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Grapefruit is rich in vitamin C, bioflavonoids, fiber and low in calories. Grapefruit is a fruit with innumerable beneficial properties for the body. It is the only citrus supposedly not from Southeast Asia, but from Central America. A glass of grapefruit juice would bring, in addition to vitamin C, potassium, lycopene, calcium, sugar and phosphorus. Drinking it for breakfast would allow you to start the day with a great energy charge.
The vegetable chemicals contained in the grapefruit would protect against cancer and heart disease; they would help metabolism and regulate insulin levels; they would fight fatigue, fever, constipation, excess of acidity and much more. Let's explore its benefits closely.
1 – Grapefruit decreases the feeling of appetite. Already its smell would be able to reduce the feeling of hunger. The high quantity of fibers inside allows you to satisfy hunger and avoid eating too much. Stimulating cholecystokinin, a hormone that regulates digestive enzymes, grapefruit acts as an hunger inhibitor.
2 – Grapefruit is a precious ally against the flu. It helps to reduce acidity in the body. The naringin, the major flavanone glycoside that gives the grapefruit its bitter taste, is a powerful antioxidant. Grapefruit is also antiviral, antifungal, antibacterial, anticancer and anti-inflammatory. It is excellent the defense of the immune system.
3 – Acidity. After digestion, grapefruit juice creates an alkaline reaction. The citric acid contained in the fruit increases the alkalinity reaction after digestion. The juice extracted from grapefruit is useful in preventing the formation of acid and the diseases that arise due to the presence of acidity in the body.
4 – Constipation. Grapefruit juice drunk in the morning is an excellent remedy to control constipation. It stimulates the colon, inducing intestinal movement. Grapefruit can relieve flatulence and an upset stomach.
5 – Fever. Grapefruit juice and pulp help to recover from fever. It strengthens the immune system in general. Combined with water, grapefruit juice can calm down thirst and maintain hydration for a long time. To allow this benefit is the high content of vitamin C.
6 – Grapefruits are an excellent remedy against fatigue. They help to dissipate tiredness. Grapefruit and lemon juice can be a panacea to quickly increase energy levels. The nootkatone contained in grapefruit improves the body's energy metabolism through the activation of ampkinase (ampk is a kinase involved in the activation of the process of beta oxidation of fatty acids). This translates into more energy, greater weight loss and fewer opportunities to develop diabetes.
7 – Grapefruit is useful for indigestion. It works by relieving heat and irritation in the stomach. Grapefruit improves the flow of gastric juices, it facilitates the movement of the bowels, it keeps the excretory apparatus in good condition. This thanks to the fibers and the pulp.
8 – Insomnia. A glass of grapefruit juice, drunk before going to bed, would be able to promote healthy sleep and alleviate the annoying symptoms and the repercussions of insomnia. This is thanks to tryptophan, an essential amino acid, not synthesized by the human body, a precursor of serotonin, involved in the modulation of mood, melatonin, involved in the regulation of sleep rhythms, and of niacin or vitamin B6.
9 – Diabetes. Consuming grapefruit helps reduce the level of starch in the body, regulate the flow of sugar in the body and effectively manage the disease. This thanks to the flavonoids contained within it.
10 – Urinary disorders. Grapefruit juice is rich in potassium and vitamin C, and it is one of the best treatments for urinary problems. The high content of potassium works as a vasodilator: the blood vessels and arteries relax, reducing blood pressure and reducing the risk of heart attacks and strokes. Increased potassium levels are also associated with improved cognitive function due to increased blood flow and oxygen to the brain.
11 – Cardiovascular diseases. Grapefruit allows the human body to regulate cholesterol, which means that it minimizes the risk of various cardiovascular diseases caused by cholesterol problems.
12 – Grapefruit prevents cancer. The pulp and fiber of grapefruit would help bowel movements, reducing the chances of developing colon cancer. Grapefruit intake would also reduce the risk of developing prostate cancer.
13 – Grapefruit helps in the treatment of malaria. Quinine is an advantageous natural alkaloid in the treatment of malaria. Grapefruit is one of the rare foods from which it is possible to extract it.
Side effects
Despite all the benefits that grapefruit can have on health, so many that in many cases grapefruits are more effective than drugs, attention must be paid to the consumption of grapefruit when taking medicines. Some chemicals contained in grapefruits, such as naringin and other less common compounds, can negatively interact with different drugs and cause damage to the body. It is necessary to consult your doctor in terms of drug interactions with grapefruit juice in the diet before starting any new treatment.
The nootkatone contained in this citrus is able to remove mosquitoes, ticks, lice and bedbugs. A way of fighting mosquitoes more natural and less dangerous.


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04 Mar 2018 20:04
Study right after study has confirmed that referral advertising and marketing is a single of the ideal forms of advertising and marketing when it comes to sales and conversions. "Producing a organization off of documenting your life on social media is an unsustainable model, merely because of the toll it requires on your life, on your relationships, on your psyche, on your physical and emotional stability," Armstrong tells Chattopadhyay.If you want to use a single installation of WordPress to run several sites, then you can allow multi-internet site. Also, you can limit the quantity of login attempts your website will let just before a user is banned. We advise limiting failed login attempts, as it will help make your site much less vulnerable to hackers.Everybody says to create a typical posting schedule. I did not begin off by posting often even though, and I in no way have. Bloggers say to post three times a week, but often I've posted once a week for months on finish. When I got a book deal, I posted once each and every three months and my traffic nevertheless grew. Interestingly, I as soon as posted every single single day for a month and my traffic dropped! Most important of all is focusing on your high quality of writing. Do not rush to post as a lot as feasible if it is going to sacrifice the good quality of your function.Look to Network Options, GoDaddy, or are very good in US and if you are in the UK to analysis and uncover the ideal domain name for your web site. Wordpress also includes a feature whereby you can use a name that is tagged with their web site, for example, But if the name you choose is also accessible as acom, they will notify you when you sign up.The single most important thing about blogging, I'd recommend, is to post often. If you're not certain of your voice" and need to have to get a manage on the medium, practice makes ideal! Typical, constant posting makes it possible for you to attract readers and subscribers, engage them in dialogue (readers will not comment at an abandoned, or irregularly updated weblog) and extend the conversation beyond your blog via hyperlinks and trackbacks.Handful of internet solutions have far more name recognition than WordPress , the open-supply content material management resolution that powers much more than a quarter of all internet sites. The WordPress platform is so pervasive and extensive that it is type of challenging to believe the computer software is completely free of charge.And since advances in technologies mold each aspect of every day communication, it is often time to focus and make use of your efforts on executing a appropriate digital strategy. Advertising tips are essential whether you're thinking about building a new internet site, starting to engage through social media, or reviewing your present digital activities.Write about what you intended to create about. Jump appropriate in. Your weblog can turn into a diary of sorts, or it can be a place exactly where you collect fascinating articles from around the web and comment on them. Of course, it can be anything in amongst. Write or post about what makes you happy.

subset: commoncrawl


text:
A BUSINESSMAN'S challenge to transform Guildford Borough Council's current leadership structure has failed after a petition calling for a directly elected mayor (DEM) was declared invalid.
Following detailed checks, officials found the petition did not contain the 4,943 valid signatures, representing 5% of registered voters, required by law. The main reasons for invalidity were because signatories lived outside the borough, were not old enough to vote, or had failed to join the electoral register. "Mickey Mouse" was among the signatories, according to leader of the council, Andrew Hodges.
Petition organiser, Raschid Abdullah, however, denied this claim. "To the best of my knowledge this is not true," he said. Despite the blow Mr Abdullah said he would press on with his campaign to give the electorate an opportunity to decide how their council should be run and continue collecting signatures.
He claimed that the current leadership is "out of touch" and "inefficient" and said he did not feel disappointed by the petition result but encouraged by all the support he had received. "It is quite a demanding process," he said. "Examples of invalidation range from a failure for a signatory to complete his or her name or their full address."
Under the proposed system a DEM, such as Ken Livingstone, would work with a council manager appointed by Guildford Borough Council (GBC). At present the council's leadership structure comprises a leader and eight Executive members. Since the Local Government Act (2000) was introduced, 31 referenda have been held in the UK. However, the mayoral system has only been appointed in 11 towns.
If the petition had met all legal requirements, the council would have been required to hold a referendum in the next six months asking the electorate if the borough should have a DEM or stay with the current structure. Cllr Hodges welcomed the news as GBC will not have to spend £100,000 on a referendum. This cost would have been paid by taxpayers. Of the 7,067 signatures submitted, only 2,284 were valid, which meant the petition was 2,659 short of the required total. The council's chief executive, David Williams, said the names on the petition would remain valid for 12 months.
Councillors can now decide to hold a referendum even though the petition is invalid.
The council's Executive will consider a report on May 25 and will make a recommendation to the full council to pursue a referendum or to continue with the current decision-making structure.


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Start Sequence Convertkit Old List
" The cash is in the list." At the very least that is what all the leading online marketers mention.
As I look back on my history as an entrepreneur, I need to accept that a notable portion of the income coming from my businesses may be tied back to my e-mail list. In this ConvertKit review today, I'll not only share the pros and cons of this email autoresponder tool, I'll also share some of my own email strategies along the way. Start Sequence Convertkit Old List Start Sequence Convertkit Old List
Start Sequence Convertkit Old List
As you read this testimonial of Convertkit, feel free to consider that I am current client. I utilize this email advertising program every day right now, yet I've also made use of various other e-mail devices throughout the years, so I possess a point of recommendation to match up. I'll share the advantages and disadvantages of the functions, the prices, just how to successfully utilize it and much more.
And also in full acknowledgment, I've met the creator Nathan Barry a few attend person and also I presume he is actually a wonderful man! However, I do not plan on holding back any sort of strikes when I share a number of places that the resource might be enhanced. Start Sequence Convertkit Old List
In general, it's a wonderful e-mail marketing device for writers and small business owners.
Alleviate of Use – 100
Stability – 95
Customer Service – 90
Price – 90
In general, ConvertKit is a excellent e-mail advertising resource. It's SUPER easy to use and possesses a fantastic concept. Additionally, it has all the attributes it needs to have for a digital product or a serious blogger dealer.
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Pros Super user-friendly.
Great layout.
Endless top magnets for one initiative. Start Sequence Convertkit Old List
Totally free migration.
A/B screening of e-mail program titles.
Sequence Design isn't excellent.
No A/B screening for headlines in automated series.
Minimal capability to take care of cold users.
Unlimited offers for one project. You can easily use as various downloads or even free offer provides as you wish and have them all funnel in to one campaign. And also it's simple to create this happen.
Totally free transfer. If you have over 5,000 subscribers that you are bringing from a previous email company, then their movements crew are going to do it for free. This consists of changing out all your decide in base on your site, moving drip e-mail set, as well as just about anything else that needs to become relocated coming from your previous software program resource.
A/B testing of e-mail program headings. This was actually one more big factor for me to relocate. You could not perform any title screening in Infusionsoft ( not exactly sure if that's transformed currently). Managing to assess what titles function may greatly strengthen your open costs, which can straight equate to your profits.
If they never filled out their first name), auto-insert first name with fallback of no name (. Once again, it is actually the little particulars enjoy this that I like.
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You are not forced to use a visual editor. Some resources require you to accomplish a drag and also decrease as well as really draw collections between 1 e-mail and the following … I hate that type of things. I'm not a graphic designer, I simply wish to create my e-mails and have them go out in the ideal purchase. ConvertKit allows that basic style as revealed listed below.
No resource in the world in excellent, as well as neither is ConvertKit. I view a handful of regions of enhancement that might be made. Generally, I do not think these are deal fantastics, however, for others they might be a much bigger offer, so I would like to lay them out on the dining table.
I wish the list of sequences were in a grid format, not a huge box format. If laid out properly, I could easily find which sequence I want to edit without having to scroll a ton, but the way that it's laid out right now (and because I have several dozen sequences), I spend time h.
I love that I can just immediately get started writing the email copy!
Around 100,000 customers is $679/month.
( Regrettably for me, I end 100,000 clients … so I pay for moreover … however it costs it).
COMBINATIONS. Start Sequence Convertkit Old List
ConvertKit is actually set up sufficient that they automatically combine with tons of devices. This primarily indicates that you can use the ConvertKit API and plug it in to a 3rd event device as well as the tops are going to obtain attracted " amazingly" to your account as well as you can begin drip initiatives without your engagement.
For instance, I currently combine ConvertKit with Clickfunnels, Opt-in Beast, Prosper Leads, and potentially 1 or 2 others that are entering your mind right now.
Listed here's a couple of various other preferred ConvertKit combinations that folks utilize:.
Webinar Ninja.
Whishlist Participant.
CONVERTKIT EMAIL ADVERTISING: 'THE TECHNIQUE IT SHOULD Be Actually'? WE'LL Be Actually THE COURT OF THAT.
If you should buy email marketing software from ConvertKit, real ConvertKit review (2019 update) from real people on Twitter to help you make the best informed decision. Start Sequence Convertkit Old List
Start Sequence Convertkit Old List
ConvertKit is a fairly brand new item and although it does not possess all the bells and also whistles that other more noticeable e-mail advertising and marketing software program providers use, it still offers professional bloggers and small businesses whatever they need to have to deal with e-mail advertising and marketing campaigns.
Furthermore, functions including automation, tagging, and also delivering a pattern obviously emails incorporate great usability to this system. With ConvertKit, you can additionally establish as lots of kinds, landing pages, computerization guidelines, as well as auto responders.
Generally, ConvertKit is heading in the best path and if this is what the early model has to supply, it is going to be actually fantastic to see what brand-new developments potential upgrades will certainly supply very early adopters with.
ConvertKit's cheapest email marketing planning is actually 29.00 per month.
This provider was launched in 2013 by Nathan Barry. Therefore, it is actually relatively brand-new in the industry.
ConvertKit offers impressive features that rival many other long-established email marketing brands like MailChimp and Aweber.
ConvertKit's objective is actually to assist business and bloggers to increase their audience and also transform their e-mail subscribers right into paying consumers.
Their clients include a lengthy list of effective particular niche blogs including Zen Routines, Easy Delicate, Inconceivable HEADQUARTERS as well as The Ultimate Paleo Quick Guide.
Start Sequence Convertkit Old List
The email advertising and marketing software application scene is actually loaded along with autoresponders that each insurance claim to help marketers manage their projects efficiently. ConvertKit is no exception during that it tosses on its own as the best email-marketing system for qualified blog writers.
This company was released in 2013 as well as is actually fairly new in the sector. But, ConvertKit offers impressive e-mail marketing features that rivalrous a lot of other hard-shell email advertising and marketing brand names like MailChimp and Aweber.
ConvertKit gives list-based plannings, suggesting that costs rely on the size of your client listing. The starting planning is actually for companies along with up to 3,000 subscribers. Advanced plans are actually available for higher volume email senders upon demand.
Email marketing experts with less than 50,000 clients can deliver unrestricted information. This is actually a great component for marketing professionals that deliver plenty of emails frequently to a tiny checklist of subscribers. If you get on a higher plan, nonetheless, an email restriction applies.
This limitation is figured out by multiplying 15 as well as the strategy's customer limit. If your plan supports up to 60,000 subscribers, simply multiply that number by 15 and 90,000 will be your email limit. Start Sequence Convertkit Old List
Start Sequence Convertkit Old List
Along with ConvertKit, you can likewise establish as a lot of types, touchdown web pages, computerization regulations, as well as auto responders as you like.
What are drip initiatives? They are actually a sequence of emails "dripped out" one by one in a sequence to clients at pre-set time periods. These e-mails could be composed of a set of associated tutorials ( creating an email program) or even they could relevant information on your numerous companies along well-known blog posts and customer endorsements.
The process of developing these campaigns in other email advertising and marketing bodies could be downright and tedious tough. Nevertheless, ConvertKit streamlines every thing.
For example, you may produce a collection of emails to be delivered over a period of one month after new users join your checklist.
At any time, you can drag and also fall e-mails to re-order the sequence. You'll likewise have the capacity to view the entire drip e-mail system in one sight, that makes it simple to sustain really good circulation when generating your campaign notifications.
The moment a message is sent out, it's noticeable as released et cetera remain in draft until the booked delivery day.
More importantly, you can do so much more than just the traditional drip campaign.
SUPPLY VALUABLE and relevant SATISFIED ALONG WITH REAL EMAIL ADVERTISING AND MARKETING HANDS FREE OPERATION.
The true electrical power of advertising hands free operation comes into play: You can easily create "If This, at that point That" policies. Why is this vital? Due to the fact that it allows you to provide material that is even more applicable as well as valuable to the private vs. using a one-size-fits-all strategy.
You can arrange for a recipient who doesn't open one of the sequenced emails, to receive a special email to re-engage them. This is actually really various coming from the trivial, " direct" course of standard drip campaigns– where everyone receives the particular same email up until the pattern mores than.
You can easily possess an computerization series readied to induce when people sign up for your email bulletin. Those who check a box indicating a certain enthusiasm ( mention, "vegan recipes"), will receive a various series of e-mails than those who examine an additional interest package ( point out, "gluten-free recipes).
AESTHETIC HANDS FREE OPERATION: ANY INDIVIDUAL Can Possibly Do IT.
ConvertKit has actually made it extremely quick and easy to set up these automated e-mail set. You can simply click and pull possibilities onto a canvass and also you're performed. No html coding or advanced understanding is actually needed.
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What is an e-mail program? It's a set of e-mails, planned to become delivered in turn, each of which offers a session on a certain topic. As ConvertKit details, it resembles an online training program "but without all the whistles and bells .".
Email training courses have actually been made use of successfully as a free offer that attracts sign-ups, however plenty of blog owners offer their e-mail training courses at the same time.
ConvertKit makes it effortless to create e-mail programs. You may even tailor what happens at the end of the course. For example, you can give a 2nd training program, and subscribers that present enthusiasm is going to automatically begin getting that second training course.
The ConvertKit blog site possesses high quality advice on how to develop effective email training courses.
BEHAVIOR-BASED INDUSTRYING WITH HANDS FREE OPERATION As Well As TAGGING.
Allow's take a nearer examine automation.
Email advertising and marketing program like ConvertKit helps you acquire the absolute most out of your advertising and marketing initiatives by utilizing advertising automation. Advertising and marketing automation that lets you monitor, procedure and automate advertising and marketing initiatives.
Email advertising computerization is a sort of advertising hands free operation. Relying on exactly how you have actually put together your initiative, a certain email is actually sent out to your consumer relying on the activity they've taken.
Conditions, events, and actions are actually the foundation responsible for ConvertKit patterns.
If this sounds like it could be complicated to set up, software like ConvertKit helps streamline the process for you with its easy user interface.
For most marketers using ConvertKit to manage their email campaigns, automation and tagging are the sellout features. Automation makes it easy to manage subscribers based on events while tags help to segment contacts.
You can tag subscribers automatically if they click certain links in an email. This, in turn, triggers a course of action such as enrollment in a Webinar, subscription to a course, and so forth.
A big plus of ConvertKit: Move seamlessly between building your email automation sequence and working on the individual email content.
You might have heard about sales funnels if you're looking into marketing your small business. A sales funnel is the path your customer takes to purchasing from your business. It usually starts with awareness of your brand, moves onto interest and decision. The end goal is a sale or conversion.
An email marketing funnel will include sending your potential customer helpful content linked to your service or product. By setting up rules, you tell ConvertKit which emails to send (and when).
To keep it simple, ConvertKit visualizes the process so you can immediately see your funnel and all you have to do is add the content.
This approach means even if you've never built a funnel before you'll be able to craft a compelling email campaign. ConvertKit also has a free email course to get you started.
The landing page creator is a great addition that ConvertKit offers to help website owners who have little to no design skills. Using the visual editor, there's no need to write code for your landing page. You can change colors, upload images, and alter text with just a few clicks.
There are only three landing page layouts to choose from, ConvertKit offers full access to CSS for those with somec coding ability who wish to create a unique landing page design.
ConvertKit landing pages are also integrated with a large collection of textures and patterns from SubtlePatterns.com. You can be sure of finding a landing page style that matches your brand or website theme.
When it comes to hosting landing pages, ConvertKit is quite flexible. You have the freedom of hosting landing pages on ConvertKit servers or move it to your own domain using a WordPress plug-in.
Adding a lead magnet to your landing page is a great way to get customers to sign up for your subscriber list. A lead magnet is a resource you use to attract potential customers: leads.
Lead magnets can take many forms. Examples include:.
E-books (e.g. how-tos that are valuable to the reader).
Industry white papers.
Research reports.
Cheat sheets.
Gated videos (videos a viewer can only access if they give you their contact information).
In exchange for your free resource, your customer shares their email address.
ConvertKit comes with support to add lead magnets to their forms.
Here are a few ways you could use ConvertKit to achieve your business objectives.
Engage at the outset.
Automated welcome series based on users' interests. Automatically identify and retarget those who don't open emails.
Deepen the connection.
Use lead magnets to send recipients a content series that is relevant and valuable .
Develop funnels and monetized content like email courses.
An email marketing campaign would not be complete without signup forms. ConvertKit comes with a form builder as expected from any typical email marketing application. Using this tool, you can create multiple attractive opt-in forms and place them anywhere on your website.
Though basic in function, the form builder does its job well.
You'll receive a message confirming that your contacts have been successfully imported once you're done uploading the file.
The subscriber page marks contacts as confirmed, unconfirmed, canceled, or complained to describe their status on the list.
Subscribers can be tagged and segmented if you want to send targeted messages to a specific group– for example, you could create an email series to target individuals who attended a particular webinar you held, or an in-person conference you exhibited at.
As mentioned earlier, ConvertKit offers email creation features that allow you to send a course of messages automatically. You can create multiple email messages, save them as drafts and schedule when to send.
Email course scheduling can be great when sending content that is relevant to specific subscribers. Sometimes you'll need to send one email blast to everyone on your list.
To accomplish that, ConvertKit offers a broadcast function. A broadcast email is when a single email is sent to multiple people at the same time. This feature is great for announcing special offers, product launches, or time-sensitive discounts or news.
Currently, customers have no access to live chat and phone support. You can always get in touch with a customer support agent via email.
While this might not sound too assuring, one unique aspect about contacting ConvertKit is that when you submit a question or complaint, they personally look into your account.
If something is lacking, the assigned support agent will make recommendations to help you improve your results or enjoy a better experience.
In addition to email support, ConvertKit hosts workshops, offers training materials, and provide plenty of online help documentation. The knowledge-base categorizes help articles according to topics, thus making navigation easy. Alternatively, you can use key phrases to search for help articles.
ConvertKit offers analytics tools for easy campaign tracking. You can track email opens, bounces, visitors, subscribers, and conversion rates for each campaign separately. The program also tracks website statistics for different forms to help you know which signup tactics are providing the best results.
You can examine conversion rates as well as the number of subscribers that joined your email list via the pop-up modal or the ones who signed up for incentives using the sidebar form.
ConvertKit creates email courses easily.
The landing page and form creators feature intuitive interfaces.
Email scheduling and automation features save time.
Subscriber tagging facilitates list segmentation.
Migrating to ConvertKit is simple since you can import contacts via a CSV file.
Built-in reporting tools track campaign metrics easily.
ConvertKit offers the ability to create multiple sign up forms for different pages and track their conversion rates individually.
Currently, no free trial is new but available subscribers are protected by a 30-day refund policy.
No phone or live chat support is provided.
Limited third-party integrations compared to other email marketing platforms.
ConvertKit is a relatively new product and although it doesn't have all the bells and whistles that other more prominent email service providers offer, it still gives professional bloggers and business owners everything they need to manage an email campaign.
Additionally, features such as automation, tagging, and sending out a sequence of course emails add great usability to this platform.
Overall, ConvertKit is heading in the right direction and if this is what the early version has to offer, it will be exciting to see what new innovations future upgrades will provide early adopters with.
Easy Digital Downloads.
and many others.
And because they integrate with Zapier, that means there are lots of other combinations that you can use even if there isn't a directly integration.
I can't possible consider all the questions that you might ask before deciding to buy Convert Kit; however, here's a few questions that I might anticipate.
How good is the customer support?
The customer support is really good. They offer a live chat option that I've used. If they are not available via live chat, you can email them and they usually get back in a pretty timely manner. I haven't had any big issues with the support.
Will they help me migrate from my old provider?
Yes, they will move all of your subscribers for you, but they will also create all the forms you need and place on your website (as needed) to make sure your transition over is really smooth. They will also move any campaigns that you had on your old provider so that you don't manually have to recreate them.
How does the price compare to other email tools?
The pricing is pretty comparable. There are hundreds of options out there, so some may be cheaper, but there are definitely much more expensive options.
How many lead magnets can I deliver and create through ConvertKit?
You can create unlimited number of lead magnets or downloads that ConvertKit will deliver seamlessly to your customers. If you want, the way they have everything set up makes it really easy to have dozens (or more) of freebies that lead to the exact same list. This makes it really convenient to do special content upgrades, etc
. Does ConvertKit have options for double or single opt-in?
Yes, when you create your opt-in forms you have the option to require that the subscriber get a verification email first before they are added to your list. Single opt-in is likely to lead to more overall subscribers, but double opt-in is likely to lead to more verified subscribers.
Is ConvertKit worth the cost?
I've been using email automation tools since about 2006, and I'm a happy customer of ConvertKit. I think the cost of an email marketing tool is a necessary expense for an online business, and ConvertKit is a great option at a fair price.
At the end of the day, ConvertKit is a great email and marketing automation tool that has all the features that a serious blogger and business owner would need to help automate their business.
With an unlimited amount of campaigns and lead forms that you can create and an easy to use interface, you can spend more time growing your business than worrying about how to make your email provider "work".
The best part is that you can start a 14 day free trial (with no credit card required) so you can check out what ConvertKit looks like on the inside.
Start Sequence Convertkit Old List
I use this email marketing software on a daily basis right now, but I've also used other email tools over the years, so I have a point of reference to compare. Overall, ConvertKit is a great email marketing tool. These emails might consist of a series of related tutorials (forming an email course) or they might information on your various services along popular blog posts and customer testimonials.
A broadcast email is when a single email is sent to multiple people at the same time. In addition to email support, ConvertKit hosts workshops, offers training materials, and provide plenty of online help documentation.
settings page
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Added support
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Contact Form 7 integration
Contact Form 7 integration
Contact Form 7 integration
ConverKit Service
conversion rates
Upload wp-convertkit
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Added tools tab
meta defaults
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content creation
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Full plugin documentation
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Contact Form 7 integration
affiliate products
Contact Form 7 integration
available forms
concierge migrations
edit screen
Verified functionality
plugins/ directory
Initial release
Visual automations
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affiliate program
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right funnel
Added ability
Page metabox
Restrict Content Pro
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CF7 name and email fields
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Fixes issue
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[sspostsincat category="convertkit"]


text:
Today will be slightly warmer than Thursday with a moderate breeze, warm temperatures, lower-than-normal humidity levels and partly cloudy skies, according to the National Weather Service in Brownsville.
Highs will reach the mid to upper 80s and winds will be southeast at 10 to 20 mph.
Today Brownsville will be 84, McAllen 87 and San Manuel 88. If you visit South Padre Island, expect 78-degree weather.
Saturday will be warm and partly cloudy until a cold front arrives late Saturday night with thunderstorm chances. Expect cooler temperatures to begin next week. Best chances for rain and even an isolated thunderstorm will be overnight Saturday and Sunday.
Saturday highs will be in the 80s. On Sunday, however, temps will drop into the 50s.
On Sunday, expect for showers and scattered thunderstorms along and behind the front, continuing into Monday morning.
Hazardous marine conditions are possible with strong winds and rough seas.


text:
Allelic Exchange of Pheromones and Their Receptors Reprograms Sexual Identity in
Cell type specification is a fundamental process that all cells must carry out to ensure appropriate behaviors in response to environmental stimuli. In fungi, cell identity is critical for defining "sexes" known as mating types and is controlled by components of mating type (MAT) loci. MAT–encoded genes function to define sexes via two distinct paradigms: 1) by controlling transcription of components common to both sexes, or 2) by expressing specially encoded factors (pheromones and their receptors) that differ between mating types. The human fungal pathogen Cryptococcus neoformans has two mating types (a and α) that are specified by an extremely unusual MAT locus. The complex architecture of this locus makes it impossible to predict which paradigm governs mating type. To identify the mechanism by which the C. neoformans sexes are determined, we created strains in which the pheromone and pheromone receptor from one mating type (a) replaced the pheromone and pheromone receptor of the other (α). We discovered that these "αa" cells effectively adopt a new mating type (that of a cells); they sense and respond to α factor, they elicit a mating response from α cells, and they fuse with α cells. In addition, αa cells lose the α cell type-specific response to pheromone and do not form germ tubes, instead remaining spherical like a cells. Finally, we discovered that exogenous expression of the diploid/dikaryon-specific transcription factor Sxi2a could then promote complete sexual development in crosses between α and αa strains. These data reveal that cell identity in C. neoformans is controlled fully by three kinds of MAT–encoded proteins: pheromones, pheromone receptors, and homeodomain proteins. Our findings establish the mechanisms for maintenance of distinct cell types and subsequent developmental behaviors in this unusual human fungal pathogen.
Published in the journal:
. PLoS Genet 6(2): e32767. doi:10.1371/journal.pgen.1000860
Category: Research Article
doi: 10.1371/journal.pgen.1000860
Cell type specification is a fundamental process that all cells must carry out to ensure appropriate behaviors in response to environmental stimuli. In fungi, cell identity is critical for defining "sexes" known as mating types and is controlled by components of mating type (MAT) loci. MAT–encoded genes function to define sexes via two distinct paradigms: 1) by controlling transcription of components common to both sexes, or 2) by expressing specially encoded factors (pheromones and their receptors) that differ between mating types. The human fungal pathogen Cryptococcus neoformans has two mating types (a and α) that are specified by an extremely unusual MAT locus. The complex architecture of this locus makes it impossible to predict which paradigm governs mating type. To identify the mechanism by which the C. neoformans sexes are determined, we created strains in which the pheromone and pheromone receptor from one mating type (a) replaced the pheromone and pheromone receptor of the other (α). We discovered that these "αa" cells effectively adopt a new mating type (that of a cells); they sense and respond to α factor, they elicit a mating response from α cells, and they fuse with α cells. In addition, αa cells lose the α cell type-specific response to pheromone and do not form germ tubes, instead remaining spherical like a cells. Finally, we discovered that exogenous expression of the diploid/dikaryon-specific transcription factor Sxi2a could then promote complete sexual development in crosses between α and αa strains. These data reveal that cell identity in C. neoformans is controlled fully by three kinds of MAT–encoded proteins: pheromones, pheromone receptors, and homeodomain proteins. Our findings establish the mechanisms for maintenance of distinct cell types and subsequent developmental behaviors in this unusual human fungal pathogen.
One of the most important processes that occurs in cells is the specification of cell type, and it is by this process that cells adopt the genetic state that governs their subsequent behaviors. One mechanism of cell type-specification is the expression of genes encoded only in a given cell type. For example, X and Y chromosomes determine male and female sexes because they encode different genes, such as SRY, a Y-specific protein whose expression establishes male identity [1]. In a similar fashion, the sexes of fungi, known as "mating types," are also determined through the expression of cell type-specific genes. The most well-characterized cell identity determination system is that of the budding yeast Saccharomyces cerevisiae.
In S. cerevisiae the two haploid cell types, a and α, are distinguished from one another by the actions of specific transcription factors encoded at the mating-type (MAT) locus [2]. MATa encodes the homeodomain transcription factor a1, and MATα encodes α1 and α2, an α-domain protein and a homeodomain protein, respectively. The actions of a1, α1, and α2 govern control of haploid cell behavior through the differential expression of a-specific, and α-specific genes, including pheromone and pheromone receptor genes [3]. It is through a pheromone-pheromone receptor system that cell-cell communication occurs, and cells of opposite mating types can sense one another. Specifically, a cells secrete mating factor a pheromone (MFa), which binds to a receptor (Ste3) on the surface of α cells, and α cells secrete MFα pheromone, which is sensed by a receptor on the surface of a cells (Ste2). In response to the presence of the pheromone of a mating partner, cells undergo a cell cycle arrest and subsequent morphological changes to prepare for mating. After cell fusion, a1 and α2 act in concert to regulate haploid-specific genes, specifying the diploid a/α cell, thus completing a MAT-controlled regulatory circuit [2],[3].
A similar cell-cell recognition process occurs in many other fungi, including the corn smut, Ustilago maydis. In U. maydis specific pheromones and receptors are expressed in different cell types; however, in contrast to S. cerevisiae, these genes are not under the transcriptional control of the homeodomain proteins of the traditional MAT locus [4]. Instead, distinct alleles of the pheromones and their receptors are encoded within a separate MAT locus, and these alleles are sufficiently distinct from one another to confer cell type-specificity. In this case, haploid cells expressing distinct pheromones and receptors from the pheromone MAT locus sense and respond to partners of other mating types and fuse. Once compatible mating types have fused, two transcriptional regulators, bE and bW, which are encoded at the second MAT locus, regulate a transcriptional cascade that promotes further sexual development [4]–[6].
In a related, clinically important human pathogen, Cryptococcus neoformans, the determinants of haploid cell identity are unknown. C. neoformans contains a single MAT locus that is over 100 kb in size and contains 23 genes, some of which have been found to be involved in sexual development and others that appear to be essential "housekeeping" genes [7]. This locus represents an evolutionary transition from the two separate MAT loci found in basidiomycete fungi like U. maydis, to the single MAT locus found in ascomycetes [8]; it is unclear how components in this "fused" MAT locus function to specify haploid cell type. In the C. neoformans MAT locus, there are five genes in each mating type that represent the classic MAT components found in basidiomycete MAT loci. They include the homeodomain transcription factors SXI2a and SXI1α, the six pheromone genes MFa1-3 and MFα1-3, and the pheromone receptors STE3a and STE3α. The transcription factors, Sxi1α and Sxi2a, do not appear to play any role in haploid cells, including establishment of haploid cell identity. In contrast, Sxi1α and Sxi2a are both necessary and sufficient to specify the dikaryotic state following cell fusion and ensure that sexual development continues [9],[10]. Conversely, the pheromones and pheromone receptors of C. neoformans have been shown previously to be necessary for haploid cell behaviors, such as sensing and responding to a mating partner [11]–[16]. What is not clear is whether pheromones and pheromone receptors are sufficient to confer haploid cell identity (as in U. maydis), or whether the actions of other regulators are required to establish the a and α mating types (as in S. cerevisiae).
To test the hypothesis that pheromones and pheromone receptors alone are sufficient to confer haploid cell identity in C. neoformans, we carried out a "swapping" experiment in which components from the a mating-type (STE3a and MFa1) were relocated into a strain of the opposite mating type, α (in which STE3α and all three copies of MFα had been deleted), and the effects of these modifications on mating and development were examined. The results presented here reveal that pheromones and their receptors are both necessary and sufficient to specify haploid cell identity. That is, strains that harbor a receptor and pheromone from the opposite mating type are capable of sensing and fusing with wild type cells of the same mating type. Furthermore, the fused cells are incapable of progressing through sexual development in the absence of both Sxi transcription factors; however, exogenous addition of SXI2a facilitates complete sexual development. These findings reveal that control of haploid cell identity in C. neoformans is mediated by MAT-encoded cell-cell communication components that are not mating type-specific in their downstream effects, and establishes a model for the maintenance of distinct cell types and developmental behaviors in this human fungal pathogen.
Construction of modified α strains
To test the possibility that haploid cell identity in C. neoformans is controlled by pheromones and pheromone receptors, a strain where the pheromone and pheromone receptor genes from one mating type were replaced with those from the opposite mating type was constructed. Specifically, we created an altered α strain by functionally replacing the STE3α receptor gene and the three MFα pheromone genes of an α cell with the STE3a receptor gene and three copies of the MFa1 gene from an a cell (Figure 1).
To construct the altered α strain, α cells in which all three copies of the MFα gene had been deleted (α mfαΔ), were transformed with a construct to replace the endogenous copy of STE3α with STE3a (Figure 2A) [13]. This replacement resulted in a complete deletion of the STE3α open reading frame and the precise insertion of STE3a under the control of the endogenous STE3α promoter. This strain, designated αmfαΔ, STE3a was confirmed by PCR (data not shown) and Southern analysis (Figure 2A) to contain a single, targeted integration of the STE3a gene at the STE3α locus. This αmfαΔ, STE3a strain was then transformed with a construct expressing MFa1 (under the control of the MFα1 promoter) designed to integrate randomly into the genome (Figure 2B). Multiple transformants of the resulting strain, αa, were assessed by Southern blot analysis, and several strains containing integrated copies of the MFa construct were carried forward in further analyses. All of the transformants confirmed to contain the MFa construct exhibited identical behaviors in subsequent phenotype analyses. A representative αa transformant containing three copies of the MFa1 gene is shown in Figure 2B.
To assess the expression patterns of pheromones and their receptors in the constructed strains, northern blot analysis was carried out. Haploid strains were grown on V8 medium for 24 hours, and their relative transcript levels were examined (Figure 2C). The STE3a and STE3α transcripts were detected in wild type a and α cells, respectively, and the STE3α transcript was also present in the αmfαΔ strain. However, as expected, in the αmfαΔ, STE3a strain where the STE3a ORF had replaced the STE3α ORF, only STE3a transcript was observed. In summary, the transcripts detected from the constructed strains were in accordance with the predicted expression patterns for each of the strains tested based on their genotypes.
While pheromone transcript was not detected in either haploid cell type (2C, bottom two panels, lanes 1 and 2), this result was not surprising because pheromone genes are transcribed at low levels in haploid cells grown on minimal medium [13]. Furthermore, pheromone transcript was not observed in strains where these genes had been deleted (αmfαΔ and αmfαΔ, STE3a) (lanes 3 and 4). However, when the MFa1 gene was introduced into the αmfαΔ, STE3a strain (resulting in the αa strain), high levels of MFa transcript were detected, and this high level of expression occurred in all of the MFa transformants (lane 5 and data not shown). Because MFa1, in this case, is under the control of the MFα1 promoter (defined here as 1 kb of sequence upstream of the translational start site), a likely explanation for our findings is that normal repression of MFa1 in haploid cells is disrupted. That is, the elements required for wild type levels of expression were likely not included in our construct, resulting in higher levels of MFa1 gene expression. Because pheromones expressed from a non-native promoter do not interfere with development and the mating response, the αa strain was used in our subsequent analyses [13],[14].
Altered α strains mate with wild-type α cells
To evaluate how the series of modified α strains interacted with mating partners under sexual development conditions, crosses using various test strains were carried out and assessed microscopically for the presence of fusants after 18 hours on V8 medium. The cells used in each cross were stained with either rhodamine (Alexa Fluor 594) or fluorescein (Alexa Fluor 488) prior to mixing with partner cells so that the original mating types could be discerned after cell fusion.
In crosses between wild type a and α strains, fusants were identified as "dumbbell" shaped cells, consisting of one a cell (red) connected to one α cell (green) via a conjugation tube (Figure 3A, panel 1). In contrast, no fusants could be identified in crosses between wild type a cells (red) and any modified α cells (green) (panels 2–4). Specifically, in crosses between a cells and αmfαΔ cells, the αmfαΔ cells responded to the presence of a cells by forming conjugation tubes; however, in the absence of α pheromone production, the frequency of fusion with a cells is predicted to be very low, and consistent with this expectation, we did not observe any fusants. Accordingly, in strains no longer harboring the STE3α receptor (αmfαΔ,STE3a and αa), the cells appear to neither form conjugation tubes (in the presence of a cells) nor fuse with a cells (panels 3 & 4). These results indicate that modified α strains, in which the STE3α receptor gene has been deleted, do not respond to or mate with a cells.
In parallel experiments, wild type α strains were crossed with the modified α strains. In a control cross, differentially labeled α cells (either red or green) were crossed and evaluated for the presence of fusants. As expected for wild type cells of the same mating type, α cells did not respond to one another or fuse (panel 5). Fusants were also not detected in crosses between wild type α cells (red) and the modified αmfαΔ and αmfαΔ,STE3a (green) strains (panels 6 & 7, respectively). It was not until a wild type α cell (red) was crossed with the altered αa strain (green) that fusants could be recovered at roughly wild type a x α levels (panel 8). Subsequent quantitative fusion assays confirmed that α x αa fusion levels are comparable to those of wild type a x α crosses (data not shown). This finding indicates that, as expected, pheromones and pheromone receptors are required for wild type fusion between a and α cells. It also reveals that the altered αa strain fuses only with wild type α cells, indicating that by simply replacing the α pheromones and receptors with those from a cells, cell identify was switched from α to a. Moreover, that change was sufficient to allow the altered αa cells to fuse with wild type α cells as though they were of opposite mating types.
In addition, the introduction of pheromone and pheromone receptor from a cells into α cells changes not only their ability to fuse but also the initial mating response. That is, αa strains do not produce germ tubes in response to wild type α cells, instead appearing to behave like wild type a cells (Figure 3B). These findings indicate that pheromones and pheromone receptors specifically control both the ability to sense a mating partner and the subsequent morphological response.
Complete sexual development between αa and wild-type α cells occurs only in the presence of both Sxi1α and Sxi2a
To assess the ability of the series of modified α strains to undergo sexual development, crosses were carried out and assessed for the production of filaments, basidia, and spores. In crosses between wild type a and α strains, florid filamentation, basidia formation, and sporulation were all visible at the periphery of the cross (Figure 4A, panel 1). However, when the modified α strains (αmfαΔ, αmfαΔ,STE3a, and αa) were crossed with either wild type a or α cells, none of the combinations resulted in wild type sexual development (panels 2–8). The a x αmfαΔ cross (panel 2) revealed a significant reduction in sexual development, and this result is consistent with previously published data [13]. In addition, aberrant filaments were observed in the α x αa cross (panel 8). This limited, mutant filamentation was very close in appearance to crosses between strains containing deletions of SXI2a (panel 9). In crosses between wild type α strains and sxi2aΔ mutants, the strains fuse at wild type levels; however, only aberrant filaments are formed, and they do not progress through sexual development [9]. This phenotype occurs because both Sxi1α and Sxi2a must be present to specify the fused state and initiate either diploid-specific or dikaryon-specific developmental programs. Because the products of an α by αa cross contain only the Sxi1α developmental regulator (and not the Sxi2a regulator found only in a cells), such crosses would not be expected to progress through sexual development and would be expected to exhibit a sxi2aΔ cross phenotype. This is, in fact, the case. Conversely, if the Sxi proteins, pheromone, and pheromone receptors were necessary and sufficient to specify cellular identify (dikaryon vs. haploid), then simply supplying the SXI2a developmental regulator to an α by αa cross would result in complete sexual development.
To test the hypothesis that α x αa crosses were simply in need of SXI2a to continue through sexual development, we carried out crosses in which exogenous SXI2a was provided. Because strains containing both SXI1α and SXI2a are self-filamentous under sexual development conditions, we crossed the αa strain with an α sxi1αΔ strain carrying an integrated copy of SXI2a under the control of a constitutive promoter (GPD1). This cross resulted in the restoration of complete sexual development (panel 10). In addition, fluorescence and high-resolution microscopy show that dikaryotic filaments basidia, and spore chains were formed in αa x α + SXI2a crosses (Figures 4B, 4C). Because complete sexual development occurred between cells with nearly identical genomes (αa x α + SXI2a), no additional information from a cells is required for this process.
This finding was further supported by the results of assays for diploid formation. Previous studies have shown that a/α diploids are self-filamentous on V8 medium, undergoing sexual development [17]. To test the ability of the αa strain to form diploids, crosses of αa by wild type a or α cells were carried out. αa cells fused to form mononucleate cells (presumed diploids) with wild type α cells, but not with wild type a cells, consistent with αa cells exhibiting a cell behavior. In this assay, αa cells (ura5, NEOR) were mixed with either wild type α or a cells (URA5) and incubated on V8. After 24 hours at room temperature, the co-cultures were plated under double selection (Ura− + G418) at 37°C to select for mononucleate diploids [17]. No colonies grew from the a x αa crosses, indicating that no a/αa diploids were formed. In contrast, abundant colonies were recovered from the α x αa crosses, indicating that αa/α diploids were formed. The resulting αa/α strains were determined to be mononucleate (data not shown) and were evaluated for the ability to undergo sexual development by incubation on V8 at room temperature (Figure 4D). As in α x αa crosses, sexual development was not observed (panel 2); however, the addition of the SXI2a gene to the αa/α strain resulted in full sexual development on V8, similar to that of a wild type a/α diploid (panels 3 and 1, respectively). Taken together, these results demonstrate that cell identity and sexual development are controlled entirely by three kinds of genes in C. neoformans: pheromones, pheromone receptors, and homeodomain proteins.
The αa strain undergoes α fruiting
Monokaryotic or α fruiting is another form of sexual development that takes place in C. neoformans; however, this process is specific to α cells, which form filaments, basidia, and spores under severe nutrient limitation and desiccation [10],[18],[19]. Although fruiting is known to be an α specific process, the factors responsible for α fruiting are unknown. To assess whether changing haploid cell identity influences α fruiting, fruiting assays were carried out with wild type a, wild type α, and αa strains. Strains were incubated on filament agar for 14 days at room temperature in the dark and evaluated for the production of filaments and spores. We observed that the αa strain undergoes α fruiting that is indistinguishable from wild type α cells (Figure 5), indicating that this α-specific process does not require α-specific pheromones or pheromone receptor, and is not influenced by the presence of a-specific pheromones and pheromone receptor.
Sexual development is an essential part of the life cycle for most eukaryotic organisms and requires mechanisms to ensure that development takes place between compatible partners. In both ascomycete and basidiomycete fungi, mating type is specified by components of the MAT locus [20]; however, the mechanisms by which MAT components act to confer cell identity vary widely among fungi [21],[22]. In this study we discovered that the basidiomycete C. neoformans establishes haploid cell type through the expression of mating type-specific pheromones and pheromone receptors. By replacing the pheromone and the pheromone receptor genes of α cells with those from a cells, we created an α cell with the haploid identity of an a. This altered strain exhibited the mating behaviors of an a cell, including the ability to fuse with an α mating partner. This result shows that no additional mating type-specific factors are necessary to denote haploid cell type. Furthermore, the addition of the a-specific factor SXI2a was then sufficient to facilitate filamentation and spore formation. Through these studies we have identified the factors that determine haploid cell identity (pheromones and their receptors), thus answering the question of how the MAT locus of C. neoformans controls cell type-specification.
Cell identity in fungi
There is a long history of investigating pheromone/pheromone receptor function in fungi, particularly in S. cerevisiae, and myriad experiments have been carried out to understand the roles of two phylogenetically unrelated receptors STE2 (α factor receptor) and STE3 (a factor receptor). Through altered expression experiments it was found that mating type is controlled by the identity of the expressed surface receptor. Experiments where the receptor expression patterns were reversed in a and α cells led to changes in mating behaviors. For example, α cells that express only STE2 (and not STE3), can sense and respond to α pheromone and therefore mate as a cells and vice versa. Thus, in S. cerevisiae, both a and α cells contain the STE2 and STE3 genes (and both pheromone genes), and it is the actions of transcription factors that impart differential expression patterns that determine cell identity [23],[24].
This is in sharp contrast to other fungi, like U. maydis, in which different cell types encode distinct pheromones and pheromone receptors. Experiments in U. maydis where the pheromones and pheromone receptors have been "swapped" between mating types demonstrate that mis-expression of pheromones and pheromone receptors is sufficient to alter cell identity. In this case, the pheromone receptors have distinct binding specificities but have descended from a common ancestor [4]. This is also the case for other basidiomycetes, including S. commune and C. cinerea. Within these fungi, different mating types encode distinct alleles of pheromones and pheromone receptors; pheromone and pheromone receptors do not govern mating, and cell fusion occurs independently of pheromone signaling. Instead, pheromones and pheromone receptors control post-fusion sexual development [25]–[27]. These examples speak to the wide variety of strategies employed by pheromones and pheromone receptors to control sexual development in fungi and emphasize the need to investigate pheromone/pheromone receptor function on a case-by-case basis.
C. neoformans pheromones and pheromone receptors
In C. neoformans, both a and α cells contain mating type-specific pheromone genes (MFa1, 2, and 3 in a cells, and MFα1, 2, and 3 in α cells), but the regulation of these genes differs between mating types. In a cells, MFa pheromone is highly induced on V8 agar (or under conditions of nutrient limitation) in the absence of a mating partner. In addition to induction under nutrient limiting conditions, MFα pheromone expression is also activated by factors secreted by a cells (MFa pheromone) [13], and expression of the MFα genes is dramatically upregulated upon exposure to synthetic MFa pheromone [12],[13].
In the experiments carried out here, expression of the MFa1 gene in αa cells is driven by the MFα1 promoter. The MFa1 construct was originally engineered in this fashion to avoid any cell type-specific regulation that might be imposed upon an a-specific promoter in an α cell. However, in αa strains where MFa1 gene expression was driven by the MFa1 promoter, identical phenotypes and expression patterns were observed.
Attempts to test the roles of the pheromone receptors were not as straight forward, and initially, the STE3a cassette was transformed into a wild type α strain to replace the STE3α gene at its native locus. However, after multiple attempts, no transformants were recovered (Ekena, Giles, and Hull, unpublished data). Transformants were recovered only from strains in which the pheromone genes were deleted previously. These findings, while far from conclusive, intimate that cells expressing both STE3a and α pheromone may engage in autocrine signaling that affects cell growth.
A similar observation has been made in S. cerevisiae, in which a strain that expresses both a pheromone and its cognate receptor undergoes G1 cell cycle arrest [24]. In S. cerevisiae this arrest is transient; however, if a similar arrest is occurring in C. neoformans, the cells do not appear to recover. This is in contrast to U. maydis where cells that have been engineered to express both receptor alleles do not arrest, are fully viable, and are mating competent. It remains to be determined if and how autocrine signaling may be functioning in C. neoformans.
Receptor identity influences mating morphology but not fruiting morphology
In C. neoformans, a and α cells carry out distinct functions to initiate the mating process. For example, in response to nitrogen limitation, a cells secrete MFa pheromone, and α cells respond to the presence of the MFa pheromone by formation of germ tubes. This morphological response is specific to α cells, and only these cells (and not a cells) form germ tubes as an initial mating response [11],[28],[29]. Interestingly, αa cells not only recognize and fuse with α cells (as would be expected for an a cell), but they also exhibit the morphology of an a cell, and do not produce germ tubes. This finding indicates that the morphological response to pheromone is mediated by the pheromone receptor (not downstream signaling events) and is independent of other mating type-specific factors. Therefore, pheromones and pheromone receptors alone dictate both the detection of a mating partner and the initial morphological response of haploid cells in C. neoformans. Interestingly, however, changes in mating identity did not influence the α-specific process of α fruiting.
Monokaryotic (or α) fruiting is a process that is specific to α cells and was recently shown to be a form of sexual development [19]. While the mechanisms underlying α fruiting are relatively undefined, it is well established that the α fruiting pathway is independent of Sxi1α and Sxi2a, and that this process is attenuated in strains in which STE3α or the MFα genes have been deleted [12],[13],[19]. Our data reveal that although α fruiting is primarily an α-specific process, it occurs regardless of whether pheromones and pheromone receptor are derived from a or α cells.
In summary, while the α fruiting process occurs despite the identity of pheromones and their receptors, these factors are wholly responsible for determining the initial mating response and haploid cell identity. The findings presented here establish a complete cell identity determination profile for C. neoformans and enhance our understanding of the myriad strategies by which fungi specify mating types and undergo sexual development.
Materials and Methods
Constructing modified α strains
To generate the αmfαΔ, STE3a strain (CHY1900), the αmfαΔ (WSC17) strain was biolistically transformed with a fragment containing STE3a and a nourseothricin resistance (NATR) cassette that was flanked by ∼1 kb of sequence from upstream and downstream of the STE3α open reading frame (ORF) [13]. Nourseothricin resistant transformants were screened by PCR for proper integration of the deletion/insertion construct into the STE3α locus, and putative deletion/insertion strains were confirmed by Southern blot analysis [30]. To generate the αa strain (CHY1901), the αmfαΔ, STE3a strain was biolistically transformed with a randomly integrating fragment of DNA containing MFa1 (under the control of the MFα1 promoter) and a neomycin resistance (NEOR) cassette. Neomycin resistant transformants were screened for copy number of the insertion using Southern blot analysis [30]. The αa/α strain (CHY2049) was generated by crossing the 5-FOA resistant αa strain (CHY1517) by wild type α cells (JEC21) and selecting for fusants at 37°C on minimal medium containing neomycin. To generate the αa/αSXI2a strain (CHY2096), 5-FOA resistant αa/α cells were biolistically transformed with a randomly integrating fragment of DNA containing the SXI2a (under the control of the GPD1 promoter) and URA5 genes.
Northern blot analysis
RNA was prepared from C. neoformans cells using a hot phenol extraction [30]. Strains were grown on solid V8 medium for 24 hours at room temperature. Northern blots were carried out according to standard protocols with 10 µg of total RNA used for each sample [30]. The glycerol-3-phosphate dehydrogenase gene (GPD1) probe was PCR-generated using CHO651 (CGTCGTTGAATCTACCGGTG) & CHO652 (CACCAGCAATGTAAGAGATG). All other probes were generated by PCR using the following oligonucleotides: CHO2030 (CCCCGACTATCCCTTTTGGAATCTCACTGC) & CHO2031 (GGCGAACAGTTCTTCGGGATATTGTGATACC) for STE3a, CHO2032 (GCACGACCTCAGCCTCGTCATTTTCAGCGG) & CHO2033 (CCGTATCCAGCAGCAATGATCGTCAGC) for STE3α, CHO2052 (GACTTACTCTTGCGTTATTGCTTAAAGTGGG) & CHO1917 (GAAAAGAGGTACGAGTAGAT) for MFa1, and CHO2053 (TTGTGTCATCGCCTAGACCCAACGTCCCC) & CHO2054 (CCATCTAAACAAGTCCCATACGCTTCGTTACC) for MFα1. Radiolabeled probes (Decaprime II kit; Ambion) were used in hybridization reactions as described previously [31]. Hybridizations and washes were carried out at 65°C as described previously [30].
Strain manipulations and media
All strains used were of the serotype D background (Table 1) and were handled using standard techniques and media as described previously [32],[33]. Sexual development assays were conducted on 5% V8 medium at room temperature in the dark for 2–4 days and were evaluated by observing the periphery of test spots. The mating tester strains used were JEC20 (a) and JEC21 (α), and crosses were photographed at 100X magnification [18]. Spores were photographed at 400X magnification. Fruiting assays were carried out by growing cells on filament agar (0.67% yeast nitrogen base (without amino acids or ammonium sulfate) containing 100 nM (NH4)2SO4 at room temperature in the dark for 14 days. The periphery of test spots were photographed at 100X magnification.
Microscopy and staining
Fluorescent microscopy was carried out on a Zeiss Axioskop 2 fluorescent microscope fitted with an Axiocam MRM REV3 digital camera and corresponding AV4 software. Light microscopy was carried out using a Zeiss Axioplan microscope fitted with a 10X long working distance objective. Photographs were taken with a Nikon Coolpix 5400 camera mounted on the microscope. To label fusants, a and α cells were grown to stationary phase in yeast extract peptone dextrose (YPD) liquid medium and were suspended in 1X phosphate buffered saline (PBS). Carboxylic acid, succinimidyl-ester Alexa Fluor 488 or 594 dyes (Invitrogen, Carlsbad CA) were coupled to a or α yeast cells in 100 mM potassium phosphate buffer pH 7.5 for 30 min at room temperature. After washing, differentially labeled cells were mixed in 1X PBS and spotted onto V8 agar. Spots were incubated at room temperature for 6 hours and were then resuspended into a mounting solution of 1X PBS containing 0.4 µg/ml calcofluor white MR2 (Sigma-Aldrich). For filament staining, crosses were incubated on a thin layer of V8 media on a microscope slide and incubated at room temperature for 24 hours. Cells were fixed with 2.5% gluteraldehyde/4% paraformaldehyde for 30 minutes. Cells were washed twice with 1X PBS containing 0.1% Triton X-100, once with 1X PBS, and incubated in 1X PBS containing 0.4 µg/mL calcofluor white to stain septa, and 1nM Sytox green (Invitrogen) to stain nuclei for 20 minutes. Cells were washed in 5% glycerol with 20% DABCO antifade (Sigma) prior to visualization.
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2002 Molecular analysis of CPRα, a MATα-specific pheromone receptor gene of Cryptococcus neoformans. Eukaryot Cell 1 432 439
13. ShenWC
2002 Pheromones stimulate mating and differentiation via paracrine and autocrine signaling in Cryptococcus neoformans. Eukaryot Cell 1 366 377
14. McClellandCM
2002 Isolation and characterization of the Cryptococcus neoformans MATa pheromone gene. Genetics 160 935 947
15. DavidsonRC
2000 Characterization of the MFα pheromone of the human fungal pathogen Cryptococcus neoformans. Mol Microbiol 38 1017 1026
16. ChangYC
2003 Importance of a developmentally regulated pheromone receptor of Cryptococcus neoformans for virulence. Infect Immun 71 4953 4960
17. SiaRA
2000 Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic. Fungal Genet Biol 29 153 163
18. WickesBL
1996 Dimorphism and haploid fruiting in Cryptococcus neoformans: association with the α-mating type. Proc Natl Acad Sci U S A 93 7327 7331
19. LinX
2005 Sexual reproduction between partners of the same mating type in Cryptococcus neoformans. Nature 434 1017 1021
20. FraserJA
2003 Fungal mating-type loci. Curr Biol 13 R792 795
21. VaillancourtLJ
1996 Pheromones and pheromone receptors as mating-type determinants in basidiomycetes. Genet Eng (N Y) 18 219 247
22. KronstadJW
1997 Mating type in filamentous fungi. Annu Rev Genet 31 245 276
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Sprague JGF
1986 Yeast peptide pheromones, a-factor and α-factor, activate a common response mechanism in their target cells. Cell 47 929 937
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1987 Common signal transduction system shared by STE2 and STE3 in haploid cells of Saccharomyces cerevisiae: autocrine cell-cycle arrest results from forced expression of STE2. Embo J 6 249 254
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1998 A large pheromone and receptor gene complex determines multiple B mating type specificities in Coprinus cinereus. Genetics 148 1081 1090
26. VaillancourtLJ
1997 Multiple genes encoding pheromones and a pheromone receptor define the Bβ1 mating-type specificity in Schizophyllum commune. Genetics 146 541 551
27. WendlandJ
1995 The mating-type locus Bα1 of Schizophyllum commune contains a pheromone receptor gene and putative pheromone genes. EMBO J 14 5271 5278
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2000 The G-protein beta subunit GPB1 is required for mating and haploid fruiting in Cryptococcus neoformans. Mol Cell Biol 20 352 362
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2000 Morphogenesis of Cryptococcus neoformans. Contrib Microbiol 5 217 238
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ŠtítkyGenetika Reprodukční medicína
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PLOS Genetics
2010 Číslo 2
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- Nuclear Pore Proteins Nup153 and Megator Define Transcriptionally Active Regions in the Genome
- Uncoupling of Satellite DNA and Centromeric Function in the Genus
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AusBiotech strengthened its international partnerships in regenerative medicine, with a memorandum of understanding (MoU) signed with the peak Japanese industry body.
AusBiotech signed the MoU with Japan's peak regenerative medicine body, Forum for Innovative Regenerative Medicine (FIRM) last week) under the MOU between Australian Trade Commission (Austrade) and FIRM, in relation to the collaboration in advancing regenerative medicine and cell and gene therapies within Australia, Japan and worldwide.
FIRM is a Japanese general incorporated association with a mission to accelerate research and commercialisation of regenerative medicine in Japan. FIRM was formed in 2011 and has more than 220 members of Japanese companies from various fields with diverse expertise in such as pharmaceutical, biotechnology, manufacturing, transportation, insurance and related supporting areas for regenerative medicine.
FIRM's goal is to establish a social structure to safely and continuously deliver the regenerative medical products. FIRM works closely with the Japanese government and academia for the goal.
AusBiotech and FIRM will collaborate in advancing their individual and common missions by sharing information, encompassing technology, policy, laws etc., and assist each other in co-sponsoring meetings or through other cooperation based on common concerns.
In particular, AusBiotech sees FIRM as an important international partner and the representative of the regenerative medicine industry in Japan, and FIRM sees AusBiotech as an important international partner and the representative of the regenerative medicine industry in Australia.
AusBiotech CEO, Mr Glenn Cross said: "This memorandum mark an important milestone in building the foundations for strong industry engagement between Australia and the key industry organisations in Japan."
"The MoU will drive our already aligned goals with FIRM to devise tactics that will deliver real-world, positive outcomes to the Australian regenerative medicine and pharmaceutical sectors. Congratulations to all concerned.
Mr Cross said: "Regenerative medicine is a growing area of research, offering the potential to treat disease and disability that have remained off-limits to intervention and may one-day become treatable."
The FIRM MoU builds on commitments with Korea's Council for Advanced Regenerative Medicine (CARM), signed last year, to advance regenerative medicine capabilities and indicates that collaboration is essential to leveraging our combined innovative potential."
AusBiotech has been working with Austrade over several years to promote the Australian biotechnology around the world, with particular focus on regenerative medicine, clinical trials, medical devices and diagnostics, biotechnology and pharmaceutical sectors.


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You will need 1 cup, 1/3 cup, 1/4 cup and 1tsp.
You can use a mixer or you can mix it by hand.
This is what you can use to mix the ingredients with.
First you will add 2 1/4 cups of flour.
Then you will add 1tsp of baking soda.
Then put 1tsp of salt in.
Then put 2 sticks of softened butter in.
Then put 1/3 cups of white sugar in.
Then add 1/3 cups of brown sugar.
Then add 1tsp of vanilla extract.
Then add 2 large eggs.
Then add 1/4 cups of walnuts.
Then add 2 cups of chocolate chips.
Preheat oven to 325 degrees.
Then mix
Until it looks something like this.
Then spry pan with olive oil.
Then scoop onto pan.
Then put into oven and cook them for about 13 minutes.
Then take them out and set them down to cool of.
Let them cool for 5 to 7 minutes.
Then enjoy!!!
First you will need 2 1/4 of flour


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Around London – Notting Hill Carnival; Paralympics comes to town; Albert Adams at the IWM; and, exploring the anatomy of athletes at the Hunterian…
August 23, 2012
• The Notting Hill Carnival – the largest festival of its kind in Europe – takes place in London's west this Sunday and bank holiday Monday. More than a million people are expected top attend to see the floats, listen to the traditional steel drum bands and sample some of the food found at the hundreds of stalls along the streets of Notting Hill. Sunday, when the costume prizes are awarded, has been designated as Children's Day and Monday, when the main parade takes place, as Adult's Day. The festival has been held every summer since 1966. For more, see www.thenottinghillcarnival.com.
• With the memories of the Olympics still fresh in our minds, it's time to turn our attention to the impending Paralympics and associated events including the 24 hour Torch Relay which hits London next Wednesday, just before the Opening Ceremony. The torch enters London at Watford and and then moves south through the city, taking in many of London's most famous landmarks as it visits all six boroughs before arriving at the Olympic Stadium. For more on the route, see www.london2012.com/paralympics/torch-relay/route/. Meanwhile, as with the Olympics, those who don't have tickets to the Paralympics will be able to watch on a giant screen at Trafalgar Square daily between 11am and 10pm where there will also be live music and activities including the chance to try out a range of Paralympic sports. For more, see www.btlondonlive.com/trafalgar-square. Tower Bridge, meanwhile, is undergoing a makeover with the installation of the Paralympics symbol, Agistos, tomorrow following the earlier removal of the Olympic Rings. Historic projections, meanwhile, will once again appear on the Houses of Parliament. Other events taking place around the Paralympic Games include Surprises: What You Will: Pop-Up Shakespeare – which will see Shakespearean characters like Juliet, Hamlet and Puck suddenly appearing and performing at "cultural hotspots" around the city (the exact time and location of the performances will remain a surprise until the day but you can register for updates at www.molpresents.com/surprises or follow @molpresents and @London2012Fest.
• A series of artworks by South African-born Expressionist Albert Adams are on show at the Imperial War Museum this summer. The works on show include the last painting he completed before his death in 2006 – title Abu Ghraib, it was inspired by reports of abuse perpetrated at the Abu Ghraib prison in Iraq – as well as a series of etchings Adams created between 2001 and 20064 which address conflicts like that Iraq War and Darfur. Admission is free. For more, see www.iwm.org.uk.
On Now: Anatomy of an Athlete – Elite sport, surgery and medical art. This exhibition in the Qvist Gallery at the Royal College of Surgeons' Hunterian Museum in Lincoln's Inn Fields features new artworks by world-leading medical artists which explore the anatomy and physiology of elite athletes. The art works come in a variety of forms – watercolour, video and sculpture – and represent the human body in a selection of sports and para-sports. Admission if free. Runs until 29th September. For more, see www.rcseng.ac.uk/museums/.
Around London – Bells…and more bells; Olympic memorabilia; free wifi in the West End; The Arch returns to Kensington Gardens; and, Impressionists at the RA…
July 26, 2012
• It's all about the Olympics in London this week and many of the events – like the Opening Ceremony and Torch Relay (see last week's post) – are well covered elsewhere, but we thought we'd mention a couple of things in relation to the Games:
The first is the 'All the Bells' project which will see bells across London being rung at 8:12am on Friday to "ring in" the first day of the Olympic and Paralympic Games. Work No. 1197: All the bells in a country rung as quickly and as loudly as possible for three minutes, commissioned as part of the London 2012 Festival, is the brainchild of Turner Prize-winning artist and musician Martin Creed and will involve thousands of bells across the nation. Speaking of bells, the City of London has announced that some of the City's churches will be ringing continuously during the three Olympic marathon events – the men's, women's, and Paralympic events. As many as 57 of the country's most experienced bell ringers, co-ordinated by the Ancient Society of College Youths (a ringing society created in London in 1637) will be working for three to four hours continuously at churches including St Paul's Cathedral, St Mary le Bow, St Lawrence Jewry, St Magnus the Martyr, St Vedast and St Katharine Cree. During the women's marathon, an all-female band will be attempting a peal at St Paul's, the first all-woman attempt on the bells. (Apologies, this article had originally had the time for the bell ringing at 8.12pm – it is in the morning, not the evening!)
A new exhibition exploring London's Olympic history has opened at the British Library. Olympex 2012: Collecting the Olympic Games features a range of memorabilia including a swimming costume and the finishing tape broken by – later disqualified – marathon runner Dorando Pietri from the 1908 London Games (see our earlier post for more on him) as well as posters and artworks, stamps, letters and postcards. The exhibition also features audio interviews with Olympians including William (Bill) Roberts, a relay runner in the 1936 Berlin Olympics, and Dorothy Tyler, a medal-winning high jumper who competed in the 1936 and 1948 Olympics. Presented by the British Library and International Olympic Committee, the exhibition runs until 9th September at the library in St Pancras. Entry is free. For more, see www.bl.uk. PICTURE: Rare 1948 postcard by an unknown artist (c) Private collection/IOC
• A new free wifi network has been launched in London's West End. Westminster City Council and telco O2 launched the network this week. It will initially cover Oxford and Regent Streets, Trafalgar Square, Leicester Square, Piccadilly Circus and Parliament Square with further areas in Westminster and Covent Garden the next to be included in the network. A once-only registration process is required to join.
• Henry Moore's famous sculpture, The Arch, has been returned to its original home in Kensington Gardens. The six metre high work was presented to the nation by Moore in 1980 and was positioned on the north bank of the Long Water until 1996 when the structure became unstable and was placed in storage. In late 2010, the Royal Parks began a project with The Henry Moore Foundation to see if the work could be returned to the gardens. Work began to restore the piece – which consists of seven stones weighing 37 tonnes – to its original location earlier this year. For more on Kensington Gardens, see www.royalparks.org.uk/parks/kensington-gardens.
• On Now: From Paris: A Taste for Impressionism. This Royal Academy of Arts exhibition at Burlington House features 70 works from the Sterling and Francine Clark Art Institute in Williamstown, Massachusetts, and includes works by Manet, Monet, Pissarro, Degas, Sisley, Morisot and Renoir as well as those of post-Impressionist artists Corot, Théodore Rousseau and J-F. Millet, and 'academic' paintings by Gérôme, Alma-Tadema and Bouguereau. Runs until 23rd September. Admission charge applies. See www.royalacademy.org.uk for more.


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Rich reds, burgundies and gold are the ultimate colors of romance. They also happen to be our client Leigha's favorite colors. So for Leigha and Leo's late June wedding reception design at The Brick, we created a glam + romantic look complete with tall, sweeping fresh floral arrangements and hints of gold on the flatware, glassware and chargers. Bring on the summer romance!
Design + Coordinating + Fresh Flowers : Merry Me Events
Escort + Place Cards : PaperThread (a division of Merry Me Events)
Chargers + Glassware + Flatware : Aays Event Rentals
Cake + Fresh Desserts : Macri's Bakery
Venue : The Brick
Catering : Navarre Hospitality Group


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StartupGrindSH2020-09-20 14:48:51
Howard Donaldson is CEO of Lunar Owl Studios, a new venture focused on video games. He has over 20 years' experience as a senior operating and financial executive at global entertainment and technology companies including Disney Interactive, Electronic Arts Canada, McGraw-Hill Education Group and Vanedge Capital (Venture Capital).
Howard Donaldson 是Lunar Owl 工作室的CEO,专注电竞游戏。过去20多年,他在全球著名的娱乐及技术公司,担任资深运营及财务高管,包括在迪斯尼互动娱乐、加拿大电子艺界、麦格劳希尔教育集团、以及风投基金Vanedge Capital。
Most recently, Howard was a Partner and CFO for Vanedge Capital in Vancouver. The VC fund achieved a superior track record of company exits and investment returns. Prior to Vanedge, Howard was Vice President of Studio Operations for Disney Interactive with general oversight for internal studios. He also co-founded Propaganda Games in Vancouver which was later sold to The Walt Disney Company.
Howard 曾担任温哥华Vanedge 基金的CFO及合伙人。该风投基金有着极佳的退出及回报记录。在此之前,Howard是迪斯尼互动娱乐的工作室运营的副总裁,运营内部的工作室。他还是温哥华Propaganda 游戏公司的共同创始人,该公司被迪斯尼收购。
Howard was previously Vice President and CFO for Electronic Arts Canada. As a business partner and active member of the executive team, he was responsible for directing strategic business plans that resulted in annual double digit growth in revenue to almost $1 billion, creating the largest interactive studio in the world.
At 7:15, Dhruv Adhia, the Co-founder & CTO of H+Technology will lead a workshop on Holographic platforms and Human Adaptive Interfaces . H+ was founded in 2012 by Dhruv Adhia, Vincent Yang, and Yamin Li in Vancouver, BC. H+ designs and creates Human Adaptive Interfaces that closely understand human behavior through gestures, emotions, speech and other sensory input. Prior to H+, Dhruv was Technical Director of D-Sign Touchless Interactive, Designer and Developer of Microsoft, and TransLink.
晚7:15,H+ Technology的CTO和共同创始人 Dhruv Adhia 将为大家介绍全息平台以及自适应界面。H+是由Dhruv Adhia, Vincent Yang, 和Yamin Li于2012年在温哥华创立。H+专注设计及开发自适应界面,可以通过人的动作、表情、语言、及其他传感输入,实时了解人的行为。在创立H+之前,Dhruv是D-Sign Touchless Interactive的技术总监,也是微软和TransLink的设计开发师。
H+'s product Holus is a tabletop platform that converts any digital content from a computer, tablet or smartphone into a 3D, holographic experience.
7:00pNetworking, wine, and light dinner
7:15p Workshop with Dhruv about Holographic platforms and Human Adaptive Interfaces
8:00p Fireside Chat and Q&A with Howard
晚8点 开始采访Howard
9:00p Informal discussion and networking
晚9点 会后交谈
Time & Address 时间地点
11/23, Wed, 7p-10p
@ CaoHeJing Hi-Tech Park Innovation Center, Building 16, SBP Phase 3, No.1036 Tianlin Rd (Line 9, HeChuan Rd) 田林路1036号漕河径科技绿洲三期16号楼国际孵化联合体 (9号线,合川路)
90 Seconds Pitch
In 90 seconds, pitch your startup to our audience and speakers, for recruitment/ funding/ or identifying partners. To apply, please fill in the survey below (Long press and extract the QR code to start).
Tickets 门票及支付方式
Early Bird before 11/15 RMB 80/p; Full Price RMB 100/p online, RMB 150/p at the door
Each ticket includes free flow beer and light dinner
Visa Payment Link 信用卡
Alipay 支付宝
Click "Read More" at the bottom of this post to pay with Alipay
Special Sponsors 特别赞助商
Shanghai Caohejing Hi-tech Park Innovation Center was solely invested and run by Shanghai Caohejing Hi-tech Park Development Corporation. As a business incubator, Innovation Center has committed itself to cultivating and supporting technology-intensive enterprises and promoting commercialization, industrialization and internationalization of scientific and technological achievements.
Partners 合作伙伴
October Event Recap 十月活动回顾
Our Values 我们的信念
About us
Startup Grind is the largest independent startup community, actively educating, inspiring, and connecting 400,000 founders in over 200 cities. We nurture startup ecosystems in 85 countries through events, media, and partnerships with organizations like Google for Entrepreneurs.
The cornerstone of our global community are monthly events featuring successful local founders, innovators, educators and investors who share lessons learned on the road to building great companies.
Founded in Silicon Valley, Startup Grind has now hosted 2,000 fireside chats since its founding in 2010. To date, Startup Grind has helped millions of entrepreneurs find mentorship, connect to partners and hires, pursue funding, and reach new users.
Since 2013 Startup Grind Shanghai Team has been trying to build up a community for entrepreneur in this amazing city. What make us glad is that we see many familiar faces regularly at our events! Yes, now we have a community and we are making it better!


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It's that time again! Let's all get together via the Internet and write some mail with one another, to one another, for one another! The Virtual Social will last the whole day of July 5th with a Live Video from the L.W.A. Clubhouse happening at 3pm CST. That's this Sunday. According to Kathy's time zone calculations*, this should be an hour when most of the world is awake, so hopefully most of our International members can join us for the video. Either way, you can participate all day long with #LWASocial on Twitter or Instagram.
Kathy built a page specifically for the Live Video Social that has the video, chat window, and the Instagram feed for#LWASocial all in one place. No more multiple windows open; just time to write, listen, and chat with your fellow members. If you can't join us for the Live Video, you can create your own Virtual Social by watching the recording on YouTube. Either way, we hope we can share a day of letter writing with you.
Remember, there are some free commemorative downloads for virtual socials (and other fun stationery too) in our Downloads section. Members have access to even more downloads. Be sure to log-in so you see the full roster of goodies. See you on the Internet!
* Kathy's Time Zone Calcualtions for the Live Video
We chose random cities that some of member's reside in to get a good spread of time differences. Please verify with your own time zone if your city isn't listed.
(NZDT) Aukland, New Zealand: Monday, Feb.9th at 10am
(MSK) Moscow, Russia: Midnight of Feb 8th (going to Monday the 9th)
(GMT) London, England: 9pm
(PST) Portland, OR, USA: 1pm
Looking forward to writing with you soon! ~Donovan & Kathy


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Electrifying color, bold design, and exotic style define Treska's Gallery Collection! Stunning hi-low pendant necklace with exotic black metal beads and colorful glass beads to add that WOW FACTOR to any look... the perfect pallette for exciting Fall & Winter style! This versatile piece can be separated into two single-strand necklaces - so you have 3 looks in one stylish piece! Complete the look and pair with Gallery Collection bracelets and earrings, some of which are shown here, sold separately. Check out our entire Gallery Collection!
All Treska jewelry is hand designed in Fort Worth, TX and comprised of a lead & nickel-free base metal. Each collection is available for a limited time depending on trends and seasons. Common stylish components include glass, stone, acrylic and resin.
Gallery Collection Hi-Low Pendant Necklace by Treska
- Treska Gallery Collection hi-low two-strand pendant necklace
- Style #: TRE-0917NK-GAL0047
- Colors: Black/blues/greens/red/multi
- Measures: 21-1/2" including pendant
- Lobster claw clasp
- Material: Metal, glass
- Lead & nickel-free
- Designed in USA


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At Key Stage 5 we offer provision at level 2 and level 3 alongside opportunities to resit GCSE English and Maths where appropriate.
At level 2 students follow a BTEC Travel and Tourism course with a choice of either BTEC Health and Social Care or OCR Cambridge National Business Studies. Students are also expected to participate in a work experience placement.
At level 3 most provision is AS and A level, with a small number of alternative courses such as BTEC and OCR Cambridge Technicals.
For full details of the courses offered please refer to the Sixth Form pages of our website and the 6th Form Options booklet.
Our dedicated Sixth Form team will support you as an individual in choosing appropriate courses which allow you to follow your areas of strength and interest, whilst also being open to discovering completely new subjects too. We help you enjoy, flourish and succeed on your chosen programme.
Committed and experienced Post-16 teachers employ a range of teaching styles and approaches which make the most of outstanding, extensive and state of the art facilities and ensure learning in this superb environment is personalised and fun!
More detailed information on the Sixth Form curriculum is available in the Sixth Form section of the website.


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About project
ROSA Media Player is a media player based on Mplayer/Ffmpeg technologies. It has a simple interface and many powerful functions:
- Possibility to get an audio track from video clip;
- Possibility to get a screencast;
- Possibility to trim video;
And many many others.
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Contact Us
Get in touch today! Book your dates or contact us for a tour.
By the way…. we are so excited to unveil our barns after renovations are completed! The wonderful images of our family and friend's weddings in the gallery were all taken before any of our barns transformed into what will now be Bluebird Manor. When you come visit, things will look a little different!
Bluebird Manor
1765A Greenfield Rd.
Adamstown, MD 21710