text:
Hostage for a Hood and The Merriweather File by Lionel White - book review
In two top-notch crime capers from the 1950s, a car accident steers an innocent woman into the thick of a violent armoured car robbery, and dark marital revelations surface after a dead body is found in the trunk of a salesman's car.
Tuesday, 24th July 2018, 4:52 pm
Updated Tuesday, 24th July 2018, 4:57 pm
Hostage for a Hood and The Merriweather File by Lionel White
Influential American crime writer Lionel White is often described as the master of the big caper. His 1955 novel Clean Break was adapted by Stanley Kubrick as the basis for the film noir classic The Killing and a number of White's other 35 novels have been made into films.
His twelfth novel, Hostage for a Hood, originally published by Gold Medal Books in 1957, is a high-suspense, heist-gone-wrong tale that is finally back in print in this newly released double-novel from Stark House Press.
Tense and briskly paced, it begins with a car accident involving two hoodlums and Joyce Sherwood, a distracted, twenty-something housewife who is mysteriously returning from the bank with a cashier's cheque for $2,600.
When the bumper of her seven-year-old sedan punctures the radiator on a fast but unobtrusive stolen car, it puts a severe dent in the crook's plans to steal a quarter of a million dollars from an armoured car due to arrive in 12 minutes' time.
Desperately needing a replacement vehicle, the thieves commandeer Joyce's old Chevy and take her and her poodle hostage, keeping her chained to a bed in an old vacant mansion in the distant town of Cameron Corners.
The hasty decision proves to be their undoing as Joyce's dog escapes and arouses suspicion, and her husband, refusing to believe she has left him for another man and taken their life savings, begins an unrelenting search for her, determined to track her down and discover the truth.
White assembles a volatile band of vicious and dangerous thugs to heighten the drama. Among the more interesting characters are the 'extremely meticulous and precise' gang leader Harry Cribbins, the 'mean and crazy' Santino, who is a thin, puny junkie with 'a sharp, evil mind and an uncontrollable temper,' and Santino's untrustworthy girlfriend, Paula, who inadvertently causes friction between the two men.
Exciting, carefully plotted, and with a perpetually ominous tone, Hostage for a Hood keeps the reader on the edge of the seat, right up to the thrilling final pages.
The second novel, The Merriweather File, is a startling tale of bloodshed, murder and violence that was first published by Dutton in 1959 and filmed as an episode of the TV series Thriller two years later.
It is narrated by New York City attorney-at-law Howard Means Yates, a neighbour and friend of Charles Merriweather and his wife Ann who unwittingly becomes 'a major actor' in a complicated murder investigation.
Ann, 'a lovely, fragile, soft-spoken girl in her mid-twenties,' approaches Yates seeking his advice regarding a series of disturbing incidents that have made her fear for her life. First, her dog, Puddles, is inexplicably killed by an injection of atropine and then she discovers somebody has broken into her home in the night and turned on one of the gas burners on the stove.
A tragic accident in her past that resulted in the death of her child has led her to question her own sanity, and she refuses to report the incidents to the police to avoid worrying her husband.
Weeks later, her husband gets a puncture on his way to work and while receiving roadside assistance, a crumpled-up dead body is discovered in the trunk of his car.
Yates suddenly finds himself caught up in a perplexing murder investigation and in over his head. Appointed to defend his neighbour, he grows increasingly uncertain about his client's honesty, as well as his innocence.
Utterly compelling, shocking, and with an ingeniously twisted plot, The Merriweather File is a remarkably well written and deeply fascinating crime story that will leave the reader wanting to discover more of Lionel White's exceptional novels.
(Stark House Press, paperback, £15.95)


text:
Unreleased State Department review blames Trump for 'delayed' pandemic response: report
by Alex Williams May 12, 2021 May 12, 2021
An unreleased State Department report on the Trump administration's handling of the coronavirus pandemic reportedly blames the former president for "delayed" warnings to Americans and a "void of U.S. international leadership."
According to excerpts of the State Department's draft COVID-19 Interim Review obtained by Politico, diplomats and other career agency officials expressed frustrations with former President Trump's response to the virus.
The draft document reportedly argues that Trump's withdrawal from international forums weakened U.S. global leadership in responding to the health crisis.
Politico reported that State Department officials "identified the void of U.S. international leadership" as early as March 2020 and pushed for greater global engagement, referencing the country's role in responding to Ebola in 2014.
However, the draft report notes, "Instead, an over-emphasis on China's responsibility for the pandemic led to a backwards-looking focus on assigning blame for the virus, which Department research later showed was an ineffective approach to messaging, as the United States repeatedly appeared unwilling to take on greater international leadership to confront the pandemic."
According to Politico, the draft report also references Trump's decision to downplay the virus in the early months of the pandemic to avoid a "frenzy," as revealed in tapes of interviews with Washington Post journalist Bob Woodward.
"[T]he decision to reassure Americans of their safety and avoid damaging the economy delayed warnings to American citizens about travel during a pandemic until well after airlines were cutting flights and borders were closing around the world," the document says, according to Politico.
The draft report goes on to say, "The United States' struggle to contain the virus, strident criticism of the World Health Organization (WHO) and other multilateral institutions, and the politicized internal debate on science and mitigation measures undermined international trust in U.S. leadership."
The report comes just shy of one year after the former Trump administration pulled the United States out of the World Health Organization. The move came after criticism from Trump that the WHO was too slow to sound the alarm on the virus, and accused the health body of being too trusting of China and its handling of the disease.
Trump has also attacked China over its handling of the virus, blaming the outbreak on the country. He continuously referred to coronavirus as the "Chinese virus" among other names.
Critics and members of the Asian American and Pacific Islander community have condemned Trump for these remarks and say that his rhetoric surrounding the virus has lead to an outbreak of violence against Asians in America.
President Biden earlier this year reentered the U.S. into the WHO.
When contacted by The Hill, State Department spokesperson Ned Price declined to comment "on a purportedly leaked report covering the previous administration, especially one that remains in draft and awaits broader input."
Price added that Secretary of State Antony Blinken "from his first day in office has prioritized the health and safety of our global workforce, and the State Department has been at the center of the administration's efforts to combat the virus around the world."
He went on to say the State Department has provided nearly 200,000 vaccine doses to its workforce, and has also "provided significant life-saving assistance – on a multilateral and bilateral basis – to countries around the world, recognizing that as long as the virus is spreading anywhere, it poses a threat to people everywhere, including Americans here at home."
However, Price admitted that "as a Department, we recognize the importance of identifying and accounting for lessons learned – and that includes from episodes that pre-date this administration."
"Doing so is part and parcel of our commitment to the security, health, and safety of our workforce," he added.
The Trump administration has been criticized for a bungled early response to the virus. Audio interviews released from the journalist Woodward revealed that Trump privately called the coronavirus "deadly," while comparing it to the flu publicly.
As early as February 2020, Trump told Woodward that the virus was transmitted through germs in the air.
"You just breathe the air and that's how it's passed," Trump said in a Feb. 7 call with Woodward for his book, "Rage."
At the start of the pandemic, Trump also claimed publicly that by Easter of 2020 the pandemic would disappear.
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Mum dies just months after giving birth to 'miracle' baby girl during cancer fight

redpajama_set_name: RedPajamaCommonCrawl


text:
require File.expand_path(File.dirname(__FILE__) + '/../spec_helper')

describe 'Commands.part' do
  before :each do
    @tester = CommandTester.new
  end

  context 'given valid expression' do
    it 'should assign primitives to given label under @env[ENV_EXPRS]' do
      notes = [ Note::Half.new([C2]), Note::Half.new, Note::Quarter.new([E2]) ]
      tgt = Part.new(notes: notes, dynamic_profile: Profile.new(Dynamics::MF))
      @tester.part("hello","mf","/2C2 /2 /4D2+2")
      @tester.env[Commands::ENV_PARTS]["hello"].should eq(tgt)
    end
  end
end

redpajama_set_name: RedPajamaGithub


text:
freebooksonline.com
HOME | Billionaire | Recommend | Top View | Bookslist
By: Sam Skyborne
THREE CRIME-FIGHTING WOMEN
ALL FOR
LOVE, LUST AND JUSTICE
RISK Synopsis
Toni Mendez, an ex-cop turned private investigator in London, takes on a case to find the truth behind the violent murder of the young wife of a successful entrepreneur, Lucille Ransom-Evans. Little did she know that this case would bring to her door Maxine Bennett, a feisty, opinionated, American detective, on the hunt for a serial killer.
Maxine not only infiltrates Toni's working life presenting a sore reminder of a profession she once loved, she also invades her private life by seducing her best friend, Lizbeth Du Cannon. As the investigation continues, Toni is immersed more and more, both professionally and privately, into a subculture she knew only from a distance - the LGBTQ+ world. She is forced to reassess everything she thought she knew about herself and the foundations of her long-term friendship with Lizbeth.
Thank you to all the women who participated in the Whipped Cream Production "lesbian erotica investigations" that ultimately lead to the creation of RISK. It was a load of fun and luckily the investigations continue.
Thank you to all my wonderful friends and family who have lovingly and tirelessly helped and supported me on this journey.
I love and thank you from the bottom of my heart!
"…and then the day came
when the risk to remain tight,
in a bud,
became more painful
than the risk it took to blossom…"
Lassie Benton1 1979
Toni ran her hand through her long dark hair. Her frustration was bubbling very close to the surface. She flicked through the next folder.
"Ransom-Evans. Ransom-Evans," she muttered, grabbing the next file from the pile. She'd been a Private Investigator long enough that already her filing cabinets, like her small office, were bursting at the seams with paperwork all filed in precise chronological order.
Toni prided herself on having a particularly good memory and somewhere in the middle of this mountain of paper she was positive she had an article about the Ransom-Evans family from a few months earlier. If only she could remember the damned date.
Detective Maxine Bennett strode into the office carrying two coffees and a large box of doughnuts. "Didn't think doughnuts would be so hard to find, especially in a 'world city'," she said in a soft but recognisably American accent as she scanned the room for an empty surface.
Maxine had apparently come over from New York earlier in the week, but her accent was definitely not broad enough to have originated solely from there, Toni thought. She also suspected Maxine knew London a lot better than she'd let on.
Unable to find a suitable clear surface, Maxine nudged aside the small silver plaque that read "Toni Mendez PI" on top of one of the more stable looking paper piles on Toni's desk.
It's only been three days and she already acts like she owns the place. Toni slammed another cabinet drawer closed and was about to reprimand Maxine when she caught sight of the teddy-bear tucked under her arm. The incongruity of it froze the acid remark on the tip of her tongue.
"Many things are quite different in London, Detective Bennett." Though Toni kept the irritation out of her voice, her intent was clear.
"Let's hope that goes for this case too." Maxine sipped her coffee and studied the large freestanding whiteboard that stood crammed into the corner of the small office.
"She looks sorta sad." Maxine indicated the photo of an attractive young woman in her thirties with dyed red hair and dark green eyes.
Toni glanced over. Erika Ransom-Evans did indeed look sad in a troubled, wistful way. Fragile at the very least.
She gave up on the article hunt and retrieved another headshot of a more masculine looking older woman with short greying hair wearing a man's collared shirt and a v-neck jumper and pinned it next to Erika. She grabbed the blue marker pen, ripped the lid off with her teeth and wrote "Lucille Ransom-Evans" under the photo and drew an arrow from the photo of Erika to that of Lucille and labelled it "wife of".
Toni stood back and studied her handy work. "They were together for thirteen years." She shook her head slightly.
"What?" Maxine asked.
"No, nothing," Toni nodded at Lucille. "I just feel for her. It can't be easy losing one's wife while having to be in the public eye like that."
"No, it can't be."
"I'm sometimes very glad I'm not gay," Toni continued.
Maxine almost spilt her hot drink. "You make it sound like a birth defect."
"No, not at all, I just don't think anyone would choose that life if they had a choice. It must be so much harder. Would you?" Toni asked frowning.
Also By Sam Skyborne
· Risk
Previous1 234..75 Next
SupportMail To:[email protected]


text:
Welcome Guest, if you want to disable advertisements, please login at EAB with "Remember Me" option.
Artist is : James D. Sachs (Jim Sachs)
Artist name James D. Sachs (Jim Sachs)
Birth date 1949
Website http://www.fish-byte.com/
Mother country USA
Notes Born in California in 1949 and grew up in the San Fernando Valley, north of Los Angeles, USA. At college, Jim concentrated mainly on architecture. After college, he spent 6 years in the U.S. Air Force as a pilot flying C-141 Starlifters. In the early 1980s, he got into computers with the Commodore 64, bought some books on 6502 machine language, and began writing games. His first game, SAUCER ATTACK, sold quite well via mail order, but rampant piracy drove him away from the C64 market and he turned to the Amiga as soon as it was released. By his own admission, Jim had no actual training in programming and although he'd been forced to do quite a lot of it, it is the artistic side of computers that has always captivated him.
The Amiga was the first computer that allowed Jim to express his love affair with a computer palette. After a few months experimenting with the early graphics packages on the Amiga, Cinemaware were sufficiently impressed with his ability and hired him to do the graphics for their first game, the graphically stunning DEFENDER OF THE CROWN. While working on DOTC, he got involved in freelance art and graphics work, designing covers for both magazine and book covers. Jim went on to work for Commodore, drawing the internal graphics and user interfaces for the CDTV. He also spent 2 years on DOTC II for the CDTV, re-programming the original DOTC, doing some new graphics, recording actors narrating in 5 languages, re-working the plot, and writing a new musical score. Unfortunately, it ended up being released for the CD32 shortly before Commodore went bankrupt, which significantly affected sales and distribution around the world.
In 1995, Jim began the biggest project of his career, the creation of 3D software for a bicycle training device called the CompuTrainer. He spent 3+ years on it. People ranging from Robin Williams to the U.S. Olympic Cycling Team have used it for their indoor training. More recently, Jim has worked on a virtual 3D aquarium screensaver (see website above) for SereneScreen.
Artist photo
Self-Portrait of Jim Sachs
This artist page has been viewed : 6996 times
#-9 A B C D E F G H I J K L M N O P Q R S T U V W X Y Z
NAME CHIP YEAR DEVELOPER PUBLISHER
Centurion: Defender Of Rome ECS OCS
1991 Bits Of Magic Electronic Arts
Defender Of The Crown OCS 1986 Master Designer Cinemaware (Master Designer)
Mindscape
Mirrorsoft
Defender Of The Crown CDTV 1991 Cinemaware CDTV Publishing (Commodore)
Defender Of The Crown II CD32 CDTV
1993 Master Designer Commodore
Défenseur De La Couronne II CD32 CDTV
Defensor De La Corona II, El CD32 CDTV
Difensore Della Corona II CD32 CDTV
Ports Of Call OCS 1987 Aegis [Oxxi]
Disc Company, The
Verteidiger Der Krone II CD32 CDTV
Who Framed Roger Rabbit OCS 1988 Silent Buena Vista
Total of game listing generated: 7,296,878
Coded by RCK of abime.net - Content © 1998-2019 HOL TEAM - Online amiga database © 2002-2019 HOL TEAM - Gen. in 1.05s


Yes. Rebecca uses individually packaged one-time use only disposable and sterilized steel needles. There is no risk of transmitting a communicable disease using such needles.
For acupuncture sessions, please wear comfortable clothing that can easily roll up to the knees and elbows. For Sotai/Tuina sessions please wear light comfortable clothing that moves freely.
Yes. Rebecca offers a 20% payment at time of service discount if you pay for your treatment in full at the time of service (prices listed on website reflect this discount), as well as additional 10% discounts to Portland Classical Chinese Garden members, Poekoelan Tjimindie Tulen practitioners, residents of South Park Square, PSU Students, Faculty and Employees. Rebecca also provides periodic time sensitive special offers. Check the All Ways Well Blog to see if there are any time sensitive offers currently available. Discounts and special offers do not apply when billing insurance.


text:
Q: Laravel Job Property of Non Object I have a redis queue set up with a simple job. The job code is below. I keep getting the following error -
ErrorException: Trying to get property of non-object in /app/Jobs/ResolveOrders.php:42

If I take this same code and move it to my controller it works. However, when I move it here it does not even run. Here is my ResolveOrders.php - it seems like Laravel is not recognizing my Sync db model
<?php

namespace App\Jobs;

use Illuminate\Support\Facades\Auth;
use App\Sync;
use App\Helpers\getOrdersHelpers;
use Carbon\Carbon;
use Illuminate\Bus\Queueable;
use Illuminate\Queue\SerializesModels;
use Illuminate\Queue\InteractsWithQueue;
use Illuminate\Contracts\Queue\ShouldQueue;
use Illuminate\Foundation\Bus\Dispatchable;

class ResolveOrders implements ShouldQueue
{
    use Dispatchable, InteractsWithQueue, Queueable, SerializesModels;

    public $tries = 1;

    public $timeout = 660;

    /**
     * Create a new job instance.
     *
     * @return void
     */
    public function __construct()
    {

    }

    /**
     * Execute the job.
     *
     * @return void
     */
    public function handle()
    {
        $job = Sync::create(['user_id' => Auth::user()->id,
                    'last_updated_date' => Carbon::now(),
                    'status' => 'running']);

        $getOrdersHelpers = new getOrdersHelpers();
        $return = $getOrdersHelpers->getOrders(1, $job);
        $pageNum = $return[0];
        $totalPages = $return[1];

        if ($totalPages > $pageNum) {
            while ($pageNum <= $totalPages) {
                $getOrdersHelpers->getOrders($pageNum, $job);
                $pageNum++;
            }
        }

            $job->status = "complete";
        $job->save();

    }

}

A: The Point is that there is no Auth::user() when the Job is handled by the queue. You have to store the user id in the constructor.
<?php

namespace App\Jobs;

use Illuminate\Support\Facades\Auth;
use App\Sync;
use App\Helpers\getOrdersHelpers;
use Carbon\Carbon;
use Illuminate\Bus\Queueable;
use Illuminate\Queue\SerializesModels;
use Illuminate\Queue\InteractsWithQueue;
use Illuminate\Contracts\Queue\ShouldQueue;
use Illuminate\Foundation\Bus\Dispatchable;

class ResolveOrders implements ShouldQueue
{
    use Dispatchable, InteractsWithQueue, Queueable, SerializesModels;

    public $tries = 1;

    public $timeout = 660;

    protected $user_id;

    /**
     * Create a new job instance.
     *
     * @return void
     */
    public function __construct()
    {
        $this->user_id = Auth::user()->id;
    }

    /**
     * Execute the job.
     *
     * @return void
     */
    public function handle()
    {
        $job = Sync::create(['user_id' => $this->user_id,
                    'last_updated_date' => Carbon::now(),
                    'status' => 'running']);

        $getOrdersHelpers = new getOrdersHelpers();
        $return = $getOrdersHelpers->getOrders(1, $job);
        $pageNum = $return[0];
        $totalPages = $return[1];

        if ($totalPages > $pageNum) {
            while ($pageNum <= $totalPages) {
                $getOrdersHelpers->getOrders($pageNum, $job);
                $pageNum++;
            }
        }

            $job->status = "complete";
        $job->save();

    }

}

redpajama_set_name: RedPajamaStackExchange


text:
package com.example;

import com.github.ksoichiro.ability.Ability;

public class User {
    private Ability ability;
    private long id;

    public User() {
        ability = new Ability();
        ability.addRule(Book.class);
    }

    public User(long id) {
        this();
        setId(id);
    }

    public long getId() {
        return id;
    }

    public void setId(long id) {
        this.id = id;
    }

    public boolean can(String action, Object subject) {
        return ability.allowed(this, action, subject);
    }

    public boolean canReadBook(Book subject) {
        return ability.allowed(this, Book.Actions.READ.name(), subject);
    }
}


text:
Women's eNews (https://womensenews.org/2017/04/pro-ana-blogs-continue-despite-new-online-policies/)
'Pro-Ana' Blogs Continue Despite New Online Policies
By: Sarah Emily Baum | April 24, 2017
More on Teen Voices
Subscribe to Teen Voices
Andrew_Writer on Flickr, under Creative Commons
MARLBORO, N.J. (WOMENSENEWS) –Heather's goal is not to recover from her eating disorder. It's weight loss "at any cost," said the 16-year-old Texan.
As part of the pro-anorexia, or "pro-ana," community, Heather runs a Tumblr blog posting diet tips, photos of dangerously skinny models (called "thinspiration") and requests for "ana-buddies," or online friends who make a mutual weight-loss pledge. She's been pro-ana for about three years.
While Heather knows society condemns such behavior, she doesn't think it's a problem. "I've been diagnosed with an illness but I don't really have one," she said. "I know there are health risks in [being anorexic], but it's worth it. I've lost so much weight."
Teen Voices talked to three such pro-ana bloggers, who agreed to be interviewed if only their first names and states were used, through video chats. In general, the girls didn't see their behavior as harmful, they joined the movement as a way to find community.
"Losing weight makes me feel I'm worth something," Heather said. "No one liked me before. I was fat and ugly. My 'friends' treated me horribly. But being pro-ana means I belong somewhere. My friends no longer say I'm an embarrassment."
While pro-ana may feel validating, it's harmful, said Claire Mysko, chief executive office of the National Eating Disorder Association. "Eating disorders can be isolating. Young women often turn to pro-ana communities as a place to make connections with people who understand what they're going through."
To prevent online posts that encourage extreme, unhealthy weight loss like Heather's, Facebook recently implemented artificial intelligence to detect harmful content and streamlined mental health resources by connecting distressed users with in-app support from crisis professionals. But that doesn't prevent girls from starting or visiting pro-ana sites.
'Not a Choice'
For Lee, 16, her four months of being pro-ana has helped pacify feelings of inadequacy. Though she can feel her bones through her skin, she said she is fully healthy.
"I don't need to 'get better.' I'm fine like this," said the Indiana native.
"It's not a choice," Lee added, when talking about her eating habits. "I HAVE to get to a certain weight, otherwise I hate myself. Even if my heart hurts and I'm cold and tired. Pro-ana is all that matters, the only thing making us want to live."
Disordered eating behavior isn't just about losing weight, said Mysko. "Eating disorders are tied with depression and self-harm. Many gravitate to harmful coping mechanisms and want to relate to others who go through similar struggles."
The rejection pro-ana teens feel in their real lives fuel their online behavior.
"I've tried talking with real life friends," said Heather. "They don't understand. Last year, they told a school counselor about me, who called my mom. It made me angry. I was doing this for myself. It wasn't anyone else's business."
Facebook announced in March "updates to tools and resources offer[ed] to people suffering" from mental illness. This puts power to censor pro-ana material in every internet user's hands; with the push of a button, anyone can report a post to be reviewed for deletion. Most social media platforms have similar systems directing users to mental health resources, like crisis hotlines, and offer content warnings on potentially triggering pages.
Heather said social media staff censoring posts is commonplace for pro-ana accounts. But that doesn't stop her. "We just make new blogs, new posts and new tags," she said.
One way blogs subvert censorship is by making accounts "private" so only approved users can see posts. Another tactic is using special codes known as "secret families": "Debbie" is depression, "Mia" is bulimia and Ana, of course, is anorexia.
"I know pro-ana is stupid," said Emily, 19, from Washington state, "but I use the hashtag because I need people to see my posts, hear my voice."
Social media platforms are working to be a more positive outlet for girls like Emily. "Pro-ana content is limited, often through trigger warnings or outright bans," said Mysko. "Facebook, Instagram and Pinterest are working with NEDA on tools that foster positive communities and make it easier to get help. Though this won't end pro-ana content, it's a start in limiting the trend."
Only the Start
However, Mysko added, regulating pro-ana is only the start of addressing disordered eating.
"While social media policies can be helpful tools for change, more must be done on a societal level to instill confidence in young people. Pro-eating disorder content won't disappear until our culture's focus shifts from prizing thinness to encouraging health at every size," she said.
Corporations know an insecure demographic is more likely to spend money on products claiming to "fix" them, like weight-loss pills or waist trainers. So companies have a financial incentive to make women hate their bodies.
"The diet industry makes billions each year, said Mysko. "Disordered behaviors often masquerade as attempts to get healthy and are encouraged."
Moreover, in a society that "equates a woman's value with her weight," as Mysko put it, self-hate becomes "normalized."
This universal normalcy, said Emily, justifies pro-ana's existence. "I'd never say things I say to myself to another…[But] when I post about hating myself, someone out there is reading that and thinking the exact same thing."
This story is part of Teen Voices at Women's eNews. In 2013 Women's eNews retained the 25-year-old magazine Teen Voices to continue and further its mission to improve the world for female teens through media. Teen Voices at Women's eNews provides online stories and commentary about issues directly affecting female teens around the world, serving as an outlet for young women to share their experiences and views. Learn More.
About Sarah Emily Baum
Sarah Emily Baum is a young Gryffindor from New Jersey passionate about activism. When she isn't writing, Sarah can be found engaging in rigorous Facebook debates, playing the ukulele, or asking strangers in the street if she can pet their dogs.
More by Sarah
Puberty, Pressure Lead to Less Exercise for Teen Girls
By: Sabrina Abrams | December 2, 2016
Puberty can make "exercise awkward," says teen Kiera Wright. This is one of the reasons teen girls fail to meet their physical activity recommendations.
Teen Ties Her Eating Disorder to Cultural Identity
By: Angela Hui | July 27, 2016
From our partner Proud2Bme
As an Asian American girl with an eating disorder, Angela Hui examines how the "sociocultural environment in which I was raised played a part in the development of my illness."
2 thoughts on "'Pro-Ana' Blogs Continue Despite New Online Policies"
Joanna on April 26, 2017 at 2:35 pm said:
To Lee who said, "It's not a choice:" this language confirms you have a disorder. Plus if your heart hurts and you're always cold and tired, you are not healthy. I sincerely hope you get better.
To Heather who feels rejected by her "real life friends:" They have your best interest in mind. Anorexia has the highest fatality rate of any psychiatric disorder, and even if you don't die, you could suffer from brain shrinkage due to lack of nutrition (resulting in a lower IQ), damage to your skeletal system, loss of bone density, infertility, and heart disease.
kira on December 7, 2017 at 5:48 pm said:
This article really describes the outcomes and reasons of pro-ana blogs.


On behalf of Radford J. Smith, Chartered posted in divorce on Tuesday, March 27, 2018.
If you are going through a high-asset Nevada divorce, you may suspect that your spouse is hiding assets from you in an attempt to skew the property division. Hiding assets before or during a divorce is nothing new, and thanks to Bitcoin and other cryptocurrencies, it now is easier than ever. If you have never heard of Bitcoin and similar digital currencies, you should research them and also alert your attorney to their existence. (S)he needs to add them to the possible ways in which your spouse could be hiding assets.
Bitcoin and the other cryptocurrencies are currencies, but not money as that word is commonly understood. They are virtual money that exists in "wallets" such as a file stored on a computer, external hard drive, thumb drive, smartphone or online in the cloud. They are easy to buy, easy to sell and easy to use in payment for an ever-growing number of goods and services. They also are easy to get out of the country since a person can have multiple wallets, some of which are not in the United States.
New digital currencies come and go and the value of all cryptocurrencies fluctuates from day to day, sometimes hour to hour. In some ways they are like a stock in that anything can happen. Some investors "make a killing" while others lose most or all of their investment.
While few, if any, digital currencies are 100 percent anonymous, all are pseudonymous. What this means is that a person does not need to use his or her real name or give any personal identification in order to buy, sell or trade them. Theoretically, all U.S. Bitcoin accounts must be linked to the owner's bank account or credit or debit card, but cash-for-Bitcoin trade opportunities are not difficult to find.
Not surprisingly, asset-hiding spouses are not the only people who love Bitcoin, et al. So do money launderers, particularly drug dealers, darknet marketeers, ransomware scammers and other cybercriminals. Since cryptocurrencies are virtual money, they leave no paper trails and their virtual trails are difficult if not impossible to find and follow.
If you suspect that your spouse is hiding assets but you and your attorney have yet to discover what, where and how, this is a strong indication that your spouse may have one or more Bitcoin accounts and/or wallets. Now that you know what to ask about in interrogatories, production of document requests and depositions, you are more likely to find them.
Related Posts: When might a forensic accountant help in a divorce case?, What happens to home equity when you divorce?, 3 key benefits of divorce mediation, What happens to the family business when you divorce?


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Evaluation of Escherichia coli Membrane Preparations of Canine CYP1A1, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26 with Coexpressed Canine Cytochrome P450 Reductase
Charles W. Locuson, Brian T. Ethell, Michael Voice, David Lee and Kenneth L. Feenstra
Drug Metabolism and Disposition March 2009, 37 (3) 457-461; DOI: https://doi.org/10.1124/dmd.108.025312
Charles W. Locuson
Brian T. Ethell
Michael Voice
Kenneth L. Feenstra
The preparation of bacterial membranes ("Bactosomes") containing expressed canine (beagle) hepatic cytochromes P450 (P450s) is described. cDNAs from seven canine P450s were subcloned into inducible expression plasmids and, for the first time, cotransformed and expressed with a canine P450 oxidoreductase in Escherichia coli to produce active, full-length, native sequence P450s. Enzyme expression levels, although variable, were generally sufficient to enable short incubation times and to limit the total protein present in enzyme incubations. Steady-state kinetics of CYP1A1, 2C21, and 2D15 Bactosomes demonstrated similarities with dog liver microsomes or Baculosomes. However, 3A12 lacked substrate inhibition in the formation of 1′-OH midazolam, and 2B11 displayed non-Michaelis-Menten kinetics, suggesting possible differences in protein interaction effects. In monitoring the metabolites of common P450 substrates, phenacetin deethylation, temazepam demethylation, and bufuralol 1′-hydroxylation were shown to be relatively selective reactions catalyzed by CYP1A1, 2B11, and 2D15, respectively. 1′-OH midazolam was formed in higher quantities by CYP2B11 and 2C21 than by 3A12, raising questions about the use of midazolam as a CYP3A12 probe in vivo. In summary, a panel of recombinant P450s was produced to make up for the lack of commercially available canine P450 isoforms. The Bactosomes are expected to facilitate reaction phenotyping and metabolic drug-drug interaction assessment in canine drug development and to enable the study of interspecies differences in P450-mediated drug metabolism.
The cytochromes P450 (P450s) are well known for their role in the metabolism of drugs and other xenobiotics. Whereas hepatocytes and other liver tissue preparations are a rich source of the P450 isoforms that tend to be involved in xenobiotic metabolism, use of recombinant, expressed P450s (rP450s) has grown over the last decade. Several recombinant human P450 isoforms are expressed from cDNA and partially purified as membrane preparations and are commercially available. Common uses of rP450s in the pharmaceutical industry include the assessment of drug-drug interactions (Crespi and Stresser, 2000), reaction "phenotyping," metabolite generation, and estimating the in vivo clearance of drugs (Venkatakrishnan et al., 2001).
Currently, nonhuman rP450s are not widely available. Yet, preclinical species used in human pharmaceutical research, as well as veterinary species, could arguably benefit from the availability of an expanded panel of rP450s. For instance, dogs are an important nonrodent species used in scaling pharmacokinetics and evaluating toxicology in drug development research. Yet, several notable differences have been described in the ligand selectivity and expression levels of human P450s and their canine homologs (Eguchi et al., 1996; Tibbitts, 2003). As a patient population, dogs are also increasingly being considered by pharmaceutical companies (Petkewich, 2007). For all of these reasons, canine and other nonhuman rP450s would prove to be a valuable tool to the pharmaceutical scientist.
To expand the number of canine rP450s available for research, we describe the plasmid constructs and bacterial expression methods for seven of the major beagle hepatic drug-metabolizing P450s. Shou et al. (2003) previously described the insect cell expression of the canine P450s with human P450 reductase. In the current work, we have cloned the hepatic beagle P450 reductase, coexpressed it with the dog P450s, and evaluated the activity of common substrates with a completely dog-specific enzyme system for the first time.
Materials. Chemicals were obtained from Sigma-Aldrich (Poole, Dorset, UK, or Milwaukee, WI), except for some metabolite standards obtained from BD Gentest (Woburn, MA), modified terrific broth powder (Merck, Darmstadt, Germany), isopropyl-β-d-thio-galactopyranoside Melford Laboratories, Ipswich, UK), and δ-aminolevulinic acid (Molekula, Gillingham, UK). Solvents were purchased from Honeywell Burdick & Jackson (Meskegon, MI).
Isolation of the cDNA Coding for Seven Canine P450 Enzymes and Canine NADPH P450 Reductase. The cDNA coding for the seven canine P450s were isolated by reverse transcription-polymerase chain reaction (PCR) from mRNA made from isolated cultured beagle hepatocytes. Before the isolation of the mRNA, the hepatocytes were treated in culture with 1 mM phenobarbital, 25 mM 3-methylcholanthrene, and 25 mM rifampicin for 48 h. Reverse transcription was performed using random primers and Superscript Reverse Transcriptase III (Invitrogen, Paisley, UK) following the manufacturer's instructions. The PCR was performed using Phusion DNA polymerase (NEB, Ipswich, MA), using conditions recommended by the manufacturer.
The primer pairs used for the PCR reactions to isolate the cDNAs coding for the P450s and P450 reductase are as follows: CYP1A1, GCTCCGATGATGTCCATGTTCAGACTCTC and GCGAATTCTAGGCTGCAGGGCTCTCAG; CYP2B11, ATGGAGCTCAGCGTCCTTCTCC and GTGAATTCAGCACCCTCCACGAGACAG; CYP2C21, ATGGATCTCTTCATAGTTCTGGTGAT and GCTCTAGATCAGACTGGAACAAAACAGAGCTTATAG; CYP2C41, ATGGATCCAGTTGTGGTTCTGGTG and CATCTAGATCACACGGGAATGAAGCAGAGC; CYP3A12, ATGGACTTGATCCCAAGCTTTTCCAC and AGTCTAGATCAGGCTCCATTTACACTCCCATC; CYP3A26, ATGGACTTGATCCCAAGCTTTTCCAC and CATCTAGATCAGGCTCCACTTACACTCCCA; CYP2D15, ATGGGGCTGCTGACCGGGGACAC and TGTCTAGACTAGCGAGGCTCCACACAGAGCTGG; and reductase, ATGAAGAAGACGGGCAGGAACATC and CTGAATTCTAGCTCCACACGTCCAGGG.
The accession numbers for the sequences are as follows: CYP1A1, L77459 (full-length sequence supplied by Dr. Magang Shou, Amgen, Thousand Oaks, CA); CYP2B11, M92447; CYP2C21, XM543973; CYP2C41, AF016248; CYP3A12, X54915; CYP3A26, AF547269; CYP2D15, AB004268; and reductase, XM852956.
The full-length cDNAs coding for the canine P450s were cloned into a pCW vector under the control of a tac tac promoter, fused in frame with the sequence coding for the leader peptide of the bacterial ompA protein. This system has been used previously to express native mammalian P450s in Escherichia coli (Pritchard et al., 1998). An Ala-Pro insertion was made between ompA and the CYP1A1 N terminus to enable more efficient cleavage in the bacterial membrane by the signal peptidase. The full-length cDNA coding for canine NADPH P450 reductase was cloned into a derivative of pACYC184 in frame with a tac tac promoter element (designated pCY070). A CYP1A2 isoform is known to be expressed in dog liver (Uchida et al., 1990), but it has not been cloned nor its expression relative to CYP1A1 characterized.
Coexpression of Canine P450s with Canine NADPH P450 Reductase. The expression plasmid for each canine P450 was cotransformed into E. coli JM109 (Promega, Southampton, UK) with the canine P450 reductase plasmid, pCY070. Next, a 3-ml starter culture was grown overnight at 37°C, and 2 ml was used to inoculate 200 ml of modified terrific broth in a 2-liter conical flask. The culture was grown at 30°C with shaking at 150 rpm until the OD600 reached 0.8 to 1.0 when expression of the recombinant proteins was induced by the addition of 0.5 mM isopropyl-β-d-thio-galactopyranoside and 1 mM δ-aminolevulinic acid. The induced cultures were incubated for an additional 48 h at 30°C with shaking at 150 rpm. The cultures were harvested by centrifugation, and the harvested cells were resuspended in 10-ml of buffer [100 mM Tris acetate (pH 7.6), 500 mM sucrose, and 0.5 mM EDTA]. A 0.5-ml sample was taken and diluted 1:1 with deionized water, and 1.25 ml of 20 mg/ml lysozyme was added to the remainder of the resuspended cells. Ten-milliliter deionized water was added to the resuspended cells, and the suspension was incubated at 4°C for 10 min. The lysozyme-treated cells (sphaeroplasts) were harvested by centrifugation, and the pellet was resuspended in 4-ml of buffer [100 mM potassium phosphate (pH 7.5), 20% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 1 μg/ml leupeptin]. The sphaeroplasts were disrupted by sonication, and the cell debris was removed by centrifugation at 12000g for 20 min. The bacterial membrane fraction was collected by centrifugation at 100,000g for 1 h and resuspended in 2-ml of buffer [500 mM Tris acetate (pH 7.6), 250 mM sucrose, and 0.25 mM EDTA]. Large-scale cultures were grown in 10-liter fermenters (Brighton Systems, Brighton, UK) under standard conditions and subsequently treated the same as the shake flask cultures above with volumes that were scaled appropriately. After the final resuspension in 500 mM Tris acetate (pH 7.6), 250 mM sucrose, and 0.25 mM EDTA, the membrane suspension was centrifuged at least once at 12,000g for 20 min and filtered through a 0.22-μm syringe filter to remove any intact cells.
The P450 content of the whole cells after harvesting and the membrane preparations were determined using Fe2+-CO versus Fe2+ difference spectra (Omura and Sato, 1964). P450 reductase content was estimated using a spectrophotometric assay to determine the rate of NADPH-dependent reduction of cytochrome c (Strobel and Dignam, 1978). The total protein content of the membrane preparations was determined using a Bio-Rad DC protein assay following the manufacturer's instructions (Bio-Rad, Hemel Hempstead, UK).
Kinetic Assays. Incubations of P450 drug substrates with the Bactosomes prepared from fermenter cultures were conducted to assess their activity. Reactions (0.1 ml) were conducted in 20-mm-tall 96-well polypropylene plates with 1 mM NADPH in 0.1 M potassium phosphate buffer (pH 7.4) containing 2.5 to 5 pmol of P450 (0.5 pmol/ml) across a range of substrate concentrations encompassing Km (1–1000 μM phenacetin, 1–500 μM temazepam, 1–1000 μM diclofenac, 0.1–100 μM bufuralol, and 0.2–200 μM midazolam). After 5 min at 37°C, reactions were quenched by the addition of 0.1 ml of formic acid (25%) containing an internal standard. Acetonitrile and dimethyl sulfoxide used to solubilize and dilute substrate stock solutions did not exceed 1.0 and 0.1% (v/v) of the final incubation volume, respectively. All experiments were controlled by using the same proportion of solvent in all reactions. Reactions were conducted in triplicate, except those containing CYP3A26 due to lower expression and need for higher levels of Bactosomes. Linearity of metabolite formation was established with time (up to 20 min) and with P450 concentrations (up to 50–100 pmol/ml).
Quantitation of metabolite formation was performed with ultraperformance liquid chromatography (UPLC) tandem mass spectrometry methods. In brief, an ACQUITY UPLC system (Waters, Milford, MA) with a Waters BEH Shield RP18 column (1.7 μ, 2.1 × 50 mm) or a Phenomenex Synergi Polar RP column (4 μ, 2 × 50 mm) (Phenomenex, Torrance, CA) was used for separation. Typical chromatography conditions began at 20 to 40% methanol in water containing 0.1% formic acid and used as a gradient from 0.2 to 0.4 min that reached 60 to 95% methanol with flow rates between 0.5 and 0.7 ml/min. Retention times of analytes ranged from 0.39 to 0.97 min. Detection was accomplished with a Sciex 4000 (Applied Biosystems, Foster City, CA) triple quadrupole mass spectrometer (Foster City, CA) operating in positive mode. The transitions used for quantitating metabolites were reported previously (Aidasani et al., 2008), with the exception of 4-OH midazolam (342 → 297). All methods were developed to ensure that substrate did not coelute with the monitored metabolites to reduce suppression, because coelution of midazolam suppressed the signal of 1′-OH midazolam and gave the impression of substrate inhibition. In addition, the two hydroxy midazolam metabolites required separation because both are detected with the m/z 297 transition.
The 4′-OH diclofenac peak, as verified with the synthetic 4′-OH diclofenac standard, had a retention time of 0.80 min in diclofenac incubations. However, this metabolite peak appeared in chromatograms as an early eluting shoulder to a larger peak at 0.82 min using UPLC separation (data not shown). This larger peak was dependent on the presence of NADPH and was present under varying liquid chromatography conditions so that it did not appear to be an artifact or a matrix effect. The unknown peak detected with the m/z 312 → 231 transition may suggest that CYP2C21/41 forms more 3′-OH diclofenac than human CYP2C enzymes. Quantitation was carried out with the merged peaks.
P450 Phenotyping. Reactions containing 50 μM phenacetin, 5 μM temazepam, 30 μM diclofenac, 10 μM bufuralol, and 4 μM midazolam were conducted near Km with all seven constructed canine Bactosomes in quadruplicate using the same conditions, volumes, and sample preparation as described above.
Kinetic Analysis. Enzyme kinetics for each substrate was defined using a single site (Michaelis-Menten) or two-site saturation model in SigmaPlot version 9.0 (Systat Software, Inc., San Jose, CA). Appropriateness of the kinetic model was assessed by the R2 resulting from nonlinear regression and the linearity of Eadie-Hofstee replots (V/[S] v V). Velocity of CYP2C21 slightly decreased at saturating diclofenac concentrations, but fitting with a substrate inhibition model greatly overestimated V. CYP2C41 demonstrated some biphasic nature, but regression failed with the two-site model. Therefore, diclofenac hydroxylation by both CYP2C enzymes was fit with the Michaelis-Menten model.
Expressed recombinant P450 preparations have proven to be a useful research tool in the drug discovery environment for the in vitro assessment of inhibition, reaction phenotyping, or to generate metabolites for identification purposes. However, species differences in the regioselectivity and turnover rate of drugs (Turpeinen et al., 2007; Fink-Gremmels, 2008) by P450s may complicate the scaling of preclinical pharmacokinetics and metabolite exposures from animals to humans. Therefore, a method that could be used to produce more rP450s from preclinical species was desired. Common expression hosts for mammalian P450s include insect ovary cells (Supersomes and Baculosomes), and human lymphoblastoid cells, but E. coli expression of P450s (Barnes et al., 1991; Parikh et al., 1997) and reductase (Shen et al., 1989; Blake et al., 1996) have also seen progress. Bacterial P450 preparations have even demonstrated surprisingly similar results to hepatocytes in the assessment of drug-drug interactions (McGinnity et al., 2008). Because beagle dogs are an important preclinical and veterinary species, a method used to express the major hepatic canine rP450s in E. coli (Pritchard et al., 1998), along with the canine P450 reductase, was shown to be a reliable approach to produce multiple P450 isoforms using the same conditions.
Expression of Canine rP450s. All but one of the tested canine hepatic P450s (CYP3A26) was produced well in E. coli by the addition of a bacterial sequence leader (Table 1). Moreover, this modification has been shown to be cleaved off in the bacterial membrane to yield the native P450, and no modification of the native canine P450 sequences was necessary. An important step in the present work is that a hepatic canine P450 reductase transcript was identified, cloned, and coexpressed with the rP450s from a separate plasmid. However, initial E. coli expression experiments carried out in shake flasks did give a large spread in the expression levels achieved for each P450 (Table 1). The levels of coexpressed P450 reductase as measured by NADPH-dependent cytochrome c reduction were less variable than P450 content between preparations.
Canine P450 and reductase expression yields using shake flask and fermenter cultures
All of the P450s were next scaled-up to expression in 10-liter fermenters, but, again, there was a wide variation in the yield of P450 between the P450s with the yield of CYP1A1 being the highest and CYP3A26 being the lowest (Table 1). The protein concentration tended to be lower (8.4–11.9 mg/ml) in membrane suspensions made from the 10-liter fermenter cultures than shake flask cultures (8.3–19 mg/ml) because of the extra centrifugation and filtration steps. CYP3A26 and CYP2D15 preparations were difficult to filter through the 0.22-μm syringe filters, so they were centrifuged more than once at 12,000g after resuspension of the membrane fraction. The reason for the poorer expression of CYP3A26 is not yet known, and it is likely that further optimization of expression conditions will be needed.
Activity Characterization. The activity level of the Bactosomes prepared from fermenter cultures was suitable for typical rP450 applications, requiring relatively low P450/protein levels and short incubation times. Catalytic efficiencies (V/Km) calculated from Table 2 were similar to the diclofenac (CYP2C21), bufuralol (CYP2D15), and midazolam (CYP3A12) values described previously in dog liver microsomes (Aidasani et al., 2008) and were also similar to those of rP450s expressed in insect cells in the cases of phenacetin, temazepam, and bufuralol (Shou et al., 2003; Lu et al., 2005). The apparent affinities (Km) and maximum velocities (V) for three substrates (diclofenac, bufuralol, and midazolam) determined with the beagle rP450/reductase Bactosomes were comparable with those described for both canine liver microsomes and beagle rP450s with human P450 reductase (Shou et al., 2003; Aidasani et al., 2008) (Table 2). Using the same three studies above as a comparison, velocities for phenacetin metabolism were higher with Bactosome rCYP1A1 than with microsomes or rCYP1A1 with human P450 reductase, and the Km for temazepam was lower with Bactosomes. The higher catalytic efficiency of CYP2C21 over CYP2C41 and of CYP3A12 over CYP3A26 was consistent with previous findings (Shou et al., 2003; Carr et al., 2006).
Canine rP450 Bactosome kinetic parameters
Some other notable findings were the nonhyperbolic kinetics of temazepam demethylation by CYP2B11 and midazolam 1′-hydroxylation and 4-hydroxylation by CYP3A26. In both cases, kinetics followed a biphasic response with substrate concentration. In addition, there was no indication of substrate inhibition on 1′-OH midazolam formation at up to 200 μM midazolam, which was surprising given that substrate inhibition is observed with canine liver microsomes (Aidasani et al., 2008) and CYP3A12 Baculosomes (Carr et al., 2006). This is the first observation of atypical kinetics displayed by rCYP2B11. Differences in protein-protein interactions caused by the presence of multiple P450s in microsomes, the species of P450 reductase, or the P450/P450 reductase ratio may explain these findings.
The P450/P450 reductase ratio will certainly affect velocities and, conceivably, might subtly modulate drug affinity, regioselectivity, and kinetic profiles via changes in P450 conformation. As mentioned previously, the canine rP450 content/cytochrome c reductase activity ratios were variable, but, on average, they were the same as the ratios characterized for human Bactosomes (Cypex Ltd., Dundee, UK) (data not shown). The CYP1A1, 2C21, 2D15, and 3A12 levels exceed P450 reductase by 2- to 9-fold (Table 1). It is interesting to note that this is the case with the physiological ratios, where total P450s outnumber P450 reductase ∼10:1 in purchased human liver microsomes and ∼27:1 in beagle liver microsomes according to cytochrome c reductase activities (XenoTech, LLC, Lenexa, KS). Although increased P450 reductase levels may enhance rP450 activity, what gives optimal reaction velocities in vitro may have fewer physiological characteristics of P450 catalysis in vivo where P450 oligomerization is probably important.
Phenotyping of P450 Substrates. The seven rP450 panel was screened to characterize the turnover of phenacetin, temazepam, diclofenac, bufuralol, and midazolam (Fig. 1). Such phenotyping experiments used to define the selectivity of substrates and inhibitors for specific P450s are now commonly conducted with panels of rP450s (Zhang et al., 2007). Phenotyping experiments with four of the studied substrates demonstrated that the P450 isoforms with the highest turnover were identical to that previously described with baculovirus-expressed P450s (Shou et al., 2003; Lu et al., 2005). The monitored metabolites of CYP1A1, 2B11, 2C21, and 2D15 were produced at levels 9-, 5.3-, 1.9-, and 3.8-fold greater than the enzymes producing the second highest levels of the metabolites.
Reaction phenotyping with phenacetin (A), temazepam (B), diclofenac (C), bufuralol (D), and midazolam (E) to determine the specificity of metabolite formation by seven hepatic canine rP450s. The monitored metabolites were 4-acetamidophenol, oxazepam, 4′-OH diclofenac, 1′-OH bufuralol, and 1′-OH midazolam, respectively. Experiments were performed in quadruplicate and normalized to the isoform with the highest specific activity (±S.D.).
1′-OH midazolam formation by the major hepatic canine rP450s is presented for the first time. A common CYP3A probe, midazolam, was not selective for canine CYP3A12 or 3A26 and, instead, CYP2B11 (56-fold) and CYP2C21 (16-fold) were larger contributors to 1′-OH midazolam formation. Considering that CYP2B11, 2C21, and 3A12 constitute ∼10, 34, and 15% of canine liver total P450 content (Eguchi et al., 1996), the relative in vitro turnover rate for midazolam by CYP3A12 would be estimated to be responsible for ∼2% of 1′-OH midazolam formed in the liver in the absence of any intersystem normalization methods (Proctor et al., 2004). Again, the Bactosome P450 reductase levels could be important, although the sheer magnitude in isoform differences might argue against this as the sole explanation. We previously found that common CYP3A ligands did tend to inhibit midazolam hydroxylation in dog liver microsomes (Aidasani et al., 2008), but the number of inhibitors identified for canine CYP3A was low considering the documented promiscuity of CYP3A enzymes. Taken together, the findings suggest that midazolam may not be the most sensitive in vivo canine CYP3A probe.
In summary, increasing the number of canine rP450s should give more opportunities to answer specific questions related to drug metabolism in dogs. In addition, the novel use of canine P450 reductase is expected to have an advantage over other commercial enzyme preparations that do not match P450s with P450 reductase from the same species. Based on the findings, we are working to make all seven canine P450s available for research purposes.
We thank Matthew Zaya for helpful discussions and the manuscript reviewers for comments.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.108.025312.
ABBREVIATIONS: P450, cytochrome P450; rP450, recombinant P450; PCR, polymerase chain reaction; UPLC, ultraperformance liquid chromatography.
Received October 23, 2008.
Accepted December 5, 2008.
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Drug Metabolism and Disposition March 1, 2009, 37 (3) 457-461; DOI: https://doi.org/10.1124/dmd.108.025312
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The reason we're talking a lot about videos here is that they have the highest potential to generate viral marketing. A stunning, interesting video is more likely to be watched and shared by people within a relatively short period of time, delivering that much-needed viral effect.
Without a doubt, the visual component of viral marketing is a great approach to appeal to the subjective, emotional factors that impact the decisions a typical real estate customer. As a real estate agent, you know perfectly well how important these emotional factors are for people who contact you, so let's give you the tips on viral marketing that you need to get everything right.
Unless you happen to be in the right place at the right time with a camera, you have to follow a number of specific rules to create a video that has the potential to go viral.
Let's be honest here: not every marketing video goes viral, so you need all your creativity to achieve your goal. That's why we've collected this list of ideas for a viral video to get your creative juices flowing and encourage original thinking.
Record a series of videos about the job a real estate agent.
Whatever option you choose, keep in mind that your video should be viewer-focused, e.g. have useful tips and takeaways for them, so avoid being too promotional.
Your business has a specific target audience, so chances are you've studied it to gain the insights into their lifestyle, buying behaviors, preferences, and interests. This information will serve you well now because you need to craft your video around the people you're targeting.
Using the language they're using. For example, in most cases, it's recommended to use natural, conversational language to connect with your target audience because it's the easiest one for them to understand, therefore, relate to. Online tools such as Hemingway Editor, A-Writer, Grammarly, and College Paper can help with getting your tone and style right.
Use a celebrity if possible. Your target audience certainly has some favorite actors, athletes, and other celebrities, so if you have a chance to recruit them, do so.
If you watch videos on a regular basis, chances are that you've noticed that the many popular videos are made in a mini-movie format. The main reason for that is that people are sick and tired of hard sell advertising, so they crave something authentic and non-salesy.
And that's why storytelling has become a hit in viral marketing today. It involves presenting content in the form of story to make it easy for the viewers to relate to the message and become loyal followers.
Take the following video for example.
It tells an incredible story which is also very engaging because it's extremely relatable. The creators of this video did a great job because they showed a kind of life that customers can have in the home that's available. And they did so by connecting with the viewers on an emotional level by showing family and their happy life. Everyone can relate to that.
Improving branding. If you use the elements of your personal brand in the viral video, you can raise awareness of your business and build a positive reputation.
Highlighting your professionalism and creativity. Buyers don't just want homes, they want perfect neighborhoods. By showing such important parts of the area as a local park, restaurant, or school demonstrates your ability to research and tell the story in a creative way.
Helping buyers connect with you on a personal level. This happens when you share videos of some important moments from your life, e.g. a successful project that resulted in helping a family to find a perfect home in the area they loved.
In an effort to attract maximum attention, many businesses use an approach to presenting themselves that is a bit light-hearted. While some think that's a risky road to take – and let's be honest here, it kinda is – but many people like when businesses do that because it makes them stand out among the others.
In order to make an "unexpected" video or another content type that has the potential to go viral, you have to step outside the box a little bit. For example, the video below, made by Josh Altman, a luxury real estate agent, is an excellent example of how a business tries a light-hearted approach to present itself, and does so in a very humorous way.
The unexpected and humorous nature of the video helped the video to get 1.1 million views so far. As the number of views as well as the comments under this video show, a piece of viral content is inevitably going to be discussed by many people, and the creators aren't going to get positive reviews from everyone. But that's totally okay because the main purpose here is to get people talking about you and your services.
Viral marketing is something that you as a real estate agent should definitely try because it can attract a lot of attention to you and your business. We focused on videos in this article because they're among the high-performing content types but keep in mind that a lot of tips here apply to infographics, articles, and other content as well.
Lucy Benton is a marketing specialist, business consultant and helps people to turn their dreams into the profitable business and currently works at Proessaywriting. Now she is writing for marketing and business resources. Also Lucy has her own blog Pro Writing where you can check her last publications.


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Premier calls for more efforts to promote China-US economic ties
Updated: Sep 20,2016 10:45 AM Xinhua
Premier Li Keqiang on Sept 19 met with US President Barack Obama, calling for stepped-up efforts to promote relations between the two countries.
The meeting was held on the sidelines of the ongoing 71st session of the United Nations General Assembly that opened earlier last week.
Enhancing China-US cooperation accords with the fundamental interests of the two peoples and the common wish of the world community, Premier Li said.
China is willing to further deepen bilateral, regional and global cooperation with the United States in a variety of fields, and expand pragmatic cooperation in economy and trade, military, law enforcement and judicial affairs, counterterrorism, cyber-security, people-to-people exchanges and local-level cooperation, the Premier said.
China is also willing to enhance coordination with the United States on major international issues including the Korean Peninsula and climate change, and work with the US to facilitate the implementation of the results of the latest G20 summit in Hangzhou, China, he added.
Premier Li urged the two sides to "inject new impetus" to carry forward the China-US cooperation agenda and promote the sustained, healthy and stable development of China-US ties.
CORNERSTONE & PROPELLER
Economic and trade cooperation is the "cornerstone" and "propeller" of China-US relations, Premier Li said.
The two sides should work together to ensure an early conclusion of negotiations for a China-US bilateral investment treaty (BIT), expand market access to each other, and create a better business environment and better cooperation prospects for enterprises from both sides, the Premier said.
The two sides should also properly handle economic and trade frictions to prevent bilateral economic and trade ties from suffering unnecessary distractions, he added.
Meanwhile, the Premier urged the US side to relax restrictions on high-tech exports to China.
Obama said economic relations are the stabilizing factor of US-China ties.
The US side supports China's reform process, the US president said, hoping the two sides can make further progress in BIT negotiations.
China supports the UN Security Council to make further reaction to the latest nuclear test conducted by the Democratic People's Republic of Korea, the Premier said as he met with Obama.
At the same time, China expects all relevant parties to refrain from any action that may lead to escalation of tensions, and opposes the US deployment of the THAAD missile defense system in the Republic of Korea, Premier Li said.
Premier Li reiterated China's position on the nuclear issue of the Korean Peninsula.
"China is committed to the denuclearization of the peninsula, to maintaining peace and stability there, and to settling the issue through dialogue," he said.
During the meeting, Premier Li also reiterated China's principles and stance on issues concerning Taiwan and Tibet, urging the United States to properly handle related issues.
Obama reiterated that the United States remains committed to the one-China policy.
On the Chinese currency, Premier Li said the exchange rate of RMB will stay basically stable at a reasonable and balanced level, adding that there is no basis for a sustained devaluation of the currency.
The two sides also exchanged views on global issues including sustainable development, refugees and peace-keeping, and agreed to maintain and enhance close communication, conduct effective cooperation and provide more public goods for the international community.
It is of great significance to the world for the US-China relations to stay stable, said Obama.
The United States hopes relations between the two countries can develop in a strong and sustainable manner, and praises the cooperation the two sides have conducted on international and regions issues, Obama added.
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Seth Baylis participates in "ice cross downhill" at Fenway Park in Boston, Mass.
Creating an Instagram, Seth Baylis never thought his work on social media would lead him to Fenway Park.
Baylis, junior in accounting and former member of Auburn University's hockey team, began an Instagram account years ago that he dedicated to hockey, which has led him to many opportunities as a popular influencer in the hockey world.
He is considered a major influencer and was recently invited to Boston for an all-expenses-paid trip to the Red Bull Crashed Ice 2019 event in Fenway Park that took place Feb. 8 and 9.
Baylis said he considers this Red Bull-sponsored trip to be the most rewarding opportunity he has experienced. This trip comes as a result of his growing Instagram page, @hockeytonky that is solely dedicated to hockey with a following of over a quarter of one million Instagram users.
The technical name of the sport being played at the event in Fenway Park is "ice cross downhill," which is an extreme winter sport involving downhill ice skating on a walled track, featuring sharp turns and high vertical drops.
Baylis, along with four other popular social media hockey influencers, got to experience that track for himself and the video of them doing so accumulated over 5 million views in one week.
That group of five is part of a larger group of hockey influencers called "welive.hockey," which was founded by Baylis' close friend Lee Elias. Between all of them, they have a social media following of 2.5 million users.
Red Bull had invited the group to film behind-the-scenes footage of the Crashed Ice event and demonstrate to the hockey audience what ice cross downhill is.
The group of influencers that Baylis attended the event alongside flew in from Birmingham; Philidelphia; Nashville; Cambridge, United Kingdom and Chicago.
Baylis said it is a special experience being part of a community that has many diverse backgrounds and hometowns but who all share the same love for hockey.
The event at Fenway Park brought hockey fanatics together from many different cities and countries, and Baylis said it was extremely rewarding and well worth the effort he put in over the years to grow his Instagram account.
Baylis became interested in hockey during the 2000s when his grandfather would take Baylis and his brother to East Coast Hockey League and Southern Professional Hockey League games in Biloxi, Mississippi.
Baylis did not know anyone at his small, private high school who knew much about hockey, so he started his hockey Instagram account in October 2012 as a way to connect with more people who shared his interest in hockey.
Baylis said he realized the future of social media was in the person behind the screen and not the memes, videos and other "share page" content, so he archived about 5,000 posts and switched to a more personalized brand of himself.
Baylis now uses his Instagram account to document his personal hockey pictures and experiences of hockey.
Baylis has also made some money by posting sponsored advertisements on his Instagram accounts for various companies and brands, but it was not until summer 2018 that Baylis went on a trip because of his Instagram account.
For that trip, Vincent Stellato, creator of The Original Hockey Sauce Kit flew Baylis up to Chicago to spend the weekend meeting other hockey influencers and filming content.
Baylis' most recent cross-country hockey adventure took place this year on March 8 and 9 when he was flown to the Let's Play Hockey Expo in Saint Paul, Minnesota, to play a pickup game with the top influencers and business owners in the hockey world, represent the businesses Hockey Wraparound and Bladeshades and attend the Single A State championship game.
Baylis has also met and interacted with many NHL players because of his Instagram account, including Dominik Kahun of the Chicago Blackhawks, who Baylis considers a close friend.
His large following consists of many big names in the NHL, including Marcus Pettersson and Niclas Almari of the Pittsburgh Penguins, Nico Hischier of the New Jersey Devils, Carter Hutton of the Buffalo Sabres, Canada women's hockey gold and silver medalist Natalie Spooner and Akil Thomas, one of multiple younger NHL prospects that follow Baylis' account.
Baylis has connected with many former NHL players, as well, including Chris Mason and Hal Gill of the Nashville Predators and Daniel Carcillo of the Chicago Blackhawks.
Baylis said that as a member of welive.hockey, he is able to work with Bryce Salvador, former captain of the New Jersey Devils.
Baylis said since the South is a not considered a traditional hockey market, he considers his participation with the sport to be an especially rare experience for him, enabling him to brand himself as a voice of hockey in the South.
Baylis plans to move to Nashville, Tennessee, after completing a master's degree in accountancy at Auburn University, while maintaining his involvement in the hockey world.


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Sheriff Authier began his law enforcement career in 1978 with the Taylor Lake Village Police Department as a reserve officer. A year later he hired on as a patrolman with the Nassau Bay Police Department. In 1982 the Sheriff was recruited by the Texas Department of Public Safety and after rigorous training earned the position of Texas State Trooper. In 1994 he tested for and was promoted to Sergeant in the Ranger Service and assigned to Ranger Company "B" and stationed in Parker, Palo Pinto, and Young Counties. Sheriff Authier served in that capacity until his retirement in 2008. Shortly after his retirement, Sheriff Authier was hired by then District Attorney Don Schnebly as a criminal investigator for the Parker County District Attorney's Office. In 2009 Sheriff Larry Fowler appointed Authier to Chief Deputy of the Parker County Sheriff's Office, where he served with distinction until his appointment to Sheriff. Sheriff Authier graduated from San Jacinto College in 1978 with an Associate's Degree in Criminal Justice.
Sheriff Authier is the current Board President of the Freedom House and a former board member with the Children's Advocacy Center of Parker County. Sheriff Authier is a member of the Texas Ranger Foundation, Former Texas Ranger Foundation, Texas Department of Public Safety Mutual Association, Homicide Investigators of Texas, Texas Sheriff's Association, East Parker County chamber of Commerce, Weatherford Chamber of Commerce, Springtown Chamber of Commerce, and National Rifle Association.
Sheriff Authier has been married to his wife Linda for 38 years. They have two married children, Mandy and her husband Stephen and their son Simon, and Mike and his wife Lauren, and their daughters Brinlee and Kirby, all of Weatherford.
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Sometimes, It takes an ECO village
The ECO village
Research shows that while many people are willing to perform sustainable behaviour, much of this good behaviour can quickly wear off as it can be hard to see the impact of the change. People may then be inclined to drop out or quickly go back to old behaviours. To make sure that users of the ECO coin would be more intrinsically motivated, we set up a future scenario to make sustainability more tangible.
Over the past year, we have been experimenting with showing people what kind of future impact they could have when they perform actions with the ECO coin. We did this in two places, at the headquarters of L'Oréal Netherlands and at the TU/e(Eindhoven University of Technology) campus in the department of Industrial Design.Here we crafted the ECO village, a fictional community that grows as participants perform actions incentivised by the ECO coin. Different future technologies were introduced over time to make users aware of the future scenario.
Travelling by public transport during the week meant that users were rewarded with ECO coins and that the ECO village received autonomous cars or modular trains.Saving energy lead to solar panels and windmills being installed in the ECO village. New technologies were introduced in the ECO village every day as it progressed into the future.
ECO coin xL'Oréal
AtL'Oréal, we were invited to make 250 staff members more sustainable. During the one-month implementation of our sustainably currency, the vast majority of their employees fanatically earned ECO coins. Over 4000 actions were performed in total in just 20 days! One big scale model, which showed the neighbourhood around L'Oréal's office (their ECO village) was present in the canteen and was updated once every week, based on the actions of all the employees together.The employees liked seeing the landscape change and improve, but we soon learned that they found it hard to distinguish the exact impact that their personal actions had.
Next to the model, we also invited the employees to think about what the future of theECO coin might be like by introducing dilemmas about the ECO coin. An example of such a dilemma was whether airline companies should be allowed to join theECO coin network or whether the amount of ECO coins earned for an action should be changed in different locations. These dilemmas sparked several discussions about how the ECO coin might be implemented on a global scale!
ECO coin xTU/e
After learning a lot from the L'Oréal implementation, we took our ideas back to the drawing board to improve the ECO village experience. We particularly focussed on adding a personal perspective and improving the visibility of individual impact in the scale model. We then carried out a two-week trial with three groups of staff and students at TU/e. Each group of three to four students of staff received their own small-scale model and every member of the group was responsible for one quarter of the landscape.
The elements they received were based on their individual actions, and instead of once every week they received elements daily through a fictional 'drone delivery'. In this case the users could place their own parts in their landscape. The model could also be scanned in an augmented reality app for extra insight into the technologies and the inhabitants of the future village.
The participants at TU/e said that they very much enjoyed getting this story as a reward for their actions. It even motivated them more than the fact that they could spend their coins in some cases: "Having a kind of possible ethical implications being the reward to me feels nicer as a whole than getting a thing".They even wanted to use their ECO coins to buy more tiny trees to decorate their landscapes! While they did not like the augmented reality as a medium so much, they liked "the personal scenario because you already get the information and then it's nice to have a human perspective instead of something very abstract".
Could we use future fiction to stimulate even more sustainable actions? Can we up-scale the ECO village experience or make it virtual to reach people all around the world? A lot of interesting ideas came from this explorative project. Stay tuned to see what happens next at the ECO coin!
The ECO village will be on display at Dutch Design Week at the Design United exhibition in Het Klokgebouw, as well as the Embassy of Sustainable Design at the Innovation Powerhouse.
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/ Dan Fogelberg
July 15, 2019 By Joe Marchese 10 Comments
Shimmering guitars, breezy horns, smooth keyboards, and crisp vocals on well-crafted songs with catchy choruses: all of those qualities might have once described "soft rock" or "adult-oriented rock," but more recently the genre has experienced a resurgence as "yacht rock." Though the term was originally intended in a pejorative way, it's come to be accepted by many of the progenitors of the genre including Michael McDonald and John Oates. There's an entire book on the yacht rock phenomenon, a
Categories: News Formats: CD, Digital Download, Digital Streaming, Vinyl Genre: Classic Rock, Pop, Rock Tags: 10cc, Dan Fogelberg, Dave Mason, Kenny Loggins, Peter Frampton, Poco, Rupert Holmes, The Climax Blues Band, Various Artists
February 8, 2018 By Randy Fairman 4 Comments
Real Gone Music has announced that, in conjunction with Sony's Legacy Recordings, the label is adding a new title to the long-running Essential series with a volume devoted to singer-songwriter Eric Andersen. The 2-CD The Essential Eric Andersen, due March 30, features 33 songs chosen by Andersen himself which span his entire career and feature recordings from eighteen albums and ten different labels including Vanguard, Warner Bros., Columbia, Arista, Gold Castle, Appleseed, Meyer, Ryko,
Categories: News Formats: CD Genre: Folk Tags: Dan Fogelberg, Eric Andersen, Joni Mitchell, Lou Reed, Richard Thompson
November 17, 2017 By Joe Marchese Leave a Comment
Welcome to this week's Release Round-Up! Queen, News of the World: 40th Anniversary Edition (UMC (U.K.) / Hollywood (U.S.) (Amazon U.S. / Amazon Canada / Amazon U.K.) Queen's sixth album, 1977's News of the World, is being generously expanded for its 40th anniversary in a new 3CD/1DVD/1LP box set. The original album is presented on CD and LP. The first bonus CD, the entirely unreleased Raw Sessions, presents an alternate version of the original album from demos, outtakes and live
Categories: News Formats: Blu-Ray, Box Sets, CD, Digital Download, Digital Streaming, DVD, Vinyl Genre: Classic Rock, Country, Disco/Dance, Holiday, Pop, Popular Standards/Vocal, R&B/Soul, Rock, Soundtracks Tags: America, Bob Seger, Carpenters, Chuck Berry, Dan Fogelberg, Diana Ross, Garth Brooks, Green Day, Jimmy Buffett, John Fogerty, Mavis Staples, Micky Dolenz, Mike Love, Paul McCartney, Queen, The Bee Gees, The Doors, The Moody Blues, The Sweet, Whitney Houston
May 12, 2017 By Joe Marchese 2 Comments
Welcome to this week's Release Round-Up! Dion, Kickin' Child: The Lost Album 1965 (Norton) (Amazon U.S. / Amazon U.K. / Amazon Canada) Norton Records has rounded up 15 songs recorded by Dion DiMucci and his group The Wanderers with producer Tom Wilson over sessions between spring and fall of 1965. It all adds up to a trailblazing folk-rock album featuring original songs and covers of Bob Dylan, Tom Paxton, and Mort Shuman. Also available on vinyl. Read more here! Brinsley
Categories: Release Round-Up Formats: Box Sets, CD, Digital Download, Digital Streaming, Vinyl Genre: Blues, Cast Recordings, Classic Rock, Pop, Rock, Soundtracks Tags: Bette Midler, Brinsley Schwarz, Dan Fogelberg, Dion, Stephen Sondheim, The Allman Brothers Band, Todd Rundgren
May 6, 2016 By Joe Marchese 4 Comments
Welcome to this week's Release Round-Up featuring Second Disc Records' very first title of 2016, plus more fantastic releases from our partners at Real Gone Music and titles from Brian Wilson, The Cars, and more! Bobby Darin, Another Song on My Mind: The Motown Years (Second Disc/Real Gone) (Amazon U.S. / Amazon U.K. / Amazon Canada) We know that you all know about this one...it's the ultimate celebration of Bobby Darin's time at Motown Records on 2 CDs, newly remastered from the
Categories: Release Round-Up Formats: CD, Digital Download, Vinyl Genre: Classic Rock, Country, Gospel, Jazz, Pop, Popular Standards/Vocal, R&B/Soul, Rock Tags: Bobby Darin, Brian Wilson, Cactus, Cannonball Adderley, Cyndi Lauper, Dan Fogelberg, Grateful Dead, Johnny Paycheck, Ryan Adams, The Beau Brummels, The Cars, The Move, The Rides, The Staple Singers
March 17, 2016 By Joe Marchese 5 Comments
Real Gone Music has just announced its May release slate, and with rock, jazz, gospel, country, pop and beyond, it's one of the label's most diverse months yet! Naturally, we're partial to the May 6 release of Bobby Darin's Another Song on My Mind: The Motown Years from Real Gone and our own Second Disc Records (get all of the details right here!), but there's plenty more on offer, too! Bobby Darin was a remarkable singer-songwriter; another beloved titan of popular song is the late Dan
Categories: News Formats: CD Genre: Classic Rock, Country, Gospel, Jazz, Pop, Popular Standards/Vocal, R&B/Soul, Soundtracks Tags: Bobby Darin, Cactus, Cannonball Adderley, Dan Fogelberg, Grateful Dead, Johnny Paycheck, The Beau Brummels, The Staple Singers