SEO

Have you heard about the Mission Hills Trailer Fire + Peace & Healing Centers pilot program? Interested in learning more about it? Read on to learn more about this exciting initiative! It’s been around for a while, but recently gained national attention after opening in San Francisco. The program is now accepting applications! There is still time to make your donation. Please consider participating!

Hello and happy Friday; today is National SCUD Day, founded by Stephanie West Allen in 2007 as “National Savor the Comic, Unplug the Drama Day!”! I’m back in your inbox this morning to give you everything you need to know about what’s happening locally. Here we go.

The bush fire was prevented. Also, nine local communities will receive Peace & Healing centers. Eventually, the alleged kidnapper is released on bail.

Find out what’s happening in Los Angeleswith free, real-time updates from Patch.

Clouds yielding to the sun. High: 83 Low: 61.

Find out what’s happening in Los Angeleswith free, real-time updates from Patch.

🚗 The three cheapest gas stations in Los Angeles 🚗

💧 Water crisis survey! 💧Residents are limited to watering their landscapes two days a week, before 9 a.m. or after 4 p.m. What else should the city do to deal with the water crisis? Please let me know what you think! So far, 22.6% want to require drought-resistant landscaping, 17.7% want to recycle wastewater for irrigation, another 38.7% want to build a desalination plant on the coast, and 21% don’t believe residential water use is contributing to the water crisis. .

Here are five of the best stories in Los Angeles today:

Are you a Los Angeles resident looking for an investment alternative to the stock market?

Bloomberg recently asked experts where they would invest $100,000 right now, and many of them had the same answer: fine art.

Think you can’t afford a Picasso? Well, in 2012, Congress passed the JOBS Act, which allowed for the securitization of works of art. A company called Masterworks.io built the largest platform for buying and selling art securities. In the past, you could only buy stocks of companies like Apple, but with this investment app, you can unlock this powerful investment.

This means you can invest in a million dollars worth of art stocks. And in uncertain times, it turns out, fine art can be a wise investment. Here’s why:

For more information, visit Masterworks.io, where Los Angeles Daily readers can skip the waiting list.

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Now you’re in the know and ready to head out the door this Friday. See you tomorrow for another update!

About me: Sylvia Cochran works in sunny Southern California and has been a full-time freelance writer since 2005. She loves dogs, cats, books, playing best friends (don’t judge), embracing social justice, and trying to outlive Micah. 6:8.

Have news tips or suggestions for an upcoming Los Angeles daily? Contact me at [email protected]

The rules of replying:

Written by Jennyfer Espiritu, Journalist

2020 is a very important year for the hospitality and tourism industry due to COVID-19. At the end of the first quarter of 2020, the World Health Organization effectively declared the outbreak a pandemic, initiated the government to enforce a stay-at-home order, advising against non-essential overseas and domestic travel. Within a month, the trip became impossible. As a result, a $1.8 trillion global industry stalled, disrupting the livelihoods of more than 300 million people, including travel publishers small and large.

It’s hard for travel-focused publishers to create relevant content when people aren’t prepared to go out and risk their health and safety. However, despite the unprecedented challenges, small publishers like Local Adventurer, Be My Travel Muse, and MarcoFeng.com are seeing massive growth, garnering more than 10 million views in organic search in 2021 alone. The team’s willingness to adapt to the current situation was at the core of their breakthrough. According to founder Hailun (Marco) Feng, when COVID-19 hit, they shifted their focus from travel to home content that surged in search queries. And most of the content is based on reviews, surveys and polls, and independent sources, which gives the site an advantage to maintain and produce travel content even when it’s nearly impossible to travel.

In 2021, the newcomer also launched faceted navigation that allows readers to search for destinations and guidance using filters such as “best time to visit”, “located in”, “suitable for”, “cost”, and “visa and vaccination requirements”. as a one-stop-shop for travel planning. While such a feature is common in eCommerce, this is the first time it has been used in publishing, offering readers new ways to discover and consume content. Also, during a pandemic, when infections and policies change frequently and uniformly, this offers publishers more flexibility in presenting information in a timely manner without a lot of manual input. Feng said, “This feature will continue to be refined to provide better results.”

While many sites are starting to cover news related to the pandemic, Feng recommends finding the right angle to keep the brand alive. Unless it’s a health site, avoid topics that worry too much about people’s lives and wealth. Google’s algorithms enforce strict rules for ranking websites in YMYL (your money, your life) categories, and you risk losing rankings to cover up that content without the right skills. “In early 2020, we removed all of our insurance and health-related content from the site, which was not working at all, resulting in a slight increase in organic traffic. A reference section is also added to the bottom of each article to signify our authority to Google.”

Another recommendation from Feng is to review content regularly to ensure the information is accurate so that readers can have the same experience as before the pandemic. For example, this section lost organic visibility because it wasn’t updated to reflect local health and safety guidelines for restaurants but saw a spike in ratings after the update. If you don’t have the resources to do it or do it quickly, an alternative is to add an announcement bar to your site to remind readers to check the venue before making a reservation.

While all of this may sound simple, Feng reveals that it takes a lot of effort, involving 15 to 25 hours researching, editing, fact-checking, and adding references and visuals for each section. Also, to stay competitive as newcomers, they have created unique formats such as grouping feature-length content by geography, type, and genre, or using a transparent scoring system to rank recommended places based on the best.

Hailun (Marco) Fenghttps://www.marcofeng.com[email protected]

COMTEX_409800311/2776/2022-07-07T04:50:46

Search Engine Optimization (SEO) is a vital tool for any drug rehab center. However, the goal of a successful drug rehab SEO campaign is to increase the likelihood that clients will find the treatment they need. Although the goal is to increase the number of patient admissions, drug rehab centers must not appear pushy or intrusive. Fortunately, the Internet has made this balance easier. SEO campaigns for addiction treatment can be very effective and increase awareness of your center.

The first step to finding a treatment center is to establish a strong web presence. Although many programs offer scholarships, a solid web presence will give patients confidence in choosing a good treatment facility. Furthermore, a solid web presence will help clients avoid spending money on a website that offers mediocre or no results. If your budget is tight and you can’t afford treatment, consider fundraising as a final payment option.

Search Engine Optimization is an art and science that can help a drug rehab website rank high on Google. Dreamscape Marketing understands how this process works and will use the most appropriate keywords to attract potential patients. They will also make sure your content is designed to inform loved ones and friends about the benefits of the treatment center. That way they can take appropriate measures. And when you increase your website visibility and organic search results, you can relax knowing that your work is done.

Traverse City, July 06, 2022 (GLOBE NEWSWIRE) — Traverse City, Michigan –

MasterMindSEO, a Traverse City, MI-based digital marketing agency that continues to lead the way in addiction treatment marketing, is on a mission to help people struggling with addiction find treatment that allows them to begin their journey to recovery. The company continues to be the best in drug rehab SEO results, from ranking Google Business Profiles in the most competitive cities like San Diego, Los Angeles and Dallas to dominating the search engine results pages (SERPS) for its clients. They want to emphasize that all these efforts are motivated by the desire to help those who are looking for a suitable treatment facility either for themselves or their loved ones.

Stephen Twomeythe, founder of MasterMindSEO, had this to say about why he entered the addiction treatment marketing space: “The rehab marketing space is very personal to us, as we have a family member who is an addict. MasterMind entered the space because we wanted people struggling with addiction to find quality addiction treatment centers. After doing lead generation for many different centers around the country, we saw the good ones and the not-so-good ones. Our main goal is to match qualified mental health treatment centers and dependencies with people matching their services.”

They want to point out that the key to successful SEO for addiction treatment centers is a multi-layered approach that includes: Facebook ads that will drive new patients into client recovery programs, usually from retargeting; Pay-per-click marketing for drug and alcohol rehabilitation; and content marketing and SEO activities to achieve Google search results.

They also want to emphasize that quality content on a website is not enough. It’s like someone going to a party and simply hoping that someone notices them. That person will need to introduce themselves to groups they find interesting in order to get noticed quickly. In online marketing, this is done through on-page SEO, which is an area of ​​expertise for the SEO professionals at MasterMindSEO. This has to do with structuring the content of the page in such a way that it is easier for search engines to understand what it is about.

Meanwhile, MasterMindSEO offers a free evaluation of a specific website and provides some recommendations for better SEO. An important aspect of SEO is having quality links. Twomey had this to say: “MasterMind creates well-written website copy aligned with the competition, creates industry-specific links and promotes locations on Google Maps in such a way that the entire entity gets a boost in search results. Links are an important factor, but not the only one factor when it comes to organic search engine optimization for substance abuse treatment facilities This is where a professional SEO company can help as they use tools that can verify that those pages have good metrics and are indeed a website that will send real traffic. “

A good SEO company will also know what to do in terms of keyword density and proper use of “no-follow” and “follow” links so that they have a positive impact on SEO rankings. And the SEO task doesn’t stop after the page reaches the number one spot. There is a constant battle for companies in the same industry or niche to come out on top. Stopping the SEO process will allow competitors to catch up and eventually reach the top, leaving your company behind in terms of search engine rankings.

Started in 2013, MasterMindSEO has a team of digital marketing experts whose work has already been featured in numerous online publications, including Forbes, Entrepreneur, tech.co and many more. They are able to provide lead generation services, content marketing, organic SEO, white hat link building outreach, Facebook ads and more. They specialize in achieving results to improve their clients’ business. They have extensive experience in launching digital marketing campaigns for national, e-commerce, local and regional business enterprises.

People who would like to know more about their services such as rehab center marketing can visit the MasterMindSEO website or contact them by phone or email.

For more information about MasterMindSEO, contact the company here:

MasterMindSEOSStephen Twomey855-983-0303827 W ​​Front St Suite 101 Traverse City, MI 49684

Opinions expressed by Entrepreneurial Contributors are their own.

People like to think simplified. We like to categorize concepts nicely and see things in black and white. We like to ignore nuances for the sake of a summary of one sentence. And when it comes to complex marketing strategies like search engine optimization (SEO), we like to boil hypothetical approaches down to a handful of action-oriented tactics.

Maybe that’s why so many modern SEO experts have tried to make SEO synonymous with link building. But here’s the thing, while link building is important for SEO, it is not the only factor you need to consider. And if you focus too much on link building, it could end up costing you your chance to reach first place.

Related: How to shake up an old link building strategy

SEO and link building

First, we need to recognize that link building is very important for SEO. Mostly it is because links have a closely related relationship with your domain authority. Your domain authority is an invisible score that represents the credibility of your site in the eyes of Google and other search engines. The higher this authority is, the more credible your site is considered to be, and the higher you will rank in relevant searches.

While the back-end link quality evaluation system is sophisticated, it is mostly true to say that the more links you have, and the more authoritative these links are, the higher your domain authority will be. Building links on sites that already have high authority will pass some of that authority on to you, accelerating the rise of your domain authority.

Related: Why is Link Building So Controversial?

But if you focus exclusively on link building, you are going to sabotage your campaign. Although it is very difficult to increase your domain authority without active link building, if you focus exclusively or too much on link building, it will only work against you.

Non-link building SEO essentials

If you focus too much on link building, you will neglect other important elements of search engine optimization, including:

The irony of excessive focus on link building

There is a bit of irony in focusing excessively on link building, because if you do not focus on these other elements, link building becomes more difficult.

While it is possible to intentionally and manually build high quality, natural looking links to your site, it is also important to naturally attract links whenever possible. Partly because Google will penalize your site if it appears that you are participating in link schemes, and partly because naturally attracted links tend to come from a multitude of different sources, which ultimately builds your authority further.

Related: 7 Reasons Why SEO Matters for Every Startup

Without content (or bad content) you will never attract these natural links. You will be completely dependent on manual link building – and even then you will not have powerful content to serve as a destination for your new links.

I’m not here to bash link building. On the contrary, I think mastering SEO is the most important element. But we need to honestly acknowledge that link building is not the only aspect of SEO that you should pursue – and if you focus too much on link building, it will work against you.

Related: How to shake up an old link building strategy

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While hiring an agency can be expensive, a high-performance action-driven agency can help your business achieve its marketing goals. These results-driven agencies have dedicated teams that focus on all aspects of internet marketing, from social to search to e-commerce development. These experts work as growth partners with their clients to find the best possible marketing strategies for their businesses. Let’s take a look at some of the top results-driven online marketing agencies that every brand should know about.

Disruptive Marketing specializes in search, Facebook and display advertising, providing direct PPC expertise and individualized strategies to achieve the goals of its customers. This marketing marketing agency also provides one-on-one experience for its clients by analyzing their business and industry goals to develop a customized plan. Their goal-focused approach to digital marketing results in a positive return on investment (ROI) for their customers.

Disruptive Advertising, based in Copley, Ohio, specializes in performance marketing for start-ups. Its team of experts implements branding strategies through search, social and local media. They create the perfect website experience for customers through dynamic digital marketing. They don’t use stupid country pages. This agency helps brands be noticed, and has over 2,500 satisfied customers. An excellent digital marketing agency will work to increase sales and generate new leads.

Avalaunch Media focuses on content and copy for customers. Their team specializes in increasing ROI. Located in Lehi, Utah, this digital marketing agency of results helps brands grow for 15 years. They are Leading Google Partners. They are not your average digital marketing agency – everyone is a specialist in their field. The team is working on developing marketing strategies for a variety of different businesses.

Whole Whale is another results-driven online marketing agency that specializes in helping customers build a digital presence. They use data and technology to make every marketing strategy as effective as possible. They provide customized solutions to their customers by incorporating social impact and ensuring high ROI. Their customer retention rate is close to 100%. Overall, they help their customers achieve growth in their target market. Once a brand has a good understanding of their target audience, they can decide how to use the digital marketing channel most effectively.

In addition to researching the agency’s record, it is important to understand their specific strengths. Look for testimonials on their website. If possible, read testimonials posted by previous clients. This will give you an idea of ​​whether they are effective or not. The agency’s reputation is directly linked to their ability to deliver results. It’s a good sign if they have a lot of happy customers.

Before hiring an online marketing agency, make sure you choose a local agency with the same standards as your company. Look for an agency that offers analysis and can give proofs of results. Lastly, make sure they are located near your area. This is important for the overall feel of the agency. Once you find a great agency that shares your goals and values, you can make the best decision for your business.

Finally, consider Power Digital Marketing, a digital marketing agency with offices in Chicago, New York and San Diego. Founded in 2012, this agency provides clients with comprehensive marketing solutions and consults with other arms of the business. Power Digital Marketing specializes in email marketing, social media marketing, internet analytics, PR and content creation. They also offer website design and development, SEO, PPC and UX / UI.

Before choosing a digital marketing agency, determine how much you are willing to spend. Decide what services you need from an agency and adjust to that budget. Receiving quotes from several agencies will help you narrow down your search results. It is best to get rid of agencies that cost more than your budget. You can also ask about the time zone and physical location of the agency, as well as about its scope of services.

The team at 97th Floor is a very creative agency. They combine market research with creative ideas to help their clients succeed. Their mission is to become a partner of your business. The agency focuses on various aspects of digital marketing, but their clients are most interested in content marketing, copywriting, email marketing and mobile marketing. With the help of their team, you can be sure that your online marketing efforts are hitting the right goals.

Not all businesses have the time, skill, or inclination to undertake in-house online marketing campaigns. Indeed, most large organizations prefer to delegate their marketing activities to specialized agencies rather than having to compete for the highest talent in a field that is not their specialty. On the other hand, many smaller businesses work with online marketing agencies because they don’t have the resources and nothing to create everything inside. And some companies with marketing departments may prefer to delegate some activities to specialized agencies rather than devote time to all the tasks necessary for successful marketing campaigns. In this post, we examine some of the best online marketing agencies you can find, some serve the entire digital marketing field, and others choose to specialize in a few narrow niches. You will see more in-depth reviews in our collection of Digital Marketing Agencies.

1. Disruptive Advertising

Disruptive Advertising provides services related to paid advertising, lifecycle marketing, creative services and website optimization.

They look at three issues of potential customers:

Disruptive Advertising helps companies grow to the next level by developing and implementing a dynamic digital marketing strategy. They emphasize that they are not a full-service agency. However, they have established reputable partnerships with other over-focused lead agencies that can help you with other aspects of your digital marketing strategy, such as SEO, content marketing, website design, branding, and affiliate marketing.

2. Brandwatch (Formerly Falcon.io)

You may have noticed a regular mention of Falcon.io while browsing through the Influencer Marketing Hub. However, the company has recently rebranded and is now a Brandwatch.

Although not a full-scale digital marketing agency, Brandwatch offers multiple digital marketing services to brands, particularly in terms of content and social media marketing. It provides everything you need to build your followers and grow your brand through your social channels.

Brandwatch’s approach to social media marketing is to break it down into what matters most. It gives social media professionals access to structured features that help them listen, participate, measure, and publish content that makes a difference.

Brandwatch works well with other agencies to smooth out your collaborative processes if you want an agency for more than just your social media. You can control all accesses and set permissions.

3. Referral Factory

Referral Factory is not a full-service digital marketing agency, but it specializes in helping brands that want to dedicate a portion of their digital marketing spend to referral marketing. Although you do a lot of the work, customers on the Enterprise plan get access to dedicated referral marketing.

You can manage your referral program from many existing business tools that you can use, including HubSpot, Shopify, Salesforce, Intercom, Zapier and more.

Reference Factory has created a collection of over 1000 templates that you can use. These cover a variety of industries and business types and even a variety of reference marketing models, so most companies will find a template to meet their needs. However, if you prove to be the exception, you can build a program from scratch that best meets your needs.

4. Loomly

Loomly also focuses on social media marketing and programming rather than acting as a full-fledged internet marketing agency. If you want your entire digital marketing covered, you’ll probably need to look at a more extensive agency on this list. However, if your focus is on social media marketing, Loomly will simplify your life.

Using Loomly facilitates brand and content management for marketing teams. It has everything you need to effectively collaborate on building a successful brand. You can manage your social media content – organic posts and ads – from one platform. And they notify you every time someone on your team updates or comments on content via email, push, slack and Microsoft Teams notifications. Loomly even lets you post ideas based on trending topics, RSS feeds, dating events, and best practices on social media. And it provides a central, intuitive library for storing, organizing, and using your assets.

5. Sendible

Sendible helps you manage your social media on a scale, whether it’s Facebook, Twitter, Instagram, YouTube, LinkedIn or Google My Business. You can customize and preview posts for each platform and save images seamlessly with Sendible’s built-in editor or draw new graphics with Canva.

Sendible puts a lot of emphasis on helping you make your social media management workflow easier. It includes valuable features like post preview, content suggestions, automatic post RSS feeds, mass planning, smart queues, and a content library.

You’ll find powerful editing features that allow you to create social media posts the way you prefer. You can schedule tailored posts individually, in queues, or in bulk, and check them in the calendar view. Add seasonal advertisements, campaign ideas and vacations as sketch posts in your calendar to pencil on critical dates.

6. Iconosquare

Iconosquare is another company specializing in a single niche market – offering easy-to-analyze social media analytics so you can make data-driven decisions for Instagram, TikTok, LinkedIn, Facebook and Twitter. It gives you a custom dashboard that quickly visualizes the most important metrics, and you can schedule reports to help you stay on top of your game. You can even get analytics for advertised Instagram content on selected plans.

However, you can use Iconosquare for more than just social analysis. It includes a powerful scheduler so you can publish your content automatically.

In addition, you can take a detailed look at selected competitors and understand how your performance is accumulating with the rest of your industry in several ways.

7. Netpeak

Netpeak offers a full range of digital marketing services, from SEO 2.0 to comparative shopping service. Their mission is to help you grow your business using the power of the internet spaces, and they now have 16 years of result-oriented work to showcase their efforts.

Their specialties include SEO, PPC, email marketing, SMM, mobile performance marketing, SERM, internet analytics and business intelligence. They use a full range of tools for pay per click and targeted digital advertising to reach the right person at the right time, in the right place.

8. Power Digital Marketing

Power Digital Marketing offers full funnel digital marketing services to reach your target audience. They identify shortcomings in your current marketing strategy and then consult you on what you need and how they can do it. The digital marketing agency views itself as an extension of your team that integrates seamlessly with the other arms of your business.

Powerful Digital Marketing can provide scalable marketing for every stage of your funnel. Their services cover SEO, PR, branded and unbranded search (PPC and Amazon), influential marketing, paid social prospecting, social media, content marketing and affiliate marketing.

9. Promodo

Promodo offers digital marketing solutions for e-commerce websites, B2B lead generation and startup projects. You’ll find full all-channel support under one roof, covering paid media and PPC, search engine marketing, design and creation, retention marketing, UX / UI, network analytics, mobile marketing and strategy.

Promodo uses a three-step strategy for rapid revenue growth when working with business ventures. The steps are:

They recognize that eCommerce companies ’websites are their main sales tool, and the only way to get prospects to find them is to build a solid online reputation.

10. Ignite Visibility

Ignite Visibility is a highly award-winning performance-based digital marketing agency based in San Diego, CA, representing leading brands. They will provide you with a strategy and team of experts to build you the best digital marketing program in your space.

You can get help from Ignite Visibility for earned media, paid media, email marketing, Amazon marketing and a wide range of custom services uniquely tailored to each business.

Ignite Visibility’s mission is to provide the most effective digital marketing solutions in the industry, a high-impact customer experience, deliver results and use profits to reinvest in customer success, employee success and the community. It has a proprietary multi-channel performance-based forecasting system and software allowing businesses to clearly hit targets.

11. WebFX

WebFX aims to be a leader in SEO, PPC, social, marketing and web design services. The digital marketing agency prides itself on driving qualified traffic, converting visitors and using cutting-edge technology to measure effectiveness to deliver perceptible customer results. So far, they have had more than 700 satisfied customers.

The agency’s team of more than 450 experts makes it one of the largest performing digital marketing agencies in the world. In addition, all clients have access to MarketingCloudFX, WebFX’s best marketing software that tracks the smallest details of each campaign.

WebFX offers a wide range of services related to organic search, digital advertising and social media. In addition, they provide special services for business enterprises, not only digital marketing such as SEO, PPC and social media advertising, but also setting up business platforms, Amazon marketing, and even enabling B2B eCommerce. In addition, they can help your company with UX and interactive designs, content marketing and development.

12. Brainvire 

Brainvire describes itself as a digital transformation agency that empowers businesses through innovation. They help many companies build digital solutions that deliver outstanding customer experiences.

Well, much of their work is more technological than marketing, with their list of services including eCommerce development (including Magento development), Microsoft development, Lamp technology, cloud services, mobile commerce and development, enterprise services, and Node.js and MongoDB. development. However, they fit into this list because they also offer a variety of digital marketing services.

Brainvire can help you with search engine optimization, paid advertising, social media marketing, SEO for e-commerce and content marketing. For example, with SEO, Brainvire’s Certified Digital Marketing specialists can help you drive traffic with controlled keywords that drive conversions higher speed than competitors ’websites. You can recruit their PPC services to place your brand at the top of search results. Their paid search specialists are working on PPC proposal strategies to improve ranking and impression.

13. Bop Design

Bop Design is a specialist B2B marketing agency offering B2B website design, content marketing and branding. They understand the B2B sales funnel and the tactics that generate and nurture ideal customer guides. B2B customer acquisition is different: a longer sales cycle, multiple decision makers, a higher price product or service, targeting a niche market with very specific priorities and pain points. Bop Design works with you to implement an ongoing digital and B2B content marketing strategy to ensure that your company is up to the right decision makers.

The agency primarily works with B2B companies in the following sectors: industry, software, consulting, technology, biotechnology, healthcare, accounting, engineering and finance.

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Headquartered primarily in Rajasthan, India, SamBlogs is an SEO consulting agency offering various tailored services for digital and social media marketing to clients in its area and the world.

Very often, the success of small businesses and online startups often depends on the ability to market themselves digitally to reach their target demographic. Failure or even falling short on this can make it difficult for any company to recover, which is why SamBlogs is here to make sure nothing resembles its customers. SamBlogs practices proven and true online marketing tactics that help companies easily reach their target audience while also offering them lasting solutions to their problems.

Working with SamBlogs gives you access to the experience and knowledge you need to effectively promote your business and products online. They have been nominated as the leading marketing company in Rajasthan by PR Expert so that you can be sure of their quality of work. The company has more than 10 years of experience working in the sector and only aims to offer services of the highest quality with the latest marketing techniques available. They are proud to be able to offer customers unique experiences with their secret techniques that give them a unique advantage over other companies in the industry. Working closely with SamBlogs guarantees that you will receive the most unique and practical marketing solutions in the digital world and you will have long-lasting results. The SamBlogs team specializes primarily in search engine optimization (SEO) and provides it as a primary service to customers looking to market and promote themselves online.

SEO is considered one of the most practical ways to promote any online business as it offers a lot of benefits that positively affect a business in the long run. One of the main objects of SEO generates natural traffic to the website which allows them to gain traffic and grow in popularity. Other things that arise from this include improving the brand’s reputation and being a long-term solution to an ongoing problem. SamBlogs promises these advantages and more, as they have been able to constantly deliver the desired results to their customers, having worked with over a thousand so far.

Aside from SEO, SamBlogs also offers a variety of services such as social media marketing, online reputation management, content marketing, link building and keyword analysis, all of which help to promote any website. and business at great heights. The team working in the business is always looking for a way to improve their services as they are committed to being able to offer the best services to all their customers and help them scale their business. They are always looking for dynamic approaches to problems and are willing to help a company no matter its size or the potential competition it has to face.

Visit the website at https://samblogs.com to learn about SEO agency.

SamBlogs can do everything you need, from promoting your online business to basic SEO service to content marketing and campaigns, to make sure your business can grow and develop. Interested customers can go to their official website for more information about their services and take advantage of them.

Contact the media Company name: SamBlogsContact person: Support e-mail: Send an e-mail Telephone: +91 98280 80195 Address: C-102 Unnati Residency, Sector 1, Vidhyadhar Nagar City: Jaipur State: Rajasthan, 302023Country: India Website: https: // samblogs.

Abstract

Background

Non-pathological cognitive decline is a neurodegenerative condition associated with brain aging due to epigenetic changes, telomere shortening, stem cell depletion, or altered differentiation. Human umbilical cord mesenchymal stem cells (hUCMSCs) have shown excellent therapeutic prospects on the hallmarks of aging. In this study, we aimed to elucidate the role of hUCMSCs with down-regulated miRNA-206 (hUCMSCs anti-miR-206) on cognitive decline and the underlying mechanism.

Methods

After daily subcutaneous injection of D-gal (500 mg / kg / d) for 8 weeks, 17-week-old male mice C57BL / 6 J were stem cells transplanted by lateral ventricular localization injection. During the 10-day rest period, the behavioral experiments applied to cognitive behavior in the hippocampus were tested. And then, the mice were sacrificed for sampling to complete the molecular and morphological experiments.

Results

Our behavioral experiments of an open field test (OFT), a new object recognition test (NOR), and a Y-maze revealed that D-galactose (D-gal) -induced aging mice treated with hUCMSCs anti-miR-206 had no evidence. spontaneous action. disorder and had recovery in learning and spatial memory ability compared with the PBS-treated group. The anti-miR-206 hUCMSCs reconstructed neuronal physiological function in the hippocampal regions of the aging mice with an increase in Nissl bodies and the overexpression of Egr-1, BDNF, and PSD-95.

Conclusion

This study first reports that hUCMSCs anti-miR-206 could provide a new stem cell-based anti-aging therapeutic approach.

Introduction

Aging is a gradual decrease in the biological functions of multiple organs in the body, among which the brain is one of the most susceptible [1, 2]. The non-pathological changes increase in the process of brain aging, characterized by a cognitive decline, manifested in the decline of learning, memory and recognition [3]. Although the neural aging mechanism remains transient, the hippocampus, which performs the learning and memory functions, is highly involved [4]. The hippocampus has a huge network of intermediate neurons. The main neurons in these regions are granular cells in the DG region and pyramidal cells in CA1 and CA3 of the Ammon’s horn, which form a one-way three-Synaptic pathway. This network has been shown to affect cognition, which in turn affects memory and mood. Hippocampal neurodegeneration is associated with decreased cellular transcription level, neuronal physiological function, synaptic plasticity [6, 7], and cognitive function [8,9,10,11,12]. Brain aging can be induced by D-gal, which causes oxidative stress-mediated superoxide free radicals. Studies have shown that long-term subcutaneous injection of high doses of D-gal in rodents has resulted in cellular swelling, metabolic disorders, and accelerated aging processes [13].

The unique tendency of MSCs to multiply and differentiate has been exploited in repair and transplantation of tissues / organs such as heart, bone and lung [14,15,16]. However, transplanted MSCs have shown low differentiation efficiency and survival rate decrease in the host [17] due to paracrine substances containing many cellular regulatory factors, and overexpressed exosomal microRNAs [18]. For example, paracrine function of stem cell therapy has been enhanced through microenvironmental control in vivo, immune regulation, inhibition of apoptosis, and nutritional support [19, 20]. Among differentially expressed microRNAs, miR-206 targets a brain-derived neurotrophic factor (BNDF), an important member of the neurotrophic factor family, mostly active in gray matter regions such as the cortex, hippocampus, and basal forebrain that protect the nervous system. Zhang et al. reported that MSCs with lower miR-206 showed improved survival in the infarcted heart [21]. In addition, J. Liu et al. showed that miR-206 overexpression reversed MSCs-Exo-mediated mitigation of chondrocyte injury [22].

Here, we explained that hUCMSCs with lower miR-206 improved neuroprotection and knowledge in a D-gal-induced aging mouse model, and behavioral tests showed upstream mechanisms by the expression of Egr-1, BDNF, and PSD-95 neurotrophic factors. in the hippocampus. . Overall, this study provides new insights into stem cell anti-aging therapy.

Results

Characterization of hUCMSCs

hUCMSCs of human umbilical cord may multiply in culture plates (Fig. 1a). In vitro differentiation experiment showed that hUCMSCs were successfully differentiated into osteoblasts or adipocytes under the special induction medium (Fig. 1b, c). Flow cytometric sign identification provided confirmatory results that hUCMSCs showed positive for CD29 (99.87%), CD44 (99.31%), CD73 (94.41%), CD90 (99.17%) and CD105 (99.17%), and negative for CD34 (1.64%). ), CD45 (0.22%), CD31 (0.23%) and HLA-DR (0%), confirming the presence of human stem cells (Fig. 1d).

Spindle-shaped morphology of hUCMSCs (bar = 50 µm). b Osteogen-derived hUCMSCs (bar = 200 µm). c fat-derived hUCMSCs (bar = 50 µm). d Fluorescence activated cell sorting (FACS) analysis of hUCMSCs with positive CD44, CD90, CD105, CD29, CD73, and negative CD34, CD45, CD31, and HLA-DR (n = 6, p & lt; 0.05).

hUCMSCs improved the learning and spatial memory ability of mice

Prolonged subcutaneous injection of high-dose D-gal increases free radical medial oxidative stress in mice, causing premature brain aging, hippocampal damage, and possibly cognitive decline [13]. The effect of D-gal on cognitive behavior was analyzed by open-field activity (OFT), new object recognition test (NOR), and Y-maze) in the hippocampus during a 10-day rest period after treatment at 18 weeks. -old male C57BL / 6 J mice. The mice were randomly divided into four groups: normal, D-gal treated with PBS (D-gal + PBS), hucMSCs (Cell), and miR-206 down-regulated hucMSCs (Cell-anti-miR-206). After daily subcutaneous injection of D-gal (500 mg / kg / d) for 8 weeks, mice were injected into the lateral ventricle with untreated hucMSCs or pre-treated with Cell-anti-miR-206, separately. During the 10-day rest period, the OFT, NOR, and Y maze were completed. Mice were sacrificed on day 10 to remove the cerebral hippocampus for molecular and morphological subsequent experiments.

The OFT was performed to test the motor skills of mice. As shown in Figs. 2a, d, the results were not statistically different, indicating no obvious spontaneous activity disorder and anxiety. The NOR test was performed to observe the short-term learning and memory ability of mice. Compared with the normal group, the new object recognition index (NOI) of the D-gal group decreased and NOI increased significantly after cell transplantation (Fig. 2b). This showed that D-gal-induced aging mice decreased learning memory that recovered after cell intervention.

OFT experimentation of spontaneous mobility and anxiety of mice. b The learning and memory ability of mice tested by NOR. c The spatial memory capacity of mice tested by a Y maze. d Spontaneous movement of mice in an open field trajectory diagram. The results were expressed as the Mean ± SD, * p & lt; 0.05, versus D-gal + PBS groups, n = 6.

The Y-maze was made to observe the short-term spatial working memory of mice. The results showed that the spontaneous alternating rate of D-gal mice decreased, and after cell transplantation increased compared to the PBS control group (Fig. 2c). It is noteworthy that although there was no significant difference in OFT between the cell and the cell-versus-miRNA206 groups; difference was observed in NOR and more significant in Y-maze (Fig. 2b, c). Overall, the huMSCs improved the learning and spatial memory ability of mice.

hUCMSCs intervention through a paracrine mechanism

To track hUCMSCs, we stained hUCMSCs with DIR paint before transplanting. Immunofluorescence staining showed highly stained cells (Fig. 3a). After lateral ventricular injection transplantation, cells were monitored for 1, 5, and 10 days. The signal decreased progressively over time and there was no detectable trace of hUCMSCs in the hippocampus after 10 days (Fig. 3b), suggesting migration of hUCMSC through paracrine exosomes to stimulate treatment.

Detection of hUCMSCs-DiR and cell nucleus stained with DAPI in the lateral ventricle. b Detection of huCMSCs-DIR in the hippocampus, Bar = 50 µm, n = 6.

BDNF is negatively regulated with miR-206

MicroRNA, as a post-transcriptional regulatory element, is involved in the post-transcriptional regulation of target gene expression [23]. The target Scan Human Database analysis showed that miR-206 targeted BDNF, suggesting that miR-206 may be associated with neuroprotection. To verify whether miR-206 regulates BDNF, we transfected hUCMSCs with miR-206 inhibitor and NC. RT-qPCR showed no significant difference in BDNF-mRNA expression (Fig. 4b). However, western blot showed that the protein expression level of BDNF was significantly increased (Fig. 4C, D). The results showed that the low expression of miR-206 promoted the high expression of BDNF in hUCMSCs, and BDNF was negatively regulated with miR-206, confirming that miR-206 could directly bind to the BDNF.

After the transfection of hUCMSCs with a miR-206 inhibitor, the expression level of miR-206 mRNA was detected by RT-qPCR. b After transfection with a miR-206 inhibitor, the expression level of BDNF-mRNA was detected by RT-qPCR. c After transfection of hUCMSCs with an miR-206 inhibitor, the expression level of BDNF protein was detected by western blot. d Quantitative analysis of BDNF protein levels. The results were expressed as the Mean ± SD, * p & lt; 0.05, against normal groups, n = 6.

hUCMSCs with down-regulated miR-206 achieve neuroprotective effects by targeting BDNF in vivo

The above results confirmed that BDNF is negatively regulated with miR-206. In addition, to study the effect of low miR-206 expression on brain aging, we performed immunofluorescence staining of anti-BDNF antibody and western blot in the hippocampus of mice. As shown in Figs. 5, the results showed that the low expression of miR-206 in hUCMSCs promoted the expression of BDNF in the damaged hippocampus. These results revealed that miR-206 subregulated hUCMSCs exhibited neuroprotection.

a BDNF protein levels were determined by western blotting. b Quantitative analysis of BDNF protein levels using ImageJ. c Representative micrographs for BDNF immunofluorescence staining of hippocampus of mice in four groups (n = 5 per group). d Quantification of BDNF-positive cells in four groups. The results were expressed as the Mean ± SD, * p & lt; 0.05, n.s., insignificant, bar = 50 µm, n = 6.

hUCMSCs with downregulated miR-206 promoted the physiological function of hippocampal neurons

Ten days after transplantation of hUCMSCs, mouse brain coronary sections were HE stained, which showed no mass cell shrinkage in the hippocampus of each group, and the cell structure was complete and orderly (Fig. 6a). The results showed that there was no large number of neuronal apoptosis that was consistent with non-pathological aging. However, more differences were observed in the Nissl staining. Nissl staining was performed to observe the changes in hippocampal CA1 and DG regions, involved in memory formation. The Nissl body staining was deeper, and neuron morphology was regular. In the D-gal + PBS group, the number of Nissl bodies decreased, the stain was superficial, and the morphology of neuronal cells was irregular. After transplantation of hUCMSCs with lower miR-206, the number of Nissl bodies increased, and the spot was deeper than that of the D-gal + Cell group (Fig. 6b, c). The increase in the Nissl bodies can be attributed to the reconstruction of protein synthesis in neurons and their normal physiological functioning, suggesting that hUCMSCs with lower miR-206 promoted the physiological function of hippocampal neurons.

a HE staining of the hippocampus of D-gal-induced aging mice. b Nissl staining of the hippocampal CA1 and DG regions of D-gal-induced aging mice. Bar = 200 µm. Enlarged images after the black frame. Bar = 50 µm. c Quantitative analysis of the number of neurons using ImageJ. The results are expressed as the Mean ± SD, * p & lt; 0.05, verses D-gal + PBS groups, #p & lt; 0.05, verses D-gal + Cell Groups (n = 6).

HUCMSCs with down-regulated miR-206 promoted the expression of learning and memory-related proteins

Postsynaptic density-95 (PSD-95) is the main component of the excitatory postsynaptic membrane is dense and plays an important role in synaptic signaling, learning, and memory. Studies have shown that PSD-95 can promote synaptic maturation and improve synaptic plasticity [24]. Therefore, a PSD-95 protein expression level was detected, which was significantly upregulated in tissue samples from an anti-miR-206 group (Fig. 7a, b). We then detected Egr-1, an immediate early gene and one of the important molecules involved in the formation and consolidation of long-term memory by synaptic plasticity of neurons, [25] and its decline impairs spatial learning and memory [26]. Interestingly, our immunohistochemical staining results showed Egr-1 expression upregulated in the dental gyrus in the anti-miR-206 group (Fig. 7c, d).

a The protein levels of PSD-95 were determined by western blotting. b Quantitative analysis of the protein levels of PSD-95 and using ImageJ. c Egr-1 in the hippocampus was detected by immunohistochemistry, Bar = 50 µm. d Quantitative analysis of the Egr-1 protein levels of ImageJ. The results were expressed as the Mean ± SD, * p & lt; 0.05, against D-gal + PBS groups (n = 6).

Discussion

Aging is an inevitable process. With the deterioration of the aging population, minimizing or mitigating aging is essential to possibly slow down age and age-related diseases. Stem cell therapy promises anti-aging due to its rapid and effective therapeutic effect, precision delivery and safety.

MicroRNAs are a class of endogenous small non-coding RNAs responsible for post-transcriptional regulation of gene expression. Recent studies have shown that miR-206 targets BDNF, which regulates anxiety-related behaviors and neuropathic pain induced by chronic contraction injury [28, 29]. The miR-206 knockdown exosomes therapy has been more effective in reducing neuronal death and improving neurobehavioral scores during early brain injury (EBI) [30]. We hypothesize that microRNAs play a key role in communication between MSCs and parenchymal cells. In this study, we focused on the relationship between miR-206 abundance in paracrine hUCMSCs and neuroprotection. Decreasing miR-206 expression from hUCMSCs, cells were transplanted into D-gal-induced mouse aging. Our results showed that the BDNF of hUCMSCs was increased after the subregulation of miR-206. Because microRNA is responsible for the post-transcriptional regulation of gene expression, we detected no difference in the mRNA level of BDNF, but the protein expression was upregulated. This is why we know that miR-206 targets BDNF and is negatively regulated. In addition, we detected that after subregulation of miR-206, BDNF expression in the mouse hippocampus was significantly increased. And Nissl staining showed that the state of neurons in the DG and CA1 regions of the mouse hippocampus was restored to varying degrees after stem cell intervention. Nissl staining may reflect the reproductive and synthetic capacity of nerve cells, indicating that the physiological state of nerve cells in the hippocampus has been better improved. Suggesting that miR-206 may be a key regulator of intervention in D-gal-induced aging.

Recently, proteins containing the PDZ motif have been proposed as a molecular scaffold of receptors and synaptic cytoskeletal elements. The prototype PDZ protein, PSD-95 / SAP-90, is a membrane-related guanylate kinase (MAGUK) concentrated at glutamatergic synapse. PSD-95 is the main component of excitatory postsynapses, which plays an important role in the formation and maturation of synapses and synaptic signaling, learning and memory functions. Meanwhile, D-gal as a common model of aging research, induced aging has been widely used in pharmacological studies. Our results also showed that after D-gal-induced aging, the protein expression level of PSD-95 in the hippocampal tissues of mice decreased significantly, which was recovered after stem cell transplantation. Egr-1 is also involved in learning and memory and synaptic plasticity, memory formation, and consolidation. We detected that Egr-1 expression was downregulated after D-gal-induced aging, then up-regulated after treated hUCMSCs with lower miR-206. Therefore, we believe that hUCMSCs with down-regulated miR-206 have a better neuroprotective effect targeting BDNF in D-gal-induced aging mice.

Upstream CREB regulates Egr-1. When p-CREB, the activated form of CREB, is continuously enhanced in the hippocampus, the expression of downstream Egr-1 will be promoted, thus influencing learning and memory ability [31]. CREB is a key transcription factor in the activation signal of TrkB, which is the binding receptor of BDNF. Overregulation of BDNF / TrkB / CREB this classical neuroprotective pathway signaling prevents neuronal apoptosis in neurodegenerative diseases [32]. Therefore, we consider that miR-206 can enhance aging knowledge by the BDNF / TrkB / CREB signaling pathway targeting BDNF in D-gal-induced aging mice.

In our study, the tracking of stem cells in the brain showed no cells in the lateral ventricle after 10 days. We also tested three short-term memory behavioral experiments related to episodic and spatial learning. However, it is also necessary to explore long-term learning and memory ability of cognitive ability, which can be explored by other related behavioral tests in future studies. In addition, we have shown that miR-206 targets BDNF to enhance cognitive decline by investigating key intermediate molecules associated with the signaling pathway, suggesting further studies on the regulatory mechanism of its internal signaling pathway.

Conclusion

Our results first report that miR-206 targets BDNF and negatively regulates BDNF expression. hUCMSCs with down-regulated miR-206 can enhance the physiological function of nerve cells in the hippocampus and promote the expression of PSD-95 and Egr-1 in relation to learning and memory. The data propose that the neuroprotective role of hUCMSCs with subregulated miR-206 is one strategy to improve the cognitive decline of aging.

Methods

Animals

In this experiment, a total of 60 C57BL / 6 J mice (Hunan Slack Jingda Experimental Animal Co., Ltd, Changsha, China) weighing 25–30 g at 9 weeks in this experiment were used. The animal model was established for eight weeks, to ensure the physical health of the animals, we selected a 9-week-old in young mice. Mice were bred in the Department of Laboratory Animal Science (Central South University, Changsha, Hunan, China) and housed individually in separate cages (SPF + IVC) and had free access to food and water and a 12 h light-dark cycle for seven days. . before the experiment. All animal experiments were in accordance with the “Guide for the Care and Use of Laboratory Animals, 8th ed., 2010” (National Institutes of Health, Bethesda, MD) and were approved by the Institutional Animal Care and Use Committee of Central South University (Changsha, China; License Number: 2020sydw0913).

hUCMSCs Culture

hUCMSCs were from the company HYS Cell Gene Engineering (Changsha, Hunan, China). The hUCMSCs were cultured using F12-DMEM (DMEM, Gibco, Grand Island, NY, USA), which contained 10% FBS (FBS, Gibco, Grand Island, NY, USA) for the primary culture (37 ° C, 5% CO2). ). ), and then passed after reaching 80-90% confluence. P5 hUCMSCs were used in all experiments and counted by hemocytometer. Cell phenotypes were analyzed by flow cytometry. The P5 hUCMSCs were characterized using CD34, CD44, CD45, CD29, CD31, CD73, CD90, CD105, and HLA-DR antibodies (Biolegend, Way San Diego, CA, USA). When hUCMSCs reached an 80–90% confluence, hUCMSCs were seeded in a six-well plate with approximately 5 × 106 cells per well. After 24 h, Mesenchymal Stem Cell Osteogenic Differentiation Kit (5011-024-K, Trevigen, MD, USA) was used to induce osteogenic differentiation of hUCMSCs. Calcium nodules formed after osteogenic induction were stained with Alizarin Red. The hUCMSCs were induced by mesenchymal stem cell adipogenic differentiation kit (5010–024-K, Trevigen, MD, USA) to stimulate adipogenic differentiation and stained with red O oil.

Cell transfection

The miR-206 inhibitors and negative control (NC) (GenePharma, Shanghai, China) were transfected into hUCMSCs at a final concentration of 50 nmol / L. After recovery, P5 hUCMSCs were cultured in the 6-well plates until their density reached 80–90%. The miR-206 or NC inhibitors and Lipofectamine 3000 Transfection Reagent (L3000001, Thermo Fisher Scientific, MA, USA) were diluted with Opti-MEM (31985062, Gibco, Grand Island, NY, USA) and cells were cultured with Opti-MEM . After 6 hours of transfection, the hUCMSCs were transferred to DMEM medium supplemented with 10% FBS for 48 h before post-transfection performance measurement by RT-PCR or Western blot.

Animal model experimentation and cell transplantation

D-gal (500 mg / kg / d) was subcutaneously injected into the back of each C57 mouse once a day for 8 weeks. The normal group injected the same volume of salt. Mice were anesthetized by intraperitoneal injection of pentobarbital (ZaoZhuang, Shandong, China) at approximately 10 mg / kg. According to the mouse brain brain stereotypical map, the anterior fontanelle is 2.0 mm backward, the sagittal suture (middle, ML) was 1.5 mm left and right, and the insertion depth (dorsoventral, DV) was 2.5 mm. 10 µl of PBS or MSCs cell suspension was slowly injected into the lateral ventricle with microliter injectors and stopped for 2 min after each injection of 2 µl. After completion, the syringe was stopped for 5 min.

The mice were randomly assigned to 4 groups: normal (n = 15) control group, D-gal + PBS (n = 15), D-gal + Cell (n = 15), D-gal + Cell-anti-miR-206 (n = 15).

After the operation, 10 days after injection of MSCs, all mice were sacrificed and the hippocampus and entire brain were removed.

Tracers after cell transplantation

P5 hUCMSCs were collected and covered in the 6-well plates or circular microscope cover. When it reaches 80–90%, cells were stained with DIR Iodine (DiIC18) (Maokangbio, Shanghai, China) at 37 ° C for 20 min according to the manufacturing protocol (7). The cells were revived, washed and collected using PBS. Then, the glass slide was DAPI stained for nuclear observation under fluorescence microscopy. The collected cells were injected into the lateral ventricle of the mouse brain, and the post-OCT embedded brain was cut into serial 15-μm-thick coronal sections and DAPI stained for fluorescence microscopy.

Behavioral experiment

The open-field test (OFT): a 40 × 40 × 40 cm cube box was used. The bottom of the box was divided into 25 squares of equal size, of which nine squares in the center were defined as the central region and the rest as the peripheral region. Each mouse in turn was placed in the central area and left to explore for 10 min.

The new object recognition test (NOR): it is divided into three periods: adaptation, training and testing. The first day was the adaptation period. The mice were positioned in the middle of the open field in turn and explored freely for 5 min. Twenty-four hours later, entering the training period, two identical objects were placed in the open field with an interval of 20 cm or 10 cm between the two objects and the side wall. Each mouse was placed from the center line attached to the wall on the side of the non-object area of ​​the open field and was left to explore for 5 min. After a break of 1 h, entering the test period, one of the objects in the open field was removed and replaced with a new object with the same material but different colors and sizes. Then, the mice were placed in the same position in the box in turn and left to explore freely for 5 min. Then calculate according to the formula: new object recognition index (NOI) = explore new object time / (exploring new object time + exploring old object time) × 100% [34, 35].

The Y-maze: three identical 30 × 5 × 15 cm gray closed arms with a 120 ° angle between the three arms (new arm, different arm and initial arm), and a triangle middle joint. There are two periods: the training period and the testing period. In the training period, the new arm was closed, and the mice were placed in the initial arm turning to the middle articular part and left to explore for 10 minutes. After a 1 h rest, open the new arm, and the mice were tested for free exploration for 5 minutes in a row. When the mouse limbs fully entered the arm, it was assessed as an arm entry. When the third bracing was different from the first two, it was considered the correct bracing. Then calculate according to the formula: spontaneous alternation percentage% = (Number of Alternations / [Total number of arm entries-2]) × 100% [36].

In all three behavioral experiments, the mice were placed in a behavioral room an hour earlier to acclimatize to their environment. Prior to the experiment, even after each mouse completed the test, the devices were cleaned and wiped with 70% alcohol and kept dry, eliminating the interference of biases and olfactory signals. Each mouse completed the test and was placed in a separate cage to avoid interaction with the untested mice.

The hippocampal-involved behavioral experiments were completed in the Department of Laboratory Animal Science (Central South University, Changsha, China). An infrared camera was installed on top of the site and connected to a computer to track the mouse. The experimental data were collected and analyzed using the Smart3.0 software system.

RNA isolation and RT-qPCR

Total RNA was extracted from cells using Trizol (Thermo, USA) according to the manufacturing protocol. RNA was inversely transcribed into cDNA using a cDNA synthesis kit (Cwbio, Beijing, China). Subsequently, the cDNA product was used as a template for PCR using SYBR green mixture (Cwbio, Beijing, China). The tests were performed in triplicate, and data were analyzed using the 2-ΔΔCt method. U6 and Actin were used as internal controls. The U6, Actin, and BDNF introductions were designed as shown in the following table.

Western blot analysis

Total protein was extracted from cultured cells or tissue using RIPA (CWBIO, Shanghai, China) and a protease inhibitor (CWBIO, Shanghai, China). Protein concentration was determined by BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Equal amounts of proteins were electrophoresed in 10% SDS-PAGE and electro-transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA), and sealed with 5% skim milk for 2 h at room temperature and incubated with anti -BDNF antibody (1: 1000, Abcam, USA) or anti-PSD95 antibody (1: 1000, Proteintech, Wuhan China) overnight at 4 ° C. After washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h at room temperature. Protein bands were detected by enhanced chemiluminescent gear (Thermo Scientific, Rockford, IL, USA). Image J software (National Institutes of Health, USA) was used to quantify the band densities.

Morphology staining

After mouse intracranial infusion with PBS followed by 4% paraformaldehyde (PFA), the whole brain was fixed with PFA solution (Sinopharm Chemical Reagent Co. Ltd, Shanghai, China) t.e. 4% PFA in 0.1 M PBS (Solarbio, Beijing, China). ) at 4 ° C for 24 h. Subsequently, it was dehydrated with 15% sucrose solution at 4 ° C for 24 h and replaced with 30% sucrose solution for 24 h the next day. Brains were then cut into serial 15-μm-thick crown sections (Leica, Wetzlar, Germany) and mounted on glass slides. Tissue sections were stained with Hematoxylin Eosin (HE) (Beyotime, Shanghai, China) or with a Nissl body staining Kit (Meilun, Dalian, China) and ultimately studied using light microscopy. Images were captured with a Nikon confocal microscope (Nikon Instruments, Inc., Japan).

Immunohistochemistry and immunofluorescence

For immunohistochemical staining, tissue sections were incubated in 0.3% hydrogen peroxide / PBST (Solarbio, Beijing, China) for 30 min. The sections were then sealed with normal horse serum (Beyotime, Shanghai, China) / PBST (1: 200) for 2 h. Subsequently, they were incubated with anti-Egr1 antibody (1: 100, PTG, USA) overnight at 4 ° C, washed with PBS, and then incubated with HRP-labeled Goat Anti-Rabbit IgG antibody (1: 500). , ZSGB-BIO, Beijing China) at 37 ° C for 2 h. Finally, the slices were washed with PBS and sealed with the lid with neutral resin (Sinopharm Chemical Reagent Co. Ltd, Shanghai, China).

For immunofluorescence staining, the sections were blocked in normal azense serum / PBST (1: 200) for 2 h. Subsequently, they were incubated with anti-BDNF antibody (1: 1000, Abcam, USA) at 37 ° C for 1 h and washed with PBS. Subsequently, the sections were incubated with secondary antibodies (Alexa Fluor Cy5, 1: 800; APExBIO, USA). Cell cores were stained with DAPI (1: 1000, DAPI, Sigma Aldrich, MO, USA) and images were captured with a Nikon confocal microscope (Nikon Instruments, Inc., Japan). Finally, the slices were sealed with the lid with glycerin (Solarbio, Beijing, China).

Statistical Analysis

The data are expressed as Mean ± SD. The Prism Graph Pad software (version 7.0, La Jolla, CA) was used to perform the statistical analysis. One-way ANOVA and the Turkey test (analysis of variance) were used to check the differences between groups. p & lt; 0.05 was considered statistically significant.

Data availability

All relevant data and materials are available from the authors upon acceptable request.

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These insights (listed below) can help you predict customer behavior and make changes to your e-commerce website accordingly:

4. Limited Reporting Capabilities Unlike AU, GA4 does not yet have “out of the box” reporting templates and options. Everything still needs to be customized and created through a multi-step process.

The platform also limits data retention to 14 months, so you’ll need to connect to BigQuery data warehouses and data streams for long-term report analysis.

The good news? Once you get used to the interface and set up your account, you’ll appreciate that GA4’s SEO analytics and reporting capabilities are much more powerful than AU’s.

Common Google Analytics Errors for ecommerce Accounts

It is highly recommended that your e-commerce brand migrate your accounts as soon as possible to preserve year-over-year data and confirm correct account setup. But don’t let your rush to transfer introduce errors into your process.

To ensure that your data is accurate and actionable, try to avoid these common mistakes that e-commerce businesses make when using Google Analytics 4:

1. Turning off UA TrackingJust because your brand is transitioning to Google Analytics 4 doesn’t mean it’s time to ditch Universal Analytics just yet. For the future, we recommend continuing to track your data through UA until its end next summer.

Given the current limitations in GA4, parallel tracking with both platforms will allow for the fullest possible data within the next year. It also serves as a backup in case your initial migration doesn’t go well.

Continue to collect your data through UA as you evaluate your GA4 setup, and then migrate the rest of your stack when you’re ready.

2. Not Connecting BigQueryBigQuery’s data warehousing capabilities used to be available only to GA360 customers, at a cost of hundreds of thousands of dollars per year. But with the data retention restrictions in GA4, that has now changed.

Today, you can start your own data warehouse to get around GA4’s data retention limitations practically for free.

Again, the earlier you set up this capability, the more data you’ll have in the future. Forget to set it up, and you’ll be severely limited when looking back at your historical data.

3. Not Using “Recommended” Event Names and Parameters As mentioned above, GA4 still has a long way to go in terms of reporting. However, by using recommended event names and parameters, you can “unlock” extended reports and features in the platform.

For example, by labeling your e-commerce transaction events as “purchases,” you’ll enable the financial and e-commerce reporting in GA4—essential to tracking the growth of your online business.

Create Your GA4 Plan Now

If you haven’t started migrating your e-commerce site to GA4 yet, you’re working on a pretty tight timeline – but it’s vital that you’re ready when UA takes a bow in July 2023.

To move your GA4 accounts on your own, check out the following helpful resources on reviewing and setting up your GA4 account:

We also recommend reaching out to your ecommerce digital marketing agency or web development team for help. They may be able to help you start or complete your migration process by this summer’s deadline.

Entrepreneur and digital marketer Mike Belasco has been the founder and CEO of the digital marketing agency eCommerce Inflow since 2007. He built his background as a web developer and SEO specialist an agency that has worked with major brands such as Amazon, Overstock.com , Dish Network and many more. Today he leads a team of more than 25 PPC, SEO and conversion optimization experts as a boutique, fully remote e-commerce marketing agency.

The internet has become a valuable resource for finding a high quality place to order your custom night guards, specially fitted for your teeth. These custom guards can be done in three to four weeks and cost around $ 150 to $ 200. Online retailers have the same quality as dentists and can provide a dedicated night guard in about three to four weeks. Here are some tips to consider before making your choice:

First, get it right. Custom-made mouth protection fits better than one that is available over the counter. It will be more comfortable and maintain its shape than an over-the-counter mouth guard, which will not protect your teeth as effectively as a dedicated one. Perfect fitting is important, and an online lab will make sure you get the right fit every time. It will also be easier to maintain mouth protection if it fits properly.

Make an impression. You can take an impression at home or order a special guard through a dentist. Tailor-made mouth guards are more accurate. However, if you do not feel comfortable with a guard made by the dentist, you can make one yourself. You can also order a specially fitted mouthguard directly from the online dental lab. There are several benefits to both of these methods, but the former is more accurate and convenient.

A suitable nightguard is made of high quality material and designed to fit well. The night guard is comfortable to wear, and protects the teeth and does not interfere with your natural bite. It will not move, swing, or put uneven pressure on your teeth while you sleep. Plus, it won’t slide out of your teeth overnight. In addition to being more durable, these specially adapted night guards will not switch or rock overnight.

A dedicated night guard can help prevent bruxism and other problems caused by grinding teeth. It can prevent TMJ, grinding teeth, and other issues associated with TMJ and jaw pain. And since dental lab night guards are more comfortable and affordable, they will last longer than the options purchased from the store. So, when you need a specially adapted night guard, look no further than the dental lab.

There are several reasons for bruxism. In addition to bruxism, a dentist may recommend the use of a night guard to correct malocclusion. By addressing all of these issues, dental appliances work more effectively. A specially adapted night guard can also help prevent tooth decay and tooth loss. Your dentist may also recommend a specially designed night guard to improve your oral health and prevent future problems related to your teeth.

Folks who lighten their teeth during sleep rely on specially equipped guards for prevention. For the sellers of those guards, a complication is the custom fitting. It requires an exact impression of the teeth, which presumably requires a visit in person.

But not if the seller is Pro Teeth Guard, an online dental lab based in San Diego, California that sells custom-made tooth guards. Its mail-in printing process facilitates custom manufacturing – and satisfied customers.

I recently spoke with JP Ji, co-founder of Pro Teeth Guard. It has addressed the challenges of production, customer service, marketing, and more.

The full audio of that interview is incorporated below. The transcript is edited and condensed.

Eric Bandholz: Tell us about the Pro Teeth Guard.

JP Ji: We sell specially fitted mouth guards for people who brush their teeth when they sleep. We send kit to customers. They take an impression of their teeth and return it to us. We then manufacture the mouth guard and send the complete product to the buyer. We offer adjustments, one year warranty, and 60 day money back guarantee.

We started the business in 2012 when my mother started a lab serving local dentists. We now have nine employees and just over $ 1 million in annual revenue. During the Covid, many dentists closed. At the time, online sales had become a minor part of the business, probably 5% of overall revenue. So we said, “Let’s focus on our e-commerce site.”

Currently, the dental laboratory only serves the e-commerce business. My mother is responsible for the technical aspect of manufacturing the product and making sure it fits perfectly. I’m in charge of marketing, customer service, and finance.

We use a scorecard for each role within the company – inspired by Geoff Smart’s “Who: The Method for Hiring”. Each position has key results. We also have what we call “stretching outcomes.” A key outcome for a customer service employee is being able to follow processes, answer questions, and provide great experience. A strict result is the discovery of opportunities for improvement, such as better efficiency.

I ask employees, “What did you suggest this year or this quarter for improvement?” They will not be negatively affected or disciplined if they do not perform a stretch goal, but it is part of the role. We refer to customer service as “customer happiness.” Everyone in our company is a manager of customer happiness.

Bandholz: You have 24/7 support. How does it work?

Ji: We use a third party live chat service called HelpFlow. It uses our internal list of frequently asked questions. We collect all the questions we received via email and phone calls, build the list, and send it to them. Over time, HelpFlow staff is improved by updating internal FAQs. We have used the service for seven years. Eighty percent of customer service questions can now be answered based on previous support tickets.

Most chat agents are foreign. Agents are assigned to multiple clients, not just us. They report on a metric called “chatted revenue.” They can document, “This customer chatted with us and then converted.” Some customers may have converted regardless, but the conversion rate for those who were involved in chats is about 10 times higher than those who were not. The service charges a monthly fee. Scale based on volume, which works out to a few dollars per chat.

Bandholz: You also use a conversion optimization firm.

Ji: Yes. The firm, Conversion Crimes, conducts user testing to detect frictional points on a website and where people are confused. Conversion Crimes sends a buyer who matches our target audience to our site – someone who needs a night watch. The firm registers the person navigating the site with specific steps and indications. For example, the steps might be, “Land on the page. Scroll. click on anything. What is this page about? ” It tests whether a page conveys the purpose of our brand, website or product – on mobile and desktop.

We send those registrations to people in our user interface agency. They will take notes and address those limitations. After living with a new design, we will repeat the process with another text.

Here’s an example. We have learned that it is difficult for people to know which of our products to buy. We have four custom night guards, all similar but with different thickness and materials. Customers want to know which one they need. So we integrated a quiz to help. Based on the answers, I will recommend the best product. That quiz has a high level of engagement.

Bandholz: What is your marketing strategy?

Ji: We started very strong on search engine optimization in 2012. It was easier then. So that has been our main channel for a long time, SEO and Google Ads. We had our ups and downs with the Google algorithm. We’ve had updates that have halved our traffic over time. Then we recovered. Now customers from organic search are about 15% to 20% of total revenue.

We still use Google Ads. And affiliates are a great channel – about 25% of revenue – because many are masters at SEO. Whether you’re looking for the best mouth guard, night guard, or the best watch for grinding teeth, our affiliates are typically the best one, two or three results.

The rest is email marketing. We never break Facebook Ads. For a while, we used to run retargeting ads on Facebook. That was good. But even then, after the iOS 14.5 updates, retargeting was not profitable for us. We probably spent the equivalent of a Honda Civic on Facebook Ads and never got a return.

We’ve nailed our content marketing process pretty well. So far, it’s almost all for SEO. But people don’t always search on Google for questions about mouth guards and grinding teeth. So we’re looking to invest in videos, like on YouTube and TikTok.

Competitor started TikTok channel. One of their videos on tooth grinding on TikTok got over a million views. We are looking to unlock TikTok and the creator market to try similar things.

Bandholz: Where can people learn more about you?

Ji: Our website is ProTeethGuard.com. I’m on Twitter and LinkedIn.

Dallas, TX, June 20, 2022 (GLOBE NEWSWIRE) — KISS PR is a Dallas-based SEO company that offers comprehensive audits and SEO services for businesses of all sizes. The company has a team of experienced SEO experts who have helped countless companies improve their online visibility and organic traffic.

Technical SEO is the process of optimizing a website for search engines. It involves improving the architecture, code, and page elements of the website to make it easier for search engines to crawl and index the website. Technical SEO also includes optimizing your site’s content and structure for the keywords you want to rank for.

Technical SEO is important because it helps search engines understand your site and index it correctly. If your site is not properly optimized, search engines may have difficulty crawling and indexing your site, which can affect your ability to rank in search results.

Examples of technical SEO include

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