clay artist making jewelry and all kinds of things, for her business KevsoKraft! She has articles on display in various galleries throughout the city!
Samantha West, Registered Dental Hygienist
"Hi, my name is Samantha and I am the newest member of the team. I look forward to meeting all the great patients in Dr. Westermann's practice and helping them maintain their oral health. I love my job, and patients tell me I am very gentle. Your comfort is always my main concern. I am excited to work with you to achieve your overall optimum health! I am an Indiana native, graduating with a degree in Kinesiology from Indiana University, and continuing on to get a Bachelors of Dental Hygiene. I love education, and will enjoy educating you about what is going on with your dental health. I feel that hygiene was my destiny because my mom has been a hygienist for 30 years and I am looking forward to following in her footsteps."
When not working I like being outside hiking, biking, boating and playing with our yellow lab, Ellie.
Cassie Witten, Patient Coordinator/ Dental Assistant
"Hi, I'm Cassie Witten. I'm usually the first person you'll talk with, and the first person you will meet when you come to our practice! It is my job to make sure you have a great first impression of our practice! I love my job because I am a people person and love the personal communication…it makes me smile! I am the patient coordinator in our practice, and have been in dentistry for 25 years. It is very rewarding for me to be able to make the appointments and financial arrangements for you, that will allow you to receive the great care you deserve! I've helped so many people maximize their insurance benefits because of my vast experience. I am also cross trained as a dental assistant and will frequently get a chance to help with your treatment chairside! I have the best of both worlds!
I am married to Tim, and enjoy spending time with my 3 children and 7 grandchildren! We enjoy boating, fishing, golf bowling and baseball!
Life is good… Westermann Family Dentistry makes it better! I'm looking forward to meeting you soon!"
Kathy Lega, Expanded Duties Dental Assistant
"Hi, I am Kathy Lega. I am the expanded duty dental assistant, who will be working very closely with Dr. Westermann on your clinical care. It is very rewarding to change a decayed or broken tooth and make it new again! It is so gratifying to create a beautiful smile, and boost someone's self esteem! I love working directly with our patients and making everyone feel comfortable. Your comfort is my top priority! Our gentle touch and caring ways will make your visit painfree. I have been a dental assistant for 38 years, receiving my dental education from Jefferson State Vocational School and the University of Louisville. I love learning, so keeping up with the latest in dentistry is a true pleasure! I am fortunate to belong to an organization, Dental BootKamp, who teach dentists and their team how to run a successful and ethical dental practice, with the patients well being the primary concern!"
"I have been married to Alan for 36 years. We have three children, three grandsons, Nathan, Dylan and Austin. When I'm not spending time with my family I love running with my " Sole Sisters". I have run marathons in 11 different states! I would love the opportunity to meet you and help you keep smiling for a lifetime!"
Alice DeWitt, Expanded Duties Dental Assistant
"Hi, I'm Alice and I'm relatively new to Dr. Westermann's practice. I am so thrilled to be working for a quality practice that really cares about the patients. Seeing the caring relationships that my team has with their patients is so inspiring! I can't wait to meet all of you, and get to know you the way that they do!"
Alice has 15 years experience as an expanded duties assistant, working chairside clinically. She also has experience working with insurance companies, scheduling, and patient management, and can help you with about anything you need here!
Bart Doerhoefer, Marketing Director
Bart Doerhoefer is the Marketing Director for Westermann Family Dentistry. He does a lot of the behind the scenes hard work, that keeps us running smoothly every day!


text:
Full day golf camp!
For novice and beginner golfers.
Ages 6 – 12
Mon-Fri 9am – 4pm
Includes lunch each day
The Academy at Piper's Heath Full Day Junior Camps 2018
Camps begin in July and August during the selected weeks below. These camps are designed for introducing young beginner golfers, ages 6-12, to the game and learning the fundamentals.
Our acclaimed junior camps are action packed from the start! We have taken the latest research from child development experts and designed our golf camps to cover the areas that are essential for the enjoyment of golf and the development of golf related physical skills. Agility, Balance, Coordination and Fun! Through our fun and engaging activities kids will learn about the game of golf and the skills that are involved.
However it won't be the boring standing on the range hitting balls like the adults do – kids will learn how to create power, control ball flight, learn swing angles with the help of baseball bats, tennis balls, soccer balls, hula hoops and of course: golf clubs.
Each camp will cover the best in instruction in a safe, professional and exciting learning environment hosted by our Canadian PGA certified staff. For safety and confidence building purposes, this program is 100% range instruction. This concept is designed to give kids the improved ball striking and lessons in etiquette before heading onto the course.
All camp students will be grouped initially by age and then further by golf ability and skill progression at the discretion of the Academy professionals during the week. Golf camp highlight topics include, but not limited to: full swing, putting, how to practice, stance, grip, balance, rules of golf, and chipping & pitching.
|Sample Lunch Menu
|Camp 1
|July 2 - July 6
|View Lunch Menu
|Camp 2
|July 9 - July 13
|View Lunch Menu
|Camp 3
|July 16 - July 20
|View Lunch Menu
|Camp 4
|July 23 - July 27
|View Lunch Menu
|Camp 5
|July 30 - Aug 3
|View Lunch Menu
|Camp 6
|August 6 - August 10
|View Lunch Menu
|Camp 7
|August 13 - August 17
|View Lunch Menu
|Camp 8
|August 20 - August 24
|View Lunch Menu
Please note menu subject to change. Piper's Heath will provide vegetarian and allergy conscious options when necessary.
Please download below (pdf) and return to email@example.com or fax 905.864.1730
- Please bring your own golf clubs (putter & a driver/iron at minimum will suffice!)
- Wear comfortable clothing (t-shirt, shorts, running shoes) and dress for the weather
- Bring a small backpack with snacks and extra drinks
- Sunscreen!
- Pack any necessary medications
- Drop off between 8:45am and 9:00am in the south-end parking lot
- Pick up at 4:00pm in the south-end parking lot
- Please remind your instructor of any allergies or medical issues on the first day
- No make up dates or partial refunds. In the event of inclement weather we will move indoors as required.
- Recommended ages 6 – 12


text:
Based on appearances, it would seem that Los Angeles developer Geoff Palmer is doing OK. This past summer, he bought a $17-million beachfront house in Malibu, with solid bronze doors, teak cabinets and his-and-hers bathrooms.
The beach bungalow could come in handy if Palmer needs a break from the French Regency-style Beverly Hills estate he bought for $21 million in 2001. That property includes a 16,400-square-foot main house, plus two guest houses, each larger than 5,000 square feet.
Given Palmer's obvious good fortune, some folks are surprised to see how vigorously he keeps fighting a city requirement that 15% of the units in the massive downtown apartment projects he builds be reserved for low-income renters, meaning for families that make up to about $40,000 a year. The buildings have hundreds of units each and grandiose names like the Medici, the Orsini and the Piero, and they have been multiplying in and around downtown Los Angeles like rabbits.
Palmer's latest fight with City Hall is over a project called the Lorenzo, and as in the past, he doesn't give up easily. His request for a waiver of the 15% requirement has been given a thumbs down by the city Planning Department, the Planning Commission and a City Council committee, but Palmer still intends to argue his case next week before the full City Council.


text:
Shared by MetroclickMetroclick New York BoothThe diamond were blended up through the diamond seller's facet, which would have cost them dollars – But we caught the error.Of course I previously assumed so!however, if it's claims within the GIA report that's it's triple exceptional it has to be reality, right?Searching for a really sparkly
Metroclick - The Ultimate Guide To Ny Photobooth
Posting by Metroclick.comRent from MetroclickAll registered and/or unregistered trademarks and/or service marks (collectively, "Marks") applied or referred to on this Site will be the assets of Gemini, unless if not pointed out.I'm quite pleased with the high-quality assistance you kindly present to us. Wanting forward with the approaching Edit
The Ultimate Guide To Photo Booth Rental
Aiming to have a number of backgrounds devoid of guest choice? Our eco-friendly monitor technological innovation can preset a cycle of backgrounds to be applied to Just about every and every photo.A broad and massive Photo Booth will let you have continual photo shoot coupled with more substantial groups in it. The more and more people inside the p
Fascination About Photo Booth
Arvid Kastermans , The Snapshots Cupboard Experienced a fantastic night time at an out of doors wedding ceremony we did! The Mirror Me Booth was a big hit with everyone! The couple absolutely beloved the custom made overlay which i developed for them. I am glad I brought a lot of business enterprise playing cards as they were going speedy!We now ha
The Definitive Guide to photo booth rental
Open is a completely new step and repeat assistance from ShutterBooth wherever your visitors pose in front of a backdrop of your respective preference after which get the instantaneously printed photographs.Get it back a number of years that has a stylish vintage filter. Pair this having a coordinating backdrop or environmentally friendly monitor a


text:
Sauvion Cart d'Or Muscadet 2007 - $11.50 (12/25/2008)
Style: W2
France: Loire
Maybe if we turn to lighter wines the weather will warm up. This medium golden straw rendering shows light apple, pear, honey and lemon aromas and just a hint of toast. The palate is lean and crisp, with tart apple and lemon flavours. Good body and length; nicely done, and would work with rich fish or surf & turf. (LCBO 143016)


text:
so now we like threads about rankings again? or are we only supposed to hate rankings posts or threads if it is from boomer? or was it a new yrs resolution?
Think on this also: UTA and the Cajuns played West Coast Conference teams, so not only do we get whatever their records will be (OWP), we also get Gonzaga and the rest of the conference (OOWP). On the flip side, those of us who played Texas Southern will get whatever their record will be, we also get the rest of the SWAC, who all currently have losing records.
Dies anyone know if our rip changed yet following the LR game?
Wisconsin wins tonight against Indiana. That's a good thing. They're 13/2.
Today's RPI update:
AState: 45
Louisiana: 81
GaSt: 117
GaSo: 127
Little Rock: 228
Coastal: 231
Troy: 236
Appy: 253
TxSt: 323
As an aside: Our RPI would also be the third best in the WCC, behind Gonzaga and St. Mary's, but ahead of BYU.
Wouldn't have guessed the top RPI team in LA is Nicholls until I looked at their schedule. They have the number 6 non-conf SOS. The RPI will keep falling now of course in conference. Their OWP is pretty high (got an ok ACC team in BC, a great ACC team in FSU, a really good UCF, a WCC team in USD, Samford with a good record and our good record on there.) FSU is the only team to beat them worse than we did.
Texas Southern is another team we played with a good RPI, though they don't help us too much because their WP isn't great. Their RPI is top 60
Minnesota as well helps obviously


text:
The development is situated within a short stroll of the seafront and Paphos harbour with many restaurants, bars and tavernas being passed along the way and local cafs which serve a great value breakfast.
Constantia Gardens is ideally located, within the heart of Kato Paphos tourist area. It is within seconds walk of bars, restaurants, clubs and cafes and within 5 minutes walking distance of other essential amenities, including the beach and harbour.
As centrally located as it is, the small private complex maintains its sense of peace and tranquillity due to the enclosed nature of the developments design.The apartment itself is tastefully decorated and is well equipped and designed throughout. All rooms have Air-Conditioning units and comfortable furnishings for your convenience and relaxation. This property is situated on the ground floor with the main entrance leading into a small hallway then in to an open plan, lounge/dining/kitchen with patio doors leading out on to a small sun terrace. This overlooks the swimming pool, which is fenced off for child safety.The apartments water is usually very hot due to the solar powered heating system and there is a back up immersion should this be needed. A supply of fresh spring water is supplied to the property.
The property benefits from private parking outside and there is a Communal Pool which is fenced off for child safety. There is Air-Conditioning provided throughout for both cool and warm air as required and free WiFi.
Public transport is excellent with lots of busses running regularly. There is a local taxi office down the road but usually you can wave down a taxi wherever you are.
Should you require cots or highchairs for your stay then these are also available
Apartment is cleaned and bed linen/towels changed before and after the arrival/departure of guests. Should you require additional cleaning and or bed linen/towels change during your stay we can arrange this for an additional cost of 20 to be paid on the premises.
When guests are not at home we kindly ask that they switch off the lights and air condition units so as to avoid wasting electricity.
Please do not flush down the toilet items such as toilet paper, nappies, sanitary pads etc to avoid blockage of the sewerage system.
This is a non-smoking apartment so please keep this in mind.
If you have small children please refrain from letting them paint on walls or furniture as we like the apartment decorated just as it is!
Finally and most importantly please respect the neighbors by keeping the noise down.
Treat the property as if its your own!
Air condition paid locally at 3.50 euros per unit. 100 Euros deposit required on booking and 100 Euros for Electricity deposit at arrival which is to be paid to the onsite property manager. You will be refunded the balance less electricity charges at departure.


text:
Maksik firmly creates the "place" as the Pacific Northwest, though his characters have a difficult time finding any kind of "shelter"—from place or from each other.
After graduating from an undemanding college, Joe March finds himself a bit lost. He works part time as a bartender and meets Tess Wolff, a free-spirited young woman with something of a wild streak. Besides developing a relationship with Tess, two things haunt Joe's life. First, he starts to feel the beginnings of bipolar disorder, a disease he characterizes with the metaphors of "tar" and "a bird" whose talons grip him fiercely. Second, Joe's mother, Anne-Marie, witnesses an act of bullying in a grocery store parking lot, and she takes action by seizing a framing hammer and killing the perpetrator of the violence. (Her defense is weakened by the fact that she delivers seven blows with the hammer, which suggests the level of her rage.) She's tried, found guilty, and given five-to-25 years. Maksik offers up all of this plot in a chronologically convoluted narrative, moving back and forth to various fragments of his characters' complicated histories. This strategy serves the narrative well, for it emphasizes the recurring significance of family ties and obligations. After an initial separation, Tess eventually finds Joe and visits Anne-Marie in prison. Along with a number of other women, Tess finds herself admiring Anne-Marie for taking a definitive stand against domestic violence, and she persuades Joe and Seymour, a bouncer at a local bar as well as a prison guard, to get involved in a wacko plot to take revenge on a local college professor who's physically abusing his wife.
On every page we're reminded of the paradox of how mysterious, thorny, and delicate family relationships can be.


text:
Join Yeuda Ben-Atar for an in-depth discussion in this video Settings and user mode, part of Ableton Push: Making Music.
- [Voiceover] A new button on Ableton Push…is the Setup button.…This will let us change the Workflow…from Scene to Clip. First let's check out Scene.…When using the arrow buttons in Note mode,…by default, it will launch an entire new scene.…(musical notes play)…(percussion effect)…Furthermore, when you hit the New button…it will duplicate all the playing clips.…(percussion effect)…And when using the Duplicate button,…it will duplicate the entire scene.…
In Clip mode,…the arrow buttons will only affect the focus track.…(percussion effect)…New will only jump to the new available clip slot…on that selected track, and Duplicate will only…duplicate the clip on the focus track.…Personally, I prefer the Clip Workflow.…(percussion effect)…Next, in the Setup menu we can change…the display brightness,…the LED brightness,…and we can also change how the pads react to our playing.…
Here we can change the pad sensitivity.…In higher settings, it will take less force…to trigger higher velocities. The default is five.…Pad Gain will boost or cut the overall velocity curve.…
First, Yeuda demystifies the many trigger pads, knobs, and buttons on the Push and shows how to map the device to Ableton Live. Next, learn to browse and load sounds and create drumbeats with the step sequencer. Humanize the sound of these beats by changing individual note velocity, length, and position and adding in quantization and swing. Then, learn to play Push like a pitched instrument, and use it to remotely control a Live set and Live devices. Along the way, Yeuda offers valuable lessons about basic music theory—concepts like notes, chords, scales, and time signature—that will make your experience with Push more rewarding.
- What is Ableton Push?
- What's new in the Push 2?
- Comparing Push 1 and Push 2
- Browsing and loading sounds
- Programming beats
- Recording drums in real time
- Adding quantization and swing
- Controlling the mixer
- Controlling Live devices
- Adding custom LED feedback
- Step sequencing melodies
- Browsing and loading third-party plugins
Skill Level Appropriate for all
Q: This course was updated on 04/29/2016. What changed?
A: Ableton released the new Push controller, Push 2. We added eight tutorials covering the new hardware and its integration with Ableton Live 9.5.


text:
Two companies file protests over small business awards
UPDATE: This story was updated to add that a second company has filed a protest against the Air Force.
Is the Air Force headed for another NetCents 2 debacle? Time will tell. So far only two losing bidders has been confirmed as a protester involving the Air Force's $960 million NetCents Application Services Small Business Companion contract. The contract was awarded June 21 to 12 small businesses to provide IT services. Missing from the list of winners was Ace Info Solutions Inc. of Reston, Va., and Insignia Spectrum LLC, a joint venture formed by Insignia Technology and Spectrum. Ace Info field its protest on July 2. A decision is expected by Oct. 10. Insignia-Spectrum filed on July 9 and a decision is expected by Oct. 17.So far other protesters have not emerged. The Air Force is still trying to recover from the protests of its $6.9 billion NetCents product contract that was pulled back for re-evaluation after 11 companies filed protests. New winners are expected to be named soon. The NetCents 2 program is a $24 billion follow-on to the Air Force's successful $10 billion NetCents 1 program. But delays and the bid protests have forced the Air Force to extend NetCents 1 several times. Once fully awarded, the NetCents 2 contracts will be the Air Force's primary source of netcentric and IT products, services and solutions.
About the Author
Nick Wakeman is the editor-in-chief of Washington Technology. Follow him on Twitter: @nick_wakeman.
Printable Format
E-Mail this page
Advanced Search
In an exclusive for WT Insider members, we are collecting all of the contract awards we cover into a database that you can sort by contractor, agency, value and other parameters. You can also download it into a spreadsheet. Our databases track awards back to 2013.
Read More
In our latest WT Insider Report, we pull together our best advice, insights and reporting on the trends and issues that will shape the market in 2016 and beyond.
Read More
Sign up for our newsletter.
I agree to this site's Privacy Policy.