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University of St. Thomas is a Comprehensive institution located in Houston, TX, in an urban setting.


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Q: How can I preserve the form values when I use MVC with Knockout I'm developing (for the first time) an MVC application using ajax call and knockout MVVM to fill 2 cascading dropdown lists.
The question is, How can I preserve the values I selected in the dropdown lists, when I post the web page?
The web page I'm developing makes calculations when is posted to the controller and the controller must return the calculated result. When the result is returned, the values of the form must be preserved.
a part of the View
    @using (Html.BeginForm("Calculate", "Entypo", FormMethod.Post, new { role = "form" }))
{
     @Html.AntiForgeryToken()
    @Html.ValidationSummary(false)

    <fieldset>
        <legend>Printers</legend>

                <select id="printers" name="printers" class="form-control" data-bind="options: printers, optionsValue: 'ID', optionsText: 'BrandModelName', value: selectedPrinter, optionsCaption: 'Choose Printer...'"></select>

                <select id="sheets" name="sheets" class="form-control" data-bind="options: sheets, optionsValue: 'ID', optionsText: 'Description', optionsCaption: 'Choose Sheet...', enable: sheets().length, value: sheet">
                </select>

    </fieldset>

                <input type="submit" value="Calculate" class="btn btn-primary" />

    }
    @section Scripts {
    @Scripts.Render("~/bundles/jqueryval")

The Controller
using System;
using System.Collections.Generic;
using System.Linq;
using System.Web;
using System.Web.Mvc;
using QVNet_v2.Models;

namespace QVNet_v2.Controllers
{
    public class EntypoController : Controller
    {
        private QVNetEntities db = new QVNetEntities();

        //
        // Calc: /Entypo/
        [HttpGet]
        public ActionResult Calculate()
        {

            return View();
        }

        [HttpPost]
        public ActionResult Calculate()
        {
            return View();
        }

        //GET PRINTERS
        public JsonResult getPrinters()
        {
            var printers = db.Printers.Select(s => new { s.ID, s.BrandModelName }).OrderBy(s=>s.BrandModelName).ToList();
            return Json(printers, JsonRequestBehavior.AllowGet);
        }
        //GET SHEETS USED FROM SELECTED PRINTER
        public JsonResult getSheets(int id)
        {
            var sheets = db.Sheets.Select(s => new { s.ID, s.Description, s.PrinterID }).Where(s=>s.PrinterID ==id).OrderBy(s=>s.Description).ToList();
            return Json(sheets, JsonRequestBehavior.AllowGet);
        }
        // GET PAPERS
        public JsonResult getPapers()
        {
            var papers = db.Papers.Select(s => new { s.ID, s.Description }).OrderBy(s => s.Description).ToList();
            return Json(papers, JsonRequestBehavior.AllowGet);
        }
        // GET WEIGHTS OF SELECTED PAPER
        public JsonResult getWeights(int id)
        {
            var weights = db.PaperWeights.Select(s => new { s.ID, s.Weight, s.PaperID }).Where(s => s.PaperID == id).OrderBy(s => s.Weight).ToList();
            return Json(weights, JsonRequestBehavior.AllowGet);
        }
        //GET SIZES OF SELECTED PAPER AND WEIGHT
        public JsonResult getSizes(int id)
        {
            var sizes = db.PaperSizes.Select(s => new { s.ID, s.Description, s.PaperWeightID }).Where(s => s.PaperWeightID == id).OrderBy(s => s.Description).ToList();
            return Json(sizes, JsonRequestBehavior.AllowGet);
        }
        //GET LAMINATION
        public JsonResult getLamination()
        {
            var lam = db.SheetLaminations.Select(s => new { s.ID, s.Description }).OrderBy(s => s.Description).ToList();
            return Json(lam, JsonRequestBehavior.AllowGet);
        }
        //Dispose db
        protected override void Dispose(bool disposing)
        {
            if (disposing)
            {
                db.Dispose();
            }
            base.Dispose(disposing);
        }
    }
}

When the user click the submit value, the controller must take the data from the form and return the results of a calculation while keeping intact the form fields.
Sorry for my English.

A: You have two possible ways.

*

*Submit your form using ajax. You will keep your form
open thus all values will be unchanged. You can use @using
(Ajax.BeginForm) or create knockout method that will call $.ajax to save your data and
assing this method to submit button.

*Second approach - make your form strongly typed. Create viewmodel
    class that will hold all values needed to construct the form. In
    your saving action (Calculate method annotated with [HttpPost])
    you shoud recreate viewmodel based on actual form values and send it
    back to the view.

redpajama_set_name: RedPajamaStackExchange


TOLEDO, OH (WTOL) - Slipping and falling on snow or ice covered sidewalks is something nobody wants to happen.
Have the sidewalks in your neck of the woods been cleared of snow? Are they supposed to be walk-able after winter storms?
For some residents, having clear paths is vital.
Whether you own a residential or a commercial property in the city of Toledo, the rule is, you have no later than 24 hours after the snow stops falling to clear the sidewalk in front of your property.
Those students who live within walking distance of schools, people waiting at bus stops and handicapped residents, just to name a few, rely on having safe walking paths where they do not have to risk falling.
South Toledo resident Brian Yarger is handicapped, as a result of a serious fall years ago, and takes issue with the icy sidewalks.
"Right here, the last time it snowed, the dog and I, my service dog and I actually slipped and fell, and that was the second time that I had called the city, and they were just changing over to the new Mayor then, and the new Mayor's been great, and his team's been great," Yarger said.
Yarger has been trekking through non shoveled sidewalks to get to the bus stop and also walk his service dog.
"There's traffic, foot traffic on these sidewalks through here, having to walk and slipping around through these commercial sidewalks and residential sidewalks not being cleared," Yarger explained.
In cases where the snow is not cleared within a reasonable amount of time, the city can issue fines starting at $75. For those unable to do so, the Snow Angels program through the YMCA may be able to connect you with a neighbor who is willing to help.
"We do have dozens of notices that have gone out so far and we continue to send them out. Actually, today we sent out a few because we received complaints through our Engage Toledo call center," Dennis Kennedy, Commissioner of the City of Toledo's Division of Code Enforcement said Tuesday.
Legal notice will be provided to the property owner, not the current tenant, through the mail if they are not in compliance with the law. Photo proof is also taken at the time the notice is issued.
If you would like to report a trouble spot, like Yarger has, you can contact Engage Toledo at 419-936-2020.


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Readers ask: How Many Greek Mythology Gods Are There?
1 How many Greek gods are there in total?
2 Who are the 14 Olympian gods?
3 What are the 14 Greek gods?
4 Which Greek gods died?
5 Who was the most handsome god?
6 Who is the oldest Olympian god?
7 Is Percy Jackson a God?
8 Who are the 6 Olympian gods?
9 Who are the 13 Olympians?
10 Who is the strongest Greek god?
11 Who is the most famous Greek god goddess?
12 What are the 3 most powerful Greek gods?
13 Why did Zeus eat his wife?
14 Who is God of Death?
15 Who killed Zeus?
How many Greek gods are there in total?
In terms of gods, the Greek pantheon consists of 12 deities who were said to reside at Mount Olympus: Zeus, Hera, Aphrodite, Apollo, Ares, Artemis, Athena, Demeter, Dionysus, Hephaestus, Hermes, and Poseidon. (This list sometimes also includes Hades or Hestia).
Who are the 14 Olympian gods?
The Olympians are the council of principal Greek and Roman deities, consisting of Zeus/Jupiter, Poseidon/Neptune, Hera/Juno, Athena/Minerva, Ares/Mars, Apollo, Artemis/Diana, Demeter/Ceres, Hephaestus/Vulcan, Aphrodite/Venus, Hermes/Mercury, and Dionysus/Bacchus or Hestia/Vesta.
What are the 14 Greek gods?
The Olympian Gods and Goddesses
Hera (Roman name: Juno) Hera was goddess of marriage and the queen of Olympus.
Poseidon (Roman name: Neptune) Poseidon was god of the sea.
Hades (Roman name: Pluto)
Aphrodite (Roman name: Venus)
Ares (Roman name: Mars)
Artemis (Roman name: Diana)
Hephaestus (Roman name: Vulcan)
Which Greek gods died?
In Greek mythology there are only a few Gods who are actually said to be dead. There is Pan, the god of the wild who dies because people forget him and stop worshipping him but this whole story is shrouded in mystery.
You might be interested: How To Get The Pomegranate In Mythology Island?
Who was the most handsome god?
Apollo was considered to be the most handsome of all the gods. He was always depicted as having long, golden hair – the same color as the sun.
Who is the oldest Olympian god?
Hestia was the first born child of the Titans Cronus (Kronos) and Rhea, making her the oldest Greek God.
Is Percy Jackson a God?
Character. In the story, Percy Jackson is portrayed as a demigod, the son of the mortal Sally Jackson and the Greek god Poseidon. He serves as the first head counselor of the Poseidon cabin at his demigod summer camp – Camp Half-Blood.
Who are the 6 Olympian gods?
In ancient Greek religion and mythology, the twelve Olympians are the major deities of the Greek pantheon, commonly considered to be Zeus, Hera, Poseidon, Demeter, Athena, Apollo, Artemis, Ares, Hephaestus, Aphrodite, Hermes, and either Hestia or Dionysus.
Who are the 13 Olympians?
The Olympians were twelve and comprised of Zeus, Hera, Poseidon, Demeter, Athena, Apollo, Artemis, Ares, Aphrodite, Hephaestus, Hermes and either Hestia or Dionysus.
Who is the strongest Greek god?
Zeus was the Greek god that both deities and man would call upon for help. Zeus would help the other gods, goddesses, and mortals if they needed help, but would also invoke his wrath upon them if he felt they weren't worthy of his help. This made Zeus the strongest Greek god in Greek mythology.
Who is the most famous Greek god goddess?
Top 10 Ancient Greek Goddesses
Nemesis.
Leto.
Rhea.
Aphrodite.
Demeter.
Artemis.
Hera. Also known as the goddess of marriage and birth, Hera was the wife of Zeus and by extension, also the queen of all gods.
Athena. At the top of the list comes the goddess of wisdom, reasoning, and intelligence – Athena.
You might be interested: Readers ask: How Much Can You Get Paid For Being An Expert On Mythology?
What are the 3 most powerful Greek gods?
Zeus. The most powerful of all, Zeus was god of the sky and the king of Mount Olympus.
Hera. Hera was goddess of marriage and the queen of Olympus.
Aphrodite. Aphrodite was the goddess of love and beauty, and the protector of sailors.
Athena.
Hephaestus.
Hestia.
Hermes.
Why did Zeus eat his wife?
Why did Zeus eat his wife? In some versions of Greek mythology, Zeus ate his wife Metis because it was known that their second child would be more powerful than him. After Metis's demise, their first child Athena was born when Hephaestus cleaved Zeus's head open and the goddess of war emerged, fully grown and armed.
Who is God of Death?
Osiris. Osiris is the god of death and the underworld but he is also regarded as the god of transition, regeneration, and resurrection. And while he is the god of death according to Egyptian mythology, he is often described as the Lord of Love in ancient times.
Who killed Zeus?
The most widely known example is probably the God of War series. The protagonist Kratos main motivation is to kill Zeus. In The episode of Xena Warrior Princess "God Fearing Child" Hercules kills Zeus when the King of Olympus tries to kill Xena's daughter.
Often asked: How Do I Play Age Of Mythology With Windows 10?
Quick Answer: What Does A Salamander Symbolize In Mythology?
Question: Painters Who Did Mythology?

redpajama_set_name: RedPajamaCommonCrawl


José Victoriano Carmelo Carlos González-Pérez (1887-03-23 - 1927-05-11), better known as Juan Gris, was a Spanish painter who lived and worked in France almost all of his life. His works are closely connected to the emergence of Cubism.
Cézanne made a cylinder out of a bottle. I start from the cylinder to create a special kind of individual object. I make a bottle a particular bottle out of a cylinder.
Painting for me is like a fabric, all of a piece and uniform, with one set of threads as the representational, esthetic element, and the cross-threads as the technical, architectural, or abstract element. These threads are interdependent and complementary, and if one set is lacking the fabric does not exist. A picture with no representational purpose is to my mind always an incomplete technical exercise, for the only purpose of any picture is to achieve representation.


We've heard this being said quite a few times about God, right?
"I tell from the beginning what will happen in the end.
There's a song called 'Made a Way', by a man named Travis Greene.
He sings, 'Nothing can catch You by Surprise', and when he sings this, he's singing it unto God.
Now how many of us truly believe that God is an All-knowing and Intentional God?
There isn't a thing that happens to us that God doesn't know about, neither is there a thing that He 'accidentally missed out on', or 'let slip past Him', about us, or the circumstances of our lives.
So in that case I guess, yes, we can all agree that He knows everything, but I want to draw our attention particularly to the fact that I also said He's Intentional.
And He surely knows how we're going to evolve from it!
Therefore whatever stage we find ourselves at in life, what should we do?
Friend, Let us yield to the Will of God.
Having confidence in a God who perfectly wrote out your life before He even made you to live in it, you should trust Him and just enjoy the journey of fulfilling His purpose for you.
Though it can be daunting to consider the fact that God can do ABSOLUTELY whatever He wants with us, we can be comforted by the fact that He isn't an abusive God, neither is He a bully (Jeremiah 29:11 says He has GOOD intentions for us).
So in entrusting Him with our lives, and living for Him, we can be confident that regardless of how deep and dark situations may get, He will always make a way, and we will surely see His perfect hand in everything.


text:
Editorial: Admin must let leadership lead
American public high schools are viewed in an iconic light: huge Friday night football games, lively dances packed with sweaty and shouting teenagers, exciting pep rallies and other spirited activities are seen as universal experiences for teens. However, Redwood strays far away from these idealizations and has become a drab and lackluster environment for many students. While it has the potential to be a tight-knit and vibrant community, the shortage of all-inclusive student events has yielded a decline in school spirit. This issue is perpetuated by the administration's frequent, unwanted and restrictive infringements on class activities proposed by leadership. Administrators must allow leadership to have more freedom and control to increase school spirit and unify students after almost a year of distance learning.
According to junior class president Gavin Green, many ideas that leadership presented to the administration to improve Redwood's overall spirit for the last few weeks of the school year have been rejected.
"Having events that really bring a community together [get] shut down is the worst thing for spirit because it makes people not want to participate in [the future]," Green said.
Although students recognize the limitations imposed by COVID-19, it feels as though Redwood is obligated to follow stricter protocols than other high schools in the district. For example, on April 30, Tamalpais High School (Tam) leadership hosted a "Tam Unity Day" where food trucks came to campus, and World Wrapps, Jamba Juice, boba tea and donuts were served as students listened to live music and made arts and crafts, all while following COVID-19 guidelines. Tam students unified once again on May 14 when Tam leadership held a similar "field day" event. These occasions were valuable to the Tam community and contributed to a sense of togetherness, a feeling currently lacking at Redwood. Like Redwood, there was initially some pushback from the admin regarding interference with cafeteria profits, according to Tam Associated Student Body (ASB) President Solomon Tolson, but unlike Redwood, a Tam leadership committee worked with the administration and cafeteria staff to strike a deal and go forward with food-related events.
Redwood's leadership class should have been able to do the same, but all propositions were denied until recently when leadership was approved to vend boba tea and burritos at lunch for senior week. Still, this food will only be available for seniors and feels a day late and a dollar short when Tam students have been holding similar occasions for months.
A study from the Connecticut Association of Schools proves why bonding events like those held at Tam are so important: school spirit is vital to a healthy learning environment. The study surveyed principals from 150 high schools across the United States who all reported that those who have a "great deal" of spirit are 91 percent more confident, 87 percent more active in their communities and 88 percent happier than schools with lower levels of spirit.
Another instance of our administration disproportionately controlling leadership events was during this year's homecoming week. According to Green, the winners of the homecoming court had to be crowned over Zoom to limit crowds despite the ceremony being outside in a large enough space for social distancing.
"[The video] was really blurry and people [didn't] even know who won homecoming court. It's just little things like that that were limited this year and in the past that have made our job harder," Green said.
Even in pre-pandemic years, the administration has made irrational vetoes to leadership initiatives. Last year, Josh Chan, former Redwood ASB president, dedicated a significant amount of time to working with World Wrapps to bring their business to the CEA year-round. After an exciting week of nori wraps and bowls, the administration shut the project down due to its violation of California Education Code (EDC) 45103.1, which requires food from outside vendors to meet very specific criteria. Chan repeatedly advertised the project and worked with the owner of World Wrapps, Matt Blair, for over a year to revise the menu to meet requirements, but no one in the school district communicated with leadership prior to the administration's unreasonable decision to cancel the project.
This all begs the question: why does Redwood fund and run a leadership program if student representatives are not allowed to lead? The purpose of leadership and ASB is to unite the student body and therefore heighten school spirits. Students ask that the administration works with leadership students to develop sensible and equitable solutions to their concerns about proposed ideas, such as how it is done at Tam. As the 2020-2021 school year draws to a close and COVID-19 restrictions lighten, the incoming leadership class and elected officers should be given the role in which they were elected to serve.
josh chan
Solomon Tolson
World Wrapps
Editorial Staff, Author
Drive for change: Be the influence instead of driving under it
Addressing misgendering within TUHSD: learning to become LGBTQIA+ allies
Modernizing Patriotism
Cub Editorial; Hate crimes won't stop with an 'End Racism' poster
Editorial: Don't let your mothers, sisters, wives and daughters be a part of the 97 percent
Editorial: Dear College Board: we need modified tests for modified learning
Cultivating engaged learning
Editorial: Vote 'yes' on Measure M to ensure the success of the TUHSD
Editorial: The district implemented a new distance learning plan; here's what still needs to be fixed
Editorial: An open letter to Tara Taupier and the Board of Trustees


The TSHA Executive Board meets annually to establish, review, and evaluate TSHA's long-range goals. The desired outcome for the meeting is to define a direction for the Association that is clear, concise, measurable, doable, and concentrated/focused on just a few priorities. This document provides a summary of the goals, objectives, and key activities adopted by the TSHA Executive Board to achieve TSHA's mission: "Empowering members to provide the highest quality, life-changing care for individuals with communication and related disorders." .


Tonight's Human Services Commission meeting is the first one where I designed the agenda (privileges of being chair). The agenda is structured around our ad hoc committees, summarized above.* We're talking about the Women's Bill of Rights in San José, ending domestic violence, protecting the rights of people with disabilities, reporting on the needs of youth with disabilities who are justice-involved, protecting environmental sustainability rights, and protecting immigrants' rights. It's a big agenda, but we're going to fly through it and if you're interested in why, if you get excited by discussions of how to structure meetings and civic committee work, check-out the #Metas at the bottom of this post.
Right now our consent calendar includes approving the minutes from the last meeting. But! The minutes haven't been posted anywhere. So, I'll ask to remove it from consent and vote against it, since, you know, I can't vote for something I didn't read.
In my Letter of Intent to run for chair, I highlighted 3 structural changes I would make as chair; one of them was sending a monthly letter to council updating them on our work, since I have have heard from staff, city council members, and my fellow commissioners that they don't often see the recommendations we send. That sort-of defeats the purpose of having volunteer commissioners advising City Council on human rights policy, but rather than drag-on about the communication issues between the City Manager, council staff, commissions, and council members, I figured sending a one-page monthly update would be a nice mix of persistent and helpful.
Ensuring there is an independent gender-based analysis of city departments, programs, and policies.
In June of 2017, the Santa Clara County Blue-Ribbon Task Force produced a report on ending domestic violence in our county. Since 1 in 2 Santa Clara County residents is a San José resident, San José has an important role to play here. Our newest commissioner, Chris Demers, is chairing this important ad hoc, bringing experience in DC and internationally doing human rights work. The ad hoc will be producing a report in January on how the city is doing with implementing the recommendations of the report. I volunteered to help with this work and I'm excited to hear what feedback the other commissioners have for it.
Thoughts: This trick with this ad hoc will be getting stakeholder meetings; thankfully, I'm medium-good at getting people to sit-down for meetings, so I will get to use my scheduler-powers for good as a member of this committee.
Here is the workplan for this ad hoc focusing on physical accessibility of city events and spaces. That is one of the issues I mentioned in my first application to be on the Human Services Commission, so I am thrilled it is the focus of this team's good work.
Thoughts: Visual evidence might be very valuable for this ad hoc — I wonder if there are some photographers out there interested in making this into an accessibility series like on r/HostileArchitecture.
A workplan wasn't submitted for this ad hoc, so I am excited to hear how they are narrowing their scope and finding just the right angle to make some substantive community change here.
Thoughts: This is the topic I know the least about and I am pretty stoked to learn more and see how I can help.
I threw a report-writing kick-off dinner for this ad hoc, bringing together teachers, social workers, youth with disabilities, and parents over a dinner of salmon teriyaki and stir-fry, trying to dig into what kinds of needs each had seen in their communities and which kinds of interventions might be best. Here's the workplan.
Thoughts: This is a huge topic impacting vulnerable young people, so a lot of care and compassion will need to go into the work.
This is my second ad hoc (here's the workplan) and it involves writing a report on the last 9 months of testimony we've heard from staff, community members, service providers and lawyers on how Immigration and Customs Enforcement (ICE)'s behavior is impacting our community. Two of us commissioners are also trained Rapid Responders and last month I attended a federal district court hearing in support of 10 men who ICE had moved in the dead-of-night from their detention facility in the East Bay as far away as Aurora, CO and Tacoma, WA. That case did not go the way we had hoped, but there is nothing to do but keep fighting.
Thoughts: I'm excited to learn from Commissioner Demers on this committee topic and to organize and present some of the powerful stories of ICE's impact on our community to city council.
The outgoing chair, commissioner Thomas Estrada, and I prepared this summary of the commissioner's work in 2017-2018. A lot of good work got done and a lot of good work remains to be done.
*These are draft ad hoc committees pending council approval, but we'll move forward with them until we hear back.
**Our commission secretary, a member the San José City Manager Dave Sykes's staff, changed the agenda to remove an item after I approved it, after some discussion with the Clerk's office, and added an extra 10 minutes to my ad hoc's time. I'm going to be removing it first thing in the meeting for fairness's sake. As we work through the agenda-setting process, I'm hoping we'll smooth out these bumps.


text:
module Collective
  module Contract
    VERSION = "0.0.1"
  end
end

redpajama_set_name: RedPajamaGithub


text:
Suspects Wanted In Young Dolph Murder Captured
Autumn Hawkins	January 12th
ATLANTA, GEORGIA - OCTOBER 05: Young Dolph attends the BET Hip Hop Awards 2019 at Cobb Energy Center on October 05, 2019 in Atlanta, Georgia.
The suspects wanted for the murder of rap star Young Dolph have been captured, according to U.S. Marshals, per Memphis news station WREG. According to the press release per the local news outlet, Justin Johnson, 23, was captured around 3:00 p.m. in Indiana on Tuesday (Jan. 11).
The second suspect, Cornelius Smith, 32, was indicted on several charges including first-degree murder, according to District Attorney General Amy P Wierich. Smith was also charged with attempted murder involving Young Dolph's brother who was with him at the time of the shooting per the press release.
Smith was in DeSoto County Jail in Hernando, Mississippi for a previous arrest and was later transferred to Shelby County Jail on Tuesday (Jan. 11). Police say Smith was arrested in Southaven, Mississippi for an auto-theft warrant, on Dec. 9, which involved the white vehicle that was believed to be the getaway car in Dolph's murder.
The white vehicle was stolen on Nov. 10 at a gas station on Kirby Road the release states. Smith is currently being held without bond.
The capture of the suspects comes days after Johnson went on social media informing his followers that he would turn himself in and declare his innocence.
Dolph's label, Paper Route Empire, recently announced that they will be putting together a compilation album as a tribute to the late rapper titled Paper Route Empire Presents: Long Live Dolph. The album is expected to drop Jan. 21.
Young Dolph was murdered outside of his hometown local bakery, Makeda's Cookies, in November of 2021.


text:
#pragma once

#include <string.h>

#include "redismodule.h"

#include "consts.h"
#include "utils.h"

/*
 * sessiongate.end <sign_key> <token>
 * Ends a session.
 * Returns OK.
 */
int EndCommand(RedisModuleCtx *ctx, RedisModuleString **argv, int argc);


text:
Ryan Gosling May Star In & Direct A Busby Berkeley Biopic
March 20, 2014 By Tim Isaac Leave a Comment
A few months ago Ryan Gosling was suggesting he was planning to take a break from acting. But he's not stopped lining films up, as THR reports that he is in negotiations to star in and produce a biopic of legendary director and choreographer Busby Berkeley.
There's also a chance it will be his second directorial outing, following the upcoming How To Catch A Monster.
The movie will be based on Jeffrey Spivak's book, 'Buzz: The Life and Art of Busby Berkeley', which tells the story of how the filmmaker revolutionized the musical and changed the world of choreography. He pioneered many conventions still in use today, and became well-know for his ability to use both the camera and dancer to create complex and beautiful formations.
Assuming he sign on, Gosling will work alongside producer with Marc Platt, who has plenty of musical experience, having produced the stage musical Wicked and the upcoming film adaptation of Stephen Sondheim's Into the Woods.
CHECK OUT THESE RELATED ARCHIVES:
ACTORS: Ryan Gosling
Ryan Gosling Shows Off An Impressive Package In A Place Beyond The Pines Deleted Scene
August 3, 2013 By Tim Isaac Leave a Comment
There are many reasons to admire Ryan Gosling, just one of which is his willingness to strip down for a role (for very artistic reasons, of course). Indeed Gosling's fleshly offerings have had more appreciation – especially his Crazy Stupid Love abs – than pretty much any actor in recent years.
We can't understand them when on earth they'd have cut Gosling stripping down in The Place Beyond The Pines and showing off, as many on the web have already pointed out, a very impressive bulge.
Now that deleted scene in question has been released via Total Film and you can watch the whole thing above. Alternatively, take a look at the GIF version of the bit that's gotten people excited below.
The Place Beyond The Pines hits DVD & Blu-ray soon. [Read more…]
ACTORS: Ryan Gosling FILMS: The Place Beyond The Pines
Zac Efron & Ryan Gosling Rumoured For Star Wars Episode VII
July 25, 2013 By Tim Isaac Leave a Comment
We'd take this with a pinch of salt at the moment, as it's merely one of many rumours swirling around the upcoming Star Wars: Episode VII. However, Latino Review is suggesting that Zac Efron and Ryan Gosling are being sought for the film. The site previously suggested Leonardo DiCaprio had come in for a meeting about Star Wars, but bailed to do the Robotech movie instead.
It is not known who they might play, but it's being said that one of the actors has already taken a meeting with Lucasfilm and Disney. The current speculation is that Ryan Gosling is being sought to play Luke Skywalker's kid, while Zac Efron will play Han Solo's child.
Disney and Lucasfilm are said to be saving their big Star Wars: Episode VII casting announcements for the D23 Expo, which happens August 9th-11th in Anaheim, California, home of Disneyland. However even that's speculation at the moment, but with an early 2014 shoot being eyed for a 2015 release, it's certainly not impossible we'll find out who's set to star at the Expo.
ACTORS: Zac Efron, Ryan Gosling DIRECTORS: JJ Abrams FILMS: Star Wars: The Force Awakens
Great New Look At Ryan Gosling In Only God Forgives With A UK Trailer
July 5, 2013 By Tim Isaac Leave a Comment
They're certainly laying on the style in this new UK trailer for Only God Forgive, which reunites Ryan Gosling with Drive director Nicolas Winding Refn.
Here's the synopsis: 'Bangkok. Julian (Ryan Gosling) runs a Thai boxing club as a front for a drugs operation. He has everything he wants for and is respected in the criminal underworld though, deep inside, he feels empty.
'When Julian's brother murders a prostitute the police call on retired cop Chang – the Angel of Vengeance (Vithaya Pansringarm). Chang allows the father to kill his daughter's murderer, then 'restores order' by chopping off the man's right hand. Julian's mother Jenna (Kristin Scott Thomas) – the head of a powerful criminal organization – arrives in Bangkok to collect her son's body. She dispatches Julian to find his killers and 'raise hell'.
'Increasingly obsessed with the Angel of Vengeance, Julian challenges him to a boxing match, hoping that by defeating him he might find spiritual release… but Chang triumphs. A furious Jenna plots revenge and the stage is set for a bloody journey through betrayal and vengeance towards a final confrontation and the possibility of redemption.'
It reaches UK cinemas on August 2nd, while it'll be out in the US on July 19th. [Read more…]
ACTORS: Kristin Scott Thomas, Ryan Gosling DIRECTORS: Nicolas Winding Refn FILMS: Only God Forgives
New Only God Forgives Trailer – Fresh look at the Ryan Gosling flick
June 17, 2013 By Tim Isaac Leave a Comment
Ryan Gosling and Drive director Nicolas Wining Refn have reunited for the upcoming Only God Forgives, which looks like it's going to be as tense as their previous effort. Now we get a fresh look with a new trailer.
Julian (Gosling) lives in exile in Bangkok where he runs a Thai boxing club as a front for the family's drugs smuggling operation. When Julian's brother Billy is killed their mother, Jenna (Kristen Scott Thomas), arrives in the city. She wants revenge and forces Julian to find the killer. Julian's contacts in the criminal underworld lead him directly to The Angel of Vengeance, a retired police officer who knows everything and who is both Judge and Punisher. Jenna demands that Julian kill The Angel of Vengeance, an act that will cost him dearly.
An August 2nd UK release is set, with the film reaching the US on July 19th. [Read more…]
Ryan Gosling Gets Slightly Neon For An Only God Forgives Poster
Only God Forgives slightly divided critics when it debuted at Cannes, with some suggesting it was a truly exceptional film that more than lived up to the idea of Ryan Gosling reuniting with Drive director Nicolas Winding Refn, while others were a little underwhelmed.
Well US audiences will be able to make up their own mind on July 19th, with UK cinemas getting it on August 2nd. And now a new poster has arrived, showing us Gosling looking very red and blue.
The movie is about Julian (Gosling), who lives in exile in Bangkok where he runs a Thai boxing club as a front for his family's drugs smuggling operation. When Julian's brother Billy is killed, their mother, Jenna (Kristen Scott Thomas), arrives in the city. She wants revenge and forces Julian to find the killer. Julian's contacts in the criminal underworld lead him directly to The Angel of Vengeance, a retired police officer who knows everything and who is both Judge and Punisher. Jenna demands that Julian kill The Angel of Vengeance, an act that will cost him dearly.
ACTORS: Ryan Gosling DIRECTORS: Nicolas Winding Refn FILMS: Only God Forgives
Two New Only God Forgives Trailers – Ryan Gosling reunites with Drive director Nicolas Winding Refn
April 19, 2013 By Tim Isaac Leave a Comment
Two new trailers for the Ryan Gosling flick Only God Forgives have arrived, selling the tension and violence of the film, which reunites the actor with Drive director Nicolas Winding Refn.
The movie reunites Gosling with Drive director Nicolas Winding Refn and follow Julian (Gosling), who lives in exile in Bangkok where he runs a Thai boxing club as a front for his family's drugs smuggling operation. When Julian's brother Billy is killed, their mother, Jenna (Kristen Scott Thomas), arrives in the city. She wants revenge and forces Julian to find the killer. Julian's contacts in the criminal underworld lead him directly to The Angel of Vengeance, a retired police officer who knows everything and who is both Judge and Punisher. Jenna demands that Julian kill The Angel of Vengeance, an act that will cost him dearly.
The film's due out this summer.
Funky New Poster For Ryan Gosling's Only God Forgives
Ryan Gosling may have suggested that he's planning to take a break from acting for a bit, but he's got The Place Beyond The Pines in cinemas and Only God Forgives, as well as Terrence Malick's currently untitled movie, coming out before then.
Now a new poster for Only God Forgives has arrived, which you can see above. The movie reunites Gosling with Drive director Nicolas Winding Refn and follow Julian (Gosling), who lives in exile in Bangkok where he runs a Thai boxing club as a front for his family's drugs smuggling operation. When Julian's brother Billy is killed, their mother, Jenna (Kristen Scott Thomas), arrives in the city. She wants revenge and forces Julian to find the killer. Julian's contacts in the criminal underworld lead him directly to The Angel of Vengeance, a retired police officer who knows everything and who is both Judge and Punisher. Jenna demands that Julian kill The Angel of Vengeance, an act that will cost him dearly.
Only God Forgives Red Band Trailer – Ryan Gosling reunites with Drive director Nicolas Winding Refn
April 4, 2013 By Tim Isaac Leave a Comment
Ryan Gosling and Drive director Nicolas Wining Refn have reunited for the upcoming Only God Forgives, which looks like it's going to be as tense as their previous effort – although there's less evidence Drive's gore in this new red band trailer.
It should be in cinemas this summer, with Gosling looking all bashed up.
New Place Beyond The Pines Poster & Ryan Gosling Images Released
The Place Beyond the Pines hits cinemas in the US on March 29th and the UK on April 12th. With the release date fast approaching a new UK poster and couple more images of Ryan Gosling in the movie have been released, which you can see above and below.
The film reunites Gosling with Blue Valentine director Derek Cianfrance, to tell the tale of Luke (Gosling), a professional motorcycle rider who turns to bank robberies to support his newborn son. However, when he crosses paths with a rookie police officer (Bradley Cooper), their violent confrontation spirals into a tense generational feud. Rose Byrne, Eva Mendes, Ray Liotta and Dane DeHaan also star.
The movie premiered at the Toronto International Film Festival to good although not rapturous reviews, and will hope for success with general audiences soon. Click on the pics below for larger versions.
ACTORS: Bradley Cooper, Ryan Gosling DIRECTORS: Derek Cianfrance FILMS: The Place Beyond The Pines


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Masato Toys is finally free!
Demetrious Johnson and Ben Askren will be indeed be switching promotions.
Daichi Abe had a short notice against Luis Santos in Myanmar.
They have the brand and the marketing, pay the big backs but they CREATE the big names.
A reported injury to Luke Rockhold has forced him out of UFC 230 .
Mas n Canuckster, you guys going to UFC 231?
Me and some friends probably will be. Its gonna be a good card. Fairly good seats going for a bit over 200 $s.
He's back. And he's coming for the title that he never lost.
Former UFC and Bellator lightweight champion Eddie Alvarez is headed to ONE Championship.
Gesias Cavalcante wants to go back to the place where he became a mixed martial arts star.
The long saga of UFC 230 added another chapter Tuesday night.
Check out the fight card and bout order for Bellator 206: Mousasi vs. MacDonald, set for September 29th in San Jose, CA.


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How to cook red quinoa on stove. How to cook red quinoa recipes. How to cook red quinoa on the stove. How to cook red quinoa in a rice cooker. Compare Search ( Please select at least 2 keywords ) Most Searched Keywords. How do i flip my screen upright 1 . Examples of giving in bible 2 . Preturi profile aluminiu 3 . Petco chinchilla cage 4 . University of michigan computer mouse 5 . What is a... The quinoa here is sprouted, not cooked and the crackers are dehydrated, (so they are technically raw), or baked on the lowest temperature on your oven. They're a delicious, incredibly healthy snack and much cheaper than store bought versions.
Directions. 1. Preheat the oven to 425°. In a small bowl, stir together cardamom, coriander, pepper and ¾ teaspoon salt. In a 9--by-13-inch baking dish, add the carrots, apple, red onion and... But now, I have learned the best way to cook quinoa using both on the stove top and my Instant Pot 6 quart Duo that leads to the perfectly cooked quinoa every single time! Over the past several years, I have begun substituting quinoa for many of my rice dishes.


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Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.
Ruba, A., Luo, W., Yang, W. Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium. J. Vis. Exp. (131), e56475, doi:10.3791/56475 (2018).
The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.
Since stated by Ernst Abbe in 1873, the resolution of conventional light microscopy has been believed to be limited to approximately 200 nm due to light diffraction from the objective1,2. Currently, super-resolution light microscopy techniques break this limitation and allow the capture of dynamic images with sub-diffraction (< 200 nm) resolution. The techniques generally fall into two broad categories: stimulated emission depletion (STED) microscopy based approaches, which generate sub-diffraction illumination volume due to nonlinear optical response of fluorophores in samples3; and photoactivated light microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)-based super-resolution techniques, which utilize mathematical functions to localize the centroids of fluorophores and then reconstitute these centroids to form super-resolution images4,5. Currently, due to the relatively uncomplicated optical setup, PALM and STORM are extensively employed by only activating a small subset of fluorophores in each frame of a long video of a biological preparation. This allows for the more accurate localization by 2D Gaussian fitting of the fluorescent spot, termed the point spread function (PSF), of fluorescently-labeled proteins in each frame of the video. The 2D location of each fluorescently-labeled molecule can then be superimposed on a single imaging plane to produce a super-resolution image of the biological preparation1,2. While these single-molecule localization, super-resolution approaches to microscopy certainly revolutionized how imaging of biological samples was performed, there are still challenges to be overcome. For example, STORM and PALM can achieve their best spatial resolutions after fixation of biological samples and thus present a static representation of the fluorescently-labeled proteins, which is a similar limitation of electron microscopy. Additionally, to achieve high spatial resolution for each fluorescently-labeled protein in live cells, samples must be imaged at very long framerates which are unable to capture protein dynamics. Therefore, it is necessary to overcome these main technical hurdles.
To obtain a high spatiotemporal resolution that is well-suited for detecting fast-moving proteins or RNAs in live cells, we have developed super-resolution SPEED microscopy in our laboratory (Figure 1)6,7,8. Several major technical advances in SPEED microscopy have previously enabled us to successfully track nucleocytoplasmic transport of small molecules, proteins, mRNA and virus through native nuclear pore complexes (NPCs)6,7,8. Briefly, the following features of SPEED microscopy will be used to track fast-moving macromolecules through sub-micrometer rotationally symmetrical structures in live cells, such as NPCs and primary cilia: (1) An inclined or a vertical illumination PSF enables the excitation of single molecules within a small diffraction-limit volume in the focal plane (Figure 1); (2) The inclined PSF can greatly avoid out-of-focus fluorescence and thus improve the signal-to-noise ratio. (3) The optical density of 100-500 kW/ cm2 in the illumination PSF allows thousands of photons to be collected from single fluorophores with fast detection speeds (> 500 Hz). (4) The fast detection speed also greatly reduces the single-molecule spatial localization error (< 10 nm) in determining the spatial trajectories of moving fluorescent molecules in live cells, because molecular diffusion is one of major factors causing imperfections of single-molecule localization for moving molecules. (5) Well-established 2D to 3D transformation algorithms enable us to provide 3D spatial probability density maps of transport routes for molecules in the NPC or the primary cilium. It is noteworthy that our conversion process between the Cartesian and the cylindrical coordination system is used to generate a 3D spatial probability density map rather than 3D single-molecule tracking (Figure 2). Previously, electron microscopy data have revealed that the NPC9,10 and the primary cilium11 both have a rotationally symmetrical structure. In principle, randomly diffusing molecules moving through the NPC or primary cilium should also have rotationally symmetrical distributions. As shown in Figure 2, a high number of randomly diffusing molecules inside the cylinder would generate rotationally symmetrical distributions at the cross-section view as that in the NPC, further resulting in an approximately uniform spatial distribution within each very small sub-region between two neighboring rings (Figure 2E). This uniform distribution leads that the spatial distribution along θ dimension in the cylindrical system is constant. Then the 3D coordinates (R, X, θ) can be simplified to be the 2D coordinates (R, X, constant). Actually, our conversion process between the Cartesian and the cylindrical systems is from 2D (X, Y) to 2D (R, X, constant). The constant θ, refers to the spatial density p in Figure 2E, is calculated by using the equation A .
Ultimately, single-molecule tracking has broad application in biological research, thus, it is natural that a plethora of techniques will be developed to fill specific biological niches12,13,14. Such is the case with SPEED microscopy. Previously, when coupled with a 3D transformation algorithm, this technique was developed to resolve 3D transport routes of transiting molecules through the NPCs, a sub-diffraction-sized and rotationally symmetric biological structure6. In this paper, primary cilia are shown to be excellent model organelles as well. Primary cilia are cylindrical, antenna-like organelles (~125 nm radius) that project from the surface of most mammalian cells15,16,17. They are responsible for receiving external signals and transmitting an intracellular response typically associated with growth and metabolism15,16. Therefore, flux of structural proteins, recycling of transmembrane receptors, and transmission of intracellular messengers are vital responsibilities of primary cilia. At the juncture between the primary cilia and the cell body is a critical selectivity barrier, called the transition zone or TZ, through which all this protein transport must occur11,18,19,20. In addition to the gating function of the TZ, at least two transport processes, intraflagellar transport and passive diffusion, are thought to be responsible for the movement of protein through this region16,21,22. From a human health standpoint, the loss of primary cilia and subsequent deregulation of downstream signaling is characteristic of many cancers. In addition, many genetic diseases, such as Bardet-Biedl syndrome and polycystic kidney disease, are associated with defective protein transport23. Both the sub-diffraction limit size and the complex process of selective protein transport through the TZ make the primary cilia a prime target for this technique. In this methods paper, we will demonstrate the tracking of a ciliary transmembrane protein, somatostatin receptor 3 (SSTR3)24, labeled externally with Alexa Fluor 647 and a component of IFT, IFT2025, labeled with a fused GFP molecule.
1.5 weeks in advance of the experiment, recover a fresh culture of NIH-3T3 cells from a frozen stock by thawing at 37 °C and transferring the cells to a 25 cm2 cell culture flask with 3 mL of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 110 mg/mL sodium pyruvate, 2 mM glutamine, 10% fetal bovine serum, and 1% penicillin/streptomycin.
Incubate the cells at 37 °C in a 5% CO2 incubator.
Split cells at 80% confluency, about every two days, at least three times before experimental day to ensure homogeneity of cell cycle. Trypsinize cells with 0.25% trypsin for 2 min at 37 °C, aspirate trypsin and replace it with 2 mL of medium. Pipette the medium repeatedly to break up cell clusters, remove the desired number of cells and bring total volume of media back up to 3 mL.
NOTE: NIH-3T3 were previously genetically engineered to express NPHP-4, a protein that localizes to the TZ26, fused at the C terminus to mCherry. mCherry is a fluorophore which can be excited with 561 nm illumination to quantitatively localize the TZ selectivity barrier and orient the primary cilia.
Two days before the experiment, plate the cells into a 35 mm glass bottom dish at 60-70% confluency with 1.5 mL of the same medium as step 1.1 and return the cells to the incubator.
One day before the experiment, chemically transfect the cells with the desired plasmid. Mix 500 - 1000 ng of the desired plasmid (see the Note below) in a 1:2.5 ratio with transfection reagent in 0.25 mL of reduced serum media without antibiotics for 30 min. Aspirate media from the 35 mm glass bottom dish and replace it with the 0.25 mL plasmid/transfection reagent mix plus an extra 1.25 mL of reduced serum media without antibiotics. Reduced serum media serves the purpose facilitating a successful transfection, inducing primary cilia growth, as well as keeping the cells alive long enough to perform experiment. Return cells to the incubator for the experiment on the following day.
NOTE: When performing single-molecule tracking of IFT20, a plasmid containing a genetically-modified IFT20 fused at its C terminus to GFP is used25. When performing single-molecule tracking of SSTR3, a plasmid containing a genetically-modified SSTR3 fused at its N terminus to an acceptor peptide (AP) domain and C terminus to GFP is used22. In addition to the SSTR3 construct, a plasmid containing the biotin ligase BirA must be co-expressed and the transfection media must be supplemented with 10 µM biotin. BirA then attaches biotin to the AP domain of newly synthesized AP-SSTR3-GFP molecules at the level of the ER. Alexa647 conjugated to three of the four biotin-binding sites on streptavidin, on average, may then be supplemented to the media prior to imaging to fluorescently label the AP-SSTR3-GFP molecules on the external surface of the cell22,27. GFP and AlexaFluor647 are used in this method; however, other fluorescent probes can be used if they have similarly high photo-stability and quantum yield.
If using the externally-labeled SSTR3 construct, remove the media from the glass bottom dish 1 h before experiment, wash the cell 5 times with 1 mL of phosphate-buffered saline (PBS), and add 1 mL of reduced serum media supplemented with 1 µM Alexa647 conjugated streptavidin.
No more than 15 min before the experiment, remove media from the glass bottom dish and wash the transfected and labeled cells 5 times with 1 mL of PBS.
Place 1 mL of imaging buffer (20 mM HEPES, 110 mM KOAc, 5 mM NaOAc, 2 mM MgOAc, 1 mM EGTA, pH 7.3) in the glass bottom dish.
NOTE: In the imaging buffer, cells are viable for no longer than 3 h. Therefore, only 2 h of experiments are performed on each dish.
Note: The SPEED microscopy setup includes an inverted fluorescence microscope equipped with a 1.4-NA 100× oil-immersion apochromatic objective, a 35 mW 633 nm He-Ne laser, 50 mW solid state 488-nm and 561-nm lasers, an on-chip multiplication gain charge-coupled-device camera and a microscope software package for data acquisition and processing (Figure 1). For individual channel imaging, GFP, mCherry, and Alexa647 are excited by 488 nm, 561 nm, or 633 nm lasers, respectively. For single molecule tracking, single point illumination is used to track individual fluorescently-labeled molecules. For epifluorescence imaging, a concave lens is placed in the laser illumination path to expand the beam into a uniform field of illumination. The fluorescence emissions are collected by the same objective, filtered by a dichroic filter (405/488/561/635) and an emission filter (405/488/561/635), and imaged with the above CCD camera operating at 500 Hz for single molecule tracking or 2 Hz for epifluorescence imaging.
Affix the glass bottom plate to the stage of the microscope and locate a cell that is properly expressing the desired constructs. Once a suitable cell has been found, align the NPHP4-mCherry spot at the base of the primary cilia with the location on the imaging plane that corresponds to the laser's single point illumination.
Capture an epifluorescence image of NPHP4-mCherry and either IFT20-GFP or AP-SSTR3-GFP using the "Snap" function in the "Camera" tab of the "Focus Controls" window if using the digital microscopy software package (see Table of Materials).
NOTE: These images will act as a reference for the subsequent single molecule locations.
Once the reference images are obtained, locally reduce the concentration of labeled single molecules. Photo-bleach the TZ with 1 mW laser illumination for 20 s or until the fluorescence intensity is close to that of background fluorescence.
NOTE: When the precise concentration can be controlled, 0.1-1 nM labeled single molecules are used.
To prepare for single molecule tracking, reduce the laser illumination power to ~0.15 mW for single molecules labeled with GFP or ~0.5 mW for molecules labeled with Alexa647.
As soon as the laser power and imaging parameters are set, maximum gain and intensification and 2 ms frame rate, for single molecule imaging, engage the appropriate illumination laser and record non-photobleached, labeled single molecules as they are transported through the photobleached region of the TZ by clicking the "Stream" button in the "Camera" tab of the "Focus Controls" window.
NOTE: No more than 2 min of video should be captured to minimize the effects of ciliary drift to a negligible level.
After capturing the single molecule video, process the videos using a 2D Gaussian fitting algorithm, such as Glimpse by the Gelles Lab, which precisely localizes the centroid of each single molecule's excitation PSF in an encompassing area of interest (AOI).
Select all single molecule locations with precision <10 nm and correct the center of cilia based on distribution of single molecule locations fitted with a 2D Gaussian function.
NOTE: By using 2D to 3D transformation algorithm, the 3D transport routes of IFT20-GFP and AP-SSTR3 routes are clearly shown on ciliary axonemal or ciliary membrane, respectively.
Once several thousand localizations for transiting molecules (signal to noise ratio > 11) in the cilium are collected, select the long axis of the cilium as the X-dimension. Make a Y dimension histogram of the locations and obtain the bin sums in 10 nm increments.
NOTE: The 2D to 3D transformation may be evaluated by hand or any software or programming language. The authors have successfully implemented the transformation in both Matlab and Python 2.7.
This section demonstrates the data obtained from performing SPEED microscopy at the TZ of primary cilia to study the transport route of SSTR3 connected by a ~15 nm external linker to Alexa647 (Figure 3A). It serves the dual purpose of verifying the 3D transformation algorithm. Alexa647 should only label the external surface of the primary cilium and therefore, the 3D transport route should reveal a high-density transport route at that location. NIH-3T3 stably expressing NPHP3-mCherry at the TZ must be transfected with AP-SSTR3-GFP and incubated according to the above protocol with streptavidin-conjugated Alexa647. Wide field imaging of the mCherry label is used to localize the TZ while the GFP is used to ensure that a cell has expressed the desired SSTR3 construct. Additionally, wide field imaging of the Alexa647 label using 633 nm illumination is necessary to confirm that the cell has properly expressed the BirA plasmid and has absorbed sufficient biotin from the media. Positive confirmation is characterized by a visible cilium distinct from the background fluorescence (Figure 3B). Efficient labeling of the AP-SSTR3-GFP construct with streptavidin-conjugated Alexa647 can only occur under these conditions. Once positive confirmation of selective labeling of the primary cilium by Alexa647 has been obtained, photobleaching and single point illumination of the TZ with a 633 nm laser will allow single molecules to be visualized. Before data analysis, superimposing the single molecule video on the wide field illumination is a useful validation step (Figure 3C). For example, if the laser is not aligned properly, no single molecules will appear to co-localize with the TZ. Another pre-data analysis validation step to ensure an adequate signal-to-noise ratio is to check the intensity at the TZ over the length of the single molecule video (Figure 3E). Such analysis can be performed quickly in ImageJ using the 'plot z axis profile' function. The peaks in Figure 3F correspond to the appearance of a single molecule at the TZ with a signal-to-noise of ~5.0. This graph is also capable of ensuring that enough photobleaching has occurred as evidenced by the well-separated frequency of single molecules. Figure 3G demonstrates a low signal-to-noise ratio of < 0.5. One explanation would be that the laser is not aligned directly on the TZ. Another reason may be that the laser illumination power is too low. Both explanations result in low excitation and, therefore, low emission of the fluorophores at the TZ. After these checks have been made, 2D Gaussian fitting of the single molecules will produce their trajectories in the TZ (Figure 3H). Superimposing many trajectories will produce the transport route of the labeled protein of interest in the TZ (Figure 3I). Since the 2D Gaussian fitting corresponds to the pixel values on the detector, the x, y single molecule data may be merged with the wide field image of the TZ and primary cilia to further illustrate the protein of interest's localization (Figure 3J).
IFT20 is a component of the IFT complex, which carries cargo along microtubules inside primary cilia25. Arl13b-mCherry, a widely used ciliary marker protein was used to label primary cilia19. Wide-field epi-fluorescence microscopy was used to image the primary cilium expressing both Arl13b-mCherry and IFT20-GFP and then switched to SPEED microscopy to track individual IFT20-GFP proteins in primary cilia after a pre-photobleaching of GFP fluorescence down to single-molecule level in the illumination area of SPEED microscopy (Figure 4A-4C).
Eventually, hundreds of single-molecule IFT20-GFP locations with a systematical localization precision of < 16 nm can be obtained from the primary cilium of a live cell (Figure 4D). According to our simulation, 250 single molecule locations with a radius of 95 nm was enough to generate a reliable 3D transport route (Figure 5). A final determination of IFT20-GFP transport route is based on thousands of single-molecule IFT20-GFP locations collected from ten cilia (Figure 4E). Since primary cilia possess a structure with rotational symmetry, the 2D to 3D transformation algorithm was applied to the 2D projected data. Interestingly, the 3D spatial probability density maps of IFT20 indicated that only a single high-density region with a ~60-nm width peaked at ~95 nm along the radius of primary cilia (Figure 4E). This high-density region likely co-localizes with the axonemal microtubules, in agreement with the known location of the IFT route (Figure 4F).
Figure 1. Simplified schematic of SPEED Microscopy. A vertical (path 1) or an inclined (path 2) point spread function is used to illuminate single fluorophore molecules in the focal plane. "d" refers to the distance between vertical illumination laser beam which passes through the center of the objective and angled illumination which is achieved by focusing the illumination laser on the edge of the objective. Two or more lasers regulated by an optical chopper to have an on-off excitation mode are used to track the single molecules transiting through the NPC or the primary cilium. The off time is at least ten-fold longer than the photobleaching time of the fluorophore label on the targeted molecules. Please click here to view a larger version of this figure.
Figure 2. Graphical demonstration of 2D to 3D conversion process. Schematics demonstrate the 2D to 3D transformation algorithm for molecules, for example, if they diffuse within the central lumen (A-F) or a peripheral region (G-L) of a tube. (A) Ideal 3D spatial locations of randomly diffusing molecules inside a tube can be coordinated in a cylindrical coordination system (R, X, θ). In SPEED, only the 3D density map is ultimately calculated from the 2D spatial locations. However, this is an idealized case to illustrate that the 3D density map is equivalent whether the 2D or 3D spatial locations are known. (B) The 3D molecular locations in A are projected onto a 2D plane in a Cartesian coordination system (X, Y, Z) by microscopy imaging. (C) A very thin slice (Δx) cut from the cylinder in A along x dimension. (D) The 3D spatial locations in the slice shown in C can be projected within a narrow 2D region. (E) Cross-section view of all the locations in the thin slice shown in C. These locations can be grouped into the sub-regions between concentric rings. Given the high-number randomly distributed molecules in the cylinder and the cut very thin slice, the spatial density of locations (pi) in each sub-region (Si) between two neighboring rings will be rotationally symmetrical and uniform. These locations can be further projected into 1D along the Y dimension. If the locations along Y dimension are clustered in a histogram with j columns. The total number of locations in each column (Aj) is equal to , which can be experimentally measured as shown in (F). (G-L) Similar as the above, the transformation process is presented for molecules diffusing within a peripheral region of a tube. Please click here to view a larger version of this figure.
Figure 3. 3D spatial location of the transport route for SSTR3 on live primary cilia mapped by SPEED microscopy. (A) Graphical representation of SPEED microscopy being applied to tracking of GFP-tagged SSTR3 through the TZ marked by NPHP4-mCherry. (B) Epifluorescence image of NPHP4-mCherry (red), AP-SSTR3-GFP (green), Alexa647 used to externally label AP-SSTR3-GFP (white), and the final merged image. Scale bar: 2 µm. (C) Tracking of a single Alexa 647-labeled AP-SSTR3-GFP (white) that passes through the point illumination field for four frames (2 ms/frame). Scale bar: 2 µm. (D) Single molecule trajectory (white) from (C) superimposed on the epifluorescence image of NPHP4-mCherry (red) and AP-SSTR3-GFP (green). Scale bar: 2 µm. (E) White dashed square highlights the area of single point illumination centered on the TZ. Scale bar: 2 µm. (F) Photon count vs Frame number graph for a single molecule video with a high signal to noise ratio determined by dividing the maximum fluorescence of the single molecule divided by the background fluorescence. (G) Photon count vs Frame number graph for a single molecule video with a low signal to noise ratio. (H) Trajectory from (E) and (D) localized in each frame by 2D Gaussian fitting and superimposed on an accurate graphical representation of the TZ, also localized by 2D Gaussian fitting. The 2D Gaussian fitting process fits a Gaussian function to the X and Y dimensions of the intensity profile of an area of interest (AOI) encompassing the single molecule's PSF.(I) 2D super-resolution spatial distribution of 220 individual Alexa Fluor 647-labeled AP-SSTR3-GFP locations collected from a single primary cilium. (J) Super-resolution single molecule locations from (I) superimposed on the epifluorescence image of NPHP4-mCherry (red) and AP-SSTR3-GFP (green). Scale bar: 2 µm. (K) By a 2D to 3D transformation algorithm, the spatial probability density distribution of Alexa Fluor 647-labeled AP-SSTR3-GFP on primary cilia along the R dimension was obtained. Based on Gaussian function fitting, Alexa Fluor 647-labeled AP-SSTR3-GFP primarily located on the ciliary membrane at a radius of 131±3 nm with a full width at half maximum (FWHM) of 24±1 nm. (L) Cross-section view of the spatial probability density distribution (green cloud) of Alexa Fluor 647-labeled AP-SSTR3-GFP on primary cilia. Please click here to view a larger version of this figure.
Figure 4. 3D spatial location of the transport route for IFT20 inside live primary cilia mapped by SPEED microscopy. (A-C) Representative image of Arl13b-mCherry (A) and IFT20-GFP (B) co-expressed in an NIH3T3 cell and the merged (C). The circle (cyan) indicates the location of single-point illumination of SPEED microscopy on a primary cilium grew on the side of a cell (white dashed lines) whose edges are determined by IFT20-GFP fluorescence in the cell body and bright field illumination (not shown). Scale bar: 10 µm. (D) 2D super-resolution spatial distribution of 286 individual IFT20-GFP locations collected from a single primary cilium. (E) By a 2D to 3D transformation algorithm, the spatial probability density distribution of IFT20-GFP inside primary cilia along the R dimension is obtained. Based on Gaussian function fitting, IFT20-GFP primarily locate at a radius of 95±1 nm with a full width at half maximum (FWHM) of 56±5 nm. (F) Cross-section view of the spatial probability density distribution (green clouds) of IFT20-GFP in primary cilia, overlaid with the schematic in H. Scale bar: 100 nm. Please click here to view a larger version of this figure.
Figure 5. Simulation was used to estimate the minimum number of 2D single-molecule locations to generate a reliable 3D spatial probability density map for the 2D to 3D transformation algorithms. (A) 100 computationally-generated single-molecule locations randomly sample from a radial density normal distribution centered at 95 nm (corresponding to the primary transport route of IFT20 determined in our experiments). Localization precision of 16 nm is simulated for each single-molecule location by sampling from a normal distribution with σ = 16 nm. As shown in Figure 2C, a very thin slice (Δx) in the X dimension is used for transformation algorithms and therefore the X dimension is not shown in the computationally-generated 2D single-molecule locations. (B) Transformation between Y-dimensional project data and 3D R-dimensional density generate histograms of 3D R-dimensional densities for five different simulated data sets (each with 100 points). The number above is the peak position ± fitting error. (C-D) Simulation results based on 250 single-molecule locations. (E-F) Simulation results based on 500 single-molecule locations. (G-I) Average 3D density histograms from 100-, 250- and 500-points simulations respectively. Error bars represent variability in the histogram bin heights while the number above the peak is the average peak position ± standard deviation. Please click here to view a larger version of this figure.
This protocol describes the application of SPEED microscopy to the primary cilium, a cellular signaling organelle that is highly reliant on efficient protein transport. SPEED microscopy can provide high resolution (< 10 nm) locations for fluorescently-labeled molecules as they pass through the single point illumination centered on the TZ. Previously it has been applied to study the protein trafficking through the NPC6,7,8. However, it may be extended to study trafficking through any sub-diffraction cellular cavity. This technique has an advantage over other high-resolution imaging techniques in that it is able to capture the dynamics of single protein transport in live cell systems at a spatial and temporal resolution of <10 nm and <2 ms, respectively. Another advantage is that one must only fluorescently-label a protein of interest and express the construct via transfection, a common molecular biology approach to studying protein localization, to begin performing SPEED microscopy. One must keep in mind that the single point illumination with a high-power laser is the feature that provides the well-separated, high resolution single molecule locations. However, this also limits the illumination area and makes the technique not suitable for wide field localization. Fortunately, there are many wide field super resolution techniques for investigators to choose from if desired. Another point to consider is that SPEED microscopy only captures locations of moving single molecules. Therefore, combination with FRAP, which can determine the fraction of total molecules that are mobile, may be a useful addition to analysis.
The conversion process between the Cartesian and the cylindrical coordination system is to generate a virtual 3D probability density map rather than a 3D view based on 3D single-molecule tracking. In detail, electron microscopy data have revealed that the primary cilia have a rotationally symmetrical structure which could generate a uniform molecule distribution along certain radius. This uniform distribution leads that the spatial distribution along θ dimension in the cylindrical system is constant. Then the 3D coordinates (R, X, θ) can be simplified to be the 2D coordinates (R, X, constant). Actually, our transform process between the Cartesian and the cylindrical systems is from 2D (X, Y) to 2D (R, X, constant). The constant θ, refers to the spatial density p, is calculated by using the equation A (Figure 2). Use a matrix calculator to calculate the vector , which corresponds with the relative areas between neighboring concentric rings in the cross-section view6,7 Aj s the total interaction sites at the position j measured directly from experiments; Thus, pi, the spatial probability density of the interaction sites at ri, can be calculated by solving the matrix equation of A .
A critical step in the protocol is to minimize any perturbation of the microscopy setup between the reference image acquisition and single molecule video. If any movement occurs, it is not possible to confidently map the single molecule locations to the ultrastructure of the primary cilia that was obtained via epifluorescence imaging. Another essential step is to photobleach the area of illumination enough to locally reduce the concentration of fluorescently labeled single molecules but not so much that the population is completely photobleached. In the case of SSTR3, the diffusion coefficient is slow compared to soluble molecules and the movement of a population of un-photobleached SSTR3 molecules back into the photobleached area will be longer than two minutes, the time beyond which the ciliary drift is a non-negligible factor. If the proper level of photobleaching is difficult to attain and a high level of background fluorescence is still present, an angled illumination laser may be used to reduce the amount of fluorescently labeled single molecules that are excited above and below the imaging plane. This will reduce the background and improve the signal-to-noise ratio for each captured single molecule.
In summary, SPEED microscopy is able to be readily implemented on conventional microscopy setups by the addition of a laser illumination source, associated mirrors, and high speed CCD camera. The main advancement over other similar techniques are the inclined laser which greatly reduces the background fluorescence and the 2D-to-3D transformation algorithm which reconstructs the 3D spatial probability density map from the 2D single-molecule locations. From a biological standpoint, no extra labeling besides a fluorophore-conjugated protein of interest is needed. These features allow SPEED microscopy to track single molecules with high spatiotemporal (5-10 nm, 0.4-2 ms) resolution as they traffic through a sub-micrometer bio-cavity or bio-channel with rotationally symmetric structures. Coupled with a 3D probability density transformation algorithm, the 3D transport routes of the tagged proteins may be determined through the cavity or channel. The application of this technique has been previously used to distinguish 3D transport routes of proteins and RNAs through the NPC and, here, we show that the 3D transport of a transmembrane protein, SSTR3, and a cytosolic protein, IFT20, may be achieved in the TZ of primary cilia. Other super resolution techniques, such as 3D-STORM, have obtained x,y,z positions for single molecules by placing a cylindrical lens in the optical path which creates an asymmetrical distortion of the single-molecule PSF depending on its position above or below the focal plane28. Another advance may attempt to implement virtual pinhole technology to reduce the width of the emission PSF and thus increase resolution even further. The above techniques could be implemented on SPEED microscopy quite readily. Overall, SPEED microscopy provides a unique approach in simultaneously determining transport kinetics at single-molecule level and 3D map of transport pathways with super-high spatiotemporal resolution for inter- or intra-organelle molecular trafficking under certain circumstances in live cell systems.
We thank Dr. Kristen Verhey (University of Michigan, Ann Arbor) and Dr. Gregory Pazour (University of Massachusetts Medical School) for providing some plasmids. The project was supported by grants from the National Institutes of Health (NIH GM097037, GM116204 and GM122552 to W.Y.).
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text:
Fight the Power
With few exceptions, Canada is not famous for its protests and demonstrations. Except for the Winnipeg General Strike and 'scratch & save day' at The Bay, Canada has been a pretty quiet country.
Face it - for three-quarters of the year Canada is just too cold a country for serious civil unrest. And the rest of the time we're too grateful for the warm weather or busy packing up for the cottage. Those perpetual general strikes that happen in the south are only possible because you can march around outside shouting slogans without freezing to death.
That kind of civil disobedience in the streets lasts about fifteen minutes up here. Pretty soon everyone gets too cold and heads over to Tim Horton's for a jumbo coffee and a jelly-filled. What that means is we have to cram a whole year's worth of protests into a few weeks. The rest of the time we express our discontent with vague grumblings and calling in sick.
And we're much too self-conscious to yell things through a microphone or do the "Hey ho, hey ho" chant with much enthusiasm. As Canadians, it makes us feel vaguely silly.
That's why we've had to import so many demonstrators for the Summit of the Americas that starts today. We just don't know how to protest.
In Toronto there have even been schools for demonstrators, teaching the fine art of going limp when being hauled off for denouncing corporate globalization without getting your Gap khakis wrinkled.
I've read that there are supposed to be five thousand delegates to the Summit of the Americas and a total of six thousand police officers. That means everyone gets their very own cop and we still have a thousand to spare. Unfortunately, with over ten thousand demonstrators expected, there will be only one cop for every two protestors, so it means they'll have to share.
Don't get me wrong - I'm no free trader. I think anything the Americans are keen on us joining is probably not that great a deal for us. We do seem to come out on the short end of the stick whenever push comes to shove with our neighbours to the south.
You have to wonder if these international summits are just a pretty big waste of time and money. Haven't the world's leaders ever heard of conference calls?
And you have to question the wisdom of putting the Summit - or anything else for that matter - in Quebec. You know the separatists are going to go out of their way to embarrass us, like having a grounded teenage son hanging around the house and bothering your dinner guests.
Quebec Premier Landry has already made a speech saying French Canadians are the "Latins of the North." Think of it. Under the PQ's leadership Quebec could be the next Paraguay.
But the Summit goes on, and so will the protests - in their uniquely Canadian way. One university has even postponed exams so its students can attend the protest in Quebec City. That's the spirit of the 60s: "Excuse me sir, but can I be excused to go to the Summit to topple the multinational cartels and defeat globalization? I have a note from the Dean."
How Canadian.
© Stephen Lautens 2001


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Homechevron_rightWorldchevron_rightErdogan urges to ease...
date_range 6 Jun 2017 10:35 AM GMT
Erdogan urges to ease Gulf diplomatic crisis
Ankara: Turkish President Recep Tayyip Erdogan held bilateral phone calls on Monday to ease the unprecedented diplomatic crisis between Qatar and other Gulf countries.
According to Deputy Prime Minister Numan Kurtulmus, Erdogan is actively involved in the process for the resolution of the problem by conducting bilateral phone calls with a number of heads of states, including Islamic and western countries.
"Turkey will discharge every responsibility for our part," Xinhua quoted him as saying.
He added that Ankara is committed to finding a solution to open up all diplomatic channels with Doha.
Kurtulmus' remarks came hours after decisions by Saudi Arabia, the United Arab Emirates, Bahrain, Egypt, Yemen, Libya and the Maldives to cut diplomatic ties with Qatar over claims of the latter's "terrorism support."
On the same day, the Turkish Foreign Ministry issued a statement on the development and expressed Turkey's "sorrow" over the decision of Saudi Arabia and other Arab nations to impose sanctions on Qatar.
"Turkey, as a responsible actor in its region and as term president of the Organisation of Islamic Conference, is ready to do its best for resolving this disagreement between friendly and brotherly countries as soon as possible," read the statement.
Turkish Foreign Minister Mevlut Cavusoglu urged that dialogue should be continued under any condition so that problems can be resolved peacefully.