text:
By Charlotte Duck
Carrie Bradshaw's back! And we're already a little bit in love with AnnaSophia Robb...
A very '80s Austin Butler and AnnaSophia Robb appear to be 'caught in the act' on the set of The Carrie Diaries in New York. We can't wait to see by whom…
Rex Features
A-ha! So the couple were stealing a kiss, eh? To say we're excited about the Sex And The City prequel The Carrie Diaries is the understatement of the year.
AnnaSophia Robb donned a blush pink coat on the set of The Carrie Diaries, along with her trademark 'Carrie' bag and some polka dot tights.
There's no mistaking who AnnaSophia Robb is meant to be playing in this shot! The actress, who is playing the young Carrie Bradshaw, put her own spin on Carrie's iconic tutu, wearing this dramatic black tulle dress while filming for the new series.
Sarah Jessica Parker's Style Evolution
With her fashionable threads and heels, AnnaSophia Robb looked spot on in The Carrie Diaries. Shame she was so dressed up just to buy a pretzel from a street vendor.
PA Photos
It doesn't seem like the pretzel went down well either!
Working those Carrie curls, AnnaSophia Robb filmed a scene for The Carrie Diaries in New York. And no wonder she's got the character's look down to perfection: AnnaSophia revealed none other than Sarah Jessica Parker had given her tips on the role. "She sent me a letter! I wrote her one back, too. She was just very encouraging about it all. It was really cool!"
She might have been swaddled in a dressing gown and Ugg boots, but there was no denying AnnaSophia Robb's star quality as she made her way onto the set of the Sex And The City prequel.
A sequinned frou frou skirt, orange heels and a green animal print cardigan? AnnaSophia Robb's certainly isn't scared of taking on Carrie's unique style.
Working Carrie Bradshaw's fashion with a modern twist, AnnaSophia Robb stepped out in these fuchsia jeans and a cute cropped jacket. We just can't wait to see more of her wardrobe!
Our stylish edit of the best winter scarves - including the style you keep seeing on Instagram
The cosier, the better.
What is the alkaline diet and why is everyone Googling it?
The intel.
Kim Cattrall just made a thinly-veiled dig at the Sex and the City reunion, after SJP confirmed it was officially going ahead on Monday
The SHADE.
Happy Birthday, Kate Middleton! Here are all the reasons why 2020 was the Duchess of Cambridge's year
The year that defined the Queen-to-be?


Norima, a Winnipeg-based technology consulting company that prides itself on its ability to interpret a client's vision and materialize it into fully functional and multifaceted solutions, and recent recipient of the 2017 Outstanding Medium Business Award through the Manitoba Chambers of Commerce, is in need of experienced MuleSoft Developers to join one of our key delivery teams. We are looking for candidates with exemplary character, competence, and consistency to join a growing team of professionals who share a passion for solving complex problems with technology-based solutions.
Please reference "Senior MuleSoft Developer" in the subject line and the job reference code in your cover letter.


Bulk Liquid Soap Dispensers are available is Electronic Touchless or Manually operated models. All units are refillable and designed to be efficient and hygienic.
The Electronic Touchless Bulk Soap Dispensers are battery operated and feature a low battery indicator light. The units offer a consistent dispense amount of 1-1.5ml to prevent soap waste. The Bulk Soap Dispensers are refillable with quality bulk liquid soaps.
The Manual Bulk Liquid Soap Dispensers are constructed from impact resistant plastic to provide durability in a variety of settings. The large pushbar meets ADA requirements. The dispensers accommodate a variety of liquid and lotion soaps, but should not be used with pumice soaps.


Pure Solar Power is an all in one energy services provider. We look at the current energy use conditions of the home or business and produce a recommendation list for the property.
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Serving all of San Diego county for all your Heating and Air Conditioning needs.
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We process, underwrite and fund all of our loans. We have very competitive pricing, our fees are low, but most of all we get the job done right the first time.
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Packaging Solutions is your source for custom and stock packaging needs: corrugated, foam, plastic & janitorial supplies. We offer design services, delivery and just-in-time vendor managed inventory.
Whether you own a home, small boutique shop, or manage numerous commercial properties, Patriot Window Cleaning is proud to provide Superior Window Cleaning and Pressure Washing services.


text:
Environmental lawyer blasts SFWMD for public records response
Lisa Interlandi, a senior attorney for the Everglades Law Center, says the South Florida Water Management District retaliated against her for submitting a public records request.
Interlandi submitted a request for the district's email list earlier this month after the district told those on the list that, "Audubon Florida wants to raise your taxes to pay for the federal government's failure to control invasive plants that are destroying the Arthur R. Marshall Loxahatchee National Wildlife Refuge."
The district sent that blast email after Audubon Florida Executive Director Eric Draper urged district board members not to roll back tax rates so the district would have more money for program work.
"I wanted to know who was receiving those communications," Interlandi said, adding that the district's emails were, in her view, "increasingly negative and were calling out people by name."
Interlandi offered examples of the district's communications. In one from May, the district told email recipients that "former Lee County Commissioner Ray Judah inaccurately alleged flawed scientific modeling was used in Everglades restoration planning."
In another, also from May, the Caloosahatchee River Watch came in for criticism.
"Today, the Caloosahatchee River Watch group is holding what was advertised as a public forum to discuss the C-43 Reservoir project," the email state. "However, this 'forum' will consist solely of one-sided detractors in pursuit of an agenda without facts to support it."
Many government agencies do keep email lists to provide media members and others information about upcoming events and summaries of agency actions.
After the district said Audubon Florida wants to raise taxes, Interlandi asked for the district's mailing list. The district complied. But it also sent out another email, this one letting recipients know their email addresses had been sought.
Under the heading "Your Privacy," the district wrote:
"The South Florida Water Management District has received a public records request from a party associated with the email address – lisa@evergladeslaw.org – seeking more than 5,000 email addresses contained in SFWMD's electronic mailing list. This agency is legally obligated under Chapter 119 of Florida State Statutes to fulfill this demand and provide these email addresses.
"As you may know, such email lists and addresses are commercial commodities that are often bought and sold. The law prohibits SFWMD from asking about the intended use for the information. Any concern you may have about a potential invasion of privacy is understandable.
"SFWMD maintains email lists with the intention of keeping the public informed about the agency's work. These email lists represents a wide range of interested parties, including individuals who requested SFWMD information, elected officials, environmental groups, businesses, scientific communities and the media.
"You may receive unwanted solicitations or correspondence as a result of this public records request."
Interlandi said she would never sell the email list, which is a public record. She said she views the SFWMD email as retaliation for asking for the district's email list."They retaliated by publishing my email address," Interlandi said. "What other purpose is served by publishing my email address? I believe it was retaliation for requesting the list."
District Spokesman Randy Smith said those on the email list were warned of Interlandi's request "in an abundance of caution."
"When faced with a mass public record demand for private and other email addresses, SFWMD, in an abundance of caution, chose to notify the persons affected of this fact," Smith wrote in an email to The Palm Beach Post. Smith said the district "has never received a mass public records demand for an email address list. The requester did not ask for communication or specific emails but rather an entire email address list, which was completely out of the ordinary."
The district, Smith said, does not know how the list will be used and believes those on the list "have every right to know that their information has been obtained by a third party without their consent."
As for telling email recipients who had requested the list, Smith said the district "never mentioned the requester by name. SFWMD never assumes an identity of an individual solely based off an email address."

redpajama_set_name: RedPajamaCommonCrawl


text:
Chicago toy store leans into the quirky and fun to thrive amidst uncertainty
PayPal Product
"Pirate Abe" watches over Rotofugi, a designer toy shop in Chicago.
Designer toy store and art gallery Rotofugi began as a small local shop in Chicago's sleepy Ukrainian Village nearly 20 years ago. Now it's a "must visit" spot in the city's bustling Lincoln Park neighborhood, drawing collectors and toy enthusiasts and regarded among the greatest toy stores in the world.
The 3,000-square-foot toy haven sells toys designed by artists and independent toymakers, and there is something for everyone in its expansive inventory—ranging from dumpster fire vinyls to mini-marshmallow figurines.
"We get new stuff in every week, and the stock is always changing," said Rotofugi manager DC Cathro.
This has helped the store garner an ever-growing fan base so devoted that two Rotofugi superfans even tattooed the store's Pirate Abe mascot on their forearms.
"One of the great things about working here is seeing the joy on people's faces when they come in and find something that is just perfect," Cathro explained. "We have a lot of stuff in here that people just really light up for … [and] you're really not going to find anywhere else."
Adapting for the holidays
The holiday season is typically the busiest and most successful time of the year for Rotofugi, and the store is usually packed with shoppers from Thanksgiving to Christmas. And like at other places with things that are fuzzy, unique or loaded with bells and whistles, people have often liked to touch the goods before buying.
But the holiday season will look a lot different this year amid the pandemic. Shoppers' grins are now shielded by face masks. Aisles that used to be overrun with children or adults embracing their inner child are dotted with hand sanitizing stations. Foot traffic has greatly diminished, and many in-store events that were normally key sales drivers for Rotofugi - like exhibit openings and toy launches - have been canceled.
Store manager DC Cathro has steered Rotofugi through the many
challenges small businesses have faced in 2020.
"There has been a real big shift with the pandemic," said Cathro, who oversees the store. "We still have the customers, [but] they're just not coming in as much, or as often. So, that's been a little bit of a struggle, but we're hanging in there," he said.
To help counter this, Cathro is working closely with the store's owner, Kirby Kerr, to introduce new and innovative toys, tools and strategies to help give Rotofugi a leg up and spread some much-needed holiday magic this season, despite the unusual circumstances. One collector's item that has been a crowd favorite during these stressful and unpredictable times is the store's blind box series. Fans choose a mystery package and are rewarded with one of many special figurines. "It's kind of like playing the lottery with toys," Cathro explained.
One of the best parts of the collection? If shoppers don't like their surprise item, they can trade it in for another toy on display. The store is also offering special, festive, holiday-inspired blind box collectibles.
Blind Box collectibles add an element of chance to the toy shopping experience.
"People need an escape," said Cathro, and "we want to keep the store running … and have people come in as long as we're able [to]" do so.
Assuaging customers' concerns about making safe payments is part of that. With help from PayPal, the store has been able to modernize its payment system and offer a quick and simple touch-free payment option through a PayPal QR code. "It's convenient, cheap, [and] easy," Cathro said. You "scan it with your phone [and] type it in," and "don't have to touch anything," he said. The technology allows the store's customers more ease and flexibility. It's "one less thing to worry about."
Rotofugi has also thought about how to reach customers who prefer not to enter the store or who'd rather shop from home. As an added convenience to them, the staff is offering curbside pickup as a temporary solution. "You can order it online, we'll take it out to your car, and you don't have to touch anything," Cathro explained. The store has also been gradually expanding its e-commerce strategy and growing its online inventory.
A PayPal QR Code makes the check out process at Rotofugi touchless.
It's a necessary pivot in these times. PayPal's own data reveals that global sales of toys, hobbies and games increased by 26% in 2020 compared to the previous year. Yet retail experts worry that this holiday season will exacerbate the ever-growing gap between large chains and small stores as increasingly homebound consumers turn to big-box shopping and shipping.
"Brick and mortar stores are having a rough time, and the pandemic has made everything a hundred times harder," Cathro said. "It's a really trying time for everybody, and we just want to make sure that we can do as much as possible to make people happy and satisfy all of our customers."
Looking ahead at 2021, and beyond
The beloved Lil' Dumpster Fire has been a 2020 best-seller.
To strengthen its business strategy for the holiday and beyond, the shop has been able to steadily shift the bulk of its business online and grow its mail order options. There's no question that the bestselling Lil' Dumpster Fire vinyl – a mini, smiling cartoon dumpster engulfed in flames designed by 100% Soft– will spark joy for many people whether they order online or get it in person.
"It's sort of been the perfect toy for 2020," Cathro said. "We've sold out three times. We've got more coming in in December, and I'm sure they're going to sell out immediately." Thanks to its widespread popularity, this unofficial mascot of 2020 has been reproduced as a pin, key chain, hat and even an LED night light. Another very on-brand and beloved keepsake to commemorate 2020 is the cartoon COVID figurine by Vincent Scala, which depicts a colorful gem in a cozy bathrobe and fuzzy slippers – a wardrobe many have embraced while in isolation.
Rotofugi recently began showing exhibits in its gallery space again, with attendees required to wear masks and adhere to social distancing measures. The store is currently showing a new exhibit from Massachusetts-based illustrator and print-maker Daniel Danger.
A look at some of the pop art on display in Rotofugi's gallery.
Looking beyond the holidays, Cathro is hopeful the shop will keep discovering new and inventive ways to appeal to audiences during and long after the pandemic. "We just have to weather the storm. It's been a rough ride … [and] everybody's struggling, but we're going to hope that the holiday season kind of comes through for us," he said. "We have to [continue to] do as much as we can to keep our clients coming in and keep people happy."
1PayPal internal data review of changes in toy shopping patterns between 2020 and 2019, excluding November and December for both years. Review only included US, UK and AU PayPal transactions.
PayPal Rolls Out QR Code Payments for a Touch Free Way to Buy and Sell In-Person
"Pirate Abe" watches over Rotofugi, a designer toy shop in Chicago
challenges small businesses have faced in 2020
Blind Box collectibles add an element of chance to the toy
The beloved Lil' Dumpster Fire has been a 2020 best-seller


text:
Q: Setting scope properties in link function See codepen.
What do I have to do to make scope.test visible in my HTML when I give my directive an isolated scope, replacing "scope: false" with "scope: {}"?
My directive:
angular
  .module("MyApp", [])
  .directive("myDir", () => {
    return {
      scope: false,
      restrict: "A",
      link: (scope, element) => {
          scope.test = 'my test';
      }
    };
  });

My HTML:

{{test}}

No Controller, only link function in my directive.

A: Earlier it had work because you had scope: false (shared scope).
In your case adding scope: {} to directive wouldn't reflected test value changes on screen. Because when scope: {} isolated scope created in directive, it binds that scope to the directive template if it present. In your case you don't have any template in your directive.
If you wanted to see input value you could change it by

*

*Either using $parent convention like scope.$parent.test = 'my test'

*OR shift the {{test}} inside directive template so that isolated scope will get compiled with directive template.

A: In basic words scope:false means - directive takes the same scope of controller from where it was called.
On other hand scope:{} is a isolate scope and it uses internal scope of directive and its effected on directive template only

You can try to play with ng-transclude something like:

{{$parent.test}}

and:
  .directive("myDir", () => {
    return {
      scope: {},
      transclude: true,
      restrict: "A",
      template: '

',
      link: (scope, element) => {
          scope.test = 'my test';
      }
    };
  });

Codepan Demo

redpajama_set_name: RedPajamaStackExchange


Recent media attention and scrutiny has raised many questions regarding Hockey Canada Policy 6.22. To better help our members, KMHA is posting this article to answer questions and provide a direct link to the policy, and supporting documentation from our governing bodies.
Please find a link to the Hockey Canada Policy Manual HERE. A Cut and paste of policy 6.22 is below. Please note if you have any questions regarding the interpretation of this policy, KMHA will make every effort to answer or forward your concern to the correct governing body for an answer.
6.22.1 Definition Hockey Canada and its member Branches view all leagues that operate outside the auspices/sanctioning of Hockey Canada programs to be classified as "outlaw" leagues. This currently does not include summer hockey leagues/teams, adult recreational hockey leagues/teams, high school hockey, and/or hockey schools.
• Any individual who participates (knowingly or otherwise) in "outlaw" programs after September 30 of the season in question ("the Cut-Off Date") will lose all membership privileges with Hockey Canada for the remainder of that season, and may only reapply for membership with Hockey Canada after the end of that season. "Participation" in an "outlaw" program will be considered to have occurred if the individual takes part in one game (including an exhibition, tournament, league or playoff game) after the Cut-Off Date. If a participant makes the choice to participate in these "outlaw" programs, they must understand the ramifications of that choice and that the sanctions described in this paragraph will remain in effect even if the league or team folds, or the individual is released, suspended or fired.
• Hockey Canada and its member Branches across the country will make every effort to ensure that local minor hockey and female hockey associations in areas where "outlaw" leagues exist are not supporting these leagues in any manner whatsoever. For greater certainty, "supporting" includes, but is not limited to, assisting an "outlaw" league directly or indirectly through advertisement, promotion, ticket sales, volunteer activities, assigning Officials, sharing resources or enabling such a league to participate in Hockey Canada sanctioned activities. We will withhold tournament sanctions and will preclude such associations from benefiting from any Hockey Canada/Branch/CHL sanctioned event by whatever means necessary, including the withdrawal of any such event from any community within the geographic boundaries of that association if need be to stress this point.
To help provide clarity regarding the participation in Non Sanctioned Leagues, KMHA has reached out to our governing bodies for a list of leagues that would cause policy 6.22 to come into play. KMHA also requested clarification regarding 3on3 PD day programming at the request of our members and the following is a quote from Alliance Hockey.
"In light of recent events we have received questions about 3 on 3 PD Days etc. These events are good to go and do not constitute Non Sanctioned Programming.
For a list of non sanction programs please see this Information Bulletin from Alliance Hockey. This list is kept and updated by Alliance Hockey. As a member of Hockey Canada, the Ontario Hockey Federation and Alliance Hockey; KMHA players and members are expected to adhere to these rules and regulations.
We hope that this information helps to express the regulations and policies in question. Please do not hesitate to ask any questions.


We present results from a novel strategy that enables concurrent identification of protein-protein interactions and topologies in living cells without specific antibodies or genetic manipulations for immuno-/affinity purifications. The strategy consists of (i) a chemical cross-linking reaction: intact cell labeling with a novel class of chemical cross-linkers, protein interaction reporters (PIRs); (ii) two-stage mass spectrometric analysis: stage 1 identification of PIR-labeled proteins and construction of a restricted database by two-dimensional LC/MSMS and stage 2 analysis of PIR-labeled peptides by multiplexed LC/FTICR-MS; and (iii) data analysis: identification of cross-linked peptides and proteins of origin using accurate mass and other constraints. The primary advantage of the PIR approach and distinction from current technology is that protein interactions together with topologies are detected in native biological systems by stabilizing protein complexes with new covalent bonds while the proteins are present in the original cellular environment. Thus, weak or transient interactions or interactions that require properly folded, localized, or membrane-bound proteins can be labeled and identified through the PIR approach. This strategy was applied to Shewanella oneidensis bacterial cells, and initial studies resulted in identification of a set of protein-protein interactions and their contact/binding regions. Furthermore most identified interactions involved membrane proteins, suggesting that the PIR approach is particularly suited for studies of membrane protein-protein interactions, an area under-represented with current widely used approaches.
An essential component of the goal to elucidate global biological function is the determination of protein interaction networks. Current approaches for mapping protein-protein interactions include yeast two-hybrid system (1), affinity purification procedures based on immunoprecipitation (IP)1 or a single (e.g. FLAG tag (2)) or double affinity tag (e.g. TAP tag (3, 4)) followed by protein identification with mass spectrometry, protein microarray technology (5, 6), and computational prediction methods (7, 8). Although all these approaches demonstrate great promise in mapping protein-protein interactions on a proteome wide level, the resulting large scale data sets are often associated with high rates of false negatives and false positives (>50%), and poor overlap of data sets among different approaches used for the same system are often observed (9–11). Such observations suggest that no single method is flawless and comprehensive. The strengths and weaknesses of each method have been thoroughly reviewed (12–15). For example, traditional IP-based affinity purification methods require a specific antibody for every protein of interest that is a hindrance for widespread, large scale application. Tag-based methods overcome this limitation by fusing the bait protein genetically with an affinity tag that is applicable to all proteins. One of the most successful tag-based methods is TAP technology, which fuses two affinity tags to the bait protein, and nonspecific binding is significantly reduced with two sequential purification steps (3, 4). Although tag-based methods allow bait proteins to be expressed in vivo and interact with native physiological partners, recent studies showed that tagging can also cause overexpression of the bait protein that can result in association with chaperones and improper intercellular localization (16, 17). In addition, tagging one bait protein at a time for large scale studies can be tedious and costly. Another issue worth noting is that all affinity-based methods require cell lysis prior to purification of the associated complex of the bait protein. During cell lysis, the native cellular system is disturbed, and the bait protein is present in the lysis buffer, which is very different from the intracellular milieu. As described recently by Berggard et al. (13), the fact that the affinity between interacting proteins may be substantially different in vivo as compared with in vitro has not been carefully considered in the literature. We reported the first such comparison of mapping targeted protein interactions using both intact cells and cell lysates, and our results illustrated significantly different protein interaction data, highlighting the importance of identification of protein-protein interactions under native conditions (18).
Another challenge that affinity-based methods face is related to the inherent difficulty involved in maintaining the integrity of native protein complexes while removing the nonspecific bindings during washing steps. Most transient and weak protein-protein interactions may not survive through harsh washing steps; this is particularly true for interactions involving membrane proteins. For example, a high level of detergent normally required for maintaining the solubility of membrane proteins can also disturb non-covalent associations (15, 19). Chemical cross-linking can be used to stabilize and freeze protein-protein interactions by forming covalent bonds with proteins while proteins are present in the native cellular environment (15, 20, 21). The cross-linked protein complexes can remain intact during cell lysis and stringent washes. Therefore, cross-linking strategies have been successfully combined with affinity-based methods for studies in protein-protein interactions. In vivo cross-linking applications coupled with IP (22–27) and TAP tag (28, 29) techniques have been extensively reported and reviewed (15, 20, 21, 30–32).
Another important feature of chemical cross-linking methods is the potential for mapping topology of proteins and protein complexes (for reviews, see Refs. 15, 20, 21, and 30). If cross-linked residues/peptides can be identified, this information can yield clues about the contact/binding interfaces among protein complexes. Although in vivo cross-linking coupled with affinity purification can readily allow identification of interacting protein partners for a particular protein of interest with the detection of higher bands in gels or Western blot images, identification of cross-linked peptides/residues is not trivial even for purified protein complexes available in large quantity. Improved cross-linkers such as chemically cleavable cross-linkers (such as dithiobis(succinimidyl propionate)) (33), isotope-encoded cross-linkers (34), and cross-linkers with affinity tags (35, 36) have been used to aid in assignment of cross-linked residues in mapping topological structures of purified proteins and protein complexes. Factors that have slowed or inhibited the advancement of cross-linking strategies for large scale in vivo applications stem from the complex nature inherent with the cross-linking approach. Cross-linking reactions are often carried out with a large excessive amount of cross-linkers to increase reaction rate and product yield, thus resulting in highly complex mixtures. Proteolysis performed prior to MS analysis further increases the degree of complexity, and the desired cross-linked peptides are only a tiny fraction (<0.1%) of the total theoretical peptide combinations (37). In addition, interpretation of MSMS fragmentation spectra of the cross-linked peptides is challenging because simultaneous fragmentation of the cross-linker itself with one or more peptides can result in spectral complexity that precludes peptide/protein identification. The Aebersold group (37) recently reported breakthrough results illustrating identification of cross-linked peptides directly from complex Escherichia coli cell lysates with advanced informatics software development. Their efforts resulted in identification of three interactions from homo-oligomers and two interactions from hetero-oligomers, which suggests strongly growing interest in cross-linking methods for protein interaction identification and as yet untapped potential in this challenging research area.
To help overcome the limitations of current cross-linking methods, we have pursued a chemistry-based strategy by developing a novel class of cross-linkers called protein interaction reporters (PIRs). The spacer chain of a traditional cross-linker is modified in our PIR compounds with the incorporation of two specific mass spectrometry-cleavable bonds, a mass-encoded reporter tag, and an affinity tag (see Fig. 1a). Affinity-based purification is used to enrich PIR-labeled native protein complexes; however, no genetic manipulation is required as used in FLAG tag and TAP tag methods nor is any antibody required for each protein of interest because the affinity moiety is part of the PIR cross-linkers. This feature conceivably allows further extension of the PIR strategy to native biological systems other than cells, such as tissues and biological fluids. When combined with a novel two-stage mass spectrometric analysis strategy, the PIR approach allows large scale mapping of protein-protein interactions and their sites of interaction in native biological systems. Here we report the initial application of the PIR strategy to the microbial system Shewanella oneidensis MR-1 cells and the results of identification of a set of protein-protein interactions including information on contact/binding interfaces from living cells. These results represent the first such successful application of a chemical cross-linking strategy to map interactions and interaction sites from native living cells in a large scale, non-targeted way. The tunable chemistry of the PIR strategy will ultimately allow multiplexing an array of PIR cross-linkers with different structures and reporters for more comprehensive mapping of protein-protein interactions from native biological systems.
PIR structure.a, conceptual modular design of novel cross-linkers, PIRs. b, the specific fragmentation pattern of PIR-labeled peptide distinguishes dead-end, intra-, and intercross-linked peptides. The neutral mass of the precursor ion equals the sum of the neutral masses of its product ions. c, structure of a biotinylated Rink-based PIR. The singly and doubly charged reporter ions are 1122.5 and 561.7, respectively. d, structure of a biotinylated DP-based PIR. The MSMS labile bonds are indicated by the dashed lines, the reactive groups are NHS esters, and the affinity group is biotin. The singly charged reporter ion is 752.4.
All chemicals were purchased from Sigma unless otherwise noted. Fmoc-protected amino acids and HMPB-MBHA resins used for PIR synthesis were purchased from Novabiochem. Water used for preparing buffers and solutions was 18-megaohm deionized water produced with a Barnstead Nanopure Water System.
The PIR cross-linkers were synthesized using a 431A peptide synthesizer (Applied Biosystems, Foster City, CA). Glycine was coupled to HMPB-MBHA resin, and then the biotin group was introduced in the form of N-α-Fmoc-N-ε-biotinyl-l-lysine. The second lysine in the form of N-α,ε-di-Fmoc-l-lysine was coupled to the biotinylated lysine and was then used as the branch point for the cross-linker. The Rink groups (for Rink-based PIR) or aspartic acid and proline (for DP-based PIR) were coupled to lysine in forms of Fmoc-amino acid. Carboxyl groups were then introduced by coupling the primary amines of the Rink groups or aspartic acid with succinyl anhydride. Subsequently the two carboxyl groups were activated by forming the esters with N-hydroxysuccinimide (NHS). The crude product was cleaved using either 0.5 or 1.0% TFA in chloroform and then neutralized with pyridine. The chloroform and TFA pyridine salts were removed under vacuum. The crude product was purified using reversed phase HPLC, and the final product had a purity of about 90%.
To fully utilize the advanced features of PIRs, a two-stage mass spectrometric approach was developed (see Fig. 2). Stage 1 analysis was carried out with avidin capture of in vivo labeled proteins followed by digestion and shotgun two-dimensional LC/MSMS for protein identification as described previously (38).
Diagram of two-stage mass spectrometric strategy. After in vivo labeling, protein extraction, and affinity capture of labeled proteins, enriched PIR-labeled proteins are divided into two parts. Stage 1 involves digestion and shotgun LC/MSMS for protein identification to constitute a restricted protein database. Stage 2 is performed by digesting PIR-labeled proteins first followed by another affinity enrichment of PIR-labeled peptides. Analysis of the labeled peptides is carried out with multiplexed LC/FTICR-MS. Accurate masses of the labeled peptides are measured and used for protein identification by searching against the restricted database compiled from stage 1. 2D, two-dimensional.
Stage 2 analysis was carried out by labeling 0.2-g cell pellets suspended in 4 ml of PBS buffer (150 mm sodium phosphate and 100 mm NaCl (pH 7.5)), and the cross-linkers were added to the suspended cell pellets to produce a final concentration of 1 mm. The cross-linking reaction was carried out at 4 °C for 1 h and quenched by 1 m ammonium bicarbonate. Extensive washing steps with PBS after cross-linking reactions were used to eliminate most nonspecific contamination as reported previously (38). Then cells were lysed in 2% Nonidet P-40, PBS solution by sonication for 2 min. The cell lysates were centrifuged at 15,000 × g at 4 °C for 45 min. The pellets were discarded, and supernatants were collected in four clean tubes with a total amount of ∼4 ml. Protein concentration of the supernatants was determined to be ∼5 mg/ml by a Bradford assay (Bio-Rad). The PIR-labeled proteins were enriched by adding a 50-μl slurry of monomeric avidin beads (Pierce) to each tube, and the reaction was incubated for 2 h with gentle mixing at room temperature. Then the avidin beads were washed three times with 500 μl of 100 mm NH4HCO3 (pH 7.8). After the last wash, the avidin beads were suspended in a 100-μl solution of 8 m urea and 100 mm NH4HCO3 (pH 7.8). Both enriched PIR-labeled proteins and avidin were reduced directly on the beads with 10 mm DTT at 56 °C for 1 h and alkylated with 20 mm iodoacetamide for 1 h at room temperature. After reduction and alkylation, additional 10 mm DTT was added to neutralize the unreacted iodoacetamide for another 1 h at room temperature. The bead suspension was then diluted 4 times with 100 mm NH4HCO3 (pH 7.8) buffer, and 10 μg of trypsin (Promega, Madison, WI) was added for overnight digestion at 37 °C. After digestion, 1 mm PMSF was added to quench the trypsin activity. A 50-μl slurry of monomeric avidin beads was added to the digestion mixture and gently mixed for 2 h at room temperature. After beads were washed three times with 100 mm NH4HCO3 (pH 7.8), PIR-labeled peptides were eluted from avidin beads with 200 μl of 70% acetonitrile and 0.5% TFA solution four times. Finally the volume of the combined eluent was reduced to ∼30 μl using a speed vacuum, and ∼10 μl was used for multiplexed LC/FTICR-MS analysis.
A Bruker Daltonics Apex-Q 7-tesla FTICR mass spectrometer (Billerica, MA) was used for multiplexed LC/FTICR-MS experiments. The nano-ESI source, nano-LC systems, reversed phase nanocolumn, and LC gradient methods were the same as those used with the ion trap mass spectrometer as described previously (39). Ions formed by the nano-ESI source entered the instrument through a glass capillary and then passed through a hexapole followed by a quadrupole and then a second hexapole, which was used for ion accumulation and PIR activation. After the second hexapole, ions were guided through a series of ion transfer optics and entered the ICR cell where ions were detected after excitation of cyclotron motion, resulting in accurate mass measurements. Xmass 7.0.6 and HyStar 3.0 software programs were used to acquire LC/FTICR-MS data. All data sets were acquired with 131,072 points, and each spectrum was resultant from averaging four scans. The optimized collision energy, −22 V, which efficiently fragmented the labile bonds in PIR molecules but not peptide bonds, was applied during alternating scans throughout the LC elution. External calibration of the instrument was performed to a mass accuracy of less than 1 ppm with direct infusion of BSA tryptic peptides immediately prior to the LC/FTICR-MS experiments.
FTICR-MS data were analyzed using the program ICR-2LS. The time domain ICR signals were apodized with a Welch function, zero-filled once, and then Fourier transformed before calibration. The spectrum was calibrated with six or more peaks to about 1 ppm or less, and then the calibration equation was applied to the instrument. All masses were reported in neutral mass forms in a .PEK text file that was further used by the program X-links (40). Lock mass calibration using reporter ions was performed to correct all the neutral masses produced in the .PEK file to further improve mass measurement accuracy (MMA). All possible tryptic peptides of the restricted protein database were produced by X-links with the constraint of at least one internal lysine in the sequence (40). 10-ppm error tolerance was used in X-links searches for both cross-link type identifications (using the relationship between the precursor ion in the MS scan and the released peptide and reporter ions in the subsequent multiplexed MSMS scan) and cross-linked peptide sequence identifications (using the measured accurate mass of the released peptide ions in the multiplexed MSMS scans). MS2 and MS3 validation experiments were performed using a quadrupole ion trap mass spectrometer (Esquire HCT, Bruker Daltonics) as described previously (39). GPMAW (General Protein/Mass Analysis for Windows; version 6.21) was used to calculate MSMS fragmentation. The whole genome sequence of S. oneidensis MR-1 was completed in 2001, and the database containing the entire genome (4,854 ORFs) was downloaded in July 2006 from The Institute for Genomic Research.
The PIR strategy is enabled by incorporating two labile bonds in the spacer chain of the cross-linker that can be cleaved with high specificity in the mass spectrometer resulting in release of a mass-encoded reporter ion (Fig. 1a). This allows the detection of the cross-linked peptide or peptide complex during a low energy precursor scan and then detection of the intact peptide masses released in the subsequent high energy scan with accurate mass and/or additional MSMS analysis. The conceptual mathematical relationships that exist between PIR-labeled precursors, released peptides, and reporters are illustrated in Fig. 1b, and these relationships can be used to facilitate differentiation of cross-link types and identification of cross-linked peptides with informatics software tools. The tunable chemistry of PIR structure allows cross-linking a variety of different proteins by incorporation of different labile bonds, affinity tags, and reactive groups. For the current study, we used a biotin group as the affinity tag, NHS esters as the reactive group, and the Rink group (Fig. 1c) and Asp-Pro (DP) bond (Fig. 1d) as low energy MSMS-cleavable bonds.
The relatively large size of PIR cross-linkers has been a concern for obtaining useful structural constraint information (21). However, our previous studies using the model RNase S noncovalent complex showed that the distance (14 Å) between two cross-linked residues was much smaller than the maximum length (43 Å) of PIR (39). Furthermore Rinner et al. (37) recently reported identification of the same intercross-link in RNase A using the smaller cross-linker disuccinimidyl suberate. These results suggest that in solution the PIR is constrained to give rise to a shorter distance between the two reactive groups than the fully extended length; this is in agreement with the simulated estimates of the realistic lengths of 32 commercial cross-linkers in solution reported by Green et al. (41). Finally it is also worth considering that within the cellular environment many potential labeling sites that would allow identification of interactions span a wide range of distances. Therefore, flexibility in the cross-linker structure that can allow labeling sites over a range of distances is most critical to enable identification of larger numbers of interactions. Once identified as an interaction that exists in cells, these complexes can be studied for more detailed structural information using a wide variety of conventional molecular biology, analytical techniques, and computational methods.
The NHS was used as the reactive group in PIR cross-linkers. The NHS is the most frequently used group in protein labeling reagents (21) such as iTRAQ (isobaric tags for relative and absolute quantitation) (42), one of the most widely used protein quantitation reagents, and chemical cross-linkers such as disuccinimidyl suberate, dithiobis(succinimidyl propionate), bis(sulfosuccinimidyl) suberate, etc. (Pierce). The reason for this is multifold. First, the most common targets for labeling proteins are the primary amine groups, which are present on the large majority of proteins due to the high occurrence of lysine side chain ε-amines. Second, NHS forms a stable amide linkage to the primary amines in proteins at physiological condition (pH 7.0–7.5), which is important for in vivo labeling. Finally the NHS ester reaction has very rapid reaction rates with primary amines (43). Our studies (38) and those of others (23, 25) showed that a 5-min reaction time was sufficient for completion of labeling. The fast reaction rate is critical for in vivo experiments because it can quickly "snapshot" or "freeze" the protein complexes with stable covalent bonds before living cells are extensively perturbed.
To fully utilize the advanced features of the PIR strategy, a novel two-stage mass spectrometric approach was developed (Fig. 2). Intact cells were labeled with PIR compounds under near physiological conditions. After labeling, proteins were extracted, and PIR-labeled proteins were enriched by avidin capture. Then the enriched PIR-labeled proteins were divided into two parts for the subsequent stage 1 and stage 2 analyses. Stage 1 analysis was carried out with digestion of PIR-labeled proteins followed by protein identification with shotgun two-dimensional LC/MSMS (44). The goal of stage 1 analysis is to construct a candidate protein pool as complete as possible that provides a "lookup" table for stage 2 analyses. We previously used the Rink-based PIR compound as a general biotinylation reagent for labeling intact cells (38), and the ∼400 identified labeled proteins were used to constitute the restricted database for the current study. Stage 2 analysis was performed by further digestion of enriched PIR-labeled proteins on avidin beads followed by another affinity capture of PIR-labeled peptides. These peptides were then subjected to multiplexed LC/FTICR-MS experiments to measure both the intact PIR-labeled peptide masses in the low energy precursor scan and the masses of the intact peptides in the following PIR activation scan. Finally we used an informatics software tool, X-links (40), to identify cross-links in two steps: step 1 involved characterization of the precursor-product relationships and cross-link types based on the equations depicted in Fig. 1b; and step 2 involved assignment of cross-linked peptide sequences/proteins of origin with accurate peptide mass-based protein identification strategy.
Fig. 3 shows an example data set from a multiplexed LC/FTICR-MS analysis of stage 2 sample, including the base peak chromatogram and the extracted ion chromatogram (EIC) for the expected reporter ion. Fig. 3a, inset, shows the overlaid EICs for an identified PIR-labeled precursor ion, the released intact peptide ion, and the reporter ion. The observed complementarities of the peak intensity patterns and the high degree of overlap of the LC elution profiles between these traces strongly suggest a precursor-product relationship. This information can supplement multiplexed LC/MS analysis to help define precursor-product relationships and cross-link types and can be used to further constrain our search results.
LC/FTICR-MS chromatograms of PIR-labeled peptides with alternating low energy (−4 V) and high energy (−22 V) applied in the collision cell.a, base peak chromatogram (BPC). b, EIC of reporter ion. The inset in a is the overlaid EICs of a PIR-labeled precursor ion (dotted line) and its released peptide ion (dashed line) and reporter ion (solid line).
Because MMA is the most important factor in identification of both cross-link types and cross-linked peptide sequences, we routinely calibrated the FTICR-MS instrument externally to ∼1-ppm MMA with BSA tryptic peptide mixture immediately prior to LC/FTICR-MS analysis. However, space charge effects and variation of ion populations (45) can severely reduce MMA for LC/FTICR-MS experiments. Introduction of a standard mass used as an internal lock mass calibrant via dual spray has been used to improve MMA for LC/FTICR-MS analysis (46). The PIR strategy provides an inherent lock mass, i.e. the known mass of reporter ion. Thus, we calibrated the LC/FTICR-MS data set using reporter ion as lock mass, and MMA for reporter ions was corrected to ∼3 ppm (supplemental Fig. 1). However, the observed MMA for detected peptide ions can be worse than the lock mass because our approach works with all detected ions irrespective of their detected S/N. Low S/N peaks are more prone to error because of the effect of noise on the extraction of the detected peak centroid. Thus, to be conservative, 10 ppm was chosen as the mass tolerance limit for all X-links searches in the current report. Future work will implement automatic gain control as used by the LTQ-FT instrument (47) to further improve MMA.
The description of the program X-links and the estimated false discovery rate are reported elsewhere (40). Briefly the program first searches the observed neutral masses for relationships between precursors and released peptide and reporter species to assign dead-end, intra-, and intercross-links using the equations defined in Fig. 1b. Second the PIR-labeled peptide sequences and proteins of origin are identified using the measured accurate peptide masses to search against the restricted protein database. Fig. 4 shows an example relationship identification resultant from X-links searches. Fig. 4a illustrates a plot of overlaid EICs of an intercross-linked precursor (m/z 9785+), released peptide 1 (m/z 12432+), released peptide 2 (m/z 1281+), and reporter (m/z 1122+) ions, respectively. The precursor ion was observed with an alternating intensity pattern opposite to that of the reporter and released peptide ions. Fig. 4b shows superimposed spectra extracted from a low energy precursor ion scan and subsequent high energy product ion scan with corresponding precursor and product ions labeled schematically. To confirm the putative X-links assignment, we repeatedly performed LC/MSMS with a quadrupole ion trap mass spectrometer, isolated the precursor ions (m/z 9785+), and fragmented them with CID. All intense fragment peaks observed in the MSMS spectrum were accounted for by the fragmentation of one or two PIR labile bonds (Fig. 5a), validating the relationship identified with X-links and further illustrating the high cleavage specificity of PIR bonds. More validation examples of relationship identifications are provided in supplemental Figs. 2, 3, and 4.
An example of intercross-link determination from multiplexed spectra based on the mathematical relationship (precursor = peptide 1 + peptide 2 + reporter).a, overlaid EICs of m/z 9785+ (precursor 9785+; dotted line), m/z 12432+ (peptide 1 12432+; dashed bold line), m/z 1281+ (peptide 2 1281+; dashed line) and m/z 1122+ (reporter 1122+; solid line) ions, respectively. b, overlaid FTICR mass spectra extracted from scan 765 (Low Energy; solid line) and scan 766 (High Energy; dotted line). All major products resultant from cleavage of one and two labile bonds in PIR are assigned and labeled schematically.
An example of validation of intercross-link determination and peptide sequence identification for the precursor ion with neutral mass of 4884.3531.a, isolation and MS2 of precursor ion (m/z 9785+). All peaks produced from cleavage of one and two PIR labile bonds are labeled schematically. b, isolation and MS3 of the released peptide ion with m/z 12432+ and 1280+, respectively.
Peptide sequences and proteins of origin were identified using accurate peptide masses by searching against the restricted protein database. Proteins identified in the restricted database represent only that fraction of PIR-labeled proteins with highest abundance and PIR reactivity/accessibility because of the limited dynamic range of detection. However, it should be noted that stage 2 samples were similarly constrained, and thus the detected peptides were likely from the most abundant and PIR-reactive proteins present in the stage 1 database. Statistically stage 1 analysis expected a much greater probability of identifying proteins because any of the many tryptic peptides of a given protein can be used for identification. Stage 2 identification required analysis of the specific PIR-labeled peptides. Thus, although the stage 1 database was likely not comprehensive, it was much more so than the stage 2 data sets, and therefore most PIR-labeled peptides were expected to originate from proteins identified in stage 1 studies.
Table I shows the summary of identified intercross-linked peptides using Rink-based PIR. A selected list of dead-end labeled peptides and intracross-linked peptides is shown in supplemental Tables 1 and 2, respectively. It is worth noting that most identified proteins are membrane proteins; this is consistent with our previous reports that PIR molecules preferentially label membrane proteins (38). Our results also show that several identified proteins were labeled with multiple peptide sequences indicating their high abundance or accessibility. For example, SO1778 (MtrC), SO0404 (hypothetical protein), and SO1779 (OmcA) were identified by four, four, and seven different peptide sequences, respectively. On the other hand, the same peptide sequence can be labeled multiple times; this was observed previously in our efforts (39) and those of others (35, 36) to cross-link purified protein complexes. For example, FNPAKSELTYYLSNNFYDAK (The bold and underlined K was used both in the text and Table 1 to indicate the labeled lysine residue.), a peptide from SO0404, was detected in two different intercross-links and one dead-end labeled peptide, suggesting that this residue/region is highly exposed and accessible. In addition, several identified intercross-linked peptides in Table I were resultant from the same protein, e.g. OmcA, MtrC, SO0404, and SO4410 (glutamine synthetase, GlnA), which may be produced by intracross-link within the same protein sequence or intercross-link from subunits of homo-oligomers. It is worth noting that the current mass tolerance (10 ppm) used in this report is not sufficient to uniquely identify all the cross-linked peptides as demonstrated in the previous simulation study (40). Many PIR-labeled peptide masses resulted in more than one hit (supplemental Table 3). Further improvement of MMA or performing another MSMS scan on released peptides will help increase the ability to produce unambiguous protein interaction identification.
All the intercross-linked peptide masses listed in Table I were also used to search against the unrestricted database with a 10-ppm constraint (supplemental Table 4). In agreement with the previous simulation, most peptide masses generated multiple protein hits at 10-ppm MMA. At this mass tolerance, a restricted database and/or MS3 analyses are required to further differentiate between multiple possibilities.
Although the accurate mass-based strategy for protein identification is highly reliable (40, 48), validation of a few examples was necessary for the initial application of PIR technology. This required MSMS of isolated PIR-labeled precursor and another isolation and fragmentation of the released peptide, thus MS3. Fig. 5b illustrates an example of MS3 validation of intercross-linked peptides found in X-links analysis and identified by accurate mass to be peptides from the proteins MtrC and SO0404. These data were obtained by isolating the intercross-linked precursor ion (m/z 9785+), activating the PIR labile bonds, releasing the peptides, isolating the intact peptide ions at m/z 12432+ and 1280+, respectively, and then fragmenting each. It is worth noting that two MS3 mass spectra in Fig. 5b did not generate comprehensive fragment ions with decent S/N because of the dominant neutral loss of the precursor ions and sensitivity limitations of MS3 analysis. To further validate the peptide sequence identification by accurate mass and MS3, a medium sized peptide, LVYTTTKDLK, with the expected tag on internal lysine was synthesized, and the singly charged precursor ion was subjected to tandem mass spectrometry (supplemental experimental material and supplemental Fig. 8). These experiments resulted in spectra that showed the same neutral loss and backbone fragmentation peaks (MH+ − H2O, y7, y4, and y4 − NH3) as were observed with the endogenous PIR-labeled peptide shown in Fig. 5b, supporting the fragment assignments. These results confirmed the accurate mass-based identification and MS3 validation analysis. Furthermore this approach with synthetic peptides represents an additional, general, and useful approach that can be used to help confirm all identified PIR-labeled proteins and peptides. Additional MS3 sequence validation examples are included in supplemental Figs. 2, 4, and 5. It should be noted that MS3 analysis is manually obtained during repeated LC separation of stage 2 samples, and thus both throughput and sensitivity of current validation experiments are low. Only those precursors with relatively high peak intensities in MS and MS2 scans can generate sufficient signals in MS3. On the other hand, these results illustrate the benefits of using high throughput multiplexed LC/MS and accurate mass-based protein identification because none of the identified products need ever be among the most abundant species to be identified by accurate mass so long as they are detected.
The primary advantage of PIR technology that distinguishes it from other available methods is the capability of concurrently mapping protein-protein interactions and their binding interfaces in native biological systems. Two essential components enable PIR applications at the systems level: novel PIR cross-linker structures and novel two-stage mass spectrometric analysis. The first application of Rink-based PIR with the use of S. oneidensis cells at proteome wide scale resulted in identification of over 30 different intercross-linked peptides; 24 of them with unambiguous identification are included in Table I. 17 peptide pairs were derived from interprotein cross-links, whereas seven were resultant from either intraprotein cross-links or intersubunit cross-links of homodimers. Importantly although our initial PIR applications do not show the hundreds to thousands of interactions one might detect with other leading methods, such as yeast two-hybrid and TAP tag technology, these results are distinguished by the fact that the obtained information is relevant to interactions detected in the native cellular environment and by the fact that the regions of close proximity of the interacting proteins can be determined. This constitutes information not readily achieved by any of the leading strategies. Furthermore it is important to consider that such chemistry-based strategies are eminently extendable to include an array of chemical structures that can label and identify a wider range of protein interactions in cells. For example, the initial application of the more hydrophilic DP-based PIR molecules generated more than 10 intercross-linked peptides, most of which were different from those identified with Rink-based PIR. An example of an identified intercross-link between ribosomal protein L10 (SO0222, RplJ) and ribosomal protein L7/L12 (SO0223, RplL) using DP-based PIR is shown in supplemental Fig. 6. PIR cross-linkers with different structures and different physical properties can be used to simultaneously label and identify interactions among many different classes of proteins. As a consequence, the PIR strategy is scalable to enable multiplexed application of an array of PIR compounds with variable physical properties, e.g. hydrophobic character, charges, reactivity, etc., and independent reporter masses to enable even larger numbers of protein interactions to be identified from cells in a single experiment.
Among the identified cross-linked interactions, most involved outer membrane proteins. These results suggest that the current Rink-based PIR is particularly suitable for studies of membrane protein interactions, which is challenging with currently available techniques. In addition, the PIR approach allowed us to recapitulate some previously known interactions. For example, both in vitro (49) and in vivo (18, 50) studies showed that outer membrane cytochromes OmcA and MtrC form a complex to facilitate electron transport in S. oneidensis. The identification of intercross-linked peptides (precursor mass 3793.8837 in Table I) between OmcA and MtrC further confirmed the direct interaction of these two proteins (supplemental Fig. 7) and provides insight into the regions of protein sequences that are in close proximity in cells. Furthermore outer membrane proteins SO0404, SO2001 (5-nucleotidase UshA), and SOA0110 (lipoprotein, putative) identified in the previous targeted OmcA-MtrC interaction network study using IP methods (18) were also identified in the present results in the intercross-links (two from MtrC-SO0404, one from OmcA-SO2001, and one from SO0404-SOA0110). In the previous report, the OmcA-MtrC interaction network was mapped from protein identification of a gel band that appeared above 250 kDa. This high molecular mass gel band was the result of cross-linking protein complexes of three or more components and did not directly identify connectivity among interacting proteins. Identification of intercross-linked residues/peptides produced direct evidence of pairwise protein-protein interactions and enables one to pinpoint contact regions of the interactions.
Taken together, our initial results using PIR technology on living cells of S. oneidensis have demonstrated the capability for probing protein-protein interactions and protein topology concurrently without the need of specific antibodies and genetic manipulations of the system. This strategy can be further used as an in vivo tool to investigate, snapshot, and compare potential binding sites of proteins and protein complexes in cells at different growth phases, culture conditions, cell cycles, external stresses, etc. Future work will include further improvement of the throughput, efficiency, and MMA for stage 2 analysis and application of a library of PIRs with different reporter ions and isotopic labeling that will ultimately allow large scale quantitative profiling of protein-protein interactions and protein topologies in living cells.
↵ 1 The abbreviations used are: IP, immunoprecipitation; TAP, tandem affinity purification; PIR, protein interaction reporter; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HMPB, 4-hydroxymethyl-3-methoxyphenoxybutyric acid; MBHA, 4-methylbenzhydrylamine hydrochloride; NHS, N-hydroxysuccinimide; MMA, mass measurement accuracy; EIC, extracted ion chromatogram; S/N, signal to noise.
↵* This work was supported, in whole or in part, by National Institutes of Health Grants 1 R01 RR023334-01A1, 1S10RR017805-01, and 1S10RR022538-01 from the National Center for Research Resources. This work was also supported by Office of Science (Biological and Environmental Research), United States Department of Energy Grant DE-FG02-04ER63924.
↵§ Both authors made equal contributions to this work.
Revision received September 3, 2008.


I stayed at a lot of really amazing hotel properties during my time in South Africa, and this hotel in Plettenberg Bay was no different. After checking out the view from "The Heads" I drove down the Garden Coast of South Africa to the town of Plettenberg Bay where I stayed at the impressive Beacon Island Resort. The hotel is basically right out on the rocks and every single room on the property has an amazing sea view.
After dropping off my gear at the hotel, I headed to one of the most popular attractions in Plettenberg Bay, Robberg Nature Reserve which is famous for it's amazing coastline, beautiful landscapes, and fur seals. And well I didn't get to see any of the fur seals, I did have a perfect time wandering around the reserve.
The day was capped off with some photography around the Beacon Island Resort, and parts of Plettenberg Bay.
Stay tuned for the next episode of the video series when I head even farther along the coast of South Africa to explore some of the Wild Coast.


A service center for the American Automobile Association is the latest addition to the park.
For two decades Seaway Business Park in Gulfport has leased commercial office and warehouse space to a growing number of tenants in an expanded footprint. The developers currently are working with a major tenant to introduce its new business concept to the Mississippi Coast.
Owner and developer Steve Goldin, said the thriving family-owned business opened in 1997 with 12,000 square feet of warehouse space, with Fastenal as its original tenant. Office and warehouse space has grown to more than 150,000 square feet in three locations – two on Seaway Road and one near downtown Gulfport.
Goldin is working with the American Automobile Association to build a service center that will serve local members with automotive services. The membership organization is best known for its emergency roadside assistance.
"Now when you call AAA, a local towing company helps with flat tires or a dead battery." said Goldin. When the new service center is constructed, the services will be provided by employees of AAA.
From the beginning, the Goldins wanted Seaway Business Park to accommodate various business types in a common setting. Key to their success has been the central location close to all of the converging thoroughfares in the Gulfport/Biloxi area. That includes Interstate 10 and U.S. 90 for east-west traffic and U.S. 49's north-south route.
Tenants include Sherwin-Williams, RJ Young, Siemens, Primerica and Rotech Healthcare.
The Goldins are developing final plans for AAA's new 3,000-square-foot facility on a .5-acre parcel.
"Once we get the OK on their floor plan, we will start construction right away," said Steve Goldin.
He said AAA will hire 10 to 12 employees and spend $500,000 on tow trucks alone.
The new AAA project is the latest expansion activity at the business park, Steve Goldin said. Sherwin-Williams grew from 3,000 square feet to 12,000 in the last few years and Fastenal, their original tenant, is gearing up for another remodeling.
The Goldins maintain a strong relationship with their tenants, starting from their first one, and recognize the importance of each tenant to their success. "Fastenal got us to where we are today," said Steve Goldin.


Cromwell having died in 1658, the English, most of whom were still greatly attached to the royal family, soon begged Charles II. to come back and take possession of his throne. He gladly returned to England, where he punished no one for the revolution, except the men who had condemned his father, Charles I., to death. A few of these king-killers, or "regicides," as they were called, fled from England as soon as they heard the king was coming, and three took passage for America.
Two of these men, Goffe and Whalley, after some trouble, reached the New Haven colony, where Puritan friends helped them to hide. The king sent orders to arrest them, and magistrates began to search every house to secure the regicides. For about eighteen years these two men lived in constant dread of being caught; but, thanks to their many friends, they always escaped. They dwelt for a while in a deserted mill, then in a cave, and once hid under a bridge while their pursuers galloped over it, expecting soon to overtake them.
The fact that the New Haven people had sheltered some of his father's judges, added to the complaints of the Quakers and discontented colonists, displeased Charles II. greatly; and he finally declared that New Haven should cease to form a separate colony, and joined it to Connecticut, which received a new charter (1662).
It is also said, however, that these two colonies were united mainly to please the Connecticut people, because they had won the king's favor by sending him a pretty message to welcome him back to the throne. The charter he gave them was the most liberal ever granted the colonists, although the one Roger Williams secured for Rhode Island also granted many privileges.
You doubtless remember the treaty made between the Indian King Massasoit and Governor Carver, when the Pilgrims first came to Plymouth. This treaty was kept forty years, and Massasoit and his tribe faithfully helped the colonists to fight the other Indians. But when Massasoit died, his two sons, who had received the names of Alexander and Philip, began to rule in their turn.	.
Alexander knew, by the wampum belts which were the history books of his tribe, that nearly all the land of his Indian fathers had been sold to the white men, piece by piece. It had been given in exchange for beads, kettles, blankets, etc., and now very little was left. But the Indians fancied that, although they had sold the land, they could still hunt and fish there as much as they pleased. The colonists, however, would not allow them to do so, and drove the Indians farther and farther off, until they began to feel cramped for space.
It is said that when one of the colonists once came to bid an Indian chief to remove still farther from the white settlements, the red man invited him to take a seat beside him on a log. Crowding nearer and nearer his guest, the chief bade him move again and again, until he forced him to the very end of the log. But when the colonist declared he could not move another inch without falling off, the chief calmly answered: "It is just so with us. We have moved as far as we can go, and now you come here to ask us to move farther still."
This feeling of unfair treatment made Alexander so angry, at last, that he formed a secret alliance with the Narragansett Indians to kill all the white men. But the Plymouth governor, hearing of this, promptly sent for him, bidding him come and clear himself of the accusation of treachery. Then, as the Indian did not obey at once, Winslow quickly set out, with his men, to bring him by force.
Alexander, furious at being thus compelled to mind, fell seriously ill from fever. The colonists then allowed his followers to carry him home; but on the way back, the Indian chief breathed his last. Ever after, his people were in the habit of saying that he had gone to the Happy Hunting Grounds, where the palefaces could never come to crowd him out.


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Warning: The information on this page refers to version 1.1.0 (file format 1.7) onwards. Bluetooth Low Energy was not available on earlier versions of phyphox.
Phyphox can communicate with almost any Bluetooth Low Energy (BLE) device to receive and send data. It comes with several prepared experiment configurations, which you can use without any additional knowledge. On top you can add support for additional devices through our editor or by directly creating/editing a phyphox-file with a text editor. If you are just looking for devices with existing support, you can check out our Bluetooth device database instead.
This page describes how the Bluetooth Low Energy integration works in phyphox files, which applies to both, the editor and direct editing of the phyphox-file. It also explains in detail the syntax in the phyphox file and the protocol how a device can offer its own phyphox-file to the app, so you can for example program an Arduino so it can be used without the necessity to manually transfer a phyphox-file.
If this is the first time that you use the editor, you should start with our introduction to our editor and if you just started to learn about our file format (without using the editor), you should start on the main wiki page about our file format. Also, if you are new to Bluetooth Low Energy, you should look for a short introduction, so you know what the terms "GATT", "UUID", "service", "characteristic" and "notification" mean.
In order to write a phyphox-file for a BLE device, you will need to know how its communication works. If the device is supported by phyphox out of the box, you can open the existing phyphox-file in our editor to see how we communicate with it. If this is not the case and you try to communicate with the device for the first time, you can often use some documentation by the manufacturer or some similar source of information. Finally, you can try to figure out the communication yourself by using apps that list the services and characteristics and capturing the communication from other apps (please make sure that you are legally allowed to do so).
If you create support for a device which is of interest to a wider audience (or need help to do so), please contact us as we might want to include it with the next phyphox version (with concent by the manufacturer).
Also, if you want to add support for your own sensors (for example Arduino-based) you can find an example on the page on Micro-controller based sensors.
1 BLE integration in phyphox
1.1 Accessing BLE devices
1.2 BLE as input or output
1.3 Notifications and polling
1.4 Accessing characteristics as input, output or config
2 BLE-related syntax in phyphox-files
2.1 BLE device matching
2.2 BLE as input
2.3 BLE as output
2.4 config, input and output block
2.5 config conversion functions
2.6 output conversion functions
2.7 input conversion functions
3 Sending phyphox-files from a device
BLE integration in phyphox
Accessing BLE devices
There are two methods to access a BLE device from the user interface.
The first one is the one that is probably most familiar to many users. If you tap the plus button on the main screen (bottom right corner on Android, top right on iOS) you can select the option "Bluetooth" which initiates a scan for such devices. This lists every device, but will show most of them in grey unless phyphox has a matching experiment in its collection. A matching experiment needs to be included with phyphox and cannot be achieved by transferring a phyphox-file. The only exception is if the device brings its own phyphox-file, which is a rather advanced method for your own hardware, described below. Also note that these experiments will only work with a single BLE device as you first scan for one device and pick matching experiments afterwards.
The other method works just like opening any other custom eperiment in phyphox. You transfer your file to the phone (as a .phyphox-file, in a zip file or through a QR code) or pick an experiment from the list on the main screen (after you have chosen to add the transfered file or an included BLE experiment to the collection). The experiment is opened in phyphox and it will try to connect to all devices required for the experiment, which in many cases is a single device but you could also use many different devices in the same experiment.
BLE as input or output
BLE devices can be either used as an input to phyphox or as an output to receive data acquired by phyphox.
When used as an input, the characteristics are read from the device, their data will be interpreted and then written to a phyphox buffer. When used as an output, the values from a phyphox buffer are converted to a binary representation and then written to a characteristic on the device.
Notifications and polling
Most BLE devices support notifications, which means that phyphox can subscribe to a characteristic and gets notified if the device offers a new value. This is the most efficient way to retrieve data and should be used when using a BLE device as input (it does not apply to writing to the device).
However, when the device does not support notifications or if you want values at a fixed rate (i.e. not having "gaps" if there is no new value for a long time or not collecting many data points if only few are sufficient), the mode of the input device can be switched to "poll". In this case you also need to define a rate at which values are being read from the device.
Phyphox also supports indications, which are similar to notifications, but most devices typically do not support them for simple data values.
Accessing characteristics as input, output or config
When a BLE device is used as input, it needs to define its outputs to the phyphox buffers. Therefore, a BLE device that is an input to phyphox, sets up buffers to which it outputs its data. Each of these output buffers needs to be associated with a characteristic and multiple output buffers may be associated with the same characteristic. Each time the characteristic is read (through polling or because of a notification), the same value is fed to all buffers, which then interpret the data differently according to a conversion function which needs to be set as well (the conversion functions are listed below). Many conversion functions offer to set an offset and/or a size, so they only interpret a part of the incoming data. This way different values within a single characteristic can be split to different buffers.
Output devices work similar, but instead of defining output buffers, they define input buffers, which also use slightly different conversion functions.
In case of an output buffer (for an input device) the conversion function can be left out if instead the "extra" field is set to "time", in which case the output buffer does not recieve values from the characteristic, but the time (in seconds since the start of the experiment) at which it has been read.
Both, input and output devices, can also define config settings for a characteristic. These do not associate the characteristic with a buffer but with a constant value. This value will be written to the device once after a connection has been established. This is usually used for configuration like enabling a sensor or setting a data acquisition rate.
BLE-related syntax in phyphox-files
BLE device matching
Whenever a BLE device is defined in phyphox, you will need to set up how it is matched against scan results. You can define a text that is compared to the name of the device and/or the UUID of an advertised service.
<bluetooth name="..." uuid="..." id="..." autoConnect="..." mtu="...">
</bluetooth>
If this name is contained in the name of a device found while scanning, the device matches the criterium.
This is the UUID of a service advertised by the device. Note that even if the device advertises a short 16-bit UUID according to the Bluetooth specification (for example 0x1812 for a Human Interface Device service), you need to specify the 128-bit version (in this case 00001812-0000-1000-8000-00805f9b34fb) using the base-UUID 0000xxxx-0000-1000-8000-00805f9b34fb.
The id entry combines two functions to manage multiple BLE devices in one experiment. It acts as a string shown to the user to identify the use of a device during scanning (for example, if two sensor boxes are used, the could be labeled as "wheel" and "distance" if one is attached to a wheel and the other one measures distance). For this purpose the text is translatable. The second function is that phyphox treats devices with the same ID as a single device, so multiple bluetooth entries (even input and output blocks) can refer to the same device without asking the user twice to pick it from a scan.
autoConnect
If set to true (default false), phyphox will not show a scan dialog, but instead tries to connect to the first devices that matches the other criteria. You should only use this, if you can be sure that only a single matching device will be present, typically when you developed your own unique device.
Can be set to request a specific MTU size. This is ignored on iOS which has no method to control the MTU size and always requests the maximum. Note that this will always fail on all Android devices before Android 5.0. Available since phyphox file format 1.9 (phyphox 1.1.5)
BLE as input
If data should be read from a BLE device and displayed in phyphox, the device should be defined within the input block of the phyphox file:
<bluetooth name="..." uuid="..." id="..." autoConnect="..." mode="..." rate="..." subscribeOnStart="...">
<config ...>...</config>
<output ...>...</output>
</input>
When using the "bluetooth" tag within an "input" tag, values are read from the device and written to buffers specified in output tags (more on these below). Additionally, "config" tags can be used to set up the device by writing configuration values to the device after connecting.
In addition to the device matching tags "name" and "uuid" (see above), there are tags to define how and when data is read from the device:
Mode can have on of three values:
phyphox requests new values at a rate given by the attribute "rate".
New values are received when the device notifies that there is a new value. The characteristic needs to support notifications. For most devices, this mode should give the best results.
New values are received when the device indicates that there is a new value. The characteristic needs to support indication, which is very uncommon for simple values. In most cases you probably want to use notifications instead.
This attribute is only used when the mode is set to "poll" and gives the rate in Hz at which new values are polled.
subscribeOnStart
Usually, phyphox subscribes to notifications as soon as a connection to the device has been established. This minimizes latencies when starting the measurement. However, some devices should react to a subscription by starting to send buffered data or triggering something in the experiment, so if this attribute is set to true, phyphox will subscribe to notifications only when the measurement is started. true or false, default: false
BLE as output
If data should be transmitted to a BLE device (i.e. a characteristic), the device should be defined within the output block of the phyphox file:
<bluetooth name="..." uuid="..." id="..." autoConnect="...">
<input ...>...</input>
</output>
In contrast to using the tag "bluetooth" within an input tag, you cannot specify a mode or a poll rate. The values are read from buffers defined in input tags and written to the device when the analysis process of the experiment has finished. Config tags work as usual.
config, input and output block
There are three different tags within a bluetooth block:
The config tag writes a configuration value to the device after the connection has been established. This is usually used to enable a specific sensor, set a data acquisition rate or set a measurement mode. The config tag always takes a constant value.
An input tag can only be used in an output block. The name "output" of the outer block refers to phyphox as it outputs data from phyphox, while the name "input" of the inner block refers to the BLE device, as it is the input to the device. This defines a buffer from which data is written to the device.
Output tags can only be used in an input block. The name "input" of the outer block refers to phyphox as it represents a data input for phyphox, while the name "output" of the inner block refers to the BLE device, as it is the output of the device. This defines a buffer to which data is written from the device.
All three types have to be connected to the UUID of a characteristic on the BLE device through its UUID. This is defined through the "char" attribute. On top of this, a range of conversion functions are available, which need to be defined through the attribute "conversion". These are different for input, output and config tags and may imply additional attributes. They are discussed separately below.
This sets the a HID mouse to BIOS mode by writing 0x00 to the characteristic 0x2a4e after connecting to the device:
<config char="00002A4e-0000-1000-8000-00805F9B34FB" conversion="hexadecimal">00</config>
These two outputs read from the same characteristic but use different conversion functions (or rather different parameters to the same function) to get x and y movement of the mouse and write it to the buffers dx and dy:
<output char="00002A33-0000-1000-8000-00805F9B34FB" offset="1" length="1" conversion="singleByte">dx</output>
<output char="00002A33-0000-1000-8000-00805F9B34FB" offset="2" length="1" conversion="singleByte">dy</output>
The following line defines buffer "char" as an input, so its values are written to the LED character output of a BBC Micro:bit.
<input char="E95D93EE-251D-470A-A062-FA1922DFA9A8" conversion="singleByte">char</input>
config conversion functions
Config tags take a constant string and convert it to a binary value to be written to a characteristic. Currently, the following conversion functions are defined:
The string is interpreted as a string and written directly to the characteristic
The string is a hexadecimal representation of the binary value that should be written to the characteristic. (Note that there is no trailing "0x", so 0x1f is simply written as 1f.)
singleByte
The string is parsed as a decimal number and encoded as a single byte value (same as uInt8)
The string is parsed as a decimal number and encoded as a signed 8bit integer
The string is parsed as a decimal number and encoded as an unsigned 8bit integer
int16LittleEndian
The string is parsed as a decimal number and encoded as a signed 16bit integer with little endian byte order (that is starting with the LSB)
uInt16LittleEndian
The string is parsed as a decimal number and encoded as an unsigned 16bit integer with little endian byte order (that is starting with the LSB)
int16BigEndian
The string is parsed as a decimal number and encoded as a signed 16bit integer with big endian byte order (that is starting with the MSB)
uInt16BigEndian
The string is parsed as a decimal number and encoded as an unsigned 16bit integer with big endian byte order (that is starting with the MSB)
float32LittleEndian
The string is parsed as a decimal number and encoded as a 32bit floating point number with little endian byte order (that is starting with the LSB)
float32BigEndian
The string is parsed as a decimal number and encoded as a 32bit floating point number with big endian byte order (that is starting with the MSB)
output conversion functions
The input tags in an output block take a number from a phyphox buffer and convert it to a binary value to be written to a characteristic. Some functions can also take the whole content of the buffer instead of a single value. Currently, the following conversion functions are defined:
The value from the buffer is converted to a string representation of a decimal number
byteArray
All values from the buffer are taken, individually converted to bytes and then written as sequence of bytes to the characteristic
The value from the buffer is converted to a decimal number and encoded as a single byte value (same as uInt8)
The value from the buffer is converted to a decimal number and encoded as a signed 8bit integer
The value from the buffer is converted to a decimal number and encoded as an unsigned 8bit integer
The value from the buffer is converted to a decimal number and encoded as a signed 16bit integer with little endian byte order (that is starting with the LSB)
The value from the buffer is converted to a decimal number and encoded as an unsigned 16bit integer with little endian byte order (that is starting with the LSB)
The value from the buffer is converted to a decimal number and encoded as a signed 16bit integer with big endian byte order (that is starting with the MSB)
The value from the buffer is converted to a decimal number and encoded as an unsigned 16bit integer with big endian byte order (that is starting with the MSB)
The value from the buffer is converted to a decimal number and encoded as a 32bit floating point number with little endian byte order (that is starting with the LSB)
The value from the buffer is converted to a decimal number and encoded as a 32bit floating point number with big endian byte order (that is starting with the MSB)
input conversion functions
The output tags in an input block take a byte sequence from the characteristic and convert it to a number to be written to a phyphox buffer. Most conversion functions (unless noted otherwise) can use the attributes "offset" and "size" to define a subsequence of bytes. The start is defined by "offset" (in bytes, starting at zero) and the length is set by "size" (in bytes).
Instead of defining a conversion function, you can also set the attribute "extra" to "time" (extra="time"). This means that the associated buffer does not receive the value from the characteristic, but the time at which the characteristic has been read in seconds since the beginning of the experiment.
Currently, the following conversion functions are defined:
The value from the characteristic parsed as a decimal number
formattedString
This function always parses the whole data sequence from the characteristic as a single string (i.e. there is no "offset" or "size" attribute) and tries to split it into several values at the sequence set by the "separator" attribute. Each output tag can then specify which chunk corresponds to the buffer by either setting the "index" attribute, which gives the n-th entry (starting at zero), or by setting the "label" attribute, which is another character sequence which prepends the entry. Each entry is then parsed as a decimal number (labels are stripped before that happens).
A simple comma-separated value string
An output tag with separator=";" and index="0" returns 42 and a second output tag with separator=";" and index="1" returns 23.
A string with simple labels:
U42;I23
An output tag with separator=";" and label="U" returns 42, an output tag with separator=";" and label="I" returns 23.
A string from the characteristic with a line break and labels:
U = 42
I = 23
An output tag with separator="\n" and label="U = " returns 42 and an output tag with separator="\n" and label="I = " returns 23.
The data from the characteristic is interpreted as a single byte value (same as uInt8)
The data from the characteristic is interpreted as a signed 8bit integer
The data from the characteristic is interpreted as an unsigned 8bit integer
The data from the characteristic is interpreted as a signed 16bit integer with little endian byte order (that is starting with the LSB)
The data from the characteristic is interpreted as an unsigned 16bit integer with little endian byte order (that is starting with the LSB)
The data from the characteristic is interpreted as a signed 16bit integer with big endian byte order (that is starting with the MSB)
The data from the characteristic is interpreted as an unsigned 16bit integer with big endian byte order (that is starting with the MSB)
The data from the characteristic is interpreted as a 32bit floating point number with little endian byte order (that is starting with the LSB)
The data from the characteristic is interpreted as a 32bit floating point number with big endian byte order (that is starting with the MSB)
Sending phyphox-files from a device
If you create your own device (for example an Arduino-based sensor with a BLE module), you can also store experiments on the device and send them to phyphox.
To do so, your device needs to advertise and implement the phyphox service, which uses the UUID cddf0001-30f7-4671-8b43-5e40ba53514a. When the user picks the Bluetooth scan from the "+" button on the main screen, phyphox will list devices that advertise this UUID in white like any other device for which it has an internal phyphox-file.
When connecting to this device, phyphox will look for a characteristic with the UUID cddf0002-30f7-4671-8b43-5e40ba53514a and subscribe to its notifications. Your device needs to react to this subscription by starting to send the experiment file (preferably in a zip file) through this characteristic.
Sending the experiment file starts with a header. So the first package you send needs to consist of (at least 15 bytes):
phyphox[size][crc32]
The first seven bytes need to contain the keyword "phyphox", followed by four bytes for an unsigned 32-bit big endian integer giving the size of the experiment file (not including this header) and the last four bytes must give the CRC32 check value of the whole experiment file (again, not including this header). Remaining bytes in the first message are ignored and can be set to zero.
The experiment is transmitted sequentially in subsequent messages. If there is space left in the final message, it can simply be filled with zeros as phyphox ignores any bytes after the size set in the header has been reached.
Retrieved from "http://phyphox.org/wiki/index.php?title=Bluetooth_Low_Energy&oldid=20810"


BIA office will send out an email outlining everything to happen in the next few weeks so businesses are prepared.
Pay By Phone meter Parking coming to Hamilton in 2018 – meters will remain but choice to pay for meter will be added sometime this calendar year by the City of Hamilton. Ability to top up meters will be capped to safeguard against people "feeding the meters" all day.
Not being used. Dirty, eye sore, Businesses between E21st and E22nd have specifically requested to have it removed (initiated by Speedy Subs). Strategy is to move the waste receptacle in front of 560 Concession closer to the corner of E22nd when the phone booth is removed. This will add to accessibility to the bus stop – which has proven to be problematic in the winter months with snow banks & dodging the garbage can when riders are getting on/off the bus (particularly strollers and elderly passengers).
Busy time with cheques for Streetfest and Sidewalk Sounds. Full report to come in June.
Concerns expressed with conditions of sidewalks (chewing gun, stains, deterioration from salt on parts of the concrete. Street renovations not proving to show longevity in looking nice.
Visit Hamilton ad is well placed but we were missed again this year in the Festivals & Events section of the magazine. We have been promised a 200-word write up in the A&E page of the Summer Issue and increased promotion of our events through their social media. No discount was offered on the cost of our page ad 2018.
5 music performers confirmed – headliner: Born in the Eighties. Chalk artist to work on a piece in the parking lot of 555 Concession (will wash away with the next rainfall). Vendors all contacted and advised that spaces are "first come first serve" from an approved list of addresses along Concession. Hamilton Bellydance will be performing in the parking lot of 555 Concession. KLITE will bring their cruiser. Y108 will bring their cruiser. All businesses encouraged to stay open, participate or at least ramp up their window displays to promote their business to the visitors that evening.
Easy Lemon, Scott McIntosh Trio, PIP and Sharon Musgrave scheduled to perform under the pergola at CAP. Vendor spaces being offered at no cost. Momentum Fitness is doing a stair climb at 1pm. Scavenger Tour @2pm to start at BIA office. Gift baskets will be promoted to participants of the scavenger hunt. After party for all BIA members from 5-7pm at The Zoetic to show montage of Try!Concession Street Event, DJ Disc and food/drink. All members welcome to come out for this Wrap Up Party.
100 In One Day event: Dog Parade to celebrate our pets & promote responsible dog ownership with our community Suggestion to engage Deanne and Carlin to take the lead on this initiative.
Officially sold out of vendors spaces and entertainment. Rest/shade stations to be set up along Concession. Vendors will be placed along the center of the Street so ALL business owners will have access to their storefront/sidewalk. Festival to run 10am to 8pm. Final preparations will be happening over the next few weeks.
Tendered and selected REPRODUX will craft alupanels that will be attached to corner property walls. Image (to be selected in conjunction with the property owners) will be digitally printed onto a grouping of 4x8 or 5x10 panels – a grouping of 4 panels – that will showcase photography and vintage pictures of Concession Street. EG: We would like to do a 10x20 foot mural of the incline railway on the eastbound wall of 555 Concession. The Mountain Heritage Society will be sponsoring a portion of the cost of this mural ($500) – the BIA budget will cover the remaining costs. Approximate cost of mural and its installation $2000/each. Funding to come from Beautification budget. Although these installations will look beautiful they are also intended to act as deterrents to graffiti and tagging.
The remaining walls (in addition to any other property owners who come forward with interest in this project) will be considered for 2019 PHASE 2.
Pop Up Patios – BIA gaming patio at 565 Concession (Library) - very well received. Collaboration of the library appreciated to manage the signing out of the board pieces -or people can bring their own.
Lura is partnering with 100 In One Day and conducting a workshop at Mohawk College on Tuesday, May 22 from 6:30-8pm – everyone welcome to attend.
Chalmers Presbyterian Church is now vacated. Unclear on the future of this property.