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A good number of service providers lock their Huawei phones to their network, preventing you from making use of your phone on some other networks. Basically , they want to keep your business as the Huawei phone will only work only on their Network. Huawei Phone unlocking is the way of taking out the Network lock from the Huawei phone. Huawei G7300 Unlocking may be done by either purchasing an unlock code that is calculated from your Huawei G7300 unique IMEI number or by downloading unlocking computer software to unlock your Huawei phone directly.
Will unlocking my Huawei G7300 void the warranty ?
In the event that the service provider who locked the Huawei G7300 phone agrees as well as supplies you with the code to unlock your Huawei G7300 then is totally acceptable but will not influence your warranty. If however you unlock the Huawei G7300 by any other strategy you are going to totally void the warranty and void any potential the help of the carrier or manufacture.
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There are a number of methods to get your Huawei G7300 phone unlocked.
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PUK is short for Personal Unblocking Code. You must enter it if you have entered your personal pin code incorrect. This acts as a security feature on your Huawei G7300 phone. Should you not have your PUK code , you should make contact with your service provider who can supply the PUK code after several security checks.


text:
Scheibel Succeeded in Pension Rights Appeal for New York…
Attorney Articles
Legal Analysis
Scheibel Succeeded in Pension Rights Appeal for New York Supreme Court Justices
Eliza M. Scheibel (Associate-White Plains) prevailed in the appeal before to the New York Appellate Division, Third Department on behalf of three New York Supreme Court justices denied retirement benefits under a November 2013 policy adopted by the Administrative Board of the Courts. While required to retire at age 70, justices can be "certificated" for three additional two-year terms by the board. However, the 2013 policy denies certification to any justice already collecting retirement benefits for prior judicial service in the New York state court system unless the justice agrees to forfeit those benefits. Wilson Elser commenced an action against the Administrative Board on behalf of three justices who were subject to the policy. Eliza took a position based on two provisions of the New York State Constitution, and the statutes implementing those constitutional provisions, one regarding the standards for certificating Supreme Court justices for post-retirement service and the other establishing the sanctity of earned retirement benefits. The Appellate Division declared the Administrative Board's policy to be "illegal and contrary to law" on three grounds: (1) the policy was preempted by a the New York statute protecting earned retirement benefits, (2) the policy imposed a limitation on certificated justices that did not apply to non-certificated justices, and (3) the policy added a requirement for certification that is not found in the constitutional or statutory provisions that authorize the certification of retired justices. The Administrative Board of the Courts has publicly stated that it will appeal the Appellate Division order to New York's court of last resort, the Court of Appeals.
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Wayne Travers, Jr.
wayne.traversjr@wilsonelser.com
Eliza M. Scheibel

redpajama_set_name: RedPajamaCommonCrawl


text:
Region restrictions
Activates within United States
Will NOT activate in the following countries:
Albania, Armenia, Belarus, Brazil, China, Cuba, Georgia, Iran, Islamic Republic of, Kazakhstan, Korea, Democratic People's Republic of, Kosovo, Kyrgyzstan, Macedonia, Moldova, Republic of, Russian Federation, Serbia, South Sudan, Sudan, Syrian Arab Republic, Turkey, Turkmenistan, Ukraine, Uzbekistan.
1992. George H. W. Bush and Boris Yeltsin officially put an end to the Cold War. Barcelona held the summer Olympic Games. The Tonight Show aired its last show with Johnny Carson…
… and Lucas Fairfax disappeared from his bedroom in Kingdom, Virginia.
Virginia is a single-player first-person thriller set in a small town with a secret. Experience a missing person investigation through the eyes of graduate FBI agent Anne Tarver.
Together with your partner, seasoned investigator Maria Halperin, you'll take a trip to idyllic Burgess County and the secluded town of Kingdom, Virginia, where a young boy has vanished and nobody seems to know why.
Before long Anne will find herself negotiating competing interests, uncovering hidden agendas and testing the patience of a community unaccustomed to uninvited scrutiny.
As your investigation takes a turn for the sinister, and the list of suspects grows ever larger and stranger, you will make decisions which irrevocably shape the course of Anne's and agent Halperin's lives.
Cinematic editing that immerses players in a story told in the style of film and TV A dreamlike journey punctuated by intense drama and populated by a memorable cast of curious characters
A stirring soundtrack composed by composer Lyndon Holland and recorded live by the Prague Philharmonic Orchestra at the renowned Smecky Studio (Lost Highway, Mulholland Drive)
An original detective noir story in the tradition of Twin Peaks, Fargo and True Detective
A striking, painterly art style, steeped in the magic of small town America and the vibrant Virginia countryside.
©505 Games 2016. All rights reserved. Developed by Variable State Ltd. Published by 505 Games. 505 Games, Virginia and the 505 Games and Virginia logos are trademarks and/or registered trademarks of 505 Games S.p.A. throughout the world.
Mac OS X 10.11 Download the MacGameStore App to compare your Mac's information in real-time.
64bit Support Unknown
CPU Speed 2 GHz
Drive Space 5 GB
Video RAM 256 MB
Requires Steam and a Steam Account to download & play.
A strange vibe
I know that's the kind of game that is loved or hated. For it's unique storytelling, that's a win for me.
by Prizorgi, Belgium - Aug 8th 2020
Story over gameplay
If you are looking for a point-and-click adventure, or anything with a lot of gameplay really, this might not be for you. It's first and foremost telling a story in the veins of Twin Peaks. If you like films and games, then this could be a perfect crossover for you.
by cleaner, Germany - Feb 22nd 2020
An interactive movie disquised as an adventure
A short(1.5-3hrs) storytelling game which can be confusing due to lack of speech. I did like it and would recommend for a heavy discount but i don't think it's a must try.
by Theocharis M., Greece - Aug 4th 2018
Read MoreSubmit a Review
More By 505 Games
The Guest$9.99
Indivisible$39.99
Joe Dever's Lone Wolf$14.99
Joe Dever's Lone Wolf HD Remastered


text:
Mundial - TCR
MUNDIAL - TCR AUSTRÁLIA
Quarta, 15 Maio 2019 04:11 | Actualizado em Terça, 14 Janeiro 2020 16:03
Chelsea Angelo locks in Kelly Racing Opel TCR drive
Chelsea Angelo has secured a deal with Kelly Racing to contest the inaugural TCR Australia Series in an Opel Astra.
Angelo, 22, will drive the #37 Astra amongst a four-car strong Kelly Racing TCR outfit. Alex Rullo will drive the other KR-prepared Astra entry, while Molly Taylor will drive one of the Subaru WRXs.
The driver of the second Subaru entry will be announced this week ahead of the opening day of practice on Friday.
The announcement makes Angelo the third female driver to confirm her place on the TCR Australia grid alongside Taylor (Subaru) and Alexandra Whitley (Volkswagen).
It also secures the 14th driver on the grid for the historic Sydney Motorsport Park TCR opener.
Angelo is one of Australian motorsport's most experienced female racers, having cut her teeth through the ranks in Formula Ford, Super2 Supercars and Porsche racing.
Angelo was at the recent Winton TCR test, sampling one of KR's Subarus before finalising her deal.
The first round of the TCR Australia Series starts at Sydney Motorsport Park next weekend, held alongside the Shannons Nationals Championships. CLICK HERE to secure tickets to the race meeting.
Every round will be broadcast live, free and HD broadcast coverage on SBS. Saturday's race (May 18) will be shown from 2-3pm, while Sunday's two races (May 19) will have a two hour broadcast from 1-3pm.
The event will also be live streamed across the day and will be available on the TCR Australia website.
Chelsea Angelo will join Alex Rullo in the two Opel Astras for the 2019 TCR Australia Series. Click on image for high-resolution options.
Chelsea Angelo
Driver, Kelly Racing Opel Astra
"It's incredible to confirm that I will race in TCR this year," said Chelsea Angelo.
"I wasn't sure what my racing options were at the start of the season, but when I saw that TCR was coming here, I always felt that it was a great category for myself.
"There's a lot of ex-Supercars drivers in there, plus Porsche drivers, rally car drivers, a few females. It's a real mixed bag.
"I've never raced a front wheel drive car before and I'll be trying to get my head around it as fast as I can. It will be interesting. When I tested at Winton last week, one of the things I found tricky was trying to get the temperature of the rear tyres up. I've been speaking to a lot of people to learn as much as I can before we get to Sydney.
"I'm confident of getting my head around it all, but I love a challenge. I'm excited. I have absolutely no expectations, so it's nice to go there with an open mind set.
"TCR is a great category. It's known world wide and it can open up doors to overseas racing, so to be able to bring that here is a great thing. And it will be an even playing field. I'm excited and can't wait to get my bum back in seat."
Todd Kelly
Owner, Kelly Racing
"We have been speaking with Chelsea for some time and it's great to have her locked in with our team," said Todd Kelly.
"Chelsea is still very young, but she has been racing at the top level of Australia's national categories for some time. We have been keeping an eye on her so it's great to see here progress her career into TCR with us.
"The Opel performed extremely well at the test and we really feel that it is going to be a strong contender in the series with Alex and Chelsea as the drivers."
CONFIRMED TEAMS & DRIVERS FOR ROUND 1, SMSP
CAR NUMBER
NOMINATED DRIVER
Kelly Racing
Garry Rogers Motorsport
Jimmy Vernon
Alliance Autosport
Ashley Seward Motorsport
Dylan O'Keeffe
HMO Customer Racing
Nathan Morcom
Melbourne Performance
Rik Breukers
Wall Racing
Will Brown
Chris Pither
Magane RS
James Moffat
Alexandra Whitley
Tony D'Alberto
Alex Rullo


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text:
Red Raiders focused on fun, finishing
Spaulding must go through No. 3 Concord to win first state championship
Mike Whaley
mwhaley@fosters.com
ROCHESTER — The Spaulding High School softball team made a big step Tuesday night to beat No. 1 Londonderry in the Division I semifinals, 4-3. Now the No. 4 Red Raiders are hoping they can put their best foot forward one more time to win the program's first state title.
Standing in their way is No. 3 Concord (17-3). Spaulding (16-4) meets the Crimson Tide tonight, 7, at Southern New Hampshire University for the D-I crown.
"It's definitely a nice thing," said Spaulding coach Sheila Colson. "We've been telling the girls they really need to finish. After winning on Tuesday, we talked about just not being happy to be here. The goal is to finish. They are certainly capable."
Concord has the top pitcher in the state in junior Abby Rochette, the Gatorade Player of the Year. Rochette tossed a one-hitter when the two met on May 25 in Concord, a 1-0 game that the Crimson Tide won with a run in the bottom of the seventh. Rochette whiffed 10.
"We need to be aggressive at the plate," said senior all-state catcher Geana Torres, who had Spaulding's one hit against Rochette. "If she's going to give us a strike, we need to attack early. Work the count deep, too. That and be aggressive."
Spaulding has been to four final fours in five years and this trip to the championship is its second. The Red Raiders lost in the 2014 final to archrival Dover, 8-5.
"There's a first time for everything," Torres said. "We've had each other's backs because we want to be the first. That's been our goal."
Spaulding edged No. 5 Keene in the quarters and jumped out on Londonderry 4-0 and then held on for the 4-3 win in the semis. It as the first time the top-seeded Lancers trailed all season.
Concord stopped No. 6 Pinkerton in the quarters and upended No. 2 Winnacunnet in the semis, 6-2. The Tide have won five titles, the last one in 2009.
"They have the faster pitching," coach Colson said. "If we can solve that, we should be fine. We need to lay off pitches."
Colson said Concord plays solid defense, like the Red Raiders, and has "a couple quick little girls."
Spaulding's defense has been very good behind sophomore Izzi Eames, who earned her way to the No.1 spot on the staff after starting the season as the No. 2. Torres might be the best two-way catcher in the state. Around the infield it's Aleigh Toof (third base), Brittany Faucher (shortstop), Hannah Winship (second base) and Emma Proulx (first base).
The fleet-footed outfield, from left to right, includes Lilly Laganiere, Katie Raymond and Hailey Tapia.
It was Tapia's key two-run bunt single in the sixth inning that widened the Red Raiders' lead from 2-0 to 4-0 vs. Londonderry.
Faucher knocked in both runs against Keene. Her walk-off single to center in the bottom of the seventh scored Tapia to break a 1-1 tie and touch off a victory celebration.
"We need to focus and have fun, that's what coach likes to say," Torres said. "When we have fun, we play our game. That's how we win."
Now the Red Raiders are looking to have fun and keep the mojo going — and make a little history in the process.
"From March when we step into the gym inside till June, our eyes are on the prize for the big game," Toof said.
Tonight they don't get any bigger.


Notched bolt hole deign enables anchor bolt to self-thread and maintain a clean grout pocket that Is free of water and dirt.
Ribbed design provides for greater pullout strength.
Enables quicker and easier final bolt alignment for heavy equipment and structural steel.
Allows final bolt alignment to be completed with lower costs and less labor.
Eliminate the problems of custom prefitting, welding, pipe cutting, forming, packing and flame cutting in oversize mounting holes.
Made of high-density polyethylene that's lightweight, non-conductive, and non-corrosive.
Available In 15 standard sizes for bolt sizes from 1/2" to 5".
Copyright© 2004. Wilson Anchor Bolt Sleeve Company.


We are thrilled and honored to be nominated for Best Chiropractor (#121) and Best Massage Therapist (#126)! If you like what we do, please take moment and click on the link above to vote for us and all your favorite local businesses.
We would like to welcome Nicole Dolan, RMT, to our team at Stouffville Healthworks! She is looking forward to meeting our valued clients.
Nicole will be available Mondays 4-830pm, Wednesday 9-1, and Saturdays 9-12.


text:
Q: When using self in the completion block, does it always have a memory leak in Swift? I've been working on Swift for about half a year, and now I struggle with understanding when to use the [weak self] to avoid a memory leak. TBH, it was too difficult for me to understand from the document or other people's articles, so I used this video to figure out when to use it.
I kinda understood when to use the [weak self] but not 100% sure its usage.
In the video, for instance, the guy created an alert controller in the view controller.
class TestVC: UIViewController {

    override func viewDidAppear(_ animated: Bool) {
        super.viewDidAppear(animated)

        let alert = UIAlertController(title: "title", message: "message", preferredStyle: .alert)
        alert.addAction(UIAlertAction(title: "done", style: .cancel, handler: { _ in
            self.doSomething()
        }))

        present(alert, animated: true)
    }

    func doSomething() {
        // do something
    }
}

He explains that

*

*the view controller creates the alert

*when the done button is pressed, in the handler closure, it references self, which is a view controller.

So in the above, I get I must use [weak self] to deallocate the view controller.
However, now I'm confused with when not to use the weak self.
For instance, say I have a view controller with a function to animate the view.
class TestVC: UIViewController {

    let testView = TestView()

    override func viewDidAppear(_ animated: Bool) {
        super.viewDidAppear(animated)
        changeViewHeight()

    }

    func changeViewHeight() {
        UIView.animate(withDuration: 3) {
            self.testView.heightConstraint?.constant = 40
            self.view.layoutIfNeeded()
        }
    }
}

So in the code above, since the TestVC has a reference to changeViewHeight function, and in the completion block of the animate function, it points back to TestVC using self, should I change it to [weak self]?
Also, I think any function with a completion block has a chance to use self in the completion block, but that time is the retain cycle is happening and I should consider using [weak self] whenever I use self in the completion block? (which I guess it's not, but cannot explain why, so if someone can explain please let me know.)

redpajama_set_name: RedPajamaStackExchange


text:
Sana'a In The Dark For The Seventh Day
charging Mobile Phones In Yemen
Asma al-Mohattwari
"I am watching my mobile phone in its charger, like a mother watches her son as he grows up," Fuad says of his fifth day without electricity. His friend Ashraf replies to Fuad's comment: "I ask Allah give you health and the long age to see it reach 100%."
For seven consecutive days, the Yemeni capital of Sana'a and other major cities in Yemen are living in dark caused by Saudi-led Arab Coalition aircraft that have targeted the country's power transmission lines.
Decisive Storm's Operation entered its twenty-fourth day on Saturday, in an operation that has thrown Yemen's citizens into deprivation of many of the basic requirements of life. Most economic activity has stopped; citizens suffer from lack of food and petroleum, and electricity is becoming a distant memory.
The funny thing is that Yemenis have stopped thinking of anything except how to charge their phones. Ahmed, a Yemeni citizen, said that phones are the only way to know the learn news about the country and to enquire about relatives in other cities.
Ahmed charges his phone in a carpentry shop near to his home. "I only charge it [in the carpentry shop] one day per week; the other days I charge it in a shop selling drinking water; I pay 50 Riyals per hour."
Sana'a, Yemen's capital, and other major cities have been plunged into darkness. Aden in the south is also without electricity. The Saudi bombing of diesel depots and interference in oil delivery has left people without the ability to run back-up generators.
The cutoff of a Marib power station led to power cuts for Sana'a and other provinces, but the Ministry of Electricity has mitigated the impact on the capital by reallocating power loads. Stations with diesel needed to provide the country with power run for only one hour each day; meanwhile, the government warns citizens not to use water heaters, refrigerators, or washing machines. Unfortunately, these measures are only temporary: these two stations will also stop in a few days when they exhaust the diesel fuel needed to run them.


text:
Q: Bokeh - Apply color map to set of lines I have a piece of code that uses matplotlib to plot a set of lines and applies colors to those lines using a color map. The code excerpt and result are as follows:
cm = plt.cm.get_cmap('jet')
step = 15
xi = np.linspace(data[data.columns[0]].min(), data[data.columns[0]].max(), 2)
colors_l = np.linspace(0.1, 1, len(state_means[::step]))
for i, beta in enumerate(state_means[::step]):
    plt.plot(xi, beta[0] * xi + beta[1], alpha=.2, lw=1, c=cm(colors_l[i]))

The relevant part of the code here is
c=cm(colors_l[i])

which is within the plt.plot() function. Here its is possible to index the color map using a parameter (i in this case).
However, if i try to accomplish something similar using bokeh, with its ColorMapper and line() glyph, i run into and error. The relevant code lines and output are
call_color_mapper = LinearColorMapper(palette="Viridis256", low=min(call_strike_vals), high=max(call_strike_vals))
call_lines=dict()
call_chain_plot = figure(y_axis_label='Call OI', x_axis_label='Dates', x_axis_type = 'datetime')
for strike in call_strikes:
    call_lines[strike] = call_chain_plot.line('TIMESTAMP', strike, line_color=call_color_mapper(int(strike[2:])), source=callSource)

TypeError: 'LinearColorMapper' object is not callable

Is there a way to color a set of line glyphs using a color mapper in bokeh?

A: LinearColorMapper does not compute colors in Python. Rather, LinearColorMapper represents a color-mapping that happens in the browser, in JavaScript. If you really need the colors in Python, you will have to map them by hand, there are lots of ways to do this.
But you probably don't, so the best way to do this in Bokeh would be to use multi_line instead of repeated calls to line. This partially for performance reasons, Bokeh is optimized to perform best over "vectorized" operations. But also, it allows you to use the linear_cmap convenience function to make a color mapper for any data column you like. Here is a complete example:
from bokeh.io import output_file, show
from bokeh.models import ColumnDataSource
from bokeh.plotting import figure
from bokeh.transform import linear_cmap

output_file("cmap.html")

p = figure()

source = ColumnDataSource(data=dict(
    xs=[[0,1] for i in range(256)],     # x coords for each line (list of lists)
    ys=[[i, i+10] for i in range(256)], # y coords for each line (list of lists)
    foo=list(range(256))                # data to use for colormapping
))

p.multi_line('xs', 'ys', source=source,
             color=linear_cmap('foo', "Viridis256", 0, 255))

show(p)

A: While @bigreddot 's solution does provide a great alternative to the line() glyph to plot a set of lines using a linear_cmap(), it does not provide a way to capture handles for the individual lines should the handles be needed for further processing (e.g. plotting a secondary y-axis for some of them). Which is the reason why i collect the handles to each line in a dictionary in my OP.
Well, another way to plot the lines one at a time while looping through a list is as follows
from bokeh.palettes import viridis #here viridis is a function that takes\
#parameter n and provides a palette with n equally(almost) spaced colors.

call_colors = viridis(len(call_strikes))
color_key_value_pairs = list(zip(call_strikes, call_colors))
color_dict = dict(color_key_value_pairs)

Now, the dictionary color_dict can be used to access colors based on the values in the dictionary. So, I run the code from the OP has follows:
call_lines=dict()
for index, strike in enumerate(call_strikes):
call_lines[strike] = call_chain_plot.line('xs', strike, color=color_dict[strike], source=callSource)

I guess this is what @bigreddot meant when he wrote, 'If you really need the colors in Python, you will have to map them by hand, there are lots of ways to do this'.


Copyright: © 2016 Nilsson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by The Swedish Research Council to AESE, AO; Parkinsonfonden to AO; Insamlingsstiftelsen vid Umeå Universitet to AO; Alzheimerfonden to AO; Åhlénstiftelsen to AO; J.C. Kempes stiftelse to AO, KB; Hjärnfonden to AO, KB; Magn. Bergvalls stiftelse to KB; O.E. och Edlas stiftelse to AO; Torsten Söderbergs stiftelse to AO; Västerbottens Läns lasting (ALF-medel) to AO; Patientföreningen FAMY/AMYL to AO; and The Medical Faculty of Umeå University to AO.
Transthyretin-related hereditary amyloidosis is linked to more than 100 known heterozygous mutations in the gene coding for transthyretin (TTR) , a plasma protein responsible for transporting thyroxine (T4) and holo-retinol-binding protein . The mutated TTR misfolds and misassembles into various intermediate species and amyloid fibrils causing systemic organ dysfunction, frequently including peripheral neuropathy, cardiomyopathy, and ophthalmopathy . TTR-related hereditary amyloidosis is divided into two categories depending on the tissue that is predominantly affected: familial amyloid polyneuropathy (FAP), which mainly affects the peripheral nervous system, and familial amyloid cardiomyopathy (FAC), which predominantly affects the heart . In addition, a late-onset variant of TTR amyloidosis, senile systemic amyloidosis (SSA), is associated with wild-type protein resulting in deposits mainly in the heart .
From a mechanistic point of view, the mutations in TTR frequently result in destabilization of the native tetrameric structure of TTR and increase its dissociation into monomers . These monomeric species then misfold and aggregate into amyloid fibrils within various tissues depending on the mutation [7,8].
Since the 1990s, liver transplantation has been used as a treatment for patients with TTR-related hereditary amyloidosis and as a result eliminating the main source of mutated TTR . This treatment is however only suitable for a subset of patients, mainly those with the V30M FAP mutation, and in some cases the disease still progresses post-operatively due to a continuous deposition of the wild-type TTR . In addition, a liver transplant is a major surgery and is associated with many risks due to the life-long immunosuppression treatment that follows transplantation.
The discoveries that dissociation of TTR into monomers is required for amyloid fibril formation [7,8] and that small-molecule ligands increase the activation energy associated with tetramer dissociation [11,12] paved the way for a novel therapeutic approach. Small-molecule ligands that bind within the T4 binding sites of TTR can efficiently stabilize the tetramer , and the effect on T4 metabolism is minor because the primary carrier of T4 in plasma is thyroxine-binding globulin and only a very small portion of TTR (<1%) binds to T4 . In 2011, the drug tafamidis, a compound with high ability to bind and stabilize the tetrameric fold of TTR, was approved by the European Medical Agency for treatment of TTR-related hereditary amyloidosis patients with stage I polyneuropathy . However, a beneficial effect could not be confirmed in patients with a more advanced stage of disease . In 2013, diflunisal, a non-steroid anti-inflammatory drug (NSAID) was evaluated, and it was shown that the drug managed to reduce the rate of progression of neurological impairment and preserve the quality of life compared with placebo . Due to the potential side effects, e.g. gastrointestinal bleeding, renal dysfunction, and hypertension frequently observed from long-term use of NSAIDs [18–20], there is still a need for more effective drugs without adverse side effects. Furthermore, there is at present no drug approved for treatment of FAC or SSA, and the therapy for most of these patients is constrained to symptomatic relief.
We have previously shown that the flavonoid luteolin is a highly selective binder to TTR in human plasma and that it can attenuate a TTR-mediated cytotoxic effect in cultured neuronal nerve cells and can rescue the phenotype of a Drosophila melanogaster FAP model . Luteolin, commonly found in plants and fruits, has a low incidence of side effects . However, in analogy to many drugs luteolin is biotransformed in vivo, mainly by phase II enzymes, with glucuronidation and sulfation as the main metabolic pathways . UDP-glucuronosyltransferases (UGTs) are the enzymes responsible for the glucuronidation reaction in which a glucuronic acid moiety is conjugated to the hydroxyls of luteolin. The 7-hydroxy group position is the preferred site for glucuronidation by the human liver (Fig 1) .
Fig 1. Chemical structures showing luteolin, luteolin-7-O-glucuronide, and the modified variants of luteolin where the 7-OH has been substituted with a chlorine (7-Cl-Lut) or a methoxy group (7-MeO-Lut).
Our previous studies have shown that a bulky modification in position 7 of luteolin renders an inactive molecule regarding the ability to stabilize TTR . A structural study further confirmed that the 7-hydroxy group is buried at the deep end of the substrate-binding cavity at a site incompatible with incorporation of substantially larger groups . Our aim is to introduce a substituent inert to modification by the UGTs. This limits the options of substituents since frequently used moieties such as carboxyls, thiols and amines all are subjected to glucuronidation [25–27]. In the present work, we have synthesized and analysed in vitro, including crystal determinations, two analogues where the hydroxy group at position 7 has been exchanged for a chloro group (7-Cl-Lut) and a methoxy group (7-MeO-Lut), both substituent inert to glucuronidation (Fig 1).
Using an in vitro model based on human liver microsomes, we show here how the modifications increase the persistence of the drugs. The crystal structures of TTR complexed to the two analogues show that 7-Cl-Lut binds TTR in a similar manner as luteolin. The larger MeO substituent cannot be accommodated in the same space as the chlorine and as a result 7-MeO-Lut binds in the opposite direction. 7-Cl-Lut and 7-MeO-Lut bind to TTR with a high affinity, however, compared to luteolin both analogues have lost a significant proportion of the high selectivity for TTR in human plasma and display a significantly higher non-specific binding to other components of the plasma.
In this work we also demonstrate how stability measurements in the presence of human plasma, in combination with a liver microsome assay, provide a simple method to evaluate the putative efficacy of TTR-stabilizing drugs in vivo.
All reactions were carried out under an inert atmosphere with dry solvents, unless otherwise stated. Thin layer chromatography (TLC) was performed on Silica Gel 60 F254 using detection with UV light and staining with potassium permanganate or cerium molybdate. Silica gel chromatography was performed with normal phase silica gel (60 Å, 230–400 mesh, Merck, grade 9385). Microwave heated reactions were carried out in sealed EmrysTM process vials with an InitiatorTM microwave instrument (Biotage). Temperatures were monitored with an IR probe. The 1H and 13C NMR spectra were recorded at 298 K with a DRX-400 spectrometer (Bruker) and calibrated using the residual peak of solvent as the internal standard [DMSO-d6 (DMSO-d5 δH 2.50 ppm, DMSO-d6 δC 39.5 ppm)]. Low-resolution mass spectrometry was conducted on a Micromass ZQ mass spectrometer with ES+ ionization. Preparative HPLC was performed using a VP 250/21 Nucleodur C-18, HTEC, 5 μm column (Macherey-Nagel) on a 333/334 Prep-Scale system with a flow rate of 20 mL/min, detection at 230 nm (Gilson 151), and an CH3CN (0.005% HCO2H)/H2O (0.005% HCO2H) eluent system.
α,α-Dichlorodiphenylmethane (870 mg, 705 μL, 3.67 mmol) and N,N-diisopropylethylamine (DIPEA) (451 mg, 608 μL, 3.49 mmol) were added to a solution of luteolin (1.00 g, 3.49 mmol) and dioxane/NMP 3:1 (20 mL) in a capped vial, and the reaction mixture was heated in a microwave oven for 30 min at 180°C (Fig 2). A second dose of α,α-dichlorodiphenylmethane (870 mg, 705 μL, 3.67 mmol) and DIPEA (451 mg, 608 μL, 3.49 mmol) was added and the reaction was heated for an additional 30 min at 180°C. The resulting mixture was concentrated, and the residue was taken up in CH2Cl2, washed with NH4Cl (sat), NaHCO3 (sat) and brine, dried over Na2SO4, and concentrated. Purification by column flash chromatography (EtOAc: heptane 1:4 to 1:1) gave 2 as a pale yellow powder (1.30 g, 2.78 mmol, 83% yield).
Reagents and conditions: (a) 2.1 equivalents Ph2CCl2, 2 equivalents DIPEA, Dioxane/NMP, 2 x 30 min, microwave 180°C. (b) 1.2 equivalents MeI, 1.4 equivalents K2CO3, DMF, 18 h, RT. (c) AcOH/H2O 4:1, 1 h, microwave 190°C. (d) 3.3 equivalents POCl3, DMF, 5 h, 130°C.
1H NMR (500 MHz, DMSO-d6) δ 12.86 (s, 1H), 10.87 (s, 1H), 7.78 (d, J = 1.7 Hz, 1H) 7.70 (dd, J = 8.3, 1.7 Hz, 1H), 7.57–7.53 (m, 4H), 7.49–7.43 (m, 6H), 7.23 (d, J = 8.3 Hz, 1H), 6.89 (s, 1H), 6.51 (d, J = 2.1 Hz, 1H), 6.19 (d, J = 2.1 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 181.8, 164.3, 162.8, 161.4, 157.3, 149.5, 147.3, 139.1 (2C), 129.6 (2C), 128.7 (4C), 125.8 (4C), 125.0, 122.2, 117.5, 109.2, 106.9, 104.2, 103.8, 98.9, 94.1. LRMS (ESI) m/z: 451.1 (M+1).
To a stirred solution of 2 (100 mg, 0.22 mmol) in 25 mL dry dimethylformamide (DMF), K2CO3 (42 mg, 0.30 mmol) was added followed by methyl iodide (37 mg, 16 μL, 0.26 mmol). The resulting solution was allowed to stir for 18 h at 20°C. The reaction mixture was diluted with 100 mL EtOAc and washed with 3 x 100 mL H2O and 1 x 100 mL brine. The organic phase was dried (Na2SO4) and concentrated to give 3 as a crude product which was used in the next step without further purification.
To the crude product 3 from previous step was added a mixture of acetic acid/water (4:1, 10 mL) in a capped vial. The reaction mixture was heated in a microwave at 190°C for 1 h. The resulting solution was portioned between EtOAc (25 mL) and H2O (25 mL), the organic phase washed with NaHCO3 sat. (3 x 10 mL), dried with Na2SO4 and concentrated. The crude product was purified with HPLC using a gradient from 30–100% CH3CN/H2O (0.005% HCO2H) over 40 min, to give 4 as a yellow powder (36 mg, 0.12 mmol, 55% yield over two steps).
1H NMR (500 MHz, DMSO-d6) δ 12.98 (s, 1H), 9.98 (s, 1H), 9.38 (s, 1H), 7.45 (dd, J = 8.3, 2.3 Hz, 1H), 7.44 (d, J = 2.3 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 6.73 (s, 1H), 6.72 (d, J = 2.3 Hz, 1H), 6.37 (d, J = 2.3 Hz, 1H), 3.86 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 181.8, 165.1, 164.2, 161.2, 157.2, 149.8, 145.8, 121.4, 119.1, 116.0, 113.5, 104.7, 103.1, 98.0, 92.6, 56.1. LRMS (ESI) m/z: 301.1 (M+1).
POCl3 (51 mg, 31 μL, 0.33 mmol) was added dropwise to a cooled solution of 2 (45 mg, 0.1 mmol) in DMF (0.5 mL). The reaction mixture was heated to 130°C for 5 h using a microwave before being diluted with EtOAc (10 mL) and washed with H2O (3 x 10 mL) and brine (10 mL), dried with Na2SO4 and concentrated. Filtering through a plug of silica using 1:1 EtOAc:heptane gave a crude product that was used in the next step without further purification.
A mixture of acetic acid/water (4:1, 5 mL) in a capped vial was added to the crude product 5 from the previous step. The reaction mixture was heated in a microwave at 190°C for 1 h. The resulting solution was portioned between EtOAc (15 mL) and H2O (15 mL), and the organic phase washed with NaHCO3 (sat., 3 x 5 mL), dried with Na2SO4, and concentrated. The crude product was purified with HPLC using a gradient from 30–100% CH3CN/H2O (0.005% HCO2H) over 40 min, to give 6 as a yellow powder (15 mg, 0.05 mmol, 50% yield over two steps).
1H NMR (500 MHz, DMSO-d6) δ 13.09 (s, 1H), 10.10 (s, 1H), 9.42 (s, 1H), 7.48 (dd, J = 8.4, 2.3 Hz, 1H), 7.44 (d, J = 2.3 Hz, 1H), 7.33 (d, J = 1.8 Hz, 1H), 6.91 (d, J = 1.8 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.87 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 182.3, 170.4, 165.0, 160.7, 156.0, 150.3, 145.8, 121.0, 119.5, 116.0, 113.8, 111.2, 108.9, 107.9, 103.6. LRMS (ESI) m/z: 305.0 (M+1).
Expression of TTR was performed as described previously . Briefly, after transformation of the plasmid into Escherichia coli BL21, the cells were grown until an OD600 of 0.6, followed by induction with 0.4 mM isopropyl thiogalactopyranoside at 37°C. Cells were harvested after 18 h and lysed by sonication. The cell debris was removed by centrifugation at 20,000 × g for 30 min, and the supernatant was collected and loaded on an anion exchange column (Q-sepharose, Amersham Biosciences) and eluted with a NaCl gradient. Fractions containing TTR were combined and concentrated using Centriprep filter units with Ultracel YM-10 membranes (Millipore). The concentrate was loaded on a gel filtration column (Superdex G75-16/60, Amersham Biosciences) equilibrated with PBS.
We have recently developed an assay in which the relative stability as a function of drug concentration can be evaluated in complex solutions . TTR is found within the human plasma, and it is thus of interest to analyze drug efficacy in TTR's natural environment.
The analysis was performed as described previously with some modifications . Human plasma from a healthy donor was centrifuged at 20,000 × g for 10 min. The plasma was filtered using a 0.45 μm syringe filter (Filtropur S, Nümbrecht, Germany). A solution of 1 M sodium phosphate buffer at pH 7.0 was added to a final concentration of 40 mM. Luteolin, 7-Cl-Lut and 7-MeO-Lut were dissolved in dimethyl sulfoxide (DMSO) and added to the plasma at the indicated concentrations with a final DMSO concentration of 5%. The reactions were incubated for 2 h at 20°C. Denaturation was initiated by addition of 4 M urea, 40 mM sodium phosphate buffer pH 7.4, and 150 mM NaCl. The samples were incubated for 18 h at 20°C followed by separation by gel electrophoresis using tricine-based SDS-PAGE with 0.025% SDS in the running buffer and 0.2% SDS in the loading buffer. SDS at this concentration does not denature TTR tetramers but does prevent re-association of monomers. Gel electrophoresis was followed by western blotting using rabbit anti-TTR antibody (anti-prealbumin, DAKO, Denmark) followed by a horseradish peroxidase-labeled antibody (anti-rabbit HRP, GE Healthcare, Sweden). Blocking of nonspecific binding on the membrane and incubation of the antibodies were performed in 5% fat-free powdered milk containing 0.3% Tween-20. All washing steps were performed in 40 mM Tris, 150 mM NaCl, and 0.3% Tween-20. Bound antibodies were visualized on photographic film using enhanced chemiluminescence (ECL-prime, GE Healthcare, UK). The employed rabbit anti-transthyretin antibody, however, does not detect the native fraction of TTR very well, and as a consequence only the change in monomers, which reflect the level of dissociation, was monitored . The presence of monomers on the photographic film was quantified through a densitometric analysis using the ImageJ 1.48 software (National Institutes of Health, Boston, US), which also ensured that all analyses were performed within a linear range.
Pooled female human liver microsomes (n = 10) were purchased from XenoTech (Lenexa, US) and stored at −80°C and rapidly thawed before use. The microsomes, 0.1 mg/mL, were activated by incubation with alamethicin (Santa Cruz Biotechnology, US), 50 μg/mL microsomal protein, in 0.1 M sodium phosphate buffer using a VWR Mini Shaker (Radnor, US) at 37°C and 550 rpm for 10 min. Luteolin, 7-Cl-Lut or 7-MeO-Lut was added to give a final concentration of 10 μM and 5% DMSO, and these were pre-incubated with the activated microsomes for 5 min at 37°C and shaking at 550 rpm. The reaction was initiated by addition of 5 mM uridine 5'-diphosphoglucuronic acid, (Santa Cruz Biotechnology, US). The incubation time ranged from 0 min to 5 h. The reactions were quenched by flash freezing and stored at −80°C. The microsomes were heat inactivated using a VWR UnoCycler (Radnor, US) for 5 min at 95°C. All experiments were repeated three times.
Microsome-treated samples were diluted in 50% DMSO. Analytical HPLC was performed using a Nexera UHPLC system (Shimadzu, US) connected to a diode array detector (SPP M20A). Samples were analyzed using a Nucleodur C18 HTec column (EC 150 × 4.6, 5 μm, Macherey-Nagel) with a flow rate of 1 mL/min. Aliquots of 20 μL of each sample were injected, and detection was performed at 345 nm. The mobile phase was composed of solvent A (H2O in 0.1% TFA) and solvent B (acetonitrile in 0.1% TFA). The binary gradient profile was as follows with solvent B as the reference: 0–2.5 min, 17% B; 2.5–7.5 min, 17–25% B; 7.5–15.5 min, 25–35% B; 15.5–20.5 min, 35–50% B; 20.5–25.5 min, 50–100% B; 25.5–30.5 min, 100% B; 30.5–31 min, 100–17% B; 31–35.5 min, 17% B. Before first injection, the column was equilibrated at 17% B for 5 min.
We used MST to measure the affinity between the different ligands and TTR. TTR was labelled with the Monolith NT protein labelling kit (Nanotemper Technologies, Germany) using the sulfhydryl-reactive red fluorescent dye NT-647 Maleimide according to the manufacturer's instructions. Labelled TTR was kept at a constant concentration of 300 nM, and luteolin, 7-Cl-Lut and 7-MeO-Lut were serially diluted yielding 13, 16 and 12 respectively different concentrations ranging from 0.24 nM to 1 μM for luteolin, 0.24 nM to 8 μM for 7-Cl-Lut, and 0.61 nM to 1.25 μM for 7-MeO-Lut. Experiments were carried out in phosphate-buffered saline and 5% DMSO with a Monolith NT.115 instrument using standard-treated capillaries (Nanotemper Technologies, Germany). Fluorescence was detected for 5 s, and the laser was switched on for 30 s followed by fluorescence detection for another 5 s. The delay time between runs was set to 25 s. The LED power and the MST were set to 80% for all drugs. Kd values were determined using NTanalysis software (NanoTemper Technologies, Germany) by fitting the data representing the mean ± S.E.M. from three independent measurements to the Kd fit model.
The TTR protein was crystallized as described previously . Briefly, purified TTR was dialyzed against 10 mM sodium phosphate buffer pH 7.6 and 100 mM KCl and concentrated to 5 mg/mL using an Amicon Ultra centrifugal filter device (Millipore, 3 kDa molecular-weight cutoff) and co-crystallized at room temperature with a 5 molar excess of 7-Cl-Lut or 7-MeO-Lut using the vapor-diffusion hanging drop method. A drop containing 3 μL protein solution was mixed with 3 μL of the reservoir solution and equilibrated against 1 mL of reservoir solution containing a range of 1.3–1.6 M sodium citrate and 3.5% v/v glycerol at pH 5.5 in 24-well Linbro plates. Crystals grew to dimensions of 0.2 × 0.1 × 0.4 mm3 after 5 days. The crystals were cryoprotected with 12% v/v glycerol.
The X-ray diffraction data of the TTR-7-Cl-Lut and TTR-7-MeO-Lut complexes were collected under cryogenic conditions to 1.25 Å and 1.35 Å resolution respectively at the European Synchrotron Radiation Facility in Grenoble, France, on beamline ID29 using Pilatus 6M detectors. The diffraction data were processed with XDS  and scaled using AIMLESS from the CCP4 software suite . The X-ray model of TTR (PDB ID: 1F41)  and X-ray data from 40.0–2.5 Å resolution were used in molecular replacement searches with the program PHASER . The 7-Cl-Lut and 7-MeO-Lut molecules are best defined in the B-B´ hormone binding site cavity. The models were refined against all the diffraction data using PHENIX REFINE . Manual map inspections were performed with COOT . During refinement, isotropic B-factors were first applied followed by anisotropic B-factors at the end of the refinement using all data. Molecular graphics were produced using CCP4mg . See Table 1 for details of the data collection and refinement statistics. Structure factors and coordinates of the TTR–7-Cl-Lut and TTR–7-MeO-Lut complexes have been deposited at the protein data bank (PDB ID: 5EN3 and 5IHH, respectively).
Table 1. Data collection and refinement statistics for the TTR-7-Cl-Lut and TTR-7-MeO-Lut-complexes.
In order to obtain binding affinities between the drugs and TTR, we employed microscale thermophoresis (MST), which is a technique based on the directed movement of molecules in a temperature gradient. This phenomenon depends on a variety of molecular properties such as size, charge, hydration shell, and conformation. The dissociation constant (Kd) for luteolin was determined to be 150 ± 70 nM, which is in accordance with the previously published value of 100 nM determined by isothermal titration calorimetry  (Fig 3A). The Kd values for 7-Cl-Lut and 7-MeO-Lut were determined to be 620 ± 150 nM and 390 ± 40 nM respectively (Fig 3B and 3C).
Fig 3. Binding of luteolin, 7-Cl-Lut and 7-MeO-Lut to NT-647–labeled TTR.
Various concentrations of the compounds were titrated against 300 nM of NT-647–labeled TTR. Kd values were determined by averaging three independent experiments (mean ± S.E.M.) using the NT analysis software. The thermophoresis signal was plotted against luteolin (A), 7-Cl-Lut (B) and 7-MeO-Lut (C) concentrations. The Kd values were determined to be 150 ± 70 nM for luteolin, 620 ± 150 nM for 7-Cl-Lut and 390 ± 40 nM for 7-MeO-Lut.
We have recently solved the structure of the TTR–luteolin complex, which shows how the aromatic A-ring of luteolin is inserted into the deep end of the hydrophobic T4-hormone binding site . Structural determination of the TTR-7-Cl-Lut complex reveals that 7-Cl-Lut binds in the same position as luteolin. The TTR-7-MeO-Lut complex however shows that the 7-MeO-Lut ligand is oppositely oriented in the binding site (Fig 4). The binding of 7-Cl-Lut at the deep end of the cavity was verified with anomalous difference maps (Fig 4B), and the reversed binding of 7-MeO-Lut is evident from difference maps (Fig 4D). Similar to the TTR-luteolin complex structure, no direct hydrogen bonds are formed between the Nz atom of Lys15 and the luteolin analogues. The methoxy group is however wedged in the hydrophobic pocket formed by Lys15, Thr106 and Val121.
A and C) The TTR monomers in the tetrameric structure are shown as ribbons and are labeled A (gold) and B (ice-blue). The symmetry-related monomers are labeled A´ and B´, respectively. Two 7-Cl-Lut molecules (shown in A) and two 7-MeO-Lut molecules (shown in C) bind at the T4 hormone binding pocket and are shown as sticks. (B) The quality of the electron density map at the BB´ dimer-dimer interface of 7-Cl-Lut. The σA-weighted (m|Fo|-D|Fc|) electron density map contoured at 3 times the root-mean-square value of the map is shown in orange. The anomalous log-likelihood-gradient (LLG) map shown in dark blue shows unambiguously the positions of the two symmetry-related chlorine atoms and verifies the modeled orientation of the 7-Cl-Lut compound in the binding site. (D) The quality of the difference Fourier electron density map at the BB´ dimer-dimer interface of 7-MeO-Lut, calculated as described in (B). To reduce model bias, the 7-Cl-Lut and 7-MeO-Lut molecules were excluded from the coordinate files that were subjected to one round of simulated annealing before calculation of the electron density map.
Exchange of the hydroxy group at position 7 of luteolin for a chloro or methoxy group has structural consequences in both cases, and for example, impairs the luteolin analogues ability to form bridging hydrogen bonds to symmetry-related Ser117 and Thr119 over the dimer-dimer interface (Fig 5).
Comparison between TTR-binding sites of A) 7-Cl-Lut, B) 7-MeO-Lut and C) luteolin in the dimer-dimer interface. The figures show binding of one compound in the AA' and BB' binding sites. The binding of 7-Cl-Lut at the AA' and BB' channel is coupled, i.e. if a 7-Cl-Lut ligand binds at the AA' site with the chlorine directed down as shown in A, the 7-Cl-Lut ligand binding at the BB' site must have an orientation so that its chlorine atom is also pointing down. For 7-MeO-Lut (B) and luteolin (C) the orientation of the ligands is not coupled. Hydrogen bonds are indicated with dotted lines. Carbon atoms from TTR and ligands are colored with blue and yellow bonds, respectively. Residue labels are only shown in A).
To follow the metabolization of luteolin, 7-Cl-Lut and 7-MeO-Lut we incubated the compounds with human liver microsomes (Fig 6). The UHPLC chromatograms show that three metabolites are formed when luteolin is treated with the microsomes (Fig 6A). The peaks correspond to the luteolin monoglucuronosyls: luteolin-7-O-glucuronosyl, luteolin-4'-O-glucuronosyl, and luteolin-3'-O-glucuronosyl, and the identification is based on previous LC-MS and NMR speciation . The chromatograms of 7-Cl-Lut and 7-MeO-Lut are shown in Fig 6B and 6C respectively. No analysis of peak 1 and 2 in Fig 6B and 6C was performed, but based on the retention times from a previous investigation  peak 1 and 2 in 6B is most likely luteolin-Cl-4'-O-glucuronosyl and luteolin-Cl-3'-O-glucuronosyl respectively. Peak 1 and 2 in 6C is most likely luteolin-MeO-4'-O-glucuronosyl and luteolin-MeO-3'-O-glucuronosyl respectively. Complete determination of the metabolic peaks was not necessary because only the peaks of luteolin, 7-Cl-Lut and 7-MeO-Lut were used when calculating the depletion kinetics. The identification of luteolin, 7-Cl-Lut and 7-MeO-Lut was determined by comparison with the retention time for the untreated drug. The chromatograms in Fig 6B and 6C indicates, as expected, that the chlorine and the methoxy group at the 7-position are inert to glucuronidation.
Fig 6. UHPLC chromatogram of luteolin compounds incubated with 0.1 mg/mL human liver microsomes after 15 min incubation at 37°C.
Detection was at 345 nm. A) Luteolin and its metabolites. 1: luteolin-7-O-glucuronosyl, 2: luteolin-4'-O-glucuronosyl, 3: luteolin-3'-O-glucuronosyl , 4: luteolin. B) 7-Cl-Lut and its metabolites. 1: luteolin-Cl-4'-O- glucuronosyl and 2: luteolin-Cl-3'-O-glucuronosyl. 7-Cl-Lut is peak 3. C) 7-MeO-Lut and its metabolites. 1: luteolin-MeO-4'-O-glucuronosyl and 2: luteolin-MeO-3'-O-glucuronosyl. 7-MeO-Lut is peak 3. Only the peaks of luteolin, 7-Cl-Lut and 7-MeO-Lut were used when calculating the depletion kinetics.
The metabolization of luteolin, 7-Cl-Lut and 7-MeO-Lut by the microsomes can be followed by the decrease in the substrate concentration over time. The data shown in Fig 7 were fitted to a single-component exponential decay model and the half-lives were determined using Eqs (1) and (2). Luteolin was metabolized about 3-fold faster than 7-Cl-Lut with half-lives of 16 ± 1 min and 52 ± 7 min, respectively. The half-life of 7-MeO-Lut was 36 ± 8 min, about twice as long as luteolins.
Fig 7. Depletion profile of 10 μM luteolin (triangle), 7-Cl-Lut (square) and 7-MeO-Lut (diamond) after incubation in the presence of 0.1 mg/mL human liver microsomes for various times.
Each time point represents the average of three independent samples (mean ± SD) acquired through plotting the integral of the corresponding peaks from the UHPLC analysis.
The plasma concentration of TTR is approximately 5 μM , and this implies that a drug must be present at ≥5 μM to work effectively as a stabilizer. Here we used a recently developed urea-mediated denaturation assay to determine the drug concentration needed in plasma to obtain the half maximal inhibitory concentration (IC50) . In the experiment, the dissociation of TTR into monomers is monitored by the appearance of a monomeric band on a western blot (Fig 8). The fraction of denaturation was quantified by densitometric analysis using ImageJ 1.48 software. Luteolin is a highly selective stabilizer of TTR with an IC50 of ~5 μM, 1:1 stoichiometric ratio between drug and tetramer if assuming the plasma concentration to be 5 μM . 7-Cl-Lut, however, lost much of luteolins selectivity and displays an IC50 corresponding to around 65 μM (Fig 8B). 7-MeO-Lut also display a significant degree of unspecific binding as compared to luteolin with an IC50 corresponding to around 105 μM (Fig 8C).
Fig 8. Determination of IC50 for luteolin, 7-Cl-Lut and 7-MeO-Lut in human plasma.
The drug's ability to selectively stabilize TTR in plasma was evaluated using a urea denaturation assay . The dissociation of the TTR tetramer is indicated by the presence of a monomeric TTR band on a western blot. (A) Gel-shift assay of various concentrations of luteolin in human plasma. The IC50 is ~5 μM. (B) Gel-shift assay of various concentrations of 7-Cl-Lut in human plasma. The IC50 is ~65 μM. (C) Gel-shift assay of various concentrations of 7-MeO-Lut in human plasma. The IC50 is ~105 μM.
The discoveries that the dissociation of TTR into monomers is required for amyloid fibril formation [7,8] and that small-molecule ligands are able to increase the activation energy associated with tetramer dissociation [11,12] have paved the way for a novel therapeutic approach. A wide range of TTR ligands have been discovered [11,39–50], and a few have proven beneficial in the treatment of FAP [15,17]. As with all drugs, their effects can be compromised through unspecific binding to other components that both reduce their effective concentrations and trigger unwanted side effects. We have recently discovered that the plant-derived flavonoid luteolin exhibits a very high selectivity for TTR in human plasma . Taken together with its low incidence of side effects, luteolin is an interesting drug candidate for stabilizing the tetrameric structure of TTR. Unfortunately it has rather low metabolic stability in vivo, and it is rapidly biotransformed by phase II enzymes with glucuronidation and sulfation as the main modifications. The preferred site for glucuronidation by the liver is the 7-OH position, and glucuronidation at this position renders the molecule unable to stabilize TTR . In order to prevent this, we modified the 7-OH group through replacements with a chloro or a methoxy group that both are inert towards glucuronidation. TTR has two binding pockets, and both T4 and luteolin exhibit negative cooperativity in binding to TTR where the low-affinity site has a binding constant approximately 100 times above the high-affinity interaction. We therefore only focus on the high-affinity site, which in essence dominates the stabilizing effect. The results of the MST binding-assay show that both 7-Cl-Lut and 7-MeO-Lut have lost some of their binding affinity compared to the parental luteolin structure, but they still represent high-affinity binders to TTR. The crystal structure of 7-Cl-Lut complexed to TTR shows only minor differences in protein-ligand interactions compared to the structure with luteolin. Luteolin, with its hydroxyl groups at position 5 and 7, can simultaneously form hydrogen bonds over the tetramer interface (Fig 5C). However, as a consequence of exchanging the 7-OH group for a chloro group, hydrogen bonds to Ser117 and Thr119 side chains on one side of the binding site are lost, which likely explains the observed decrease in binding affinity (compare Fig 5A and 5C). Interestingly, inspection of the hydrogen bond pattern further shows that binding of 7-Cl-Lut at the AA' and BB' channel is coupled. That is, if a 7-Cl-Lut ligand binds in the AA' site, the orientation of the second 7-Cl-Lut ligand at the BB' site is restricted by the hydrogen-bonding pattern created by the first 7-Cl-Lut ligand (Fig 5A). The 7-MeO-Lut structure showed a reverse binding of the luteolin analogue in the hormone binding pocket (Fig 4D). This is consistent with the orientation observed for several glucuronidated polyphenol metabolites . However, whereas the A ring and the sugar moieties were flexible in the previous structures , the A ring with its 7-methoxy group is well defined in the electron density (Fig 4D). The two hydroxy groups at the C ring of 7-MeO-Lut interact with Ser117 and Thr119 but do not provide stability over the tetramer interface (Fig 5B). However, the methoxy group forms hydrophobic contacts with Thr106 and Val121 in the symmetry-related dimer, and in combination with the hydrogen bonds formed, stability over the tetrameric interface is provided also by bound 7-MeO-Lut (Fig 5B).
The persistence of a drug against metabolic activity in the body to large extent determines its efficacy. Luteolin unfortunately has a rather short half-life of around 8 hours  mainly due to modification of the 7-OH group of its aromatic A-ring. To study whether the relative persistence in the presence of liver enzymes can be modified as a result of exchanging the 7-OH group for an inert atom, we used an in vitro assay based on human hepatic microsomes. Through this approach, the relative resistance of the modified luteolin to glucuronidation by the UGT enzymes could be compared to luteolin . The results showed that chlorination of the 7 position of luteolin increased the metabolic stability of luteolin approximately 3-fold relative to luteolin while addition of a methoxy group doubled the resistance. The activity of the luteolin analogues is still lost over time because the OH groups at the 3' and 4' positions are also subject to conjugation by the UGT enzymes in the liver, they are just not modified as rapidly as the OH group at position 7. Based on these results, the half-life of the 7-Cl-Lut analogue is within an interesting range for a therapeutic ligand. However, the prime reason why luteolin has garnered interest as a TTR-stabilizing agent is its remarkable selectivity in plasma. Addition of an essentially stoichiometric amount of luteolin, relative to the tetrameric concentration of TTR in plasma, results in an almost complete stabilizing effect, which is significantly more efficient compared to several other well-known TTR ligands that sometimes require a more than 40-fold excess to achieve the same stabilizing effect . Using this assay with 7-Cl-Lut showed that the exchange of the 7-OH group for a chlorine affected the selectivity in plasma, and compared to luteolin 7-Cl-Lut requires a 13-fold stoichiometric excess relative to TTR in human plasma to achieve the same stabilizing effect. The 7-MeO-Lut also displays a high degree of unspecific binding to other plasma components and requires 21-fold stoichiometric excess relative to TTR in human plasma to achieve the same stabilizing effect.
The results of the present study show that it is possible to improve the metabolic stability of luteolin by exchanging the OH group at position 7 of the aromatic A-ring with an inert atom. Although the binding strength can be largely preserved, the selectivity in human plasma is diminished to a large extent. We have recently shown how the enthalpic component of binding largely affects selectivity , and the finding that removing the ability to form hydrogen bonds between the 7-OH group on luteolin and Ser117 and Thr119 of TTR also lowers the selectivity is therefore in good accordance with the previous study. In this context, it is of interest to discuss the effect of different modifications, i.e. exchange of a hydroxyl group for an inert atom. Considering the 7-OH group, we are limited to a confined space within the structure. Introducing a fluorine would give the opportunity for hydrogen bonding but the C–F group is a very weak hydrogen bond acceptor [55,56]. A structural study on complexes between TTR and ligands having a fluorine within hydrogen-bonding distance of Ser117 and Thr119 shows that the fluorine is not involved in hydrogen bonding but rather is outcompeted in favor of hydrogen bonds between the side chains of Ser117 and Thr119 [57,58].
Based on the structure of TTR, a glucuronid modification of position 3' and 4' in the aromatic B-ring is also not compatible with the interaction between luteolin and TTR. Flavonoids hydroxylated in different positions of the aromatic B-ring have previously been investigated regarding their ability to bind TTR , and it can be concluded that this also can have a large influence on the binding strength. According to this previous study, the flavonoid chrysin that lacks the -OH groups in the B-ring and apigenin which only retains a single -OH group in position 4' exhibit 17 and 5 times lower affinity, respectively, compared to luteolin, and this supports the additional importance of the -OH groups in the B-ring of the molecule . Exchanging the hydroxyl groups in the B-ring for a fluoride atom would, however, most likely not result in the generation of new hydrogen bonds due to its poor properties as a hydrogen bond acceptor. Iodine analogues were not pursued at all due to their potential risk to become a thyroxine agonist.
Taken together, the present work pinpoints the flavonoid luteolin as an interesting molecule in the quest to find ligands with the ability to stabilize TTR. We show how luteolin's resistance against glucuronidation can be modulated and how its half-life in vivo might be improved. We specifically show that the luteolin analogues only modestly affects the binding affinity to TTR but has a considerably larger effect with respect to drug selectivity. The ability of Ser117 and Thr119 to accommodate a wide range of ligands by a simple change of their side-chain rotamers, provides one explanation to the maintained high affinity of the luteolin analogues to TTR in vitro. We also demonstrate how MST can be used as a simple approach to studying TTR-ligand interactions and how the combined use of liver microsomes and a urea denaturation gel-shift assay provides an efficient method to screen for novel TTR ligands and to test their potential efficacy in vivo.
This work was supported by the Swedish Research Council, Parkinsonfonden, Insamlingsstiftelsen vid Umeå Universitet, Alzheimerfonden, Åhlénstiftelsen, Kempe stiftelserna, Hjärnfonden, Magnus-Bergvalls stiftelse, O.E. och Edlas stiftelse, Torsten Söderbergs stiftelse, Västerbottens Läns lasting (ALF-medel), Patientföreningen FAMY/AMYL, and the Medical Faculty of Umeå University. We thank Tobias Sparrman for helpful discussions regarding the depletion kinetics.
Conceived and designed the experiments: LN AO AESE. Performed the experiments: LN II KB AESE AB AL MC. Analyzed the data: LN KB AESE AB AO II MC. Contributed reagents/materials/analysis tools: AL MC. Wrote the paper: LN AO AESE AL MC.
2. Raghu P, Sivakumar B (2004) Interactions amongst plasma retinol-binding protein, transthyretin and their ligands: implications in vitamin A homeostasis and transthyretin amyloidosis. Biochim Biophys Acta 1: 1–9.
18. Epstein M (2002) Non-steroidal anti-inflammatory drugs and the continuum of renal dysfunction. J Hypertens Suppl 20: S17–23.
20. Trelle S, Reichenbach S, Wandel S, Hildebrand P, Tschannen B, Villiger PM, et al. (2011) Cardiovascular safety of non-steroidal anti-inflammatory drugs: network meta-analysis. Bmj 11.
38. Maetzler W, Tian Y, Baur SM, Gauger T, Odoj B, Schmid B, et al. (2012) Serum and cerebrospinal fluid levels of transthyretin in Lewy body disorders with and without dementia. PLoS One 7: 25.
44. Florio P, Folli C, Cianci M, Del Rio D, Zanotti G, Berni R (2015) Transthyretin Binding Heterogeneity and Anti-amyloidogenic Activity of Natural Polyphenols and their Metabolites. J Biol Chem.
51. Iakovleva I, Begum A, Pokrzywa M, Walfridsson M, Sauer-Eriksson AE, Olofsson A (2015) The flavonoid luteolin, but not luteolin-7-O-glucoside, prevents a transthyretin mediated toxic response. PLoS One 10.
55. Harrell SA, McDaniel DH (1964) Strong hydrogen bonds. II. The hydrogen difluoride ion . Journal of the American Chemical Society 86: 4497.
56. Dunitz JD, Taylor R (1997) Organic fluorine hardly ever accepts hydrogen bonds. Chemistry—A European Journal 3: 89–98.
57. Mairal T, Nieto J, Pinto M, Almeida MR, Gales L, Ballesteros A, et al. (2009) Iodine atoms: a new molecular feature for the design of potent transthyretin fibrillogenesis inhibitors. PLoS One 4: 6.
Is the Subject Area "Microsomes" applicable to this article?


text:
The Split in the western alliance - Can Europe's opposition prevent war?
The western alliance is now deeply fractured on the question of war with Iraq. Divisions within the Security Council are growing. France and Germany have pledged to try to prevent a war, even if that means not supporting America in a UN vote. Does this negate what we wrote concerning the position of France and Russia? Not at all. It was always clear that the French, Russians and Germans were opposed to the policy of the USA in relation to Iraq. They have different interests and therefore different policies. That was never in doubt. But the point is that this will not deflect the American imperialists from carrying out their declared aims.
The news of the moment is that the western alliance is now deeply fractured on the question of war with Iraq. As the moment of truth draws closer, the divisions within the Security Council are growing. On January 22, France and Germany both pledged to try to prevent a war, even if that meant not supporting America in a UN vote. Germany has declared that it would not back a resolution proposing military action against Iraq. "Our people can count on the German and French governments combining our powers and efforts to keep the peace, prevent war and maintain security," Germany's chancellor, Gerhard Schröder, wrote on January 22 in the Berliner Zeitung. Later, Mr Schröder stood next to France's president, Jacques Chirac, at an event in Paris, where Mr Chirac said that France agreed with Germany's position on Iraq.
In an angry response, Donald Rumsfeld attacked France and Germany as "old Europe". In his extraordinary outburst, Rumsfeld stated that the centre of gravity of Europe was moving East - thus drawing attention to the fact that the USA is manoeuvring to get the support of the Eastern European states - Poland, Hungary and the Czech Republic - as a pro-American Trojan horse within the EU. This has further incensed the French and Germans. It tells them in so many words that the USA intends to do what it wants - with or without the Europeans.
Does this negate what I wrote in my last article concerning the position of France and Russia? Not at all. It was always clear that the French, Russians and Germans were opposed to the policy of the USA in relation to Iraq. They have different interests and therefore different policies. That was never in doubt. But the point is that this will not deflect the American imperialists from carrying out their declared aims. It merely creates additional complications for Bush and co., which they would prefer not to have, but in the last analysis, will brush aside as a man brushes an irritating fly. Moreover, in the last analysis, the French and Russians will have to withdraw their opposition - or else be exposed before the whole world as impotent and irrelevant people who talk a lot and do nothing. In addition, there is the little matter of post-war oil contracts to consider…
To believe that this will be enough to prevent a war would be naïve in the extreme. The decision to attack Iraq was taken long ago in Washington and will not be reversed because it displeases politicians in Paris, Moscow and Berlin. Of course, George Bush says the decision to launch a military strike has not yet been taken, but with more than 150,000 troops massing in the Gulf, it would be foolish to attach the slightest credence to these assertions. On the contrary, everything points to an attack being imminent. Indeed, Russian officials say they have information that the attack will begin from mid-February. We have no reason to doubt this.
Russia has warned America against commencing military action while the inspectors want to continue their searches. But Moscow is under no illusions about the effects of such warnings. A high-ranking Russian military source, quoted on Russia's Interfax news service, stated: "According to the information we have, the operation is planned for the second half of February." He added that the campaign was expected to last about a month and that the main objective was to seize control of Iraq's oilfields. Since Russia has big interests in Iraqi oil, this is a mater of considerable concern! However, since Russia has no intention of confronting the US army in Iraq, such concern does not bother George Bush or his generals very much.
A number of countries are pressing for the UN inspectors to be given more time to search Iraq for evidence of weapons of mass destruction. According to Mohamed El Baradei, the head of the International Atomic Energy Agency, the inspectors need "quite a few months" to complete their work. "I am pleading for the inspection process to take its course," he said.
France and Germany, for example, are, so far, united in their support of the inspectors. France has hinted that as one of the five permanent members of the Security Council, along with America, Britain, China and Russia, it might even use its veto against any resolution authorising the use of force against Iraq. The French say that the evidence uncovered so far by the weapons inspectors does not justify it. This is quite correct, but also quite irrelevant, since the intention all along has been to eliminate Saddam Hussein and install a pro-US puppet regime in Baghdad.
The discontent is greatest in the Middle East. Turkey, a member of NATO, has profound misgivings and is hosting a regional meeting of foreign ministers from countries that include Syria, Egypt and Saudi Arabia. Given the extremely unstable situation in Turkey, the government in Ankara is deeply worried about a war in Iraq, but at the end of the day will support the USA - in return for certain concessions.
The Turkish bourgeois, as we have explained, have their eyes on the oilfields in Kirkuk and Mosul. The fact that the Kurds claim this as part of their own territory does not concern them. If the Kurds occupy the oilfields, the Turkish army will be sent to seize them. That will be just a small part of the fun and games that will follow an American invasion of Iraq. It is a finished recipe for chaos throughout the region. That is why the Arab regimes (and also Iran) are unhappy. But then, their happiness or the lack of it is not a determining factor in the calculations of the Pentagon.
The splits in the camp of the "allies" are, of course, very serious, reflecting a growing sense of panic in Arab capitals. Panic, yes - but also a weary sense of fatalism. The conference of Middle Eastern states in Turkey has laid bare the disarray, confusion and impotence of these regimes to decide anything. They make a lot of noise but in reality they know that their destiny is no longer in their own hands. Everything is being decided in Washington.
Mr Bush's impatience for military action grows more palpable by the day, if not by the hour. "How much more time do we need to be sure he is not disarming? This looks to me like the re-run of a bad movie and I'm not interested in watching it," he snarls. The conclusion is self-evident. If the Americans think they will not get a satisfactory vote in the Security Council (satisfactory to them, of course) they will not even try to get a UN mandate for military action, but will instead lead their own campaign to topple Saddam Hussein with the support of Britain and any other allies prepared to back them.
Hans Blix, the chief inspector, is supposed to present a progress report to the Security Council at the end of January. Mr Blix has been hoping that he will be allowed to present another report to the Security Council in February. But if the Russians are right, the weapons inspectors do not have much time left. After two months of searching, the inspectors have not uncovered any clear evidence, which would be necessary to convince the world that Iraq still has nuclear, chemical and biological weapons programmes.
To imagine that the USA would be prepared to waste time waiting for Mr. Blix and his friends to find "evidence" of something which the Americans regard as an established fact is absurd. Washington already considers Iraq to be in breach of its UN commitments, a theme which Mr Bush will reiterate in his State of the Union address on Tuesday January 28. The following day, the Security Council will convene to consider its response to the inspectors' report. None of this will have a decisive effect on the plans of the USA.
The requirements of diplomacy are now entering into conflict with the demands of the military. And in such conflicts, the military tends to win. It does not require much intelligence to see why. To keep 150,000 soldiers in a state of enforced inactivity in the heat of the desert for months on end is not a serious proposition because of the damaging effect on morale. Therefore the prediction that hostilities will commence some time in the next month is probably accurate. The only reason they have been prepared to wait as long as they have is that they had not yet got all their troops into position. Once the military build-up in the Gulf has reached a critical mass, the time for diplomacy will be over and the services of Mr. Blix will no longer be required.
Two days after the Security Council meeting, Mr Bush will hold discussions with his ventriloquist's dummy, Tony Blair, at his Camp David retreat. Since ventriloquists' dummies do not tend to argue very much, it should be an amicable affair. Blair, who is faced with growing opposition at home and would probably himself prefer to avoid a war, is now claiming that the pressure being piled on Iraq is already having an effect on Saddam's regime. "They are rattled, they are weakening," he told the British Parliament. The British are walking uncomfortably on a tightrope. They have sent a large number of troops - one quarter of the British army - to the Gulf and, as usual, are slavishly following the dictates of the latest occupant of the White House, although the war is deeply unpopular with the British public and especially within the Labour Party.
Despite everything Blair remains publicly firm in his support for Bush. This openly bourgeois politician, who every day sounds more like a Tory on all questions, has tied himself to the chariot of US imperialism and cannot extricate himself. Really, the capacity for self-delusion of this man knows no bounds. He talks of a "special relationship" with America, when the real relationship is between master and lackey. He has even deluded himself that he, and not George W Bush, is the real leader of the coalition! It is, of course, the case that a master will sometimes listen to the advice of his servant. However, such advice must be strictly limited to such matters as the colour of his tie, but never extends to big investment decisions - or wars with Iraq. As one political commentator put it: "Here we have a charging rhino with Blair on its back, shouting: 'I'm riding this!' But instead of riding, he is just being bumped up and down."
As America and Britain continue with their massive military build-up, the Camp David meeting will clearly be a council of war in which Bush will decide everything and Blair will say: "Amen". But all is not well for Mr. Blair. Support for the war in Britain is steadily falling. At least a hundred Labour MPs are threatening to revolt on the war issue. Tony Blair is beginning to feel the fire under his backside. Once the war breaks out there will be huge opposition in Britain as in the rest of Europe and the USA itself.
It is necessary to mobilise all the forces of the international labour movement against the war! Already train drivers in Britain have refused to move arms shipments for Iraq. In Belgium there is a movement to prevent the use of ports for weapons destined for the Gulf. Mass demonstrations have been organised from Washington to Rome. The movement must be generalised and intensified!
No to the war against Iraq!
No war but the class war!
Social explosion on the agenda in Iraq 24 Oct 2019
Interview with Iraqi revolutionary activist 17 Oct 2019
Iraqi Kurdistan: "don't privatise the hospitals, we will run them!" 18 Sep 2019
Iraqi Kurdistan: performance prevented by security forces 2 Apr 2019
Wave of protests in Iraq 10 Jan 2019


Our last meeting of the year will be this Thursday, December 20. We will be voting on next year's board of directors nominees and conducting our annual gift exchange. Please bring a wrapped gift of $20-25 to give as we enjoy and remember the blessing of each other.
If you're willing and able to donate blood on December 23rd in Torrance, Outback and Whole Foods will feed you and a jumper will be available for the kids. Check out the event details below for more info.
Have a wonderful week, a safe and joyous holiday, and see you Thursday!


DemeCAPRONE (Poliglecaprone 25) is a synthetic monofilament absorbable suture composed of Poly (glycolic-co-caprolactone) copolymer (PGCL) and is supplied violet and colorless. DemeCAPRONE (Poliglecaprone 25) is available in sizes 2 through 6/0.
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Progressive loss of tensile strength and eventual absorption of DemeCAPRONE (POLIGLECAPRONE 25) synthetic absorbable sutures occurs by means of hydrolysis. Absorption begins as a loss of tensile strength followed by a loss of mass. Absorption is essentially complete in approximately 90 to 120 days.
DemeCAPRONE (POLIGLECAPRONE 25) synthetic absorbable suture is available in sizes 2 through 6/0.
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inadvertent needle stick injury. Discard used needles in 'Sharps' containers.
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JHLA has been providing audio and lighting systems for over 25 years. We provide services from Fort Lauderdale,FL, as well as our satellite office in Copperhill, TN. Copperhill is centrally located to Atlanta,Knoxville,Chattanooga and Asheville NC. JHLA can provide your event in Tennesee, Georgia or Florida with a full d&b audiotechnik based audio system, Airstar Space lighting balloons, Stageright staging or the most comprehensive and well planned Event decor lighting systems. We are equally experienced in all of our areas of expertise and backed by many years of proven success.
All our audio is based around Artist performance riders so you know you are getting top quality from speaker to microphone and everything in between. Our equipment is maintained in excellent quality day in and day out. Whether we provide engineers to operate or just a system tech with your crew, you get the job done right, on time and with a friendly attitude.
With our Event lighting systems we provide utilize some of the most cutting edge wireless lighting instruments, and our nice, clean and neat installs won't hinder your event or guests. Our approach to treat the entire event as many small events means you won't have lights blaring across your event into guests eyes trying to light from afar. We give each area the treatment it deserves for a beautifully accented final look.We are also an excellent choice for providing your special event with Airstar Space Lighting ballons as we have been providing them as part of our core business for almost 15 years.
Finally, we are a company focusing largely on service, and offer the utmost in dependability and dedication to your event. Depending on the size of your event, it is likely that we will not take other events in effort to keep our attention fully focused on you.
I look forward to speaking with you about your event in the future.


It's been great to see so many of you on Christmas Tour this December. Those of you who've made it out so far have seen this little tree sitting on the piano. It's become a tradition and is part of every Christmas concert we do.
Traditions are like light. They help us when we get lost.
They're worship and they're worth repeating year after year.
What new tradition are you going to start this Christmas?
(If you want to sponsor a child, here's the link to Compassion).
All we ever hear about is how much work family is. Anything beyond survival is just a bonus.
While getting along is good, it's not ultimately what God's designed. He has so much more in mind for families.
Is family hard sometimes? Absolutely.
But is it just for survival and getting along? No way.
One of the challenges for a family can even be believing that family life can be fun and faith-filled rather than just a lot of work.
I think of it like the circus baby elephant. As soon as he's born, he has a chain put on his foot. As he grows up, every day he has this chain on his foot keeping him captive.
Once the elephant becomes an adult, he could easily break the chain and be set free. But because he has only ever known the chain of captivity, he has no idea what he's capable of!
That's a lot of families. They have no idea what they are capable of in the kingdom of God because of some kind of chain they feel they can't break!
Your family is made to break any chains keeping you from experiencing God's destiny for you!
That's what this year's All In All Out Family Worship Tour is all about.
My daughter Allison and I want families to know and experience the blessing and adventure that God has created for them.
It's not too late for your family!
God truly will take you "As Is" and start right now. Being bound by a chain of what life is like right now doesn't meant you need to stay where you're at!
The truth is: God is the only one who can take your "As Is" moment and turn it into something beautiful.
Go all in as a family seeking His Kingdom so you can discover the amazing adventure God is making in you and your family to go all out!
That's what the concert is about. That's what we believe family is about! That's what we believe God's Kingdom is all about!
Will Allison and I get to see you on the All In All Out Tour? Here's hoping it's a yes!
For concert information, head over to the tour page.
What Does Church Look Like?
Scripturally-speaking, what is church supposed to look like? What is the picture? Who is there? What are the main things you notice in a service or maybe a building?
The book of Acts comes to mind for me as well as the conversations the Apostle Paul had with the different churches. What about for you?
All of this questioning comes from an experience I just had as a presenter at an amazing conference called D6. D6 draws from Deuteronomy 6 and focuses on children and family worship. I learned a lot and hopefully offered something good. It has pushed my mind and heart to take an important look at church focus.
I'm not trying to fix anything; I'm hoping for some good conversations. Church is God's idea and I want to understand it more, not just go along with it or make any assumptions.
So, let's talk it out. Would you leave me your thoughts about what church looks like in comments section of this blog?
I hope to hear from you. Thanks for the time to talk.
Blessings and (as always) God is good all the time!
P.S. If you are looking for an amazing resource for family worship and family formation, please check out my friend Rob Rienow and his ministry Visionary Family. Awesome stuff for marriage and family!
The next day the huge crowd that had arrived for the Feast heard that Jesus was entering Jerusalem. They broke off palm branches and went out to meet Him. And they cheered: "Hosanna! Blessed is He who comes in God's name! Yes! The King of Israel!" Jesus got a young donkey and rode it, just as the Scripture has it: No fear, Daughter Zion: See how your king comes, riding a donkey's colt.
I love the word Hosanna. It's powerful. It's passionate. It's more heartfelt than we realize sometimes. The word is interpreted with the words "save us," a cry for salvation but it's also a declaration of praise! Some might find it to be a mix that doesn't easily mix but I think it mixes well.
Raised hands represent celebration, surrender, and a reaching out to touch and be touched!
Hosanna, save us, blessed is He who comes!
I think it deserves a hand raising!
If you were drowning, you would raise and wave your hands to be rescued.
At a winning score by your favorite team, you raise your hands in celebration!
This Sunday, spend some time with your hands raised to the Lord!
He is the one who saves us! He is the One who deserves all praise!
Ok, so first of all I am so sorry for this taking so long to write another blog. No excuse other than I will not try to let it happen again. I am writing this blog from about 38,000 feet above Missouri flying home from an amazing event called Behold. It's a youth conference in Alabama with over 900 students and adults. I have been so honored to do this event a number of times. It is a weekend of worship that, to me, is a taste of heaven. The people of the South are so amazing and loving. I truly feel like family even though I don't say my words correctly and I have no desire for grits and I like Auburn and Alabama (I can't and WON'T choose). The Behold team is off the charts and have become so dear to me. Oh, how I love for the people of Behold.
Every event I do is important to me but I think this weekend was in the perfect place on my calendar. In 12 hours from when I write this I get back on a plane to fly to India with my wife Sherri and my oldest son Taylor. We are traveling with a ministry that is so important to me called Compassion. We are going to see the ministry of Compassion and what they do to help release children from poverty.
The trip has stressed me a bit. Along with schedules, it's hard on Sherri and the kids with all the planning involved and it's hard on all of the wonderful families who take care of my kids while we are gone. It's one thing for Daddy to be gone in the US but another to go half way around the world.
Yet at Behold we worshipped and sang out loud that "we are not orphans" and cried to God to "do what You want with me" and that to be able to "taste and see the Lord is good" is the best of all. It was church how I hope and desire it could be every day.
It was extra special because because the folks of Behold made it possible for me to bring the band.
Oh it was fun!!! I am so blessed to play my music with the likes of Sara Renner, Tim Zhorne, Todd Anderson, Christian Ankrum, Todd Behrens, and the list goes on. You know who you are.
Hey, friends, here is the takeaway for you. Are you stressed? Overwhelmed? GO WORSHIP! Swim in the ocean of God's goodness. Dive deep, swim, just float. Whatever! I did at Behold. It helps!


text:
Mary Ann Gwinn
Moira Macdonald
A book-filled visit to Edinburgh, Seattle's sister City of Literature
Originally published July 11, 2018 at 7:00 am Updated July 11, 2018 at 1:26 pm
As a brand-new UNESCO City of Literature, what can Seattle learn from the world's first City of Literature? A writer spends four bookish days in historic Edinburgh.
Late last year, after a five-year effort, Seattle was named a UNESCO City of Literature, one of 28 world cities honored by the United Nations organization. As an admirer of Seattle's readers, writers, bookstores, libraries and all things literary I was chuffed, as the Brits would say.
But what IS a city of literature, exactly, and how will Seattle measure up? In late spring my spouse surprised me with a trip to London and a side trip to Edinburgh, the world's first UNESCO City of Literature! I braced for total immersion in words, authors and literary history in the cradle of Scottish culture. I was not disappointed — indeed, I was overwhelmed — but if I were the folks over at our local city of lit, I would be a little cowed by what Seattle has to live up to. Read along for some highlights of 72 hours in Edinburgh:
We walked out of the rail station and into Edinburgh's Old Town. A quick primer: Edinburgh's historic districts are divided into two sectors, Old Town and New Town. Edinburgh is really, really old (1,400 years older than Seattle) and Old Town is its oldest precinct, crammed with looming stone edifices stained black during the Coal Age. New Town, built when overcrowding and filth got so bad in Old Town the wealthy of Edinburgh couldn't stand it anymore, is less commercial, less crowded, and beautifully laid out, with rows of perfectly proportioned Georgian terraces marching up and down the hills. But Old Town has the deep history of Scotland's literary past, so we went out into it.
A gorgeous twilight gilded the cobblestones, and my husband, admiring the view, spotted the office of Jackson Brodie, Edinburgh writer Kate Atkinson's great detective creation, featured in the BBC version of her four Brodie books. I longed for a glimpse of Jackson, one of the great heartthrobs of detective literature, but all I found was a memorial to his possible distant cousin, Edinburgh con man, gambler and thief Deacon Brodie, who went too far when he stole the ceremonial mace of the University of Edinburgh.
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We walked downhill into the Grassmarket, an old town square and place of execution where public hangings were once all the rage. Our tour guides at the Edinburgh Literary Pub Tour put on a "Clart and McBrain" show, which features a literary bohemian and a "clean-hankied intellectual" arguing with each other. They discussed and disputed about Robert Burns and Sir Walter Scott, two writers I have not thought about in almost 40 years, and there were wink-wink references to "hoorin' " (whoring) and other non-literary activities. There was beer — a beer at each stop! I asked "McBrain" if she had heard of Kate Atkinson. "Kate Atkinson?" she said politely. I died a little, but she did own up to having heard of Alexander McCall Smith. More on him later.
We took the double-decker circle bus tour, a great way to see a hilly city. We got off in New Town, in search of the Oxford Bar, hangout of Edinburgh writer Ian Rankin's immortal creation, Inspector John Rebus.
Rebus is one of those characters who is so authentic, some readers just have to believe he exists. This was old news to our bluff Scottish tour guide, because Rebus fans always hop off the bus at the stop nearest to the bar.
And so we did. We trudged down another cobblestone street and into a series of cool, small rooms, where three local duffers sat, quaffed their 10 a.m. beers and provided commentary on the royal wedding of Prince Harry and Meghan Markle, taking place at that moment in Windsor and airing on the establishment's TV. A sleek young bartender kept the drinks coming, but when one duffer let fly with a ribald comment, she said "John, you just ruined my life."
The Oxford Bar barely owns up to its international literary significance — you can't even buy a T-shirt. We drank our beers in a side room, flanked by a gentleman in vest and suspenders writing in a tiny journal. As we prepared to leave we were slipped a bookmark by the bartender, who could spot a Rebus fan a mile away. Then a couple with AMERICAN TOURIST tattooed on their respective foreheads burst through the door. "I want to see where John Rebus drinks!" the man demanded, while his partner examined her shoes. The barkeep gave him a cool look. "He's a fictional character," she said. They did not get a bookmark.
Saturday evening: The hour before dark Old Town seethes with spookiness creates an uncanny atmosphere that has inspired generations of writers, including the author of one of the world's most popular fantasy series.
J.K. Rowling was a young mother in Edinburgh when she started writing the Harry Potter books, and like many of her ilk, she needed to either get out in public or go mad. She camped out in Old Town coffee shops, including the Elephant House in Old Town, a tea and coffee spot now permanently on the Harry Potter world tour.
For inspiration, she would walk the paths of the cemetery behind the shop, Greyfriars Kirkyard, ground zero for Scottish spooks. Edinburgh has been burying its dead there for 500 years. There are more than 700 graves, monuments and crumbling vaults, festooned with dancing skeletons and skulls with wings. Before the age of antibiotics, Death paid frequent visits, and during the civil wars that ravaged England and Scotland, 1,200 Scottish rebels were imprisoned here; only 400 survived, and they were sent into slavery in Barbados.
Back to Jo Rowling. She has suggested that the kirkyard headstones inspired the names of some of her characters. One that has attracted pilgrimages, flowers and fan notes is the grave of Tom Riddle, an Edinburghian buried in the 19th century. As all Harry Potter fans know, Tom Riddle was the original identity of Harry Potter's arch enemy Lord Voldemort. Like Stevenson's Dr. Jekyll and Mr. Hyde, Straight-up Tom Riddle was Voldemort in disguise.
I kept company on a wall by Riddle's grave with another pilgrim at least 30 years younger than me. In my mind's eye I could see Rowling, standing in the perpetual Scottish rain in late November, writing in a disintegrating notebook and wondering if her novel-writing would ever come to anything. The human imagination is a strange and spooky thing.
We stormed the gates of Edinburgh Castle, which sits on top of a rock overlooking the city and has harbored its defenders since the 12th century. We walked by the old medical school, where Arthur Conan Doyle studied, and passed Robert Louis Stevenson's home on elegant Heriot Row, where as a sickly child he watched the streets until dawn and where the lamps that inspired his poem "The Lamplighter" still stand. We regrouped with a meal at the Cumberland Bar, a gastropub in New Town where the characters of Alexander McCall Smith's "44 Scotland Street" series hang out. I have met Smith, famous for his "No. 1 Ladies' Detective "series, and I really hoped he would show up, for he is a delightful person and being a polite Scot, he might have bought us a drink. He did not, and we had to content ourselves with eavesdropping on a conversation among three Scottish academics bemoaning the fact that real estate prices were ruining their city. I felt right at home. We looked for 44 Scotland Street and learned that the house numbers end at 43.
Day Four
Back to London on the train. As we chugged south over the Yorkshire dales I pondered what Seattle could learn from Edinburgh. Random thoughts:
• Seattle loves to help the underdog, but it needs to honor writers who have won the struggle and put the city on the map, from Theodore Roethke to Betty MacDonald, from Timothy Egan to Daniel James Brown. How about a literary map of Seattle? How about imitating London's blue plaque program, along the lines of "Richard Hugo Drank Here"?
• Build a decent website with an interactive literary map. Make it easy for a literary tourist to visit.
• Highlight and discuss the works of authors who have written splendid books about the region's deep history, the thousands of years before the whites came. Coll Thrush's "Native Seattle" and David Buerge's "Chief Seattle" come to mind, but there are many others.
I'm done pontificating. I had a great time in Edinburgh. If you love literature and don't mind crowds, the Edinburgh International Book Festival, billed as the world's largest, runs Aug. 11-27. Nine hundred featured authors. Now that's something to aspire to.
Mary Ann Gwinn writes about books and authors for The Seattle Times and other publications. mgwinn@seattletimes.com.