How can I reach more customers?
What channels should I use in my marketing campaign?
Where should I direct them to?
Reaching more customers is a goal of almost every single organization. The more people you reach with your messaging, the more opportunity you have to sell your products or services. The difficult part is determining how you're going to plan and implement a strategic marketing campaign, what your campaign strategy should be, and how to tell if it is working for you.
Marketing is never set in stone. It is a constant adaptation to your market and audience to figure out what works best. What you thought may be your biggest revenue generators could in fact be your least valuable tools. This is the importance of analytics. A well created strategic marketing campaign covers a variety of channels that your audience uses, engages them with applicable and interesting content, and will provide you valuable analytics into how each channel performs.
The next step is content creation. Without the ability to generate content that your target audience finds interesting and valuable your marketing campaign will fall flat. Mixing in all types of content will ensure you have a well rounded approach that your entire audience wants to interact with.
Finally, authenticity and consistency are key during campaign development and implementation. Creating a personal connection with your audience will turn them from potential customers to loyal brand followers. A proper marketing campaign will greatly help you to reach your target audience, through the correct channels, at the correct times.
If you have any questions about your campaign strategy or how to get started on your next big marketing campaign then give us a call!


Like Good and Evil, Sales and Marketing are locked in an eternal struggle — both sides seeing themselves as good, of course. Marketers blast sales teams for not following the plan; sales teams blast marketers for saddling them with ridiculous plans. Any B2B facing this is in a no-win situation.
Fortunately, there's a simple solution to marketing-sales tension: communication. So simple, in fact, it's usually overlooked. Here, then, are a few suggestions for getting your sales and marketing teams singing in harmony instead of screaming at each other.
Send marketers out into the field with sales reps. Let the see how the sales process works first hand. Let them get to know the customers first hand, and how sales reps use the collateral they are producing.
Involve sales reps and/or sales managers in the marketing strategy. In addition to providing great input, sales reps and managers will own a piece of the strategy and thus be more inclined to support it.
Make sure sales success stories are communicated to marketing. One of the reasons Sales thinks Marketing works in a vacuum is because Marketing works in a vacuum. But whose fault is that if Sales doesn't take the initiative and let Marketing know what's going on in the real world?
Make sure sales failures are communicated to marketing. See above.
Let sales know the why behind the what. Sales reps are highly focused on customer needs; as a result, they may overlook the internal priorities that drive marketing strategy. But, when marketers take the time to explain why a particular product is being promoted, or why a particular customer segment is being targeted, sales people are far more likely to get onboard.


text:
import {Component, OnChanges, Input, SimpleChanges} from "@angular/core";

@Component({
  selector: 'mpt-gravatar',
  styleUrls: ['./gravatar.component.css'],
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})
export class GravatarComponent implements OnChanges {

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  isLoading: boolean = true;

  @Input() hash: string;
  @Input() size: number;
  @Input() alt: string;

  ngOnChanges(changes: SimpleChanges): void {
    if (this.hash) {
      this.imageUrl = `https://secure.gravatar.com/avatar/${this.hash}?s=${this.size}`;
    }
  }

  stopLoading(): void {
    this.isLoading = false;
  }

}

redpajama_set_name: RedPajamaGithub


King Pine can be the ideal place to ski or ride. Additional adventure can be found at the Twisted Pine terrain park. King Pine at Purity Spring Resort does offer night skiing and snowtubing. Additional King Pine amenities include an indoor pool and hot tub. An alpine ticket also gets you cross-country skiing, snowshoeing, and ice skating while the ticket is valid. Share your King Pine Ski Area reviews or comments, contact us.
King Pine Ski area features 3 triples, a rope tow, a magic carpet and a tow-handle lift. Alpine and snowboard rentals and lessons are available.
This ski area is known for it's family friendly atmosphere, affordability, impeccable grooming and consistent snow conditions. Additional amenities at King Pine and Purity Spring Resort include; night skiing, snowtubing, sleigh rides, nordic skiing, Zamboni prepared ice skating, an indoor pool and fitness center.
Getting There: King Pine ski area is located off Route 153 in East Madison. Take Route 16 North to Center Ossipee, take Route 25 East (5 miles) to Route 153 North (6 miles).


Suddenly the Highlanders side, who were previously soaking up possession and allowing few opportunities, were being run ragged. They couldn't organise their defensive line, and when Folau found himself one-on-one with acres of space against Liam Squire, he made the most of it, speeding past to set up Foley's second try, and the Waratahs' third strike with Naholo sidelined.
It was all too late though, as the Waratahs booked a semifinal clash against the Lions by holding on with 14 men - a missing skill that the Highlanders will be ruing all off-season.


text:
Shirley Temple's Children Remember Their Late Mother: "She Was an Amazing Person" (EXCLUSIVE)
Updated: May 3, 2018 1:33 pm·
By Closer Staff
Robin Roberts Gives Update on Amber Laign 1 Week After Father Died
Changing It Up! Prince William Receives New Title From Queen Elizabeth
She died at age 85 in 2014, but Shirley Temple's children and fans gathered in LA on Monday, April 23 to celebrate what would have been her 90th birthday. "We had a lot of parties," the late star's daughter Susan Black exclusively told Closer Weekly of her mom, who was a licensed interior decorator for most of her life.
"We had a lot of fun decorating the dining room," Susan continued. Shirley took her domestic responsibilities just as seriously as she did being a movie star or, later a US ambassador. "Being a wife and mom is the greatest of her achievements," Susan said. "We were shopping and travel buddies. We went many places together. She was one of my very best friends."
Shirley with two of her children in 1957.
Being raised by Shirley and her husband, Charles Black, "seemed very normal to us," her son Charles Black Jr. said. "The only time I noticed her star quality was when someone else would ask for her autograph. She was an amazing person." Shirley's children similarly opened up about life with their famous mom during another previous interview with Closer Weekly. "She was wonderful — and normal," Charles Jr. shared. "We had dinner at the table every night all together."
"She was devoted and generous, and she could be a little stern if we didn't behave. But she was also a lot of fun. Very inventive and imaginative. She was all about her children and her husband," Susan said.
For more on Shirley Temple and her family, pick up the latest issue of Closer Weekly, on newsstands now — and be sure to sign up for our newsletter for more exclusive news!
More in Style
Owner of Missing Shirley Temple Dress Offers Big Reward for Lucky Finder (EXCLUSIVE)
Shirley Temple's Blue Diamond Ring to Fetch $35 Million at Auction
Exclusive Shirley Temple's Oldest Friend Shares Secrets About Her Path to Stardom

redpajama_set_name: RedPajamaCommonCrawl


"Lester P. Seaver, 52, who resides 1-1/2 miles south of Milan on Route 67, was found dead shortly before 6 last evening in the cellarway at the rear of the State Furniture Co., 1134 Fifteenth avenue, East Moline.
"Neighbors reported that a noise was heard in the alley and Walter E. Crow, who lives in an apartment above the store building, investigated. He reported that he found the body of the Milan man at the foot of the stairway. Coroner Leslie Banning, investigating the death, said this morning that Seaver apparently had fallen down the 14 cement steps of the stairway. Cause of Seaver's death was not determined immediately although it is believed he may have suffered a heart attack.
"East Moline police and fire departments were called but efforts to revive the man with a resuscitator failed. It was not learned what Seaver was doing in the alley at the time of his death.
"The body was taken to the Crummy funeral home, Reynolds, where an inquest was scheduled to be held this afternoon.
"Funeral services will be held at 2 Monday afternoon in the Crummy funeral home, the Rev. Roy E. Stocking, retired Methodist minister, officiating. Burial will be in Memorial Park cemetery, Rock Island. Friends may call at the funeral home after 6 tonight.
"Lester Philip Seaver was born in Taylor Ridge, Jan. 21, 1898, a son of Jacob and Anna Seaver. He attended Rock Island schools and normal school in Moorhead, Minn. He resided in Rock Island county all of his life except for 10 years when he lived in Minnesota.
"Mr. Seaver was a veteran of World War 1. He was a member of the Rock Island Eagles and the Congregational church in Moorhead, Minn.
"Surviving are the widow; the parents in Reynolds; a brother, Douglas B. Seaver of Milan; a niece and a nephew."
"Milan Man Found Dead at Foot of Stairway in Alley," obituary, The Dispatch [Moline, Ill.], 28 October 1950, Page 3, column 1, Lester P. Seaver obituary; digital image, Newspapers.com (https://www.newspapers.com : accessed 29 November 2018).
The obituary provides his full name, birth date and residence address, does not name his spouse, and names his parents and brother. Apparently, he had no children. You would think that the funeral home would choose a more dignified name...perhaps it was named after a person.
Lester Philip Seaver was born 21 January 1898 in Taylor Ridge, Illinois, and died 27 October 1950 in Moline, Illinois. He was the son of Jacob Allen Seaver (1868-1962) and Anna Clarke Bendle (1867-1959). He married Ada Faye Wakefield (1900-1988).
I had Lester and his wife and his Seaver ancestral families in my RootsMagic family tree.
I am not related to Lester Philip Seaver - his great-grandparents, Jacob and Esther (Croberger) Seaver were born in Virginia, and settled in Switzerland County, Indiana.


text:
Vol. 12, No. 5, 2013, pp. 771-776 Bioline Code: pr13096
Tropical Journal of Pharmaceutical Research, Vol. 12, No. 5, 2013, pp. 771-776
en High Performance Liquid Chromatographic Determination of Phenolic Compounds in Propolis
Yang, Li; Yan, Qing-Hua; Ma, Jin-You; Wang, Qing; Zhang, Jian-Wei & Xi, Guo-Xi
Purpose: To develop a simple, precise and rapid high-performance liquid chromatographic technique coupled with photodiode array detection (DAD) method for the simultaneous determination of rutin, quercetin, luteolin, genistein, galangin and curcumin in propolis.
Methods: Ultrasound-assisted extraction was applied to isolate six antioxidant phenolic compounds from propolis samples. Chromatographic separation was performed using an Eclipse XDB-C18 column (150 mm×4.6 mm, 5 μm) by isocratic elution at 25 °C. The mobile phase consisted of phosphate buffered saline (pH = 4.5) and methanol (40/60, v/v). The flow rate was set at 0.8 ml/min and the effluent was monitored using a DAD set at 260 nm.
Results: Excellent linear correlation coefficient was observed in the range of 0.9977 to 0.9997. The reproducibility of the method was 1.58 % (RSD) and accuracy was confirmed with average recovery ranging from 95.71 to 104.26 %. Six antioxidant phenolic compounds in propolis samples were successfully determined within 50 min.
Conclusion: The proposed isocratic LC method gave good separation and accurate determination of rutin, quercetin, luteolin, genistein, galangin and curcumin in propolis. The proposed method showed good sensitivity, linearity, repeatability and accuracy.
Propolis; Rutin; Quercetin; Luteolin; Genistein; Galangin; Curcumin; HPLC


text:
Q: Link to an external javascript file with WordPress Excuse me, I'm very new to PHP and WordPress, but I'm trying to link to an exterenal js file called trans.js, that relies on jQuery. Here is the code at the beginning of the header.php:
  <script type="text/javascript" src="<?php bloginfo('template_url'); ?>/js/trans.js"></script>

and here is the enqueue within the functions.php

function twentytwelve_scripts_styles() {
    global $wp_styles;

    wp_enqueue_style( 'twentytwelve-style', get_stylesheet_uri() );

}

add_action( 'wp_enqueue_scripts', 'twentytwelve_scripts_styles' );

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    wp_enqueue_script(
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        get_stylesheet_directory_uri() . '/js/trans.js',
        array( 'jquery' )
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}

add_action( 'wp_enqueue_scripts', 'my_scripts_method' );

Yet it still does not link up with the trans.js — does anyone know why this is?

A: add_action( 'wp_enqueue_scripts', 'my_scripts_method' );

 function my_scripts_method() {
    wp_register_script( 'my-first-script',   get_stylesheet_directory_uri() . '/js/trans.js', );
    wp_enqueue_script( 'my-first-script' );
}

Try this, i guess you dint register the script.

redpajama_set_name: RedPajamaStackExchange


text:
Søren Peder Lauritz Sørensen, danski kemik, * 9. januar 1868, Havjebjerg, Danska, † 12. februar 1939, Charlottenlund.

Od leta 1901 je bil Sørensen vodja uglednega laboratorija Carlsberg v Københavnu. V kemijo je uvedel pomembno brezrazsežno število pH kot merilo za koncentracijo hidronijevih ionov v raztopini.

Glej tudi
 seznam danskih kemikov

Sklici

Sørensen, Søren Peder Lauritz
Člani Kraljeve švedske akademije znanosti
Danski akademiki

redpajama_set_name: RedPajamaWikipedia


text:
GIANT THIEF
"A fast-paced, witty and original fantasy, reminiscent of Scott Lynch and Fritz Leiber."
-Adrian Tchaikovsky, author of the Shadows of the Apt series
Sample Trailer Related Media Reviews
So what's a 'Giant Thief' when it's at home?
Giant Thief is my first novel, a comic fantasy adventure, published by Angry Robot early in 2012.
And what does it do?
What do you mean, what does it do? It's a book.
What I mean is, what's it all about?
Ohhh. Okay. You could have just said that.
See that dashing but shifty-looking bloke on the right there? That's Easie Damasco. The reason he has a big, suspicious-looking bag in his hand? That's because he steals stuff. He steals stuff a lot. Anything that's not nailed down and might be valuable, really. Nothing is too small and nothing's too big to attract his criminal attentions. Take, for instance a giant. Which, funnily enough, is just what Damasco does. Because the giant was there, he wasn't nailed down, and sometimes a giant is the one thing you happen to need in a pinch.
So that explains the title. I mean, this isn't a book about a giant who's also a thief. Although, come to think of, it is that too. Not that Saltlick (the giant's name is Saltlick) wants to get dragged into such things; he just wants to go home, and maybe save his people from being forced into war by a tyrannical warlord along the way.
But when you end up spending time around someone like Easie Damasco, however inadvertently, bad things start to happen...
He does look a bit shifty. But isn't it a shame to have all those words covering that lovely artwork?
Well, those words are the title, not to mention my name. So I'm going to have to say no, not really. But it is a gorgeous cover, isn't it? It's by Angelo Rinaldi, who happens to be one of my favourite artists working in the industry today.
Tell you what, here's a version without all that wacky text.
Much Better. I might trust you a little more if I knew lots of people other than you had said nice things about your book, though.
What do you know? They have. You can read a few choice extracts here. Also, there are some interviews, guest blog posts and such here that might help you make up your mind.
Point taken. But would it be too much to ask to read a bit of it?
Nothing's too much for you. Why don't you have a look at this?
Hmm. Interesting. So what's in the bag?
I can't tell you that. You'd have to actually buy the book and read the rest.
And are there any more of these so-called Tales of Easie Damasco?
There absolutely are. The second, Crown Thief, came out in late 2012, and the third, Prince Thief, was released in October 2013.
All right. Say I want to buy this 'Giant Thief' of yours. What do I do now?
Well, your local book shop (assuming you're lucky enough to have a local book shop) should be stocking it. Failing that, why not try one of these?
UK Print & Ebook Amazon.co.uk | Book Depository | Waterstones | WHSmith
US Print & Ebook Amazon.com | BarnesandNoble.com | IndieBound.org
DRM-Free Epub Ebook Robot Trading Company


Every Payments Service Provider (PSP) offers something different. As a business, you'll be choosing your PSP based on a number of factors, one of which should be how you can make your customers' lives – and payments experience – easier. This starts right at the onboarding process.
While this might sound simple, with the many PSPs out there it's important to know what type of onboarding solution will suit your business. Here's our top three features to look out for.
If a PSP's onboarding process isn't simple, then there's a good chance that the rest of the service won't be simple either. Look for a PSP who offers a quick (and no fuss) onboarding process that means you'll be able to get started almost immediately. At Bambora we don't think businesses should stop for us, so we've developed an onboarding process that means business owners can be up and running - and taking payments - within 48. No fuss and nothing complicated. If you're planning on switching payments providers, make sure you don't go through the rigmarole of signing up for a merchant account again. Lots of PSPs will require new merchant accounts which means that your valuable, historical, payments data will be lost. Win favour with your customers by choosing a payment gateway that allows them to use their existing accounts, eliminating the need to fill in duplicate forms that can be time consuming and inconvenient. Look for a PSP who has relationships with multiple banks in Australia to ensure smooth banking integration.
Business is complicated enough and any payments provider worth their salt will know that simple integration is paramount for business owners. Payments should slot smoothly into your processes to ensure that business remains as usual and your customers don't experience any delays or unnecessary admin (like re-entering pre-populated field information.) Effective integration functionality like a reliable onboarding API lets businesses set up their existing merchant accounts speedily and with as little bother as possible. An onboarding API will ensure that pre-populated fields remain the same, as the data can be retrieved easily. Your website will also be able to retain its current look and feel.
When choosing a payments provider consider who is going to take on the risk for you and your customers. For example, what happens when customer payments fail? You'll need a PSP who can take on the risks for you. At Bambora, we are Level 1 PCI compliant which means we can assume all your payments risks, as we work directly with banks and can cover all your business' PCI requirements. There's plenty of PSPs out there who push risk back onto business owners, which can lead to tricky situations if your customers fail to produce funds. You'll also need fraud tools and authorisation processes that protect your business – everyday.
Get off to a good start with a well-designed and well-executed onboarding process. A great PSP will have all the features you need to deliver better payments for your customers: easy sign-up, great onboarding API and options that give you options to your customers; multiple payment method functionality and customisable interfaces that will help you target and attract more customers.
Jump onboard now and start taking payments right away.
Fancy finding out more about Bambora's speedy onboarding process? Contact one of our team today.


text:
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Mrs. Hayes' has been teaching at Saint Charles for 2 years. Her experience includes long-term substitute teaching in both San Diego Unified and the Diocese in all grade levels from Pre-K to 5th and Special Education. She attended CSU East Bay and SDSU and completed the credential program at CSU San Marcos. She loves her family (her daughter is in 4th grade here at SCCS!), her English Bulldog, Opie, watching brains grow and being outdoors. Come visit our fabulous class!
Leave your questions and comments here.
May God bless you and your family. Hope to see you soon!


There are online GoPro videos taken from the perspective of nearly every athlete and profession. But what about a Jedi? YouTube user Bill Parker imagined what it might look like if a Jedi Knight put on a GoPro camera while battling Empirical forces.
Swiss pilot Yves Rossy is famous around the world for inventing his very own jetpack that actually works. Yes, just like The Rocketeer. It's no wonder Yves is better known as Jetman. XDubai invited Jetman and his protege Jetman Vince Reffet to fly above Dubai.


We're celebrating . . .
Drones on Demand has clocked up 100 hours of flight working on projects for our clients since the company was founded in January 2017.
We've also put in the miles, travelling from one end of the country to the other, ending up in some rather unusual places. Along the way we've conducted measured surveys on quarries, archaeological digs, large dams, MOD sites and monitored coastal erosion.
We've been commissioned to undertake visual inspection work on electricity pylons, water treatment plants, wind turbines, schools, religious buildings and photovoltaic panels.
In addition to our usual survey and inspection work we've completed several video assignments for clients, as well as sampling the on location catering with the cast and crew of sitcoms between 'takes'.
Away from the control sticks, we've been busy too – we were technical experts for Bradford City Council on the NESTA / InnovateUK Flying High Challenge, have delivered CPD sessions for several chartered industry bodies, exhibited at trade shows, became Pix4D Certified, worked on research projects with Universities and have been instrumental in driving Mountain Rescue England and Wales' drone capability for search and rescue.
As we near the end of 2018 and enter our third year of operation, 2019 is already shaping up to be a busy year with some interesting projects for clients and further developments of our operational capabilities. We also have a keen eye on the legislation changes that are due late in the year and of course any implications that Brexit may have on drone operations.


There's a great video on YouTube that is worth a watch. It gives more insight into Newman himself as well as his amazing craft that has influenced just about every current day photographer who employs that trendy photojournalism/environmental portraiture.
I was going through my own photo libraries, trying to see if I had any examples of truly environmental portraiture. And to be honest, I don't have many good examples of it. And by modifying the comment with "good" I mean environments that actually support/reveal the person being photographed. A lot of what passes these days for environmental stuff isn't really "Newmanian" environmental stuff. Take the famous example of Krupp. He looks reptilian, satanic even with the lighting, colour, mood and environment of his Nazi slave labour factory.
Every element of the photograph supports the core of who Krupp was. Its a far cry from chucking people into some random setting, just to have cool surroundings. The current trend of urban grunge photography when it comes to brides resonates with me this way. Sure, its a juxtaposed environment/subject mix. But it doesn't necessarily get at who the subject is – unless your bride is actually a homeless person who naturally lives in alleyways.
My point is, let us learn from Arnold Newman, a truly amazing gift to photography. Think about the subject. Get to know the subject. Then make the photograph in a holistic, all encompassing way that weaves together lighting, colour, gesture, mood, and an environmental setting that reveals the true essence of the subject.
This entry was posted on August 7, 2011 by schultzphotographic. It was filed under Portraits and was tagged with Arnold Newman, Context, Environmental Portraiture, Krupp, Lighting, Photography, Setting.


Connecting Finance and Natural Capital: A Supplement to the Natural Capital Protocol (the Supplement) provides a framework for financial institutions (FIs) to assess the natural capital impacts and dependencies of their investments and portfolios.
This framework has been developed by the Natural Capital Coalition (the Coalition), Natural Capital Finance Alliance (NCFA) and the Dutch SIF VBDO. The Supplement was developed against the backdrop of growing acknowledgment that the natural systems that underpin the global economy are deteriorating past the point of effective service provision, and that this will have potentially significant consequences for many businesses, and subsequently, for those who have financed or insured them.
The Supplement will guide FIs through the process of identifying, measuring and valuing material risks and opportunities as a means of informing financial decision making.
The Natural Capital Protocol framework includes: Natural Capital Protocol, Natural Capital Protocol Toolkit, Apparel Sector Guide, Food and Beverage Sector Guide, and Finance Sector Guide.


Here is a fun purse/crossbody for any time of the year!
Made with a sport or DK wt cotton blend yarn.
You will also need a zipper, 2 D rings and if you wish to add a lining a small piece of coordinating fabric.


DF in Argentina: ¡Fracásos lingüísticos!
The Spanish is, as they say, on the upswing. Oh, wait, they don't say that. We do. Or at least, I do. But as I slog through my intensive Spanish course (four hrs of daily conversazione with a real-live Porteno who is obliged to listen to me and comment on my usage and grammar), it becomes clear how many gradations (or, I've just learned, "matices") of language knowledge there are.
Which is to say: When I went to my language school's activity last night, a yoga class en Espanol, the other students, all beginners, all marveled (unwarrantedly, obv.) at the fluency of my comprehension. But the yoga teacher, who's one of my instructors, agreed wholeheartedly when I explained to the beginners (a group of Aussies taking a world tour that's limited for unclear reasons to the Southern Hemisphere) that my Spanish is far, far from perfect.
This is not false modesty, or even its obnoxious cousin, false false modesty, but rather a simple and incontrovertible fact. I have crossed a threshold where I can, more or less, converse with most (though not all) Portenos. This is something that I've long wanted--functional fluency in Spanish--yet despite, or perhaps even because of this increased language ability, it's become clear to me that one can be more or less conversational in a non-native language and still make errors all the time.
And I mean all the time. Weirdly, these errors are often very basic ones. I'll rock the pluscuamperfecto but then forget that "ley" is a feminine noun. I'll deploy the subjunctive but forget that my interlocutor is singular, not plural. And so forth. My theory on this is that those parts of foreign languages that do not exist in one's mother tongue (for English this would include, e.g., gendered nouns, the subjunctive mood, and declining nouns) are very hard to instinctively understand and require the kind of constant concentration to get right that makes it hard to have an at-ease chat with someone.
To illustrate, I thought I'd start a list of my most truly humiliating linguistic errors, or "fracasos" as I've become fond of saying, and with which word I'll be puncutating each of the following linguistic errors, largely for my own amusement, como siempre.
--In seeking to say something about religion, or perhaps America, I used the phrase "pecado original." Or at least I sought to. What I really said was, "pescado original," or "original fish." ¡Fracaso!
--For reasons unknown, I was trying to refer to "serial killers" and used a phrase that sounded like "matadores de cereal," which actually means "killer of breakfast cereal," such as your Kix and your Froot Loops and whatnot. ¡Fracaso!
--An attempt to comment on social class in America led me to desire to distinguish high-class and lower-class WASPs, though what I in actuality said was "high quality" and "low quality" WASPS, when when you think about it is also a relevant metric to distinguish them, though orthogonal to class, obvs. Anyway, ¡fracaso!
More ¡fracasos! to come, no doubt.
--Update: I was informed by a Porteno amiga that "fracaso" is far too strong a word to describe these kinds of linguistic errors, and that "error" (which is the same word in Spanish) would be more tonally appropriate. Hence the title of this post is itself an illustrative example of what this post is about. ¡Self-referential fracaso!


MesaTech is a leader in the Financial & Banking supply chain. That, combined with its Convenience Store sector, makes it one of the top distribution companies in the United States.
2019 Mesa Tech Corp, All rights reserved.


This exhibition of thirty-five prints from the collection shows the remarkable skills of two of the twentieth century's greatest portrait photographers. A relationship was established between the two photographers in 1936 when Yousuf Karsh paid his first visit to Edward Steichen's New York studio.
Working with a clientele composed of individuals celebrated for their power, wealth, or talent, both Steichen and Karsh displayed an ability to engage with their sitters on equal terms and to capture their characters without compromising their standards of artistic integrity or standards of formal excellence. Although the young Karsh clearly learned a great deal about celebrity portraiture from Steichen, a photographer whose work he studied from the days of his apprenticeship in photography, he also developed a unique portrait style that makes his work so readily recognizable to us today.


text:
Louisville Opens First Drive-Thru Coronavirus Vaccination Site
Photo: Ryan Van Velzer
City officials expect to administer about a thousand doses of the Moderna vaccine this week at Louisville's first mass coronavirus vaccination site at the Kentucky Exposition Center.
The drive-through site at Broadbent Arena is not yet open to the general public, but officials hope to scale it up as supplies grow and the city moves onto the next phase of vaccinations.
"This is a wonderful facility for administering the vaccine. Out of the weather, clean, drive through, plenty of parking space, we expect that we will be vaccinating here for weeks and months to come," said Mayor Greg Fischer.
Ryan Van Velzer | wfpl.org
A temperature check at Louisville's first mass vaccination site at Broadbent Arena on January 4, 2020.
Louisville Interim Medical Director Dr. SarahBeth Hartlage said the city received its first doses at 11 a.m. December 23rd, began vaccinating people within the first few hours and set up the drive-thru site in just 8 days.
Volunteers are operating the site. People with appointments simply drive up, undergo a clinical screening and receive the vaccination, Hartlage said. Afterwards, they'll sit in a parking lot under medical supervision for about 15 minutes before heading on their way.
"We'll be giving about 150 doses here today and about 1,000 doses here this week and increasing every week thereafter," Hartlage said.
Mayor Greg Fischer at Louisville's first mass vaccination site at Broadbent Arena on January 4, 2020.
For now, the vaccinations are by appointment only and reserved for health care personnel who fall under the phase 1a group outlined by the U.S. Centers for Disease Control and Prevention.
City officials hope to vaccinate more than 20,000 independent health care providers at the site including personnel generally working outside the hospital system such as doctor and dentist offices, said Hartlage.
Louisville expects to use the Broadbent Arena site along with hospitals and pharmacies to administer vaccines in the weeks and months to come.


As a distribution network, a question that we are often asked is "How can I get Core-Mark to distribute my product?".
It is very similar to how to submit a product to a major big box retailer.
However, most entrepreneurs only dream about getting their product distributed to major retailers, few actually do it. We have experienced a significant rise in interest since the show Shark Tank started airing on NBC. Mr. Checkout has had the pleasure of working with a few Shark Tank success stories such as KISStixx and 180 Party Cups which are now currently being distributed around the country to stores like Walmart, Target, Kroger, 7-Eleven and more.
If you envision your product would be a good fit for Core-Mark, we have a few steps to help you get your product on their shelves.
Before you read further, you may be asking yourself, "Why should I take advice from Mr. Checkout?" and "Who are you to give me advice on how to distribute my product?".
Here are 6 steps you need to take to have your product distributed by Core-Mark.
1. Start with the right questions) Before you ask Core-Mark to distribute your product, you need to ask yourself a few basic questions. Do you need to build demand for your product, or is there already a demand for it? Do you know that Core-Mark would be interested in representing your product? If you can strike a deal with Core-Mark, can you handle the production volume? Do you want to sell directly to Core-Mark, or do you want to sell direct to stores?
2. Be prepared to profit.) Does your product offer enough of a profit margin for Core-Mark? Can you sell your product at a reasonable enough profit to cover the packaging, shipping, commissions, marketing and wholesale distribution? Check Core-Mark's guidelines for other fees that you will have to build into the cost of your product in order for you to be able to turn enough of a profit to make the effort worth your while. If you work with a discount retailer, they will seek to trip your profits down to zero in order to keep their prices as low as possible.
The typical breakdown of margins are: If a products costs $1 to produce, that product will retail for $4. That product that retails for $4 will wholesale for $2 to distributors and stores that purchase direct. Big box retailers may offer to pay $1.25 to the manufacturer if the product costs $1 to produce. That is the typical profit margin.
3. Determine if Core-Mark is the right distributor or wholesaler for your product.) The relationship between you and Core-Mark starts with you browsing their store for competing products. If Core-Mark already has a similar product, it is going to be very difficult to get your product picked up. Spend some time at your local Core-Mark to see what kind of products they are selling, speak to the manager and see if he thinks your product will sell well in their store. Picture in which zone your product would best fit on the shelf and keep in mind that the most precious asset that these big box stores value are their shelf space. Keep this information in mind when you are preparing your presentation to Core-Mark.
If your company is minority or women owned, check Core-Mark's website and see if they offer specific opportunities for those designations.
4. Submit your product to Core-Mark.) Decide whether it will be you or a representative to present your product to Core-Mark. Your presentation depends heavily on your strengths as a businessperson as they will most likely ask financial questions and logistics questions.
5. Complete the required Core-Mark paperwork.) Often Core-Mark will have you go through an application process. However, before submitting the paperwork required by Core-Mark, you should contact a buyer at Core-Mark and let them know your intentions. Having a contact on the inside of the company will potentially move your application more smoothly through the process.
6. Anticipate the need for increased volume.) Having Core-Mark agree to wholesale or distribute your product will most likely mean a significant increase in volume. You should be prepared to ramp up your production and informing your manufacturer of this opportunity.
Having production, logistics and distribution to sync is not only difficult, it requires a significant amount of time invested in customer relations. Also, Core-Mark may have stipulations in the contract that may penalize you for delays in shipping and production. Have an attorney explain all contracts to you if you don't understand the terms.
If you're looking to have Core-Mark distribute your product, be sure to thoroughly assess the marketplace, carefully prepare for production growth, and take full advantage of resources available to help you grow your business.


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Is AI The Future? Or AI The Present?
Call Us At: +1 (474) 7342374 or +1 (474) 7342374
© 2021 Audiencetech Solutions
It's all about shifting gears from the eons-old manual business activities to going full-fledged digital. The struggle to keep up with the increasing consumer expectations is real. Whether it's round-the-clock customer support, face-to-face in-store transactions, or workforce productivity, businesses of today need AI to the rescue.
At present, the digital AI life has augmented human capacities and code-driven systems have taken over more than half of the world's population into a state of ambient information and connectivity. The way AI has garnered the focus of tech companies worldwide and has been considered the next significant technology shift post the evolution of mobile and cloud platforms is mind-boggling. Some industry experts foresaw it as the fourth industrial revolution.
Even Forbes predicted that by 2020, businesses that use AI and related technologies such as machine learning and deep learning will uncover new business insights and take $1.2 trillion each year from the contemporaries that don't deploy the same futuristic technologies. In 2021, this prediction came true and there's an AI surge all over the world. So let's have a clear picture of how AI is reshaping the present, the near future, and the future.
What are the applications of AI at present?
Cybercrimes are on the rise ever since businesses started going remote. The vulnerable nature of data has increased these risks. Enhancing the cybersecurity network is the only way for companies to stay alert and protected. With AI in the picture, curating threat intelligence, providing insights for businesses to fight cyberattacks, and reducing response times drastically is easy.
AI-infused microprocessors
AI has ushered in a new era of semiconductor innovation. Entrepreneurs globally have acknowledged the limitless potential and unique demands of machine learning. They have joined in the race to create chips which are purpose-built for AI to power the next-generation of computing.
Technology companies considered a keen interest in speech recognition and are working towards making voice recognition a standard for most products. These companies intend to make voice assistants speak and reply clearly with greater accuracy around context and content. Ground-breaking developments keep happening in natural language processing and neural network technology (all subsets of AI) to improve speech and voice recognition.
What's in store for AI in the near future?
Android Beings/Humanoids
Sounds straight out of a sci-fi movie but this is a huge possibility in future. With the advancements of robotics, humanoids will be the next technological advancement for generations to come. In industries such as space exploration, personal assistance and caregiving, education and entertainment, search and rescue, and healthcare, humanoids will come extremely handy. AI-powered humanoids will work closely with the humans in various ways across these verticals. There will come a time when it will be difficult to identify whether we are interacting with a human or a humanoid.
Driverless taxis
Autonomous vehicles without a human driver behind the steering wheel has been an ambitious project for many automobile companies. Self-driving car trials have happened in many cities of the world so far without any concrete results. However, with the advent of explainable AI aka XAI there's hope. The passengers sitting in these autonomous vehicles will be provided with reasons why the vehicle is taking a different route which wasn't possible before with normal AI algorithms. Simply put, XAI will put a thinking-cap to these self-driving vehicles thereby adding additional nuances or facets.
An upgrade to traditional computer systems will soon be on the horizon. The old-school way of computing with binary states – 0 and 1 will be taken to a whole new level that will be at par with quantum mechanics. Quantum systems work with qubits instead of bits and it opens the door to new possibilities and faster computation for certain tasks such as neural network optimizations and digital approximations.
What's in store for AI in the future?
AI-based video games
Every gaming enthusiast should look forward to AI-powered adaptive and responsive video gaming experiences in future. These experiences will be generated through non-player characters aka NPCs who act intelligently and creatively as if they are controlled by a human gamer. The entire process of gaming will no longer be reliant on a console or a desktop computer. It will evolve to bring video games to life, study their own patterns, and advance their own processes.
Bionic Organs
A prosthetic organ with the sensory capabilities of a human organ sounds bizarre, doesn't it? Hollywood movies have popularized this idea for a while now, however, human engineering wasn't able to deliver smarter prosthetics just yet. It's a work in progress with bioengineers looking to create human-machine interfaces embodied by a bionic organ that will really feel as if it's a human body extension.
Ever-changing and emerging algorithm-powered Artificial Intelligence is now a crucial part of day-to-day living. Long story short, AI has become our present and is leading the way to a beautiful future. Today's technological implementation appears avant-garde, but the entire business framework has become easier than before. AI has already built a strong business foundation and allows every industry to innovate.
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Day One helped Vere360 "fill skill gaps" and build a platform that would cater to their niche and diverse audience while seamlessly integrate the best of #AI and #VR technology.
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1TAM – Video Blogging Reimagined
'1TAM' was only for iOS with gesture-based controls, advanced video compression techniques, and a simple architecture that allowed actions to be completed in 2-3 taps. The real challenge for '1TAM' was to keep it distinct which bought brilliant results with all the strategies and approaches implied for best video compression techniques.
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Fit For Work – The Science of Workplace Ergonomics
Day One Technologies came with the expertise that was required and helped in building a platform that is edgy, functional, and smart, delivering engagement and conversions at every step.
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Armageddon UDP Traffic Generation with Random MAC Addresses.
Goal: Generate network traffic to a network device with one or many destination MAC addresses.
A one-sided traffic stream is used to send packets to a network device under test when round-trip reporting is not required.
On the Armageddon tab, click Create.
The connection name for this example is macgen.
Src MAC: 00:01:00:00:00:00. This is the starting point for emulated remote MAC addresses.
Dest MAC: 00:0e:fa:12:bc:3a. Use the destination MAC address of your system under test.
Max Src IP: 10.26.254.254. This emulates about 65,000 remote hosts.
Max Dst IP: 10.27.254.254. This emulates about 65,000 destination addresses.
Pps Tx: 80,000. This is a 1Gbps packet rate.
Src MAC Cnt: 65,025. This emulates 65,025 remote devices.
Select UnManaged. This allows us to fire and forget the packets.
Click OK to commit the configuration.
Src MAC: 00:01:00:00:00:00. This is the starting point for emulated source MAC addresses.
Dest MAC: 00:02:00:00:00:00. This is the starting point for emulated destination MAC addresses. This address is 4.2 billion addresses after the starting source MAC address.
Src MAC Cnt: 65,025. This emulates 65,025 source devices.
Dst MAC Cnt: 65,025. This emulates 65,025 destination devices.
In the Armageddon tab, click the Start button. Traffic will begin.


Type 2 diabetes mellitus is characterized by insulin resistance. However, the complete molecular mechanism remains unclear. In this study, zebrafish were fed a long-term high-fat diet to induce type 2 diabetes, which resulted in a higher body weight, body mass index, more lipid vacuoles in liver, increased insulin transcription level in liver, brain and muscle, and high fasting blood glucose in the high-fat diet zebrafish. Oppositely, the transcription levels of insulin substrate-2 and glucose transporter 2 were significantly decreased, indicating insulin signaling pathway and glucose transport impaired in the insulin-targeting tissues. Transcription of the autophagy-related genes, ATG3, ATG4B, ATG5, ATG7, ATG12, and FOXO3, were decreased but autophagy inhibitor gene m-TOR increased, and autophagy-flux was inhibited in liver of the high-fat diet zebrafish. Main of these changes were confirmed in palmitic acid-treated HepG2 cells. Further, in co-immunoprecipitation and subcellular co-localization experiments, the conjunction of preproinsulin with cargo-recognition protein p62 increased, but conjuncts of autophagosome with p62-cargo, lysosomes with p62-cargo, and autolysosomes decreased apparently. Interestingly, lysosomes, autolysosomes and conjuncts of p62-insulin localized at the periphery of palmitic acid-treated cells, the margination of lysosomes may mediate deactivation of proteases activity. These findings suggest that intracellular high-lipid may trigger defective autophagy, defective downstream signaling of insulin and accumulated intracellular preproinsulin, leading to dysregulation of cell homeostasis mechanism, which may be one of reasons involved in insulin-resistance in type 2 diabetes.
Type 2 diabetes mellitus is a complex metabolic disease with a high global incidence that is associated with hyperglycemia, hyperlipemia, and hyperinsulinemia. The number of patients with diabetes mellitus will reach up to 592 million in 2035 and more than 90% will have type 2 diabetes . Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus, is rapidly spreading around the globe with a prevalence estimated to be 8% worldwide by 2030 . The main etiology of this disease is β-cell dysfunction and insulin resistance, which means "the relative lack of insulin" in insulin target tissues resulting from the failure of β-cells to compensate for insulin resistance . Due to its complexity, the complete mechanism underlying type 2 diabetes remains unclear.
Currently, several mechanisms about type 2 diabetes have been identified or hypothesized. For example, lack of glucose transporters, IRSs or glucose metabolism related enzymes results in failed interaction of insulin with the insulin receptor, causing inhibition of insulin transport and function . Some local and systemic inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β and IL-6, can induce insulin resistance by suppressing the insulin signaling pathway . Obesity is also considered a causal risk factor for type 2 diabetes and commonly co-exists with this disorder [6-8]. In fact, insulin resistance is an important link between obesity and type 2 diabetes, because it can be induced by several adipokines/cytokines in adipose tissue . In addition, as a highly inducible intracellular degradation process, autophagy can maintain homeostasis of eukaryotic cells through the turnover of long-lived proteins, cytosolic components, and damaged organelles. It also plays an important role in maintaining β-cell mass and function . Dysregulation of this process causes type 2 diabetes due to the development of insulin resistance through the regulation of mitochondrial function and endoplasmic reticulum (ER) stress [11-17]. However, it is unknown how these events occur in insulin target tissues and cells. The main models used to study type 2 diabetes include mice/rats fed a high-fat diet (HFD) with or without streptozotocin treatment, transgenic diabetic mice/rats, and cellular models of insulin resistance [18,19]. Zebrafish, an established model organism for the study of developmental processes, human disease, and tissue regeneration, has similar pancreatic development and function as mammals and is reportedly a good tool for diabetic research [20-22].
In this study, we researched the correlation between autophagy and intracellular insulin gene expression induced by a HFD or palmitic acid (PA) treatment to determine the molecular events underlying insulin resistance in insulin target tissue cells. To this end, we developed a type 2 diatebes-like model in zebrafish and HepG2 liver carcinoma cells and examined the contribution of autophagic flux and preproinsulin levels in the two models. The results showed that accumulation of intracellular preproinsulin led to formation of a defective autophagic apparatus and dysregulation of glycolipid metabolism.
Liver, muscle, and brain are the main target tissues of insulin action, of which the liver is critical for glycolipid metabolism and insulin resistance in type 2 diabetes. To investigate whether a long-term HFD could induce type 2 diabetes in zebrafish, we fed adult zebrafish a HFD for 10 weeks. Then we evaluated several clinical indicators of type 2 diabetes including obesity, fasting blood glucose (FBG) levels, and mRNA expression of glucose metabolism-related genes such as insulin, glucose transporter 2 (GLUT2), and insulin receptor substrate 2 (IRS2). These zebrafish had a significantly higher weight, body mass index (BMI) and more lipid vacuoles in liver cells compared to the normal diet group (Fig. 1A-C, E), demonstrating that a long-term HFD induced obesity in zebrafish compared to those fed a normal diet. In addition, level of fasting blood glucose (FBG) and the mRNA expression level of insulin in its target organs elevated, specially, insulin in the liver and muscle were notably higher in HFD-treated zebrafish than in the normal diet group (Fig. 1D and F). However, transcription levels of key factors downstream of the insulin signaling pathway, namely, IRS2 and GLUT2, were significantly decreased in HFD-treated zebrafish liver (Fig. 1G), indicating that the signaling pathway was impaired in the insulin target cells. These data demonstrate that a long-term HFD not only induced obesity but also induced hyperglycemia-like and hyperinsulinemia-like symptoms in zebrafish, which is in accordance with a study by Oka et al . Thus, an insulin-resistant zebrafish model was established that was subsequently used for our in vivo studies.
Autophagy can be disrupted by fatty acids [24,25] and regulated by insulin signaling , and can maintain β-cell survival and function during diabetes by promoting the clearance of unfolded protein aggregates . Thus, we determined if the autophagy process was also impacted in this zebrafish type 2 diabetes model. The mRNA levels of several autophagy-related genes (ATGs) were examined by real-time qPCR; ATG3, ATG4b, ATG5 and ATG12 in liver, ATG3 and ATG5 in muscle and in brain were significantly decreased, while beclin 1 and ATG7 not notably changed (Fig. 2A-C) in HFD-treated zebrafish compared to the control group. In addition, there were decreased level of FOXO3, an upstream autophagy activator, and increased level of m-TOR, an autophagy inhibitor (Fig. 2A), indicative inhibition of autophagosome formation and autophagy. The microtubule-associated protein light chain 3B (LC3B)-II/I ratio represents the status of autophagosome development and maturation, and p62, also known as sequestosome-1 (SQSTM1), is an autophagy substrate ; their comprehensive levels indicate the status of autophagic flux. Thus we examined the expression levels of these autophagy markers, and found that lc3b transcript level was significantly reduced and LC3B-II/I ratio and total LC3B protein level decreased moderately (Fig. 2D). In contrast, there was a slight increase in p62 mRNA levels and a significant increase in protein levels in the liver of HFD-treated zebrafish (Fig. 2E). These results demonstrate that in HFD-treated or type 2 diabetes-like zebrafish, autophagosome formation was impaired due to suppression of pro-autophagy gene transcription and autophagy flux was inhibited. Thus a deficient autophagic process appears to be involved in obesity or type 2 diabetes.
To further clarify the molecular mechanism underlying type 2 diabetes development, a type 2 diabetes-like cellular model was established in HepG2 human liver carcinoma cells. Palmitic acid (PA) is frequently used to induce insulin resistance by inhibiting the insulin signaling pathway . In this study, HepG2 cells were exposed to different concentrations of PA and stained with Nile red to detect intracellular lipid content and glycolipid metabolism-related proteins. The results showed that the number of Nile Red-positive cells with orange-red fluorescence was significantly increased in PA-treated cells in a dose-dependent manner (Fig. 3A). Western blot analysis showed that the protein levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and peroxisome proliferator-activated receptor alpha (PPARα) decreased with PA treatment in a concentration-dependent (Fig. 3B), similarly, transcription levels of the two genes also decreased significantly (Fig. 3C). Previous studies have reported that PGC-1α and PPARα promote β-oxidation and fatty acid metabolism [29,30]; thus, the observed decreased levels of these two proteins indicates impaired lipid oxidation and metabolism. PA treatment also caused an increase in endogenous preproinsulin protein levels, as determined by Western blot analysis, which showed only one band at approximately 10 kD that matched the molecular weight of the preproinsulin protein; no other insulin bands (including insulin and proinsulin) were detected by the human insulin antibody. Moreover, IRS2 and GLUT2 levels declined synchronously in both transcription and translation (Fig. 3D and E), demonstrating that downstream insulin signaling was impaired by PA treatment. These results are similar to those found in the type 2 diabetes zebrafish.
Long-term high-fat diet induced Type 2 Diabetes-like symptoms in zebrafish. Adult zebrafish were fed a normal diet (Ctrl) or a high-fat diet (HFD) for 10 weeks. (A)-(C) Comparison of morphology (A), body weight (B), and body mass index (BMI; weight/length, g/cm) (C) between Ctrl and HFD zebrafish. (D) Higher fasting blood glucose induced in the HFD group compared to the normal diet group. (E) Representative images of hematoxylin and eosin (H&E) staining show lipid vesicles in zebrafish liver slices in both groups. The histogram shows the average number of fat cavities per sight field in the control group and HFD group, which were counted from ten sight fields each group. (F) Representative electrophorogram of insulin bands from RT-PCR (left) and real-time qPCR results (right) show the mRNA levels of insulin gene transcription in the liver, brain, and muscle of zebrafish in both groups. (G) The mRNA levels of insulin receptor substrate-2 (IRS-2) and glucose transporter 2 (GLUT2) assayed by RT-PCR electrophorogram (left) and real-time qPCR results (right) in zebrafish liver. *p<0.05, **p<0.01, ***p<0.001 vs the control group.
Transcription levels of autophagy related genes were suppressed and autophagic flux was blocked in the type 2 diabetes-like zebrafish. (A)-(C) The transcript levels of autophagy-related genes were assayed in liver (A), muscle (B), and brain (C) tissues in type 2 diabetes zebrafish. Representative electrophorograms of the ATGs bands from RT-PCR (left) and histograms of real-time qPCR results (right) were showed for comparison between the T2D group and the control group. (D) mRNA level of LC3B significantly decreased in real-time qPCR test, and the LC3B-II/I ratio and total LC3 protein levels moderately decreased via western blotting; (E) tested by the same methods, p62 mRNA level moderately increased and its protein levels significantly increased in the liver of HFD-treated zebrafish. *p<0.05, **p<0.01, ***p<0.001 compared with the control group.
Palmitic acid induced type 2 diabetes-like features in HepG2 cells. (A) Lipid level was increased in PA concentration-dependent manner in PA-treated cells determined by Nile Red staining. (B) Lipid metabolism-related proteins, PPARα and PGC-1α, were reduced in PA-treated cells. (C) Transcription levels of PPARα and PGC-1α were decreased by PA treatment. (D) Western blot results show that preproinsulin levels increased, and IRS2 and GLUT2 expression decreased in PA-treated cells in a concentration-dependent manner. (E) Transcription levels of insulin elevated and of IRS2 and GLUT2 declined following PA treatment. The upper panel shows representative electrophorogram of the gene bands from RT-PCR and lower panels show the corresponding histograms from real-time qPCR in C and E. *p<0.05, **p<0.01, ***p<0.001 compared with the control group.
Autophagy flux was affected by PA treatment. (A) LC3B-II/I ratio, total LC3B and p62 proteins were increased in PA-dependent manner. (B) Transcription levels of lc3b increased, but p62 was unchanged by PA treatment. The upper panel shows representative electrophorogram of lc3b and p62 bands from RT-PCR and lower panels show the corresponding histograms from real-time qPCR. (C) Co-immunoprecipitation (Co-IP) results indicate that there was decreased association of p62 with LAMP2, p62 with LC3B, and LC3B with LAMP2, but an increased association of p62 with preproinsulin in the PA treatment group. *p<0.05, **p<0.01, ***p<0.001 compared with the control group.
As mentioned above, autophagy levels decreased in HFD-treated zebrafish. Western blot analysis was performed to determine the ratio of LC3B-II/LC3B-I and p62 protein levels in type 2 diabetes-like cells. The results showed that the ratio of LC3B-II /LC3B-I, total LC3B and p62 protein levels increased in a dose-dependent manner with increasing PA concentration (Fig. 4A). Furthermore, the transcription of the two autophagy markers was examined by real-time qPCR, which shows that LC3B mRNA expression significantly increased in PA-treated cells in a concentration-dependent manner, although p62 mRNA level remained unchanged (Fig. 4B). These data illustrate that elevation of total LC3B expression resulted from its increased transcription level, LC3B-II level elevation indicates that PA can promote autophagic vacuole maturation. The increase in p62 levels may be due to accumulation of the p62 cargo loading protein. To determine if the autophagy process was functional or impaired, co-immunoprecipitation (co-ip) was performed to examine the interaction of p62 with LC3B, LAMP2 and insulin. The conjugation of p62 with LAMP2, p62 with LC3B, and the conjugation of LAMP2 with LC3B were clearly decreased in the PA-treated group compared to the control group, although the levels of p62 and LC3B proteins and ratio of LC3B-II/I were higher than those in the control cells (Fig. 4C). Importantly, the conjugation of preproinsulin protein with p62 was increased in PA-treated cells (Fig. 4C). These data suggest that PA can cause impaired autophagosomes lacking p62 cargo proteins and cause redundant preproinsulin-p62 cargos.
To confirm the results in situ, laser confocal microscopy and Pearson' correlation coefficient analysis were performed to determine the subcellular co-localization of cargo (p62 as the marker) with autophagosomes (LC3B as the marker), cargo with lysosomes (LAMP2 as the marker), and autophagosomes with lysosomes. The results showed that co-localization of p62 with LC3B presented a few regular puncta in the control group, whereas in PA-treated cells the co-localized punctas were more decreased and irregular rod-shaped structures of p62 cargos presented and did not combine with LC3B compared to the control group; correlation of autophagosome with p62 cargo was also reduced in Pearson' correlation coefficients (Fig. 5A). Co-localization experiments between p62 and LAMP2 presented similar rod-shaped structures to that in co-localization of p62 with LC3B, and co-localized puncta significantly decreased (Fig. 5B). Interestingly, the distribution of lysosomes moved from the cytoplasm to the cellular periphery, and the lysosome-p62 cargo (LAMP2-p62) conjugate also lined up together and departed from the nuclei in a PA-concentration dependent manner (Fig. 5B). Accordingly, most of p62 cargo proteins alone occupied the area between the nucleus and cellular periphery. In the experiment of LAMP2-LC3B co-localization, the co-localized punctas were not many in the control group; under PA treatment, the LAMP2-LC3B co-localized puncta was nearly disappeared and most autophagosome particles and lysosome particles were separately distributed in small pieces and rod-like structure in PA concentration-dependent manner (Fig. 6A). These evidences mean that autophagy flux and autophagy were impaired in type 2 diabetes-like HepG2 cells. In contrast, co-localization particles of p62 with "insulin" increased moderately, and other more "insulin" protein, situated in the middle area of cells, did not combine with p62 in type 2 diabetes-like HepG2 cells (Fig. 6B). The results indicate that the storage and irregular structure of both p62-cargo and autolysosomes may correlate with 'preproinsulin" protein accumulation. P62-cargo cannot enter autophagosomes and fail to fuse with lysosomes in PA-treated cells.
Analysis of autophagy flux by subcellular colocalization. (A) Combination of autophagosomes with p62-cargos was disrupted by Palmitic acid treatment. Decrease of co-localized punctas of LC3B-P62 analyzed by Pearson' correlation coefficients, the distribution and structure of autophagosomes (LC3B) and p62 were changed as rod-like shape and departing from the nuclei, separately, in Palmitic acid-treated cells. White arrows indicate conjunction of LC3B with p62-cargos; pink arrows indicate alone p62-cargo aggregates in rod-like shape. (B) Conjugation of lysosomes with p62 was decreased by Palmitic acid treatment. The marginalized distribution and abnormal structure of lysosomes were associated with the irregular p62-cargo, and more unconjuaged p62-cargos situated at the internal cytoplasm in Palmitic acid-treated cells. The open boxes in the overlay pictures are amplified at the right columns. Scale bar = 10 µm. Pearson' correlation coefficient analysis was based on 10 sight fields each group. *p<0.05, **p<0.01 vs control; ## p< 0.01 vs 250 μM PA group.
Liver and muscle are the two main tissues for the β-oxidation of fatty acids. PPARα can regulate a number of genes that function in hepatic lipid homeostasis, such as fatty acid transport protein, fatty acid translocase, fatty acid binding protein for fatty acid transfer and absorption, and so on . PGC-1α is an important transcriptional coactivator that regulates cellular energy metabolism and plays roles in the inhibition of glyconeogenesis, enhancement of fatty acid oxidation, and suppression of lipid synthesis . In this study, intracellular lipid levels were significantly increased in the liver in the type 2 diabetes-like zebrafish and in the type 2 diabetes-like cells. Meanwhile, both PPARα and PGC-1α protein levels were decreased in PA-treated cells, which demonstrate that hepatic lipid accumulation or a fatty liver induced by a HFD may be associated with deregulation of the signaling pathways involved in lipid metabolism. A study also showed that obesity in zebrafish share common pathophysiological pathways with mammalian obesity .
On the other hand, expression of endogenous preproinsulin was increased in PA-treated cells and in the insulin target tissues of HFD zebrafish with elevated FBG. IRS2 and GLUT2 were downregulated in a dose-dependent manner in the models in vitro and in vivo. These results indicate that glycometabolism disorder or type 2 diabetes-like symptoms can also be induced by long-term HFD or by long-chain saturated fatty acid treatment. IRS2 promotes glycogen synthesis in insulin target cells, but its expression can be suppressed and lipogenesis is enhanced by insulin under conditions of hepatic insulin resistance . So, in this study decrease of irs2 transcription hints an occurrence of insulin resistance. As the principal transporter for transfer of glucose between liver and blood, GLUT2 can sense changes in nutrient and can regulate sugar absorbance in insulin target tissues and β-cells . GLUT2 gene expression decreased in vivo and in vitro after HFD and PA treatment, indicating that glucose transfer was inhibited, which may be one cause of insulin resistance and high FBG in type 2 diabetes. Thus, glycometabolism and lipid metabolism are two tightly linked processes that intercommunicate and interact in insulin target cells. Meanwhile, we find that the transcription of some autophagy-related genes decreased in zebrafish, and autophagic flux was inhibited in both HFD zebrafish and PA-treated cells, which is in accordance with data from previous reports [13, 17]. Therefore, insulin resistance is involved in impaired autophagy, which is probably a common causative mechanism that links syndromes of both glycometabolism and lipid metabolism.
Intracellular insulin was involved in formation of Autophagolysosomes. (A) Formation of Autophagolysosomes was suppressed in Palmitic acid-treated cells. The fusion of lysosomes with autophagosomes was reduced and marginalized, and more aggregates of lysosomes (the red structures) or p62 (the green structures) were separately distributed in PA-treated cells. (B) The association of preproinsulin with p62 was marginalized at the cellular periphery; and some preproinsulin aggregates (the red structures) lonely accumulated in the intermediate area in PA-treated cells. The open boxes in the overlay pictures are amplified at the right columns. Scale bar = 10 µm. Pearson' correlation coefficient analysis was based on 10 sight fields each group. *p<0.05 vs control.
It is interesting that intracellular preproinsulin expression was highly induced in non-islet cells such as hepatocytes, muscle cells, and brain cells in HFD fish and PA-treated HepG2 cells relative to their corresponding control groups. We presume that the high levels of preproinsulin may be a compensatory response for insulin resistance, but failed to function in type 2 diabetes animals and in the cell model. There is an attractive relationship between non-islet intracellular insulin gene expression and autophagy in type 2 diabetes models. Recently, Chen's team  demonstrated extrapancreatic insulin gene expression in diabetic mice. Several studies reported that postprandial millions of proinsulin disulfide bonds were formed, and isomerization of improper disulfide bonds may be produced into big invalid proteins and accumulation of misfolded proinsulin protein may cause endoplasmic reticulum stress and type 2 diabetes [36, 37]. In our study, the mRNA levels of insulin were highly induced in non-islet cells such as hepatocytes, muscle, and brain in type 2 diabetes zebrafish, and its protein precursor level was higher in PA-treated HepG2 cells than in the control groups. We presume that the high level of "insulin" may be the host cellular compensatory response for the relative insulin shortage induced by high lipid conditions, but the preproinsulin could not be processed into insulin. On the other hand, we found increased particles of preproinsulin jointing with p62, although the interaction of autophagosome with p62 and lysosomes with p62, as well as autolysosomes clearly decreased. Besides, other more p62-cargos did not enter autophagosomes, which showed impaired autophagosomes and autolysosomes, and excessive useless proteins including preproinsulin were not degraded in PA-treated HepG2 cells. Thus, we infer that the sites of p62 recognizing and ligating to autophagosomes (to LC3B) might be disrupted or masked, leading to invalid assemblies of autophagosomes and inhibited autophagic flux.
Another interesting phenomenon is that the distribution of lysosomes and autoplysosomes with irregular rod-like shapes, including conjugates of p62 with insulin, were marginalized in PA-treated cells in this study. Recent studies have reported that high fatty acid levels could decrease lysosomal acidification; lysosome marginalization is a symbol of reduced acidification and deactivation of lysosmalproteolytic activity [38,39], and the subcellular localization of lysosomes is a major determinant of pH . Thus, we presume that excessive free preproinsulin and preproinsulin-containing p62 occupied the intermediate area by some unknown mechanism, which prevented lysosomes moving towards the perinucleus from the cytoplasm, leading to impaired lysosomal function in PA-treated cells.
In summary, HFD induced intracellular preproinsulin production and type 2 diabetes-like syndromes, as well as impaired autophagy in zebrafish and HepG2 cells. Accumulation of intracellular preproinsulin protein may be an important factor for insulin resistance and dysregulation of glycolipid metabolism by mediating the marginalization of lysosomes and disrupting autophagosome formation.
Zebrafish (Danio. rerio) of wild-type AB strain were maintained under standard laboratory conditions with 14-h light/10-h dark cycle at a temperature of 28.5±1°C . The fish were fed brine shrimp once daily as the control group (Ctrl), and were fed twice daily with brine shrimp (60 mg cysts per fish daily) plus 1% egg yolk as the HFD group [42, 43]. Fifteen fishes were included in each group. All of the fish were fed for 10 weeks.
After feeding for 10 weeks, BMI was analyzed; body length of the zebrafish from the front end of the mouth to the tail end was measured, and body mass was weighed using an electronic balance (BS2202S, Sartorius, Argentina, Germany). Zebrafish were fasted for 24 h, after which blood from the heart was collected using scissors and micropipette tip, and FBG was evaluated using a blood glucose meter (Accu-Check® Active, Roche, Penzberg, Germany) . Next, zebrafish liver, brain, and muscle were isolated and stored at -80°C for further use.
Fish were fixed in formaldehyde and coated with paraffin, and then cut into slices with paraffin microtome. The slices were stained with hematoxylin and eosin (H&E) and observed using a microscope.
The HepG2 human hepatocellular carcinoma cell line was cultured in MEM medium (11095-080, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (26140-079, Gibco, Grand Island, NY, USA) at 37ºC in a humidified atmosphere containing 5% CO2.
Palmitic acid (51.28 mg; P0500, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 1 ml 0.1 M NaOH at 70°C and was filtered. A total of 5% free fatty acid (FFA)-bovine serum albumin (BSA) (BAH66-500, Equitech-bio, Cotton Gin Ln, Kerrville, TX, USA) solution was prepared in double-distilled water at 55°C and filtered. The PA solution was conjugated to 5% FFA-free BSA in a 70°C water bath, after which the solution was cooled to room temperature and diluted in MEM to the final concentrations of 50 µM, 100 µM, 250 µM, and 500 µM. Cells were treated with different concentrations of PA for 12 h in 10% FBS-MEM. Cells treated without PA were considered the normal group control.
Nile Red (1 mg; 19123, Sigma-Aldrich, St. Louis, MO, USA) powder was dissolved in 1 ml DMSO to a final concentration of 1 mgml-1. DMSO was diluted in MEM to a final concentration of 50%. The nile red solution was diluted in 50% DMSO to a final concentration of 1 μg ml-1. The solution was diluted again in MEM to working concentration of 0.5 µg ml-1. HepG2 cells were exposed to PA at the indicated concentrations for 12 h and washed twice with phosphate buffer saline (PBS). Then Nile red working solution was added to the cell dishes, which was incubated in the dark for 10 min at 37°C. Then the cells were observed using Inverted Fluorescence Microscope (IX51, Olympus, Tokyo, Japan) under an excitation wavelength of 543 nm. Cells with strong orange-red fluorescence were deemed positive for lipid accumulation, and those with golden-orange fluorescence were deemed negative for lipid accumulation.
Total RNA was extracted from different zebrafish organs and HepG2 cells using Trizol reagent (15596-026, Invitrogen, Carlsbad, CA, USA). RNA samples were used to generate cDNA using the M-MLV RTase cDNA Synthesis Kit (D6130, Takara Bio Inc, Kusatsu, Japan). The reaction of quantitative real-time PCR was performed by the real-time quantification system (Roche, LightCycler® 96). The relative levels of messenger RNAs (mRNAs) were calculated with β-actin mRNA as the internal control. Relative transcript expression was determined using control sample as a calibrator and the ΔΔCT method. The PCR parameters were as follows: 95°C for 30s, 95°C for 5s, 55°C for 15s, and 72°C for 10s, for 30 cycles. The primers used in this study were shown in Table 1 and Table 2.
Briefly, total protein in the zebrafish organs and HepG2 cells was extracted using the Tissue Protein Rapid Miniprep Kit (90707-50,TIANDZ, Beijing, China) plus protease inhibitors, and as resolved on 15% or 10% SDS-PAGE gels. The protein bands were electrophoretically transferred to a nitrocellulose membrane and then blocked in Tris-buffered saline (TBS) containing 10% skim milk for 1 h at room temperature. The membranes were incubated overnight at 4°C with primary antibody including anti-human p62 polyclonal antibody (PM045, MBL, Nagoya, Japan) diluted 1:500, and anti-human β-ACTIN monoclonal antibody (TA-09, ZSGB-Bio, Beijing, China) diluted 1:1000, anti-human LC3B polyclonal antibody (PM036, MBL) diluted 1:500, anti-human LAMP2 monoclonal antibody (sc-5571, Santa Cruz Biotechnology, Dallas, TX) diluted 1:200, anti-human PPARA monoclonal antibody (AM8452b, Abgent, Suzhou, China) diluted 1:500, anti-human PGC-1α polyclonal antibody (ab54481, Abcam, Cambridge, UK) diluted 1:500, anti-human insulin polyclonal antibody (sc-7838,Santa Cruz Biotechnology) diluted 1:200, anti-human GLUT2 polyclonal antibody (sc-9117,Santa Cruz Biotechnology) diluted 1:200, and anti-human IRS2 polyclonal antibody (#4502, CST, Danvers, MA, USA) diluted 1:200. Then the membrane were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. Proteins were visualized by enhanced chemiluminescence using the Supersignal® West Pico chemiluminescent substrate (34080, Thermo Fisher, Waltham, MA) with the AlphaEase® FC Imaging System. β-actin was used as a loading control.
Cells were lysed in Nondenaturinglysis buffer (C1050, Applygen, Beijing, China). Cell lysates were separately incubated with a specific primary antibody, including anti-p62 (PM045, MBL), anti-LC3B (M186-3, MBL) and anti-insulin (L6B10, CST), overnight at 4°C; then with protein A/G-Sepharose (Santa Cruz Biotechnology) for 3 h. The immunoprecipitated complexes were collected by centrifugation at 4°C, 3000 rpm for 3 min and then eluted 5 times by the lysis buffer. The eluted protein samples were separated on SDS-PAGE and followed by immunoblotting using the three antibodies mentioned above, anti-LAMP2 (L0668) and anti-β-actin (A5441) (both from Sigma-Aldrich US).
HepG2 cells were cultured on slides and fixed, permeabilized, blocked in BSA, and incubated overnight at 4°C with primary antibodies including rabbit anti-human p62 antibody (PM045, MBL) diluted 1:500, mouse anti-human LC3 antibody (M186-3, MBL) diluted 1:500, rabbit anti-human GLUT2 antibody (sc-9117, Santa Cruz Biotechnology) diluted 1:200, and goat anti-human insulin antibody (sc-7838, Santa Cruz Biotechnology) diluted 1:200. Then, the cells were washed and incubated with fluorochrome-conjugated secondary antibodies. Finally, the cell nuclei were counterstained with DAPI to visualize nuclei. Images were captured using a laser scanning confocal microscope (LSM 710, Zeiss, Jena, Germany).
The quantitative data of immunoblot and real-time quantitative PCR are relative magnitudes that were normalized with β-actin protein and mRNA, respectively. The means and standard deviations (SD) in histograms are derived from three independent experiments.
For the fluorescence quantification of colocalization images, the free software Image J. Fiji and the Pearson's correlation coefficient were used to calculate double fluorescence correlation coefficients , and scatter diagrams were used to describe changes of co-localized fluorescence quantification, in which 10 sight fields each group were analyzed.
The Student's t-test was used for comparison between two independent groups, while one-way ANOVA was used to compare means among multiple groups. All statistical tests were two-tailed and p value was shown as *p<0.05 and **p<0.01.
T2D: type 2 diabetes; ATG: autophage related gene; FOXO3: forkhead box protein O3; IRS: insulin receptor substrate; TNF-a: tumor necrosis factor a; ER: endoplasmic reticulum; HFD: high-fat diet; PA: palmitic acid; FBG: fasting blood glucose; GLUT: glucose transporter; BMI: body mass index; SQSTM1: sequestosome-1; PGC-1a: peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PPARAa: peroxisome proliferator-activated receptor alpha.
We thank the Equipment Center of IMB and Dr Guang-Zhi Shan and Li-Min Zuo for helping in use the confocal microscope, and thank Jie Meng for fish husbandry. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
This work was supported by The National Natural Science Foundation of China (No. 81373453 to JPZ and 81603172 to BC), Foundation for Innovative Research Groups (81321004 to JDJ) and the Chinese National Key Technology R&D Program (2015BAK45B01 to JPZ).
JPZ conceived and designed the project, and wrote the manuscript. XHM and BC performed the experiments and dealt data, as well as wrote "Research Design and Methods". BC, XHM and JPZ analyzed the results.


Nat is a trauma injury specialist. What she offers is not just a massage but a physical rehabilitation treatment that works by adjusting posture and making structural changes to the body. Although she is not a chiropractor she has many patients who see her instead of or alongside a chiro or an osteopath.
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text:
Q: Astyle - how to format condition without braces to 1TBS I have a program which uses Astyle to format the code into 1TBS. So, if I have a code like this
if(condition)
    func(a, b);

it changes to this
if(condition) {
    func(a, b);
}

The problem is, when parameters in the called function are broken into multiple lines, like this:
if(condition)
    func(a,
         b);

Then Astyle is not able to add braces even if I try to force him with --add-brackets. Is it possible to do it some other way?
My command now looks like this:
astyle --style=1tbs --add-brackets  test.c

A: This may seem a bit convoluted but if you have a lot of sources
and adding braces is an operation that you just need once,
the following scheme might work.
You can stick with astyle but you need uncrustify temporarily
as well as a script I wrote called whatstyle.
In the following steps replace test1.c with your sources and keep a backup
of your sources as they will be modified.
Teach astyle the current style of your sources
whatstyle.py -f astyle --mode resilient --output astylerc test1.c

Teach uncrustify the current style of your sources
whatstyle.py -f uncrustify --output uncrustify.cfg test1.c

Tell uncrustify to always add braces to ifs
( egrep -v mod_full_brace_if < uncrustify.cfg ; echo "mod_full_brace_if = force" ) \
  > uncrustify_addbrace.cfg

Reformat your sources with as little style changes as possible with uncrustify
uncrustify --replace -c uncrustify_addbrace.cfg test1.c

The braces have been added now retransform into the original style with astyle.
ARTISTIC_STYLE_OPTIONS=astylerc astyle test1.c

Now your sources should look almost the same as before except for the added braces
although there might be more changes by the back-and-forth style transformation.


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Peter taught acting and directing classes at Burlington College and is a co-founder and Managing Director of Timely Transformations. He holds an Organizational and Executive Coaching Certificate from NYU and is a member of the International Coach Federation (ICF).


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