text:
Preventing Plaque Buildup – A Few Easy Ideas
Are you worried about your plaque buildup? It is one of the most commonly occurring health problems in the US. This is an important, but often neglected, area of your daily health.
Plaque is the mixture of hardened material such as calcium and other minerals, and acidic substances such as bile. It is the combination of substances that makes up plaque. This leads to it being regarded as a group of illnesses called hypercalcemia.
If you suffer from plaque buildup, it can seriously affect your health. It can lead to conditions such as heart disease, kidney stones, problems with blood vessels, arthritis, and even cancer. It is estimated that plaque buildup can affect almost four million Americans each year.
Unfortunately, most doctors aren't trained to spot plaque buildup. While some might suggest this as an overreaction, the truth is that plaque buildup can affect many of your most important body systems.
The problem stems from the fact that plaque doesn't just attack the body's immune system. It is also suspected to have a role in damaging your digestive system, possibly causing heart disease, or even causing cancer.
Fortunately, there are a number of ways to stop plaque buildup and regrow your healthy, natural-looking teeth. There are dietary changes you can make and herbal treatments you can use to begin to reverse the damage done by plaque buildup. There are also many natural products that can help to reduce plaque buildup and restore your teeth to their previous state.
Many of these products will include a home remedy that is believed to help to improve the condition of your teeth and make them more resistant to acid and salt damage. Another important component is the prevention of plaque buildup.
Having your teeth cleaned and whitened are common ways to help prevent plaque buildup. When these steps are taken, it is believed that plaque buildup will become much less likely.
But there are other, more subtle ways to maintain your teeth in their good shape. While dental care will certainly help reduce the accumulation of plaque, it is also true that removing plaque will help to provide you with healthy and strong teeth. As a result, dental care can even be expected to be of benefit to the very patient who suffers from plaque buildup.
Of course, prevention is better than cure. If you can, you should brush your teeth every day to ensure that you are protecting your teeth from any excess plaque buildup. If you are prone to plaque buildup, it might be a good idea to avoid alcohol and coffee.
If you find that you are suffering from plaque buildup, it may be a good idea to explore your options for a treatment. The natural products you choose will depend on your symptoms and the way in which your plaque buildup is contributing to your health.


text: Actually, QRP CW is the only thing that keeps ME involved in amateur radio. Even at that, my interest diminishes day by day. I am significantly older than you…I believe…but, unlike you, have no other significant interests to occupy my time with. But, I do enjoy reading, watching old B movies on You Tube, and listening to classical and early jazz music from the same source. Taking our new Dachshund puppy out…frequently…to relieve himself gets me plenty of fresh air. Radio wise I do make a point of getting on the air for at least an hour every day…except weekends. I don't always have a QSO but follow my signal on the RBN so I do know I am usually monitored somewhere in Europe and by a few monitors on the east coast of NA. Additionally, I read, somewhere, that fluency in Morse can be an aid in avoiding dementia. I guess I will never completely go QRT before going SK. GL es 72.73 de F8WBD

subset: commoncrawl


text:
Whether it's a financial necessity or just for fun, there are more and more moms finding ways earn an incoming from home. You can probably already think of a friend with an Etsy store, or maybe one that pushes whatever diet shake pyramid scheme is popular that month. A lot of us have tried an idea or two, and most of us have at least thought about it at some point.
If you've thought about trying the work from home thing then here are 7 home business ideas that don't involve harassing your friends about diet shakes on Facebook.
1. Become A Stock Broker Of Used Goods. Buy Low, Sell High.
This might be something you've heard about, but there is a pretty lucrative business in buying and selling used items on places like Craigslist, VarageSale, and more recently the LetGo App. Some people even hit up swap and shops and garage sales to find amazing deals to then resell online.


text:
We Value Your Referrals
If you're joining us as the result of a referral, we'd like
to thank you for putting your trust in us. We look forward to caring for you
and your family. After you have a chance to get to know us, we hope you'll tell
more of your friends and family about our office.
Finnane-Robison Dental520-B Burkarth RoadWarrensburg ,
Web Hosting powered by Network Solutions®


text:
Crossroads Home Decor
Her Smile, Remembrance of Mother, In Memory Of, Sympathy or Condolence Framed Poem, 6x12 7366
Regular price $22.99
- Picture frame poem remembering a loved one's smile after she has passed.
- Sympathy gift for someone who lost a loved one. Condolence or funeral gift for a family after a loved one has died. Remembering Mom or Grandma.
- Home decor picture frame for wall or tabletop display
- Includes glass front and easel back with wall hanger
- With a finished frame size of 6" x 12" this frame holds a 4" x 10" print
- Made in the USA


text:
Academic journal article Alcoholism and Psychiatry Research
Conference Report
Article excerpt
The Symposium on Addiction diseases was organized jointly by the Croatian Association of Alcoholism and Other Addictions and the Department of Psychiatry, University Hospital Sestre milosrdnice. It was held on 20th May 2010 in Zagreb, Croatia. Its aim was to contribute to a better understanding of problems connected to alcoholism and other addictions, as well as to improve the approach to diagnostics, treatment and rehabilitation. The lecturers are experts in the field of addictions and they discussed topics important for understanding problems connected to substance abuse, etiology, diagnostics, treatment and rehabilitation, as well as ethic, moral and forensic aspects of addiction. Easy availability of addiction substances increases all the problems connected to their intake. When in comorbidity with anxious and depressive disorders, substance abuse and addiction diseases significantly increase the risk for cerebrovascular and cardiovascular diseases, becoming so the biggest public health problem of the modem society. …


text:
See more at USA.gov
Try searching the Government Publishing Office Bookstore for books at a nominal price.
Here's a quick list of books and journal issues that might be of interest to Veterans!
The Still Picture Branch of the National Archives is excited to share more than 6,000 recently digitized photographs of U.S. Marine Corps activities in World War II and Korea! (1/25/18)
Indian Marine Fighters on Bougainville


text:
Free 4oz Bottle of Cleaner
Hurry! This FREEBIE will NOT last long!
Anderson Tuftex is offering a FREE 4oz bottle of cleaner! All you need to do is add the carpet stain and soil remover or hard surfaces floor cleaner to your cart. Make sure it is the 4oz bottle otherwise the code will not work. Fill out your shipping information and add the code "CLEAN2020" to make it totally free plus free shipping! If shipping is no longer free than this offer is over.
Note : This is not mobile friendly.
Love freebies? Be sure to claim all of the Newest Freebies here! Then, Join all of our FB Groups! Still have questions? Go to Messenger and tap GET STARTED. Then follow prompts by tapping CONTINUE and we'll walk you through it!


text:
The phony wall timepieces in Asia are made through gorgeous builder, along with the more compact elements are executed from the a variety of machines. Piaget G0a33071 With the passage of time, however, there has been an increasing appreciation for the unique charms of a mixture of one of the most practical of metals with one of the most precious, and two-tone watches are making something of a comeback both in vintage collecting circles, and in the collections of modern watch brands.
along with fastidiously manufactured sights merely continue below close, the 1st having a regulator fashion arrangement and including a totally fresh motion using the Silinvar parts and a quite uncommon conquer charge. my AP Perpetual Calendar and head out for dinner. Piaget G0a33071 Deskundig advies bij Juwelier Eugene vehicle Baal! Bestel on-line associated with kom langs bij onze winkel!? Our omega Designer watches Speedmaster moonwatch PROFESSIONAL CHRONOGRAPH; eerder vermeld). Fake John Winston replicasoldcom, rolex timepiece john winston * opus Twelve, overturns occasion | Brand new watchesrevue FH * Avril 2012The opus 12, which reinterprets moment extensively, is indeed a invention inside watchmaking.
Ben wrote a lengthy piece explaining why, and you should definitely give it a read. I think both the gold/blue and the blue/salmon color pairings look great and it's hard to argue with the proportions of the 37. How Good Are Noob Rolex Replica This season the modern patek philippe complications replicacan declare the runaway success, Even the Cartier logo, which is super discreet and almost hidden in the daylight, really pops thanks to an application of lume right on its lettering.
We see serious burrs, and a lack of defined edges in most of the engravings both on the caliber and dial side. He'd graduated from the College of Arts and Design in Lucerne, and wanted to set up a shop in the creative hub of late nineties Brooklyn.


text:
13/05/2015 | 01:05:24
The MovieByte Podcast #138: All the Good Wives
Preparando audio para descarga.
Escucha patrocinada. El audio empezará en pocos segundos...
Escucha sin esperasHazte Premium
Preparando audio para descarga.
Escucha patrocinada. El audio empezará en pocos segundos...
Escucha sin esperasHazte Premium
Join Joe and TJ in the post-apocalyptic future of 'Mad Max: Fury Road' where woman and children are property of warlords and must be set free! While that may be a plot of the most simple variety, it is by no means unsatisfying. Also join in for the discussion of three trailers: 'Supergirl', 'Pan', and 'Steve Jobs'. And did you know that Chris Pratt will be in some non-'Guardians of the Galaxy' Marvel films?Show Notes & LinksSUPERGIRL Season 1 TRAILER (2015) | New CBS Series First Look HD'Pan' Trailer: Every Kid Needs a Visually Lavish BackstoryThe story of Peter Pan is reconfigured in Pan, from Atonement and Hanna director Joe Wright.'Steve Jobs' Teaser Trailer: Michael Fassbender Invents the FutureSo far, all we've seen are photos with Michael Fassbender playing the titular Apple co-founder in Danny Boyle's forthcoming biopic Steve Jobs. But today that changes as a brief teaser trailer has just surfaced showing off the X-Men: Days of Future Past and Shame star as the late technology innovator, complete with glasses and black turtleneck.Chris Pratt Will Be in Non-'Guardians of the Galaxy' Marvel FilmsChris Pratt may be shooting down casting rumors left and right, but one thing we know for sure is that he'll be back for another Guardians of the Galaxy.Mad Max Primer: Is 'Fury Road' a Sequel or Prequel? Should You See 3D?Mad Max: Fury Road is in theaters now, and it is a triumph.The Heroic Masculinity of 'Mad Max: Fury Road'What separates the good ones from the bad ones is what they use that masculine power for.'Mad Max: Fury Road' Official SiteMad Max: Fury Road — IMDbIn a stark desert landscape where humanity is broken, two rebels just might be able to restore order: Max, a man of action and of few words, and Furiosa, a woman of action who is looking to make it back to her childhood homeland.Mad Max: Fury Road — Rotten TomatoesCritics Consensus: With exhilarating action and a surprising amount of narrative heft, Mad Max: Fury Road brings George Miller's post-apocalyptic franchise roaring vigorously back to life.Mad Max: Fury Road — Box Office MojoDomestic Total as of May. 18, 2015: $50,767,601 Distributor: Warner Bros.Release Date: May 15, 2015 Genre: Sci-Fi ActionRuntime: N/A MPAA Rating: RProduction Budget: $150 million
13/05/2015 | 01:05:24
06/05/2015 | 01:43:20
29/04/2015 | 01:19:24
22/04/2015 | 02:12:45
15/04/2015 | 01:27:56


text:
Secure Payment OptionsCheckout with the world's most secure payment methods.
Shop With ConfidenceHelping mums and bringing you gorgeousness since 2009.
Australia Wide DeliveryOrder by 1pm AEST for guaranteed same day dispatch or try our FREE 1hr Click + Collect from our Townsville store
Need Some Help?Contact our friendly team by email, phone or visit us in-store
Product Details
A great sun-safe check hat for boys! Featuring:
- Medium brim
- Detachable chin strap
- 50+ sun protection
- Reversible
- Made from cotton
Reversed side is dark blue.
2-5yrs 51cm
5-9yrs 55cm


text:
Police have released CCTV images of a man wanted over a string of armed robberies at Perth massage parlours.
The alleged robberies occurred between September 2014 and April of this year.
In the first incident in Aberdeen St, Northbridge, the man grabbed a receptionist and fled with cash.
The same month, he threated another receptionist, this time armed with a pair of scissors on Parry Street in East Perth.
He again fled with cash.
The alleged offender returned to the same Northbridge street last month and raided the front desk while the desk manager left the room.
When she returned, he threatened her with a knife and demanded money.
Finally, on April 6 this year, the same man entered another massage parlour in the city's south-east and pulled a knife while speaking with staff.
He fled on foot after demanding cash.
He is described is being olive skinned, between 30-40 years and around 175cm tall.
He is bald and has a goatee beard and moustache, and can be seen wearing a beanie during some of the alleged offences.
Anyone who recognises the man is urged to contact Crime Stoppers on 1800 333 000.


text:
Event Info > Parkinson's Unity Challenge
The 2020 Parkinson's Unity Walk is now virtual! We are thrilled to invite you to participate in a NEW way by joining our Parkinson's Unity Challenge!
The Parkinson's Unity Challenge combines all of your favorite elements of the Parkinson's Unity Walk – our PUW community, your impact, fun, physical activity & a big celebration – into one inspiring virtual experience. Even if we can't walk side by side in Central Park, our hope is we can all use the Parkinson's Unity Challenge as a way to bring this inspiring community together, which is so important now more than ever.
Starting Thursday, June 18, we will launch a series of challenges to build awareness of our fight against Parkinson's disease and raise funds for research as we countdown to our Unity Walk virtual celebration day on Saturday, July 11, at 11 a.m. (ET).
Who: Everyone is invited!
What: Help us fight against Parkinson's disease by participating in 4 weeks of social media challenges, culminating in our virtual celebration on Saturday, July 11.
Where: From anywhere!
When: Once a week! Starting Thursday, June 18, 2020 through July 11, 2020
How to join the challenge
Register for the Unity Walk Challenge so that you don't miss out on any of the fun!
|Complete the weekly challenge! Be sure to post on social media and tag @unitywalk using #PUW2020. We want to see all your smiling faces and stories!
If you're not on social media and would like to join us in this challenge, email your photo to email@example.com and we will post it for you!
We've made it to the fourth challenge and it's time to celebrate! During our virtual celebration event, we will gather together online to honor our inspiring Parkinson's community and show the impact we are able to have because of your fundraising efforts and participation.
Your challenge, this week, is to share a photo or video of you walking with us on Celebration Day!
Make sure you tag your posts with #PUW2020 so we can celebrate with you!
You can also email your photo to email@example.com, and we will post it for you!
Mark your calendar & make your plans to join us – we can't wait to celebrate with you!
For the 3rd Unity Challenge, we want to know how you are staying active! Post a video or photo of your favorite physical activity. It could be a walk around your neighborhood, your favorite yoga pose, or even some fancy dance moves.
Make sure you tag your posts with #PUW2020 so we can see your best moves!
You can also email your photo to firstname.lastname@example.com, and we will post it for you!
We are almost to the finish line. Thank you for all you are doing to support this great mission — we appreciate you!
Keep an eye out next Thursday, July 9 for the details of our 4th and final Unity Challenge!
For the 2nd Unity Challenge, we would love you to share with us "why you walk." Print our I WALK FOR ... Poster and get creative! Or, create your sign, write a message, or share a video (must be less than 60 seconds) and tell us why you love participating in the Parkinson's Unity Walk.
Download the I WALK FOR ... Poster
Tag it with #PUW2020 @unitywalk so we can share your inspiring message, too! You can also email your photo to email@example.com, and we will post it for you!
Thank you for all you are doing – we can't wait to read your story!
Be on the lookout – the 3rd unity challenge will be in your inbox next Wednesday, July 1.
We love the way the Parkinson's Unity Walk rallies this inspiring community to come together.
For your first challenge, post a throwback photo of your walk team from last year on social media – or throw it way back to a photo from the first year you walked. Be sure to tag your team members and invite them to register to join you in the Parkinson's Unity Challenge and help raise money for Parkinson's research. (Tag it with #PUW2020 @unitywalk so we can see all of your smiling faces!).
Watch your email inbox next Thursday, June 25th for your 2nd unity challenge!
Thank you for your commitment to this important mission! We look forward to seeing your photos!
Bonus Challenges
Are you ready to do more to help fight Parkinson's disease? Here are more ways to join us:
- Make a $10 donation to your own fundraiser, then ask 15 family members, friends, and co-workers to match your gift.
- Log in to your Participant Center or open our FREE mobile fundraising app, to access a host of fun and easy-to-use fundraising tools!
- Reaching out to your personal community – co-workers, neighbors, family, friends – is also a great way to ramp up your fundraising. Can you ask 5 NEW people to support you with a donation this week?
- Connect your fundraising page to your Facebook. Log in to your Participant Center and click on 'Raise more money with Facebook.' In just a few clicks, you will be connected and any donation you get from your friends and followers will be counted immediately towards your goal!
Save the Date
We are excited to host our first ever Virtual Unity Walk & Celebration Event on Saturday, July 11th starting at 11am EST.
Register or Log In today so you don't miss out on any of the fun!
Click for a printer friendly version


text:
Changeset 123900 for trunk/base/doc
- Timestamp:
- Aug 15, 2014, 9:47:47 PM (6 years ago)
- File:
- 1 edited
- Unmodified
- Added
- Removed
r123889 r123900 30 30 ----------- 31 31 These commands create OS X-native binary archives of a given port. Depending on 32 the command, either a .dmg disk image file, or a .pkg installer package is 33 created. 32 the command, 33 created. 34 34 35 *port dmg* creates an OS X disk image. *port pkg* wraps the same files in an OS 36 X installer package. In most cases you probably want to package a port and all 37 its library and runtime dependencies in a single package suitable for binary 38 redistribution. You can use a metapackage to do this. Create one using *port 39 mdmg* or *port mpkg*. 35 *port pkg* creates an OS X installer package that installs all files that belong 36 to a given port. *port dmg* wraps this installer package in a disk image. In 37 most cases you probably want to package a port and all its library and runtime 38 dependencies in a single package suitable for binary distribution. *port pkg* 39 and *port dmg* don't do that, so those are only useful if you are going to take 40 care of the dependencies separately. *port mpkg* creates an .mpkg installer 41 image that contains installer packages for each of the dependencies and is 42 suitable for standalone redistribution. *port mdmg* wraps this .mpkg package in 43 a disk image. 44 45 On OS X 10.6 and later, the generated installer packages are in "flat" format, 46 such that wrapping them in a disk image is no longer necessary for online 47 redistribution. Prior to OS X 10.6, generated installer packages use a resource 48 fork and can thus not be used for online distribution without a wrapping disk 49 image. 40 50 41 51 All packages are placed in a port's work directory, which can be located using
Note: See TracChangeset for help on using the changeset viewer.


text:
Qubool Hai 12th April 2013 Written Episode Update
Qubool Hai 12th April 2013 Written Episode, Qubool Hai 12th April 2013 Written Update
Part 1
Najma says.. Mr. Dhurandhar Bhatarwadekar…!! He enters n prononuncs his name n says.. his name is tough like him… as he is a strict person…! His assistant says.. he is called 75 and Dhurandhar says..coz he has deported 75 people this year! He asks for Zoya..! Zoya is hiding..! Dilshad looks at Asad n Asad keeps mum! Dhurandhar starts facial confirmation..with a pic..! He looks at Asad n Asad stares back n he says..why stare? He is a man of principles n rules..who knows Zoya had a hair cut..! Asad says.. great..! Dhurandar grills him n Asad says …said nothing! Dhurandhar concludes that no one seems to know it seems! His assistant offers to look around..takses Asads help to stand on the table! Tanvir deliberately looks at Zoya where she is hiding n Dhurandhar follows her stare..!
Part 2
Asad asks Dhurandhar to sit.. and he refuses.. n says. Zoya did wrong thing.. ! She lied to extend her stay here ..so by tonight 12. .Zoya has to leave India! His assistant says..that Zoya cant hide from her boss as he can see backwards too! Zoya sneaks out of her hiding place n Asad is worried..! Dhurandhar talks of knowing whats going behind his back n Zoya freezes.. but manages to leave in time! Dilshad says that Zoya is not a criminal …she is a good girl who wants to stay in her country! Dhurandhar says..surely . .but within rules..!
Dhurandhar asks his assitant if they cant start the search n she says yes! Zoya wonders what she should do n then spots sketchpen ..n gets an idea! Dhurandhar enters the room and Zoya screams dun come near.. have Chicken Pox..! Dhurandhar is shocked n asks his assistant to get the file n she gets it..! Zoya smiles at Asad! Dhurandhar says that the laws have an option that a person who is sick can stay back till she is cured.. but after that..she will be deported..! Zoya is wiping her face and overhearing..the convo..! Dhurandhar says..why Asad n family din inform him? They explain..that they wanted him to confirm! Dhurandhar says.. Atithi devo bhav.. so ok ..! Tanvir gets an idea..! Asad assures.. Zoya will be cured..! They leave..! All hug..n smile! Tanvir self thought..this happiness is for a moment only..!
Razia comes into Ayans room n finds Shireen sitting with his guitar..! She calls out to Shireen n tells her that seems Humeras dads clothes are mixed with Ayans so she needs to check n opens Ayans cupboard..! She tells Shireen that they need to find some solution to black magic! Shireen says.what can they do? Razia takes out a lighter and burns Ayans coat..! She screams n Shireen checks..! Razia says..seems someone is doing black magic on Ayan..! Shireen is scared.. and then says that the coat that is burnt is the one that Ayan wore previously during Ayan-Humeras engagement!
Zoya tells Asad that he has to agree that she is super smart..! She says that when she got the idea..she thought that she had chicken pox as a kid so how can she get now ..but she still used the idea..! So she is supersmart! Dhurandhar clapped n says.. its great he forgot his car keys.. n got to see what all Zoya has done with his own eyes..! He is lucky..! He asks..all know what he is called n AsYa say.. Aab tak Pachattar [75] .. ! Dhurandhar says.. no.. its
Aab Tak Chiaatar [76]
He tells his assitant to arrange to deport Zoya..! Asad fumes.. Zoya is sad!
Shireen tells Razia that things are out of control now.. even Baba din give solution.what to do? Razia says. .no . Baba had said that in Ayans life..some big decision is being taken n it should be taken fast.. so that the evil shadow passes off..! Razia wonders..what black magic? Shireen says.. biggest thing is Ayans engagement. .happening before Asad. .n so Dilshad is praying for all bad things for Ayan..! Razia agrees n says.. so the big thing should happen sooner for Ayan..! Shireen says..they need to do what they need to do rightaway!
The assistant says.. all is done.. ! Zoya apologises to Dhurandhar..! He says.. it wont affect him one bit..! Zoya looks at Asad..! The assistant drags Zoya out..and Najma Dilshad protest..! Asad says.. STOP ..he cant take Zoya as she has a legal reason to stay back..n the reason is that…
Shireen says.. Ayan-Humera engagement should be carried out rightaway!
Asad says.. He and Zoya are getting engaged..! Zoya is shocked.. Najma too!
Part 3
Dhurandhar.. says..engagement? Zoya says..yes.. n smiles n goes n stands next to Asad n asks if he wont wish them? Dhurandhar asks.why no one informed him? Dilshad-Najma says..that so many things happened that they couldnt tell..! Dhurandhar.. says.. she had chicken pox a while back.. n now this? Zoya says… she was panic stricken so. .n was worried that he will separate her from her UNSE .. right? Asad nods..! Dhurandhar says.. if Zoya was scared .. . .then why others din tell him about the engagement? Dilshad says.. she din wanna tell everyone! Dhurandhar says..coz they are lying..! Zoya says. .no ..they are having a small ceremony..! Dhurandhar asks Tanvir why she is not telling anything ..! Zoya says.. she came a few days back ..she is a guest…so how will she say anything..coz she doesnt know about it! Dhurandhar says.. a very SECRETIVE family this is.. but wont buy this.. ! AsYa will have to face a test! AsYa are confused!
Precap — Dhurandhar asks them Questions …
Love or Arranged marriage? Asad says Arranged ..Zoya says love n corrects Asad
He asks.. AsYa who proposed n how? Zoya says.. while jogging… n that she saw a cute puppy …
He asks color of puppy Asad says black.. Zoya says white..
Asad says. .was a Dalmatian .. n corrects Zoyas answer n says..she was too tired so din focus..!
Dilshad n Najma are worried seeing the whole madness!
Miss Farukhi will u get engaged to me? She said yes.. Story ended!
Dhurandhar congratulates AsYa on their engagement!
Update Credit to: Armu4eva


text:
Thursday's Middletown Press to include Black Friday circulars (document)
Need a guide to all of the special discounts being offered at department stores and other local retailers on "Black Friday" this year?
Thursday's Thanksgiving Day edition of the Middletown Press will be one of the largest of the year, with dozens of Black Friday circulars inside, including money-saving coupons from local and national stores.
Before sitting down for your Thanksgiving meal on Thursday, you can find a copy of the Middletown Press, full of Black Friday flyers, at any of the locations listed below.


text:
"Pay and Pensions Talk"
All staff are welcome.
This taxonomy term is used for specifically hiding nodes and manually showing contents.
All staff are welcome.
Inspired by LGBT History Month, we are delighted to announce the launch of our own LGBT+ network. This has a few aims. Firstly, we want to build a social network for LGBT+ staff and students in a relaxed, informal setting. We also want to find out what issues are important to people in the School, discover shared sources of support, and plan for future events.
The CLSRSA is inviting you to join the third seminar of our "Technique Tapas" series. Markus Posch, our CLS OMX specialist (Central Imaging Facility), will give us new insights on cutting edge fluorescence and super resolution microscopy. Find out how to use this spectacular technology best for your own research, by learning about the many possibilities this technique offers. Gain information about sample requirements, choice of the right fluorophores, where the advantages and limitations of this method lie.
Ubiquigent Ltd, Sir James Black Centre, Dow Street, Dundee, DD1 5EH


text:
Put your travel plans onto your mobile.
Create beautiful lists of restaurants and locations to visit with Everplaces. It's free, mobile and anti-roaming. Curious?
A fabulous restaurant, the staff are brilliant, funny, professional and very friendly. The food is incredible, a large menu plus specials board, so it takes a while to choose, but you can count on whatever you go for being delicious, and served with flourish. Such a great place to eat and while away an evening with friends, family, or on a romantic little date. My favourite Bath restaurant!
Save and share places you love
with Everplaces


text:
Our ServicesWe hold three regular services. They sometimes change in style from week to week, so be sure to check our calendar to find out what we've got planned! Calendar
The 8:30am
Holy Communion
A quiet service with sermon but no music, using the traditional Book of Common Prayer.
The 10:00am
Holy Communion
Holy Communion with Sunday School or All Age Morning Prayer
Please join us for refreshments in the Veranda Room, St Peter's House – exit by the north door (to the right of the baptismal font).
The 6:30pm
Evening Service
A varied programme through the month, including Choral Evensong, Evening Prayer, informal worship and, four times a year, a Taizé service of singing and meditation
Family Praise
Second Sunday of each month, 11:30am – 12pm, everyone welcome!
Children's work
Sunday School
There is special provision for children at the 10am service on the first and fourth Sundays of each month. We all start the service together, then our Sunday School meets in St Peter's House, returning in time for communion.
Toilet and baby changing facilities are in St Peter's House.
There is a children's area at the front of church where children can see and hear. Resources, particularly for pre-school children, are available to support families worshipping together.
If you need assistance before or during a service, please speak to our Verger or Wardens who will be pleased to help. We have a loop induction system, please switch your hearing aid to the T position.
If you would like to make an offering during one of our services, please consider making your contribution a Gift Aid donation – envelopes are available at your seat or at the back of church. If you'd like to make a donation online, please click below. You can also set up regular giving.


text:
- © 2014 American Society of Plant Biologists. All Rights Reserved.
Modification of anthocyanin plays an important role in increasing its stability in plants. Here, six anthocyanins were identified in peach (Prunus persica), and their structural diversity is attributed to glycosylation and methylation. Interestingly, peach is quite similar to the wild species Prunus ferganensis but differs from both Prunus davidiana and Prunus kansueasis in terms of anthocyanin composition in flowers. This indicates that peach is probably domesticated from P. ferganensis. Subsequently, genes responsible for both methylation and glycosylation of anthocyanins were identified, and their spatiotemporal expression results in different patterns of anthocyanin accumulation in flowers, leaves, and fruits. Two tandem-duplicated genes encoding flavonoid 3-O-glycosyltransferase (F3GT) in peach, PpUGT78A1 and PpUGT78A2, showed different activity toward anthocyanin, providing an example of divergent evolution of F3GT genes in plants. Two genes encoding anthocyanin O-methyltransferase (AOMT), PpAOMT1 and PpAOMT2, are expressed in leaves and flowers, but only PpAOMT2 is responsible for the O-methylation of anthocyanins at the 3′ position in peach. In addition, our study reveals a novel branch of UGT78 genes in plants that lack the highly conserved intron 2 of the UGT gene family, with a great variation of the amino acid residue at position 22 of the plant secondary product glycosyltransferase box. Our results not only provide insights into the mechanisms underlying anthocyanin glycosylation and methylation in peach but will also aid in future attempts to manipulate flavonoid biosynthesis in peach as well as in other plants.
Anthocyanins are important secondary metabolites and serve to protect plants against pathogenic attack and UV radiation and provide flowers and fruits with pigmentation to attract pollinators and seed dispersers (Winkel-Shirley, 2001). Anthocyanins are usually stored as glycosylated forms in vacuoles. The basic structure of anthocyanins is composed of an anthocyanidin aglycone and one or more sugar moieties linked to hydroxyl groups 3, 5, 7, 3′, and 5′, with the 3 position on the C-ring being dominant. The most common sugar moieties are Glc, Gal, Xyl, Ara, and Fru. With different types and/or numbers of sugar moieties attached to various positions, the structural diversity of anthocyanins increases significantly. Besides glycosylation, modifications such as methylation and acylation also contribute to the structural diversity of anthocyanins. To date, there have been more than 550 anthocyanins identified in nature (Kong et al., 2003).
The formation of glycosides is catalyzed by UDP-Glc:flavonoid glycosyltransferase (UFGT). Both monoglycosides and diglycosides are common in plants. For example, 3RT, 3GGT, and F3GGT1, responsible for further glycosylation of anthocyanidin 3-O-glycosides, have been reported in petunia (Petunia hybrida; Kroon et al., 1994), Japanese morning glory (Ipomoea nil; Morita et al., 2005), and kiwifruit (Actinidia chinensis; Montefiori et al., 2011), respectively. Glycosylation plays at least two roles in the accumulation of anthocyanins. One is to stabilize anthocyanins by attaching sugar moieties to the unstable anthocyanidin aglycones. Deficiency in UF3GT activity results in a significant reduction of anthocyanin accumulation in maize (Zea mays; Fedoroff et al., 1984) and Arabidopsis (Arabidopsis thaliana; Tohge et al., 2005), as anthocyanidins are highly unstable and easily susceptible to degradation. Another is to serve as a signal for transport of the anthocyanins to vacuoles (Ono et al., 2006). Anthocyanins are synthesized on the cytoplasmic face of the endoplasmic reticulum by metabolons, which are likely to function as a multienzyme complex (Winkel, 2004). Once synthesized, anthocyanins need to be transported to vacuoles by transporters such as multidrug and toxin extrusion transporters (Zhao et al., 2011). Vacuole localization is crucial for anthocyanins to function as pigments to attract pollinators and seed dispersers or as antioxidants to prevent photooxidation of the photosynthetic apparatus by lowering light intensity and screening out UV irradiation (Marrs et al., 1995). In short, glycosylation is essential for the accumulation of anthocyanins in plants.
UFGT belongs to family 1 glycosyltransferases, which are characterized by the presence of a conserved C-terminal domain termed the plant secondary product glycosyltransferase (PSPG) box (Yonekura-Sakakibara and Hanada, 2011). The PSPG motif is involved in binding to the UDP moiety of the sugar donor and thus plays an important role in anthocyanin biosynthesis (Caputi et al., 2012). To date, genes encoding UFGTs have been identified in a range of plant species, and they can be divided into two groups based on their specificity for sugar acceptors. The first group catalyze the glycosylation of anthocyanidins, including anthocyanidin 3-O-glycosyltransferase (F3GT; Fedoroff et al., 1984; Ford et al., 1998; Kubo et al., 2004; Tohge et al., 2005) and anthocyanidin 5,3-O-glucosyltransferase (Ogata et al., 2005). The second group are responsible for the further glycosylation of anthocyanins, including anthocyanidin 3-O-glucoside 6′′-O-rhamnosyltransferase (F3GGT; Kroon et al., 1994), anthocyanidin 3-O-glucoside 2′′-O-glucosyltransferase (F3GGT; Morita et al., 2005), anthocyanidin 3-O-glucoside 2′′-O-glucuronosyltransferase (Sawada et al., 2005), and anthocyanidin 3-O-glucoside 2′′-O-xylosyltransferase (F3GGT; Montefiori et al., 2011; Yonekura-Sakakibara et al., 2012). Among these UFGT genes, F3GTs can also catalyze the glycosylation of flavonols in some plant species, such as Arabidopsis (Tohge et al., 2005), petunia (Yamazaki et al., 2002), and grape (Vitis vinifera; Ford et al., 1998).
Methylation is known to affect both the stability and water solubility of anthocyanins; thus, it plays an important role in the accumulation of anthocyanins in plants (Sarni et al., 1995). Anthocyanin methylation is catalyzed by anthocyanin O-methyltransferase (AOMT), and AOMTs are responsible for the O-methylation of anthocyanins at either the 3′ or 5′ position (Wiering and deVlaming 1977). To date, two AOMT genes have been cloned and characterized in grapevine (Hugueney et al., 2009; Lücker et al., 2010; Fournier-Level et al., 2011).
Anthocyanin accumulation has a great impact on fruit quality, and many genes involved in the regulation of anthocyanin biosynthesis have been identified in fruit species such as grape (Kobayashi et al., 2004), apple (Malus domestica; Takos et al., 2006; Espley et al., 2007; Li et al., 2012), and pear (Pyrus communis; Feng et al., 2010). However, few studies have been reported on characterization of the UFGT and AOMT gene families in fruit trees. Functional characterization of UFGT and AOMT genes has so far only been reported in grape (Ford et al., 1998) and kiwifruit (Montefiori et al., 2011). Many questions related to the mechanism underlying the glycosylation and methylation of anthocyanins and the evolution of the UFGT and AOMT gene families in plants still remain unanswered.
Peach (Prunus persica), a member of the Rosaceae family, is the third most important of deciduous fruit trees worldwide. Peach is a diploid with a small genome size of approximately 230 Mb (Arús et al., 2012). To date, there are few reports on genes involved in the regulation of anthocyanin accumulation in peach. Recently, we sequenced peach transcriptomes using the Illumina RNA sequencing (RNA-Seq) strategy, and the results provide an opportunity to investigate genes involved in anthocyanin accumulation in peach. Here, we report on the mechanisms underlying the methylation and glycosylation of anthocyanins in peach. Our study is not only helpful for understanding the molecular mechanism of anthocyanin accumulation in fruit trees but also useful for future research to manipulate flavonoid biosynthesis in peach as well as in other plants.
The Anthocyanin Component in Red-Colored Tissues of Peach and Its Wild Relatives
Peach cultivars Mantianhong (MT), Hongyetao (HY), and Wuhong9 (WH9) produce red-colored flowers, leaves, and fruits, respectively, and these red tissues were thus selected and subjected to an analysis of anthocyanin composition. As a result, a total of six peaks were identified in these three tissues (Fig. 1A). The flowers of cv MT contained all six peaks. The leaves of cv HY consisted of two major peaks, 2 and 3, and three traceable peaks, 4 to 6. The fruits of cv WH9 contained a major peak, 2, and a traceable peak, 3. Peaks 1 and 2 had the same mass spectrometry (MS) and tandem mass spectrometry (MS/MS) spectra, and peak 2 had the same retention time with the standard of cyanidin 3-glucoside (Cy-3-glu). Thus, peaks 1 and 2 were identified to be cyanidin 3-galactoside (Cy-3-gal) and Cy-3-glu, respectively (Table I). Based on the MS and MS/MS spectra, peaks 3 to 6 were deemed cyanidin 3-rutinoside (Cy-3-rut), peonidin 3-glucoside (Pn-3-glu), cyanidin 3-rhamnoside (Cy-3-rha), and peonidin 3-rutinoside (Pn-3-rut), respectively. Among these anthocyanins, Cy-3-rha was detected for the first time in Rosaceae, while Cy-3-gal, Pn-3-glu, and Pn-3-rut were identified for the first time in peach. To validate the anthocyanins identified in this study, anthocyanins in flowers of cv MT were further hydrolyzed to identify anthocyanidin composition using HPLC-electrospray ionization (ESI)-MS. Two peaks showing the same retention times with the standards of cyanidin and peonidin were detected (Fig. 1B). This is consistent with the results of HPLC-MS/MS analysis as mentioned above.
To determine if there are other anthocyanins in peach, the identification of anthocyanin composition in flowers was further performed for six more cultivars and three wild species. As a result, only the six anthocyanins mentioned above were detected (Table II). Overall, all the cultivars showed a similarity in anthocyanin composition. The major component of anthocyanins in flowers of all the tested cultivars was Cy-3-glu, with an average content of 52.17%. The remaining components, including Cy-3-gal, Cy-3-rut, Pn-3-glu, Cy-3-rha, and Pn-3-rut, showed 1.5%, 12.43%, 12.97%, 10.93%, and 10.42% of average contents, respectively. Interestingly, Cy-3-glu also represents the major anthocyanin in flowers of Prunus ferganensis and accounts for approximately 61% of the total anthocyanins. In addition, four and two anthocyanins were detected in flowers of Prunus davidiana and Prunus kansueasis, respectively. The most abundant anthocyanin in flowers of P. davidiana is Pn-3-glu (40.92%), followed by Pn-3-rut (35.19%), Cy-3-glu (15.92%), and Cy-3-rut (7.96%). Two anthocyanins, Cy-3-rut and Cy-3-glu, account for 56.14% and 43.86% of the total anthocyanins in flowers of P. kansueasis, respectively. In summary, P. davidiana and P. kansueasis accumulate mainly two peonidin-based and two cyanidin-based anthocyanins in flowers, respectively, whereas the common major anthocyanin Cy-3-glu is synthesized in flowers of peach and P. ferganensis.
In summary, there are six anthocyanins in peach, and their structural diversity is attributed to glycosylation and/or methylation.
Identification of Genes Encoding UDP-Glycosyltransferase and O-Methyltransferase in the Peach Transcriptome
A total of 21.5 Gb of Illumina RNA-Seq reads, representing approximately 96 times the peach genome, were generated from different tissues, including red and green leaves, white- and red-fleshed fruits, and flowers (Wang et al., 2013). The RNA-Seq reads were assembled, and the consensus sequences were screened for the presence of the PSPG box, a characteristic of family 1 glycosyltransferases (UGTs). As a result, 79 genes encoding UGT were identified in the peach transcriptome (Supplemental Table S1). These peach UGT genes were named following the previously reported nomenclature system (Mackenzie et al., 1997), and they can be phylogenetically divided into eight orthologous groups according to the definition as described by Yonekura-Sakakibara and Hanada (2011). Of the eight orthologous groups, OG8 and OG23 consist of F3GT and F3GGT genes and are involved in the glycosylation of anthocyanins and anthocyanidins, respectively (Supplemental Fig. S1). Therefore, three F3GT genes, designated PpUGT78A1, PpUGT78A2, and PpUGT78B, and five F3GGT genes, designated PpUGT79A, PpUGT79B, PpUGT94A2, PpUGT94A3, and PpUGT94A5, were selected and subjected to expression profiling analysis.
Similarly, 22 genes encoding O-methyltransferase (OMT) were identified in the peach transcriptome (Fig. 2). Phylogenetic analysis indicated that two OMT genes, designated PpAOMT1 and PpAOMT2, are closely related to VvAOMT, which is responsible for the methylation of anthocyanins in grapevine (Hugueney et al., 2009). In addition, an OMT gene (ppa010674m) also shows a close relationship with VvAOMT. However, RNA-Seq and reverse transcription-PCR analysis demonstrated that ppa010674m was highly expressed in fruits that accumulate only nonmethylated anthocyanins but was not detectable in flowers (Supplemental Fig. S2), and it was not included in further analysis.
Identification of Gene Families Encoding F3G, F3GGT, and AOMT in the Peach Genome
First, we screened the genome of peach cv Lovell (Verde et al., 2013), and two more UGT78 homologs designated PpUGT78A3 and PpUGT78A4 were identified. Of the five PpUGT78 genes, four (PpUGT78A1–PpUGT78A4) are clustered in a 56-kb region on chromosome 1, and one PpUGT78B is located on chromosome 2 (Fig. 3A). All the PpUGT78 genes except PpUGT78A3 have intact coding frames. PpUGT78A3 is a pseudogene due to a frameshift mutation induced by a 5-bp insertion in the coding region. The five PpUGT78 genes share 56% to 86% identities among their DNA coding sequences (CDSs; Fig. 3B). Of the four PpUGT78A genes, PpUGT78A1, PpUGT78A2, and PpUGT78A3 share 79% to 86% CDS identity, with approximately 66% CDS identity to PpUGT78A4. PpUGT78B shows the lowest CDS identity (56%–60%) with the four PpUGT78A genes. These results suggest that the PpUGT78A cluster is likely derived from two rounds of tandem gene duplication, while the ancestor of the cluster and PpUGT78B are probably derived following a genome duplication event during the speciation process of peach (Fig. 3C).
All four genes, PpUGT78A1, PpUGT78A2, PpUGT78A4, and PpUGT78B, contain a conserved 44-amino acid residue motif known as the PSPG box (Fig. 3D). The PpUGT78A1 and PpUGT78A2 proteins are similar, with equal length and 80% identity in amino acid sequences. The PpUGT78A1 and PpUGT78A2 proteins both show higher identities in amino acid sequences with PpUGT78A4 (53%) than with PpUGT78B (47%). The PpUGT78B and PpUGT78A4 proteins show a high degree of divergence and share the lowest identity (43%) in amino acid sequences.
Second, five copies of PpUGT94A genes are clustered in a 79-kb region on chromosome 2 (Fig. 3A). All five PpUGT94A genes show an average of 86% CDS identity, and PpUGT94A1 and PpUGT94A2 share the highest degree of CDS identity (97%).
Finally, both UGT79 and AOMT gene families contain two copies in the peach genome. PpUGT79A and PpUGT79B are both located on chromosome 4 and share 58% CDS identity. PpAOMT1 and PpAOMT2 are located on chromosomes 1 and 2, respectively, and share 79% CDS identity.
Expression Profiling of F3GT, F3GGT, and AOMT Genes in Peach
Gene expression levels were initially estimated using fragments per kilo bases per million reads values (Supplemental Fig. S2). Overall, F3GGT homologous genes showed very low levels of expression in all tested tissues, including red- and white-fleshed fruits, red flowers, and green and red leaves. In contrast, three F3GT homologous genes showed high levels of expression in red tissues. Two AOMT homologous genes were highly expressed in flowers at early stages of development but not expressed in fruit tissues. These results were subsequently validated using real-time PCR analysis (Fig. 4).
PpUGT78A1 was expressed in fruits, leaves, and flowers. The expression levels of PpUGT78A1 increased during flower development and reached a maximum at full-bloom stage. Interestingly, PpUGT78A1 was expressed in both red and green leaves, and its level of expression in green leaves was higher than in red leaves. However, the transcripts of PpUGT78A1 were not detectable in red-fleshed fruits. PpUGT78A2 was predominantly expressed in flowers, and its level of expression in red flowers was significantly higher than in pale pink flowers. PpUGT78A2 transcripts were weakly accumulated in young red leaves but undetectable in green leaves and fruits. In contrast, PpUGT78B was mainly expressed in leaves and fruits, and its expression level in flowers was extremely low. PpUGT78B transcripts were significantly higher in red tissues than in nonred tissues. For example, the expression level of PpUGT78B in red-fleshed fruits of cv WH9 was 18.5-fold higher than that in white-fleshed fruits of cv Baifeng (BF). PpUGT78B transcripts were expressed at a 25.5-fold higher level in red leaves of cv HY than in green leaves of P. davidiana. In addition, PpUGT78A4 was not expressed in all analyzed tissues. In short, PpUGT78A1 was expressed in both green and red tissues, whereas PpUGT78A2 and PpUGT78B were predominantly expressed in red tissues.
PpUGT79A was exclusively expressed in flowers of P. davidiana. PpUGT79B transcripts were expressed in leaves and flowers, with high expression in nonred tissues, but were not detectable in fruits. For the PpUGT94A gene family, all three members were expressed in red-fleshed fruits, although both Cy-3-rut and Pn-3-rut were almost undetectable in red-fleshed fruits. Moreover, these PpUGT94A genes were highly expressed in flowers at full-bloom stage, but their expression levels were extremely low or undetectable in flower buds at pink and bloom stages. These results strongly suggest that the PpUGT94A gene family is unlikely to be related to the glycosylation of anthocyanins in peach.
The two PpAOMT genes were highly expressed in flowers but weakly or not expressed in leaves and fruits. The expression levels of PpAOMT1 in flowers had a maximum at pink stage and then decreased during flower development. The expression level of PpAOMT2 in flowers increased during flower development and reached a peak at bloom stage, then decreased at full-bloom stage. These results suggest that methylation of anthocyanidin would occur during flower development but prior to the full-bloom stage.
Functional Analysis of PpAOMT and PpUGT78 Genes
Enzyme activity in vitro was initially tested to determine the functionality of the PpAOMT genes. The recombinant proteins of both PpAOMT1 and PpAOMT2 were strongly induced in Escherichia coli, with an expected molecular size of approximately 29 kD (Fig. 5A). The recombinant protein was purified on an Ni2+-charged resin that binds the protein's N-terminal His tag, and the activity was then assayed using Cy-3-glu as a substrate. No product was detected for the reaction of PpAOMT1 with Cy-3-glu (Fig. 5B). In contrast, the product of the reaction of PpAOMT2 with Cy-3-glu showed the same MS and MS/MS spectral properties as Pn-3-glu (Fig. 5B). This result shows that, in vitro, PpAOMT2 works well on Cy-3-glu, leading to the generation of the methylated anthocyanin Pn-3-glu.
The functionality of PpAOMT2 was further validated by transient expression in tobacco (Nicotiana tabacum). Two other discrete anthocyanins were detected in tobacco leaves coexpressing NtAN2 and PpAOMT2 genes (Fig. 5C). HPLC-MS/MS analysis indicated that two discrete anthocyanins were malvidin 3-glucoside and malvidin 3-rutinoside. This demonstrates that PpAOMT2 is functional in vivo.
The functionality of PpUGT78 genes was also tested in vitro using cyanidin as acceptor and UDP-Glc or UDP-Gal as sugar donor (Fig. 6). PpUGT78A1 showed a very low activity toward cyanidin with either UDP-Glc or UDP-Gal as sugar donor. In contrast, both PpUGT78A2 and PpUGT78B had activity toward cyanidin, with a higher level of glucosyl transfer activity than galactosyl transfer activity.
Phylogenetic Relationship between the Peach UGT78 Genes and Their Homologs in Other Plants
The UGT78 homologs were identified in sequenced genomes of plants, including monocots such as rice (Oryza sativa), maize, millet (Pennisitum americanum), and sorghum (Sorghum bicolor), and eudicots such as grapevine, poplar (Populus spp.), castor bean (Ricinus communis), cassava (Manihot esculenta), Medicago truncatula, soybean (Glycine max), common bean (Phaseolus vulgaris), apple, strawberry (Fragaria spp.), cucumber (Cucumis sativus), orange (Citrus spp.), papaya (Carica papaya), Eucalyptus globulus, Arabidopsis, Thellungiella halophila, Capsella rubella, Brassica rapa, tomato (Solanum lycopersicum), and potato (Solanum tuberosum). Phylogenetic analysis indicates that UGT78 genes in plants are grouped into monocot and eudicot clades (Fig. 7). All UGT78 genes identified in eudicots can be divided into two groups, denoted UGT78I and UGT78II, and the UGT78I group is composed of two subgroups, designated UGT78Ia and UGT78Ib. The three peach genes PpUGT78A1, PpUGT78A2, and PpUGT78A3 belong to the UGT78Ia subgroup, while the peach gene PpUGT78B belongs to the UGT78Ib group. The UGT78Ia subgroup consists of characterized UGT78 genes showing activity toward flavonols, including VvF3GTIa-1 and VvF3GTIa-2 in grapevine (Ono et al., 2010), MtF3GTIa-1 in M. truncatula (Modolo et al., 2007), and RhF3GTIa-3 in rose (Rosa spp.; Fukuchi-Mizutani et al., 2011). However, PtF3GTIa-2, which does not accept any flavonoids as a substrate, is also grouped into the UGT78Ia subgroup (Veljanovski and Constabel, 2013). The UGT78Ib group contains characterized UGT78 genes showing activity toward anthocyanins and flavonols, including VvF3GT1b in grapevine (Ford et al., 1998), AtF3GT1b-4 in Arabidopsis (Kubo et al., 2007), RhF3T1b in rose (Fukuchi-Mizutani et al., 2011), and FaF3GT1b in strawberry (Griesser et al., 2008), but two Arabidopsis genes, AtF3GTIb-1 and AtF3GTIb-3, within the same subgroup accept only flavonols (Jones et al., 2003; Yonekura-Sakakibara et al., 2008).
Moreover, all the UGT78II genes except GmF3GT2-2 lack introns, while both UGT78Ia and UGT78Ib genes have one intron. The GmF3GT2-2 gene contains a single intron. The UGT78I genes are present in all analyzed eudicot plants, while the UGT78II genes are identified only in five eudicot plant species: poplar, castor bean, cassava, common bean, and soybean (Table III). To investigate whether specific amino acid residues within the PSPG motif could serve as sequence logos to differentiate one UGT78 gene group from another in eudicots, logos for the conserved 44-amino acid residue motif of each UGT78 gene group were analyzed using the online WebLogo program (http://weblogo.berkeley.edu). Interestingly, the amino acid residue at position 22 is very highly conserved in both UGT78Ia and UGT78Ib subgroups, whereas it is subjected to variation to Ala (A), Ser (S), or Cys (C) in the UGT78II gene group (Fig. 8). Thus, it seems that the residue Trp (W) at position 22 is a sequence logo for the UGT78I gene group.
The Complexity of Glycosylation and Methylation Leads to the Diversification of Anthocyanins in Peach
F3GTs catalyze the glycosylation of anthocyanidins. In this study, three F3GT genes, PpUGT78A1, PpUGT78A2, and PpUGT78B, are identified in the peach transcriptome. Of these genes, PpUGT78B is expressed in red-fleshed fruits, where only one anthocyanin Cy-3-glu accumulates predominantly. PpUGT78B contains the residue Gln (Q) at position 44 of the PSPG box, suggesting that it has glucosyl transfer activity (Kubo et al., 2004). This deduced functionality also has been validated in vitro. Thus, there is no doubt that PpUGT78B is involved in the synthesis of Cy-3-glu. PpUGT78A1 is closely related to the rose RhF3GTIa-1, which can use both flavonols and anthocyanins as substrates, but with extremely low activity toward anthocyanins (Fukuchi-Mizutani et al., 2011). We also found that PpUGT78A1 has very low activity toward cyanidin. Moreover, PpUGT78A1 contains the residue His (H) at position 44 of the PSPG box, which is required for recognition of the sugar donor UDP-Gal (Kubo et al., 2004). Therefore, the low activity of PpUGT78A1 for anthocyanidins is likely responsible for the traceable amount of Cy-3-gal in peach flowers.
In flowers, both PpUGT78A1 and PpUGT78A2 are expressed, but the transcripts of PpUGT78B are almost undetectable. As mentioned above, PpUGT78A1 has very low activity toward cyanidin, while PpUGT78A2 has high activity toward cyanidin with UDP-Glc as sugar. Therefore, PpUGT78A2 is responsible for the accumulation of Cy-3-glu in flowers. However, more studies are still needed to address the mechanism of functional divergence between the two clustered UGT78 genes PpUGT78A1 and PpUGT78A2. In addition, PpUGT78A2 contains the residue Asn (N) at position 44 of the PSPG box, whose activity toward the recognition of the sugar donor has not been reported. It is unclear if PpUGT78A2 also can recognize the UDP-Rha donor, leading to the accumulation of Cy-3-rha in flowers of peach.
PpUGT79A and PpUGT79B are identified in the peach transcriptome, and they are homologs of the kiwifruit and petunia F3GGTs responsible for the glycosylation of anthocyanidin 3-O-glycosides (Morita et al., 2005; Montefiori et al., 2011). PpUGT79B is not expressed in fruits, but its transcripts are abundant in leaves and flowers. Both flowers and leaves accumulate Cy-3-rut and Pn-3-rut, but they are almost undetectable in fruits. Therefore, there is no doubt that PpUGT79B is involved in the accumulation of Cy-3-rut and Pn-3-rut in peach. Moreover, transcripts of PpUGT79A are also found in the flowers of P. davidana, but its expression is not in accordance with the accumulation of Cy-3-rut and Pn-3-rut in red-colored tissues (Supplemental Fig. S3A). In contrast, the expression of PpUGT79B is well in accordance with the accumulation of Cy-3-rut and Pn-3-rut. Taken together, these results suggest that PpUGT79B is involved in the biosynthesis of Cy-3-rut and Pn-3-rut in peach. It is worth noting that functional redundancy is often observed for secondary metabolite genes in plants (Hanada et al., 2011). We cannot exclude the possibility that PpUGT79A also can catalyze the glycosylation of anthocyanins in P. davidana.
Two genes in the peach genome, PpAOMT1 and PpAOMT2, are closely related to VvAOMT, which has been shown to participate in the methylation of anthocyanins in grapevine (Hugueney et al., 2009). A significant correlation is observed between the expression level of PpAOMT2 and the accumulation of peonidin-based anthocyanins in red-colored tissues, but not for PpAOMT1 (Supplemental Fig. S3B). Moreover, PpAOMT2 has been shown to be functional in vitro and in vivo. These results strongly suggest that PpAOMT2 is responsible for anthocyanin methylation in peach. Besides PpAOMT2, PpAOMT1 also is expressed in flowers and leaves. However, the functionality of PpAOMT1 remains unclear, as it shows no activity toward cyanidin in vitro.
Taking all the results above, we propose a model underlying the structural diversification of anthocyanins in peach (Fig. 9).
Differential Expression Pattern of Genes Involved in Anthocyanin Modification in Plants
Tissue-specific expression profiles of regulatory genes involved in anthocyanin biosynthesis have been reported in previous studies. For example, four R2R3 MYB transcription factors (TFs), Anthocyanin2 (AN2), AN4, PURPLE HAZE (PHZ), and DEEP PURPLE (DPL), have been isolated in petunia. AN2 and AN4 are responsible for the coloration of floral tissues, including petal limb, flower tube, and anther (Quattrocchio et al., 1999), while PHZ and DPL mainly control anthocyanin accumulation in vegetative tissues such as leaves and stems (Albert et al., 2011). Similarly, two basic helix-loop-helix genes, B-I and B-Peru, confer anthocyanin pigmentation on vegetative tissues and seeds in maize, respectively (Selinger and Chandler, 1999). However, there are few reports of tissue-specific expression profiles of structural genes involved in anthocyanin modification.
Here, we characterize the expression profiles of gene families encoding F3GT, F3GGT, and AOMT in peach. Like the R2R3 MYB TFs in petunia and maize, two F3GT genes, PpUGT78A2 and PpUGT78B, also show tissue-specific expression. PpUGT78A2 is predominantly expressed in flowers, while PpUGT78B is mainly expressed in leaves and fruits. Similarly, two F3GGT genes, PpUGT79A and PpUGT79B, are not expressed in fruits but are expressed in leaves and/or flowers. Two AOMT genes, PpAOMT1 and PpAOMT2, are mainly expressed in flowers, but with no or low expression in fruits and leaves. Spatiotemporal control of the expression of genes involved in the methylation and glycosylation of anthocyanins results in the differences of anthocyanin composition in flower, leaf, and fruit tissues of peach. Moreover, UFGT is a key gene in anthocyanin biosynthesis, and its expression is induced by MYB TFs (Kobayashi et al., 2002). To date, seven R2R3 MYB TFs have been identified in the peach genome (Rahim et al., 2014; Uematsu et al., 2014). It is worth studying whether the expression of PpUGT78 genes is induced by specific MYB TFs, leading to the spatial and temporal patterns of anthocyanin synthesis in peach.
The UGT78II Genes Represent a Novel Branch of the UGT78 Gene Family in Plants
In this study, we have comprehensively investigated the evolutionary development of UGT78 genes in 20 sequenced plant genomes. In eudicots, UGT78 genes can be divided into two groups, UGT78I and UGT78II. It is well known that the Arabidopsis UGT intron 2 is highly conserved among the UGT78 gene family (Li et al., 2001; Paquette et al., 2003). Interestingly, a single intron equivalent to the Arabidopsis UGT intron 2 has been detected in the UGT78I group but is nearly absent in the UGT78II group. Only one gene, GmF3GT2-2, in the UGT78II group contains intron 2. Moreover, the PSPG motif of the UGT78I group contains a highly conserved amino acid residue, Trp (W), at position 22, while residue 22 has great variation in the UGT78II group. In monocots, the UGT78 genes, like the eudicot UGT78I genes, also contain intron 2 and the conserved amino acid residue Trp at position 22. This suggests that the UGT78I genes in eudicots and the UGT78 genes in monocots are derived from a common ancestor. However, the UGT78II genes can be well distinguished from other UGT78 genes in plants based on the combination of intron 2 and the amino acid residue at position 22. Thus, the UGT78II genes represent a novel branch of the UGT78 gene family in plants.
As mentioned above, four of the five characterized UGT78Ia genes show activity toward flavonols (Modolo et al., 2007; Ono et al., 2010; Fukuchi-Mizutani et al., 2011), while four of the six characterized UGT78Ib genes accept both anthocyanins and flavonols (Ford et al., 1998; Kubo et al., 2007; Griesser et al., 2008; Fukuchi-Mizutani et al., 2011). To date, two UGT78II genes, GmF3GT2-6 in soybean and PtF3GTII-2 in poplar, have been characterized to function as a 3-O-glycosyltransferase in anthocyanin and flavonol biosynthesis (Kovinich et al., 2010; Veljanovski and Constabel, 2013). This indicates that the UGT78II genes are more closely related to the UGT78Ib genes than to the UGT78Ia genes. Thus, it is reasonable to speculate that the common ancestor of UGT78I and UGT78II groups is likely to have activity toward anthocyanins and flavonols. The UGT78II group retains the ancestral function, while the UGT78I group may have undergone functional divergence, dividing genes into the two subgroups UGT78Ia and UGT78Ib. In addition, the UGT78II genes are only found in five of 10 plant species in eurosids I and are absent in the genomes of 10 plant species in eurosids II, vitales, and asterids. It is unclear if the UGT78II genes tend to be lost in eudicots.
The Difference of Anthocyanin Composition between Peach and Its Wild Relatives and Its Implications for Inferring Peach Domestication History
In this study, six anthocyanins are identified in flowers of peach and its wild relatives. Interestingly, peach and P. ferganensis show a similar anthocyanin composition in flowers. However, peach is different from both P. davidiana and P. kansueasis in terms of anthocyanin composition in flowers. These results clearly demonstrate that peach is closely related to P. ferganensis, native to Xinjiang Province of northwestern China and the Ferghana and Zeravshan valleys in central Asia (Scorza and Okie, 1990). Peach was first domesticated in the region of northwestern China between the Tarim basin and the north slopes of the Kunlun Shan Mountains (Faust and Timon, 1995). Thus, it seems likely that peach is domesticated from P. ferganensis. This finding is supported by the latest study of peach genome sequencing, which indicates that P. ferganensis represents an intermediate genome in peach domestication (Verde et al., 2013). In addition, all seven of the tested peach cultivars share a similar pattern of anthocyanin composition in flowers, which is consistent with the fact that peach cultivars have a very narrow genetic base (Scorza et al., 1985).
The peach blossom has always been a sacred symbol in Chinese culture. Peach and P. ferganensis produce red or purple flowers, while both P. davidiana and P. kansueasis have pink or pale pink flowers. In other words, the flowers of peach and P. ferganensis are more attractive than those of P. davidiana and P. kansueasis. If peach is indeed domesticated from P. ferganensis, then the question arises of the purpose of the original domestication of peach. Both P. davidiana and P. kansueasis bear edible fruits, and P. kansueasis is also native to Xinjiang Province of northwestern China. The original cultivation of peach might not have been very important as a source of fruits but could be important as a source of valuable ornamental plants due to the beauty of flowers. Moreover, our study shows that the flowers of peach and P. ferganensis are richer in anthocyanin components than those of P. davidiana and P. kansueasis. Thus, the complexity of anthocyanin composition is likely related to the diversity of flower color in peach and its wild relatives.
Peach flowers are a common ingredient used in traditional Chinese medicine. Here, we show that peach flowers accumulate a variety of anthocyanins, whereas the fruits and leaves predominantly accumulate a single anthocyanin, Cy-3-glu. Several studies have demonstrated that both peonidin-based and cyanidin-based anthocyanins can inhibit cancer cell growth (Chen et al., 2005; Ho et al., 2010). Therefore, peach flowers are a more suitable ingredient to use in medicine than leaves and fruits in terms of the anthocyanin benefits.
Methylation and glycosylation contribute to the diversification of anthocyanins in peach. Peach and P. ferganensis share a similar anthocyanin composition in flowers, suggesting that they are closely related to each other. Four UGT genes, PpUGT78A1, PpUGT78A2, PpUGT78B, and PpUGT79B, are likely responsible for the glycosylation of anthocyanins, while one gene, PpAOMT2, is involved in the methylation of anthocyanins in peach. The spatiotemporal control of expression of these anthocyanin modification genes is responsible for the different patterns of anthocyanin accumulation in flower, leaf, and fruit tissues. In addition, our study reveals the divergent evolution of both UGT78 and AOMT genes in peach.
Plant Materials
Peach (Prunus persica) accessions used in this study consisted of seven ornamental cultivars, Juhuatao, Fenhuashouxintao, Hongcuizhi, Honghuashouxintao, MT, Hongbaihuatao, and HY; two other cultivars, red-fleshed WH9 and white-fleshed BF; and three wild relatives, Prunus davidiana, Prunus ferganensis, and Prunus kansueasis. All the peach accessions are maintained at the Wuhan Botanical Garden of the Chinese Academy of Sciences in Hubei Province. Leaves were collected during juvenile stage in spring. Fruits were harvested at mature stage. Flowers were collected at pink, balloon, and blossom stages. All samples were immediately frozen in liquid nitrogen and then stored at −75°C until use.
Extraction and HPLC-ESI-MS/MS Analysis of Anthocyanins
The anthocyanins were extracted according to a previously reported method (Huang et al., 2009). Briefly, an approximately 0.5-g sample was ground in liquid nitrogen and then added to 25 mL of extraction solution (80:20 [v/v] methanol:water mixture containing 1.18 mm HCl). The mixture was incubated on a steam bath in the dark at 30°C with gentle shaking for 16 h, sonicated for 15 min, and centrifuged at 10,000g for 10 min. An aliquot of 10 mL of supernatant was collected and evaporated under vacuum at 30°C using a rotary evaporator. The residual was resuspended in acidified water (1.18 mm HCl) and added to a CNWBOND LC-C18 SPE column that was preconditioned with 5 mL of methanol followed by 5 mL of acidified water. The SPE column was then washed three times with acidified water.
Anthocyanins were eluted with 1 mL of methanol, filtered through 0.22-μm Millipore membranes, and analyzed using an HPLC-ESI-MS/MS system (ThermoFisher Scientific). The analytical column was Zorbax Extend C18, 4.6 × 250 mm, with a particle size of 5 μm (Agilent Technologies). The analytical column was sequentially eluted using mobile phase A (formic acid:water, 5:95, v/v) and mobile phase B (methanol) with a flow rate of 0.8 mL min−1. The linear gradient of phase B was as follows: 0 to 2 min, 5%; 2 to 7 min, 5% to 15%; 7 to 20 min, 15% to 20%; 20 to 25 min, 20% to 27%; 25 to 32 min, 27%; 32 to 41 min, 27% to 35%; and 41.01 to 43 min, 5%. A UV-visible light detector wavelength was set at 520 nm. Mass spectra were acquired in positive ion mode. Ion was scanned from 200 to 1,000 mass-to-charge ratio at a speed of 1,000 atomic mass units s−1. The flow rate of nebulizing gas was 1.5 L min−1, and the drying gas (N2) pressure was 0.1 MPa. The components of anthocyanins were determined according to the retention time of standards and Mr and subsequently quantified according to cyanidin equivalents.
Hydrolyzation of Anthocyanins
Anthocyanins were hydrolyzed into anthocyanidins under acidic and high-temperature conditions according to a previously reported procedure (Pinho et al., 2011). Approximately 1 g of ground sample was mixed with 10 mL of 6 m HCl and 40 mL of methanol. The mixture was flushed with nitrogen and incubated in a water bath at 100°C for 2 h. After acid hydrolysis, the mixture was cooled, mixed with 50 mL of methanol, sonicated for 15 min, and centrifuged at 10,000g for 10 min. The supernatant was purified using the SPE column as mentioned above. The components of anthocyanidins were analyzed using the HPLC-ESI-MS system. The mobile phase was composed of 3:57:40 (v/v/v) formic acid:water:methanol solution. The column flow rate was 0.8 mL min−1. The sample running time was 35 min. Anthocyanidins were recalled according to MS data, retention time, and Mr of internal standards.
Expression of the Peach AOMT and UGT78 Genes in Escherichia coli
The full complementary DNA (cDNA) sequences of PpAOMT1 and PpAOMT2 and were amplified using two pairs of primers (the XbaI and Xhol sites at the 5′ end of the forward and reverse primers, respectively, are indicated in bold), 5′-GGGAATTCCATATGATGGCAGAAGAAGGCAAACA-3′/5′-AGCCGCTCGAGCCACATTATTTGAAGACAACAGC-3′ and 5′-GGGAATTCCATATGTTGGCTCTTCTGTTCAACATGG-3′/5′-AGCCGCTCGAGCTAGTATCCTAATAGAGACGCCTGC-3′, respectively, and cloned individually into pET-28b(+) vector (Novagen). Clone authenticity was confirmed by sequencing. The constructed vector was then transferred into E. coli BL21 (DE3) cells (Transgen). The transformed cells were cultured at 37°C in Luria-Bertani medium containing 25 μg mL−1 kanamycin to an optical density at 600 nm of 0.4 to 0.6. The culture was adjusted to 18°C, and recombinant AOMT expression was induced by the addition of isopropylthio-β-galactoside to a final concentration of 5 mm. The culture was incubated for 20 h at 18°C (250 rpm) prior to harvesting the cells. Then, recombinant AOMT was purified using His•Bind Resin (Novagen). The activity of purified recombinant AOMT (5 μg) was measured in a final volume of 200 μL containing 200 μm S-adenosyl-l-methionine, 200 μm anthocyanin substrate, 0.1 m HEPES, pH 7.5, 10 mm MgCl2, and 14 mm 2-mercaptoethanol. The reaction was incubated for 30 min at 35°C and stopped with 400 μL of methanol containing 200 mm HCl. All reactions were filtered through 0.22-μm Millipore membranes and analyzed by HPLC-ESI-MS/MS. Assays were performed in triplicate, and experiments were repeated three times. Likewise, the full coding sequences of three genes, PpUGT78A1, PpUGT78A2, and PpUGT78B, were cloned into pET-28b(+) vector using three pairs of primers, 5′-GGGGATCCATGGTAGGAAACCCCAAACCC-3′/5′-GCCTCGAGCGTTTTGGACCTAGATCTAAGAAGC-3′, 5′-GGGAATTCATGGCAGGAAACCCCAAACA-3′/5′-AGCTCGAGCCTTTTTCACTGCCCATTCA-3′, and 5′-CCGAATTCATGGCACCACAACCGATT-3′/5′-GCCTCGAGGCGAGATAAATAGTAGAAGCAAAGC-3′, respectively. The activity of recombinant UGT78 was measured in a final volume of 200 μL containing 5 mm UDP-Glc or UDP-Gal, 250 mm substrate, 0.1 m HEPES, pH 7, 14 mm 2-mercaptoethanol, and raw protein extract with approximately 1 μg of recombinant protein. Enzyme assays were incubated for 30 min at 30°C and stopped by the addition of 250 μL of 0.6 m HCl.
Transient Expression in Tobacco
For Agrobacterium tumefaciens-mediated transient expression, PpAOMT2 from peach and NtAN2 from tobacco (Nicotiana tabacum) were amplified using two pairs of primers, 5′-GGGAATTCCATATGATGGCAGAAGAAGGCAAACA-3′/5′-AGCCGCTCGAGCCACATTATTTGAAGACAACAGC-3′ and 5′-CCGCTCGAGATGAATATTTGTACTAATAAG-3′/5′-GCTCTAGATCAACTGAGAAGTGGCATTTC-3′, respectively, and inserted individually into the modified binary vector pSAK277. All constructs were introduced into A. tumefaciens strain GV3101 by electroporation. Nicotiana benthamiana leaves were infiltrated with A. tumefaciens cultures (optical density at 600 nm of 0.1–0.3). Discs were punched from N. benthamiana leaves 1 week after A. tumefaciens infiltration and analyzed for anthocyanin content.
Gene Expression Analysis Using Real-Time PCR
Total RNA was extracted using the ZP401 kit (Beijing Zoman Biotechnology) according to the manufacturer's instructions and was treated with DNase I (Takara) to remove any contamination of genomic DNAs. Approximately 3 μg of total RNA per sample was then used for cDNA synthesis. A SYBR Green-based real-time PCR assay was carried out in a total volume of 20 µL of reaction mixture containing 10 µL of 2× SYBR Green I Master Mix (Takara), 0.2 µm of each primer, and 100 ng of template cDNA. An actin gene encoding glyceraldehyde-3-phosphate dehydrogenase, PpGAPDH, in peach was used as a constitutive control.
Amplifications were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). The amplification program consisted of one cycle of 95°C for 3 min followed by 40 cycles of 95°C for 30 s and 60°C for 34 s. The fluorescent product was detected at the second step of each cycle. Melting curve analysis was performed at the end of 40 cycles to ensure the proper amplification of target fragments. Fluorescence readings were consecutively collected during the melting process from 60°C to 90°C at the heating rate of 0.5°C s−1. All analyses were repeated three times using biological replicates. Primer sequences used for real-time PCR are listed in Supplemental Table S2.
Sequence Alignment and Phylogenetic Analysis
The amino acid sequences of genes encoding UGT and AOMT from various plants were used for phylogenetic analysis. Sequences were aligned using ClustalX and adjusted manually as necessary. The resulting data were analyzed using equally weighted neighbor joining. Neighbor-joining trees were sought using the heuristic search strategies of MEGA version 5. Bootstrap values (Felsenstein, 1985) were calculated from 1,000 replicate analyses, and only those values compatible with the 50% majority-rule consensus tree were recorded. A UGT gene of Picea sitchensis (GenBank accession no. ABK24936) was used as an outgroup.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Phylogenetic tree derived from amino acid sequences of genes encoding UGT in the peach transcriptome.
Supplemental Figure S2. Estimation of gene expression levels of F3GT, F3GGT, and AOMT homologs using fragments per kilobases per million reads values in peach.
Supplemental Figure S3. Correlation between the expression of the anthocyanin modification genes and anthocyanin accumulation in different tissues of peach.
Supplemental Table S1. Genes encoding UDP identified in the peach transcriptome.
Supplemental Table S2. Primers used for real-time PCR analysis of genes encoding UGT78, UGT79, UGT94, and OMT in peach.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Yuepeng Han (yphan{at}wbgcas.cn).
↵1 This work was supported by the National Program on Key Basic Research Projects of China (973 Program; grant no. 2011CB100600) and the National 863 Program of China (grant no. 2011AA10020600–02).
↵[C] Some figures in this article are displayed in color online but in black and white in the print edition.
↵[W] The online version of this article contains Web-only data.
- plant secondary product glycosyltransferase
- RNA-Seq
- RNA sequencing
- mass spectrometry
- tandem mass spectrometry
- electrospray ionization
- Cy-3-glu
- cyanidin 3-glucoside
- Cy-3-gal
- cyanidin 3-galactoside
- Cy-3-rut
- cyanidin 3-rutinoside
- Pn-3-glu
- peonidin 3-glucoside
- Cy-3-rha
- cyanidin 3-rhamnoside
- Pn-3-rut
- peonidin 3-rutinoside
- coding sequence
- Mantianhong
- Hongyetao
- Baifeng
- TFs
- transcription factors
- cDNA
- complementary DNA
- Received July 15, 2014.
- Accepted August 7, 2014.
- Published August 8, 2014.


text:
BJP MP and MLA fight video goes viral- Social Media says "Mera Boot Sabse Mazboot"
BJP MP from Sharad Tripathi from Sant Kabir Nagar in Uttar Pradesh and BJP's own MLA Rakesh Baghel who represents an area under the district today fought with shoes and fist in a public meeting. Hurling each other with abusive remarks, both quickly started puching each other in the public meeting which was being convened by top officials. The fight apparantly broke out when BJP MP got angry on his name not being included in a foundation stone for a road. The MLA said it was his decision.
The video is going viral on internet and people all over the social media are both showing their concern about such law makers and also making fun of them. One user added a remark saying: "Mera Boot Sabse Mazboot".
संत कबीर नगर से भाजपा विधायक और भाजपा सांसद ने एक दूजे पर किया सर्जिकल स्ट्राइक 🤣🤣🤣दे जूता दे चप्पल ।।मोदी है तो मुमकिन है ।।
Người đăng: Achhe Din Memes vào Thứ Tư, 6 tháng 3, 2019


text:
Statewide COVID-19 case count drops dramaticallyBy FLOYD INGRAM / THE PRESS REGISTER,
- 924 reads
The number of new COVID-19 cases in the state plummeted Monday with 357 cases reported today and only three new deaths.
But the virus is still out there and Coahoma County has reported 11 new cases over the past 11 days as the state and nation eye a possible spike in coronavirus cases and the community, state and nation try to move forward, get out from under lockdown and go back to work.
Coahoma County reported its sixth death on June 21 and 201 coronavirus cases have been reported locally since the county reported its first case of COVID-19 on March 18.
Many of those reported sick have now gotten well. The Center for Disease Control said the typical case of COVID-19 can last up to two weeks, with those suffering severe cases being ill up to three or four weeks.
Of the 31,257 cases that have been reported in the state since March 14, the Mississippi State Department of Health has reported 22,167 have gotten well. That means 70.9 percent of the people who have gotten sick have recovered.
Coahoma County has a population of 22,628 and an infection rate of 0.88-percent.
Again, while the fatality rate appears to be dropping, the disease continues to be a threat for nursing homes, the elderly and those with compromised immune systems.
As of today, there have been 2,759 cases of COVID-19 in nursing home with 542 deaths reported. That represents 8.8-percent of the state's total cases and 48.6-percent of the state's total deaths to the disease.
With massive testing ongoing across the state, Mississippi reported 870 new cases on Friday and has routinely reported above 500 cases since early June.
The Coahoma County Board of Supervisors considered making all county employees wear masks and require masks be worn by anyone entering a county building.
"Our situation is a little different in that we can't require people in the country to wear masks and it probably wouldn't do any good," Board President Johnny Newson said Monday. "I want to look at some more numbers before we do anything."
Dist. 3 Supervisor Derrell Washington said he wanted to know accurate and up-to-date percentages and now how many people had gotten sick since March.
"We've been talking about educating people since March and I go to Wal-Mart and still see people without masks," said Washington. "How are you going to get people to wear a mask unless you say, "Please wear and mask and you can go to jail if you don't."
The wearing of masks has become a politically charged issue with Republican President Donald Trump hesitant to don a mask and Democratic candidate Joe Biden sheltering in place and wearing a mask when he goes out in public.
There is no firm data saying a mask prevents a person from getting the disease.
As communities have opened up people simply don a mask and head out to shop, go to church or attend public events.
The Numbers
With a population of 2.987 million, the MSDH says 1.04-percent of Mississippians have now contracted COVID-19.
The number of new cases being reported locally also indicates many – especially young people -- may have the disease and just don't know it.
Demographics from across the state and country also show the disease is more prevalent and under reported – for a variety of reasons – in minority communities.
As of Friday, 52-percent of the fatalities have been African American, 41.2-percent have been white and 6.8-percent have been others. The total cases by race have been 58-percent African American, 31.5-percent white and 10.5-percent other races.
Health officials have warned of spikes in the case count as the state reopens businesses, restaurants, movie theaters and gym.
Gov. Tate Reeves has said he will clamp back down on the economy if he sees a resurgence of the disease. Reeves.
The statistics continue to confirm data that mid-sized communities with lower populations but adequate healthcare are handling their fight against COVID-19 better than others. It should be pointed out all cities and towns have unique factors affecting their numbers.
Gov. Tate Reeves has said not all areas of the state have reported high numbers and local officials should be willing to make their own decisions.
Across the nation 2,886,267 people or .87-percent of the nation's 328.2 million people have contracted the disease and 129,811, or .49-percent, have died
County by County
The Mississippi State Department of Health and the University of Mississippi Medical Center have brought in mobile coronavirus testing unit to Coahoma County on three separate occasions and the Aaron E. Henry Center has also conducted testing, including its most recent in Bobo on Thursday.
The results of those tests are prompting the community's infection rate to climb.
Cases reported as of today in the following Mississippi counties were: Adams (313), Alcorn (71), Amite (91), Attala (361) Benton (38), Bolivar (340), Calhoun (132), Carroll (166), Chickasaw (279), Choctaw (74), Claiborne (279), Clarke (207), Clay (251), Coahoma (201), Copiah (607), Covington (359), Desoto (1,620), Forrest (878), Franklin (45), George (89), Greene (105), Grenada (490), Hancock (130), Harrison (933), Hinds (2,536), Holmes (557), Humphreys (150), Issaquena (9), Itawamba (136), Jackson (638), Jasper (263), Jefferson (108), Jeff Davis (112), Jones (1,128), Kemper (179), Lafayette (393), Lamar (465), Lauderdale (910), Lawrence (178), Leake (578), Lee (552), Leflore (491), Lincoln (466), Lowndes (494), Madison (1,314), Marion (284), Marshall (234), Monroe (403), Montgomery (135), Neshoba (992), Newton (339), Noxubee (267), Oktibbeha (541), Panola (352), Pearl River (257), Perry (67), Pike (441), Pontotoc (292), Prentiss (108), Quitman (78), Rankin (951), Scott (773), Sharkey (38), Simpson (323), Smith (218), Stone (65), Sunflower (400), Tallahatchie (137), Tate (288), Tippah (131), Tishomingo (83), Tunica (105), Union (210), Walthall (204), Warren (516), Washington (591), Wayne (545), Webster (127), Wilkinson (94), Winston (266), Yalobusha (173) and Yazoo (518).
County case numbers change as investigations find new information on a case's home county.
The state announced its first death due to coronavirus March 20. Fatalities attributed to the disease have been reported in Adams (18), Alcorn (2), Amite (2), Attala (23), Bolivar (14), Calhoun (4), Carroll (11), Chickasaw (18), Choctaw (4), Claiborne (10), Clarke (24), Clay (11), Coahoma (6), Copiah (15), Covington (5), Desoto (16), Forrest (42), Franklin (2), George (3), Greene (8), Grenada (5), Hancock (13), Harrison (11), Hinds (40), Holmes (41), Humphreys (9), Issaquena (1), Itawamba (8), Jackson (16), Jasper (6), Jefferson (3), Jeff Davis (4), Jones (49), Kemper (14), Lafayette (4), Lamar (7), Lauderdale (79), Lawrence (2), Leake (19), Lee (19), Leflore (51), Lincoln (34), Lowndes (13), Madison (34), Marion (11), Marshall (3), Monroe (30), Montgomery (2), Neshoba (71), Newton (9), Noxubee (8), Oktibbeha (26), Panola (6), Pearl River (32), Perry (4), Pike (12), Pontotoc (6), Prentiss (3), Rankin (12), Scott (15), Simpson (3), Smith (11), Stone (1), Sunflower (8), Tallahatchie (4), Tate (10), Tippah (11), Tishomingo (1), Tunica (3), Union (9), Walthall (5), Warren (18), Washington (9), Wayne (13), Webster (10), Wilkinson (9), Winston (6), Yalobusha (7) and Yazoo (6) counties as of today.
The virus appears to be affecting the state's higher population areas such as the Gulf Coast, Jackson, Meridian metropolitan areas, Desoto County and Lee County more intensely. Communities that have had people travel to or from larger U.S. cities also appear to have a higher incident of the disease.
EDITOR'S NOTE: Your Clarksdale Press Register will keep readers posted as we learn more information and as news changes. All stories about coronavirus are being offered free to the community and will not be put behind the Clarksdale Press Register paywall.


text:
From CleanPosts
When the dragon Demonstick fell from the sky and in his death throes belly-flopped upon the river Sabik, and the last embers of hope for House Gerash had been thoroughly extinguished, Baron Bayard turned to Hovan the son of Kirodiel. And he said, "Now carry out the terms of the death combat and release my mother Queen Aurra from her captivity, and get your ships hence, for neither shall you bring Lady Talishi into a cage once again."
Then Hovan nodded his head once, and bowed, and Lord Zadkiel cut the ropes that secured Queen Aurra in her confinement.
With as much dignity as she could retrieve, she emerged from the cage and moved to within a few paces of her son, but her gratitude and pride in Bayard could not be contained, and he saw that.
Then from the air Victoria alighted on the gopherwood deck of the queen's barge and in the same movement swept into the arms of Baron Bayard Sala, overjoyed that he had prevailed in his contest with Lord Kirodiel. Gently, gently, the baron embraced Victoria, and though he winced with the pain of his wound as she squeezed him back, the fact that she did squeeze him back was a very encouraging sign. He asked her, "Victoria, is it possible for mere human beings to fall in love with one the B'nei Elohim?"
"I see no reason why not," she replied. "As I've told you before, we B'nei Elohim are mere human beings ourselves, with a few fancy tricks up our sleeves grounded in what you would call alchemy and natural philosophy."
"I am very relieved to be reminded of that," he told her. "Thank you! For now I can say with utmost confidence that I have fallen irretrievably in love with the B'nei Eloah named Victoria."
"Then there must remain only one more thing for you to say, Bayard, so let me hear it. Come on, you're among friends. Lay it out there."
"Victoria, will you consent to become my wife?"
"The thing seems to be written in the living stars," she said with no trace of hesitation. "But let me ask a question of you, Bayard, and when I have your answer, then you will have mine."
"Proceed with your question, Victoria, but beware, I cannot foresee any that would deter me from what my heart has already resolved to do."
Victoria caught the eye of the queen. "Is it safe to say, Your Highness, that with the whole House of Sala now moving at your command from their cities to many thousands of scattered faith assemblies, that there is no longer, in a real sense, any kingdom for you to rule, or for Baron Bayard to inherit?"
"Alas, Victoria" answered the queen, "that is true, and were you to marry my son and had your heart set on the title of Baroness, I'm afraid it would be little more than a figment, just an empty title. Our society is being profoundly transformed even as we speak. The irony of the situation is not lost on me, of course. All his life I have sharply rebuked my son for his taste in commoner women. I was trying to steer him to wed one of the Highborn, and now at the very end I find that the very word Highborn is a sound without meaning."
"Thank you, Queen Aurra," said Victoria. "And for my part let me say that my ability to fly, which is the biggest thing that sets me apart from humanity as one of the B'nei Elohim, is not a power that can be sustained here on Barbelo. Soon I must lay this power down again, and if I stay in this world I will be little more than a commoner woman, something that might interest the Baron of course, but I had thought you might have an intractable objection to that."
"Victoria, you are far too modest! You have killed the dragon called Demonstroke and brought an end to the world war on Barbelo. Such a deed, which will be memorialized in songs for ages to come, makes you entirely uncommon."
"Thank you once again, Your Highness Queen Aurra. And so I am led to ask my question of the man who has asked me to become his wife. Bayard, would you be willing to quit this world forever, and dwell with me on Earth?"
"Victoria I would eagerly follow you right out of this world if you consent to marry me."
"I do consent to marry you Bayard! I warn you that it will be absolutely bewildering to you at first, and you will find yourself to be what we frequently call a 'fish out of water'. For we do not merely live on the surface of the Earth, but in the skies above and in the ground below."
"I am full willing, Victoria, if the queen will give her blessing." And he turned to face his mother, who rolled her eyes and nodded her head yes. Then the Baron, filled with happiness beyond his capacity to bear, fell to the deck of the queen's barge in a dead faint.
"I have done everything I can," Talishi said after she had tended to Bayard and restored him to consciousness. "He has lost very much blood and I marvel that he slew Kirodiel. Do not permit him to stand on his feet until we have taken him downriver to Yeshua, who can provide a temporary blood substitute."
"Lady Talishi, there remains a slight problem with carrying out that order," the Fallen Angel Ambe Omphal said, pointing to Hovan Gerash and Lord Zadkiel and many of their officers who remained standing on the deck of the barge all around them.
Queen Aurra stood forth to confront them. She said, "Your dragon is dead." And the men muttered aye for the queen spoke a truth.
The queen caught the eye of Hovan just then. "You have killed my husband." And Hovan's eyes fell, and he nodded in assent and said aye.
She said, "You are in the middle of Haaretz with a greatly diminished force, and the long road home will be much longer if you must fight your way there." Then she caught the gaze of Lord Zadkiel. "The war is over, do you not agree?"
"I agree, your Highness."
"Then get yourself and your men to another one of my boats and depart this land!"
When evening fell and the queen's barge had reached the place where the rivers Sabik and Arhena became one, Talishi climbed, somehow, to the top of the highest mast where few would dare to follow her, and there she meditated on many things. Victoria, returning to the ship from a night flight she had taken for the sheer pleasure of it, saw Talishi sitting in that precarious place and asked to join her.
"By all means, Victoria, please make yourself as comfortable as you can."
Then Victoria, knowing that no one could overhear, said, "Aliwe told you, didn't she? That you and Bat-El found a way to win."
"Aliwe told me things that would have been perfectly safe for our companions to overhear on the way to the Catwalk," Talishi replied. "But I got the detailed update directly from her in the way that you and the other B'nei Elohim already know so well. Yes, we found a way to beat Mastema, both my daughter and I."
"Can you tell me about it?"
"It's a very curious thing, Victoria. It is like I have a splinter of wood that bears a tiny flame, and I must walk that small flame to a great pile of wood and kindle it to keep my loved ones from freezing to death, yet it is windy, and I dare not do the slightest thing to let the flame go out."
"I think I understand what you mean. Even after Aliwe assured me I would defeat Demonstroke, I did not relax my guard for a single instant. It still required the utmost effort."
"That is precisely what I'm trying to convey to you."
"Lady Talishi, will I meet my daughter Aliwe again?"
"You will, after that big bonfire really gets going. But if you and Bayard have a child, even a girl child, she will not be the same Aliwe. Not even if you name her that."
"Aliwe already said as much."
"I will go with you and Bayard to Earth," Talishi revealed. "It's very strange, you know. My real body is the sun that warms the Earth and drives all life and movement there, but in this body, as Talishi, I have never been there. But I must go that there may no longer be two separate locii for the El Shaddai identity."
"And will you return to Barbelo someday?"
"Just once, as an important part of that successful way Aliwe spoke of. I must save the planet from a third deluge. As Talishi I will not survive it. But Aliwe tells me the other El Shaddai, the one I must soon merge with once more, is already making arrangements to land in another body."
"Please don't make that into a habit, Lady Talishi. "We have seen what serial possession has done to Mastema."
"I will not, and Aliwe told me that I'm making arrangements to deal with that problem as well. But I knew long ago there would be no real joy in bringing Mastema to his doom. Imagine contriving the total defeat of your bitter lifelong enemy only to discover the man was in the advanced stages of dementia."


text:
Darien Library is offering a five-week coding course for adults, meeting on Wednesdays from 6:30 to 8:30 p.m., starting this coming Wednesday, Sept. 4.
Registration is now open. As of Wednesday morning, there were 10 spaces left in the course, half of the total available. The class continues through Wednesday, Oct.2.
Here's the library's description of the course:
Calling all moms, entrepreneurs, college students, or anyone interested in learning the basics of computer coding!
We will explore programming in Python. Think of it as like a new language: in addition to learning syntax, and writing scripts, we will work on developing computational thinking-breaking down big problems into simple individual tasks.
The best news? No experience is necessary! Students are required to bring a laptop for use during this class.
Looking to learn Python on your own? We've got you covered.
About the Instructor
Varghese John, Ph.D., is an experienced technologist with a scientific research background and a love for learning and teaching science, technology and mathematics.
He has led large global teams delivering innovative solutions to complex problems for leading financial service organizations.
VJ is passionate about delivering STEM education for children and adults in order to build a community of citizens who can apply logic and scientific thinking to address global challenges.


text:
2nd Lt Wendel R. Weatherly flew 5 missions, from 04/09/1945, mission #245, to 04/19/1945, mission #253. He served as a Copilot.
2nd Lt Weatherly served with the following crewman: Flt O Bernard Goldberg, Sgt Calvin S. Hallman, Sgt Henry H. Hill, Sgt Max E. Kirby, 2nd Lt Salvatore P. Paladino, Sgt Leonard V. Penny, 2nd Lt Carl W. Short and Sgt H.B. Tomkin


text:
The Bombers have landed in Bendigo for their 2020 AFL community camp, heading straight to Queen Elizabeth Oval for open training in the scorching heat on Thursday.
Training in front of hundreds of Essendon supporters, the players completed skills drills, high-intensity running and match simulation, with their Marsh Community Series opener against West Coast just two weeks away.
(View photos from the session below - click here to view on app)
The Bombers will spend the night in Bendigo, visiting schools and hospitals and running school clinics on Friday as part of the two-day camp.


text:
Before becoming residents at Judson Services, a continuing care retirement community based in Cleveland, many of its seniors were active fans of local theater. But as they've aged, it's become harder and more inconvenient for them to attend shows.
"So, we decided to bring theatre to them," Mark Corcoran, managing director of Theatre in the Circle at Judson Services, told McKnight's.
Theatre in the Circle is the resident theatre company of Judson Manor's retirement community and is run by Corcoran and his husband, Bill, who serves as the artistic director. Since being founded in 2016, Corcoran said residents and the community have supported the theatre "tremendously." Shows feature local actors. The theater is supported by the Judson Foundation and has received grants, gifts and donations from local groups and residents. The theater produces four shows each season,
"Theatre in the Circle is a tremendous asset for those of us who live at Judson Manor," resident Josephine Steinhurst said. "I simply take the elevator down and enjoy the performances."
Resident Alice said it's unique to see theater "in your home."
"I love to attend the dress rehearsal and the final performance to see how the actors have grown in their parts, and to compare my impressions with fellow theatergoers. It leads to some spirited conversations."
From the October 2019 Issue of McKnight's Long Term Care News


text:
Book Direct with the Owner at Jelcobine Australia Vacation Carnarvon Accommodation Agent in Jelcobine your location, for all your holiday and travel needs.
Address: 99 Robinson Road, Brookton WA 6306
Address: 99 Robinson, Brookton WA 6306
Address: 24 Williams, Brookton WA 6306
copyright © 2020 Carnarvon Accommodation. All rights reserved.