text:
Does Workers’ Compensation Cover Emotional Distress in Virginia?
Some work-related injuries can cause a lifetime of physical, mental, and emotional distress to employees and their loved ones.
Workers’ compensation may cover both physical and mental pain and suffering if it can be proven that this is job-related. However, emotional distress can be difficult to prove. Virginia workers compensation law generally doesn’t provide monetary compensation for mental pain and suffering, emotional distress, or psychological injuries.
If you’re struggling with this, a workers’ compensation lawyer can provide you with invaluable advice on the topic.
What Are Psychological Injuries?
When a worker suffers from a physical injury, it may also cause lasting emotional trauma. In certain cases, the worker may feel some worries and fears or emotional distress due to the incident. This may be even more aggravated if the worker expects to be out of work for an extended length of time or worries that they may not be able to pay their bills and support their family.
Some of the common signs of psychological distress are the following:
- Inability to connect with other people
- Depression or anxiety
- Mood swings, anger, or irritability
- Confusion or lack of focus
- Feelings of hopelessness
- Post-traumatic stress disorder (PTSD)
These signs may last for days, or even months, after the accident. Psychological injuries need to be treated right away so the worker can start feeling better.
Can I Get Workers Compensation for Stress?
In general, mental stress does not entitle the worker to compensation under the current system. This is true even if the impairment has led the worker to lose time from work.
Under Virginia Workers’ Compensation law, injuries from an accident or a work-related disease are compensable, but mental stress is considered neither an accident or an occupational disease. The Workers’ Compensation board has claimed that problems at work related to management and other personnel are present in all types of employment.
However, there are also exceptions to this rule, including:
- If the worker can prove that intense stress is characteristic of their employment, as in the case of emergency dispatchers.
- The stress originates from a single incident at work. An example would be a rescue worker who has developed post-traumatic stress disorder after exposure to a horrific accident.
- The worker sustains an injury or develops a disease—that is compensable under the law—which aggravates or results in stress.
How Do I Prove My Stress Claim?
A worker who claims that they’re suffering from stress, anxiety, or other psychological injuries has the burden of proving that such injury was work-related, and not caused by an external source or is pre-existing.
Some may be able to link a traumatic event to their current mental distress. For others, a direct connection may not exist. This is why it’s crucial for workers seeking compensation for work-related psychological injuries to work with mental health professionals who can help them establish a connection between their condition and their job.
To be compensable, psychological injuries must meet the following requirements:
- The conditions at the workplace are objectively stressful
- There’s believable evidence that the worker found the conditions stressful
- The stressful conditions must not be present in most other types of workplaces or roles
- There’s objective evidence establishing and supporting the worker’s claim of psychological injury
Can I Sue My Employer for Stress and Anxiety?
In rare instances, employees have sued their employer for emotional distress or mental anguish if they experience stress and anxiety that is higher than the usual amount for their job. However, this can be difficult to prove in court.
Find a Workers’ Comp Lawyer You Can Trust
You should work in a comfortable and safe environment. If your working conditions are causing unnecessary stress, trauma, and/or psychological injuries, get in touch with a qualified workers’ compensation lawyer at Slominski Law.
We can help you assemble the necessary evidence and build a strong case that increases your odds of getting compensated.
Call us at (434) 384-9400 to reach our Lynchburg Office or at (540) 554-3762 for our Roanoke Office, or book a consultation online.

domain: psychology


The cloud computing system is available in three key forms, public, private, and hybrid clouds. Among the three of them, public cloud servers are renowned to be the most popularly used variant of the standard cloud computing model. In case of the public cloud system, the relevant service providers offer an extensive range of applications storage and resources to their discerning clients over the web. All the hardware resources available in case of such a cloud are shared among diverse users, and are generally accessible over a common network.
In the modern digital economy, availing the services of public cloud servers can significantly aid organizations to put their full focus on their core business, and enables them to conduct their everyday activities without concerning themselves with any type of IT issues. Here are some of the key advantages of seeking out the services of public cloud:
- Affordable: Prominent cloud computing service providers tend to have quite a flexible and affordable price structure. These service providers usually provide their clients with the advantage of paying only for the extent of the services used by them. As a result, such public cloud servers come as a huge advantage for small or medium scale organizations that run on a limited budget. These services go a long way in enabling organizations to run their web applications quite smoothly without having to commit to any type of expenses related to their servers, software, setup, and maintenance.
- Quick set up: Public cloud services can easily be bought online, and then deployed and configured remotely through the website of the relevant service provider. This whole process typically takes only a few hours. In addition to this, the internal IT team of a company is generally able to manage the public cloud setup with just a stable internet connection.
- Reduces the staffing cost: Hiring qualified and experienced IT engineers can prove to be quite expensive for an organization. Companies can easily avoid this high expense by seeking out the services of cloud computing from a renowned service provider. As in this scenario the service provider would be responsible for managing the diverse aspects of the public cloud, companies would not be required to hire any specialized IT staff.
To know more about the advantages offered by public cloud servers, people can easily visit the website of some of the renowned companies offering the services of cloud hosting.Top Searches - Trending Searches - New Articles - Top Articles - Trending Articles - Featured Articles - Top Members
Copyright © 2020 Uberant.com700,134 total articles and counting.


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CENTEREACH COVER BANDS
Cover Bands are a fun alternative to traditional musical entertainment. You can use GigMasters to hire a Cover Band to play all your favorite hits at your event. Check out who’s available in the Centereach, NY area now!
Please note these Cover Bands
will also travel to Lake Grove, Selden, Port Jefferson Station, Farmingville, Stony Brook, Nesconset, Saint James, Coram, East Setauket, Port Jefferson, Holtsville, Mount Sinai, Ronkonkoma, Holbrook, Middle Island, Medford, Miller Place, Central Islip, Hauppauge, Bohemia, Islandia, Smithtown, Sayville, Sound Beach, Yaphank, Bayport, Blue Point, Kings Park, Oakdale, Islip Terrace

domain: artistic


text:
Risk sharing in procurement
André de Palma () and
International Journal of Industrial Organization, 2019, vol. 65, issue C, 173-220
We introduce bilateral risk aversion into the mixed adverse selection - moral hazard model of Laffont and Tirole (1986). The presence of exogenous risk interacts with the adverse selection problem in interesting ways. In particular, we show that it is never optimal to present the firm with a fixed price contract, that the efficient firm typically bears more risk than the inefficient firm, and that an increase in exogenous risk may bring about a decrease in expected cost of the project. As a by-product, we also establish that the famous ‘no-distortion-on-the top’ result in adverse selection models relies on risk neutrality of the agent.
Keywords: Regulation; Procurement; Incentives; Risk sharing (search for similar items in EconPapers)
JEL-codes: D86 L32 L51 (search for similar items in EconPapers)
References: View references in EconPapers View complete reference list from CitEc
Citations: View citations in EconPapers (1) Track citations by RSS feed
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Persistent link: https://EconPapers.repec.org/RePEc:eee:indorg:v:65:y:2019:i:c:p:173-220
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domain: literature


text:
To all the boys I've loved before. #1 / Jenny Han.
- 2 of 2 copies available at LARL/NWRL Consortium.
- 2 of 2 copies available at Lake Agassiz Regional Library. (Show preferred library)
0 current holds with 2 total copies.
View other formats and editions
|Location||Call Number / Copy Notes||Barcode||Shelving Location||Status||Due Date|
|Bagley Public Library||CD TALKING BOOK Y HAN (Text)||33500013252556||Main||Available||-|
|Moorhead Public Library||CD TALKING BOOK Y HAN (Text)||33500013252564||Main||Available||-|
- ISBN: 9781980047407
- ISBN: 1980047405
- Physical Description: 8 audio discs (8 hr., 45 min.) : digital ; 4 3/4 in.
- Edition: Unabridged.
- Publisher: Prince Frederick, Md. : Recorded Books,
- Copyright: ℗2020
In container (17 cm.).
Title from container.
|Participant or Performer Note:||
Narrated by Ali Ahn.
Lara Jean writes love letters to all the boys she has loved and then hides them in a hatbox until one day those letters are accidentally sent.
|Target Audience Note:||
12 years and up.
Search for related items by subject
|Subject:||Dating (Social customs) > Fiction.
Sisters > Fiction.
YOUNG ADULT FICTION > Romance > General.
Dating (Social customs)

domain: library


The former governor of Jigawa State, Sule Lamido, and his sons, have been transfered from Kano prison to Kuje prison in Abuja, PREMIUM TIMES has learnt.
Mr. Lamido was transferred in a preparations for a bail hearing, expected on Tuesday.
The former governor and his sons are facing corruption charges.
On Wednesday, a federal judge ordered they be detained in Kano prison till September 28 when their bail applications would be heard.
However, a prison official in Kano told PREMIUM TIMES that he saw the governor and his sons being taken away from the prison at about 10am on Monday.
He said Lamido and his sons could be transported to Abuja by road because there was no sign they were to travel by air.
When our correspondent visited the Kano prison early Monday, there were several guests waiting to see Mr. Lamido, apparently unaware they had been moved.
PREMIUM TIMES understands that the former governor’s counsel, Effiong Effiong, succeeded in having a vacation judge assigned to hear the case in Abuja.
A source said already some of the former governor’s aides had moved to Abuja in preparations for a possible bail hearing.
A spokesperson for the EFCC, Wilson Uwujaren, told PREMIUM TIMES he did not have the information about the transfer.
However, a prison official in Kuje confirmed to PREMIUM TIMES that the former governor and his sons had been moved into the facility at about 4pm Monday.
Support PREMIUM TIMES' journalism of integrity and credibility
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For continued free access to the best investigative journalism in the country we ask you to consider making a modest support to this noble endeavour.
By contributing to PREMIUM TIMES, you are helping to sustain a journalism of relevance and ensuring it remains free and available to all.
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text:
Transferring Ownership of Real Property
Although we cannot give legal advice, we can provide you with the requirements to make a document transferring ownership or real property acceptable for recording. The two most common documents used to transfer ownership are the Grant Deed or Quit Claim Deed. These forms are available at many Stanislaus area office supply stores.
When a document transferring ownership of real property is presented for recording we will look for the following information:
- Name(s) of the Grantor(s). Also known as the Seller(s), old owners
- Name(s) of the Grantee(s). Also known as the Buyer(s), new owners
- Description/identification of real property located in Stanislaus County, such as lot & tract
- Assessor's Parcel Number
- Documentary transfer tax declaration
- "When recorded mail to" address – listed in upper left hand corner of first page of document
- “Recording Requested by” – listed in upper left hand corner of first page of document
- Mailing address for future tax statements
- City where property located or Unincorporated specified
- Signature(s) of grantor(s)
- Signature(s) of grantor(s) acknowledged by a notary public
If transfer tax is paid: Fees will include fees for recording the first page of the document, a fee for each additional page, a survey monument fee, and the full amount of transfer tax. For current fee amounts, click here. For information on transfer tax, click here.
If no transfer tax is paid: Fees will include fees for recording the first page of the document, a fee for each additional page, and a real estate prosecution fraud fee. For current fee amounts, click here.
If any part of the document is other than 8 1/2" x 11" an additional fee per page for each page of the document will be charged.
Mailing address information for the Clerk Recorder Office is located on the homepage. Use the below link to return to the homepage.

domain: finance


I have a ceiling fan that had one of the four wires that feed the motor break loose. I'm having trouble getting the motor cover off the motor to expose where the wires get soldered on to the motor windings. The motor cover has four screws on the top part of the cover and another four on the bottom. I have removed all screws and tried to pry the two pieces apart with a large screwdriver but I'm not sure it this is the way to do this. Is there any trick to getting the covers off. I'm fairly certain I can soldered the one broken wire back on once I get the cover off.
The link below from this web site helped me learn what I need to do once I get inside the motor.


text:
Website: No website available.
Address: 201 WEST SECOND Ola, Arkansas 72801
Company Name: Kerm, Inc.
You are viewing the details for KARV FM 101.3 which is located in Ola, Arkansas. You can contact KARV FM 101.3 by simply calling them at their phone number listed above. You can listen to KARV FM 101.3 on the radio pretty much anywhere in Ola and the surrounding areas. So enjoy your stay at radiostation.org, your one stop site for (Karv) Country.

domain: geography


text:
Signed from Paris Saint Germain Ginola for £2.5 million was an instant hit in the Tyne side. He was a tricky winger who played forward. In his first month at the club he provided three assists and scored one goal to help Newcastle win all four opening matches and stay on top of the table.
|Name||Club||Position||Matches Played||Goals Scored||Team Wins|
|David Ginola||Newcastle United||Forward||4||1||4|
/ 2 days ago
#PLStories- Cherries chief executive Neill Blake explains why AFC Bournemouth chose Gary O’Neil to be permanent boss #AFCB
CHERRIES chief executive Neill Blake has explained why the club opted to make Gary...
#PLStories – Former Manchester United player Louis Saha explains why Cristiano Ronaldo hugged him but snubbed Gary Neville #MUFC
Louis Saha attempted to distance himself from what happened between Cristiano Ronaldo and Gary...
#PLStories- Jack Grealish sends Man City teammate Phil Foden message after Gareth Southgate England snub #MCFC #Worldcup2022
Jack Grealish has backed Phil Foden to play a key role in the remainder...
#PLStories- Jesse Marsch addresses Junior Firpo’s Leeds United criticisms amid January left-back hunt #LUFC
Junior Firpo’s technicality, passing, athleticism, intelligence and tactical awareness are all rated among the...
#PLStories- Brighton’s Kaoru Mitoma ready for next World Cup challenge #BHAFC #Worldcup2022 #Worldcup2026
Kaoru Mitoma says his feet are back on the ground as he goes for...

domain: news


text:
Biodiversity essentially refers to all aspects of variability evident in the living world, including diversity within and between individuals, populations, species, communities and ecosystems. Biodiversity trends are mainly assessed in terms of ...
declining populations and species, either individually or collectively, when manifest of loss of habitants or reduction in the area of ecosystems. The challenges to biodiversity have been explicit from different facts and figures suggesting that many species are no longer found throughout their former ranges, and may only occur in reduced numbers. Many high value fish have shown considerable decline mainly owing to over exploitation. Conservation and management of biodiversity is a priority concern in environmental as well as development policies world-wide. This is a due to the increasing growing need for maintaining ecological balance reserve. Besides, human exploitation beyond a concerning limit may pose challenges to the global wealth of biodiversity every now and then. Against this backdrop, the book attempts to capture some of the issues pertaining to biodiversity conservation and management. The book also highlights the existing and potential role of society, including different institutional arrangements in the context of conservation and management of biodiversity. The book emphasises on these aspects through various country experiences.
Number of pages: 350
Date of publication: 12/01/2012
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Ti piace Biodiversity for Sustainable Development? Iscriviti ad aNobii per vedere chi dei tuoi amici lo ha letto, e scopri libri simili!


Report: Ninjas in Pyjamas merging with eStar Gaming owner ESV5
Sweden-based esports organization Ninjas in Pyjamas is merging with a Chinese company called ESV5, with plans to be listed on the NASDAQ, according to a report from Reuters. ESV5 owns esports organization eStar Gaming and has investments from Chinese live streaming platform DouYu, among others. A “source with direct knowledge on that matter” tells Reuters […]
The post Report: Ninjas in Pyjamas merging with eStar Gaming owner ESV5 first appeared on The Esports Observer.


text:
3 Common Financial Issues to Watch Out for in a Divorce
Divorce is an act of permanent separation, and it includes more than merely separating yourself from your spouse. You’re not only turning one household into two and diverging your paths in life, but you also have to separate marital assets, and this can be a complex undertaking, especially when emotions are already high and the situation is contentious.
You and your attorneys will have to account for all shared assets and debts so that they can be fairly divided. With luck, both parties will be aware of all existing assets and debts, neither will try to hide information, and there will be no surprises after the fact.
Of course, if you don’t fully understand the process involved in division of assets, you might end up surprised by how little you get or how much you owe. Here are just a few common financial issues you should be aware of when finalizing your divorce.
1. Splitting up Real Estate
Owning a home is a key component of the American dream, but it can turn into a nightmare during a divorce, when this major asset must somehow be divided. It’s not like you can split it down the middle and each live in half. You’re going to have to find a way to divide the value of the home.
Many families with children prefer to allow one spouse, the primary caregiver, to continue living in the family home with the kids. However, this can be difficult if the parent remaining in residence doesn’t have the cash on hand to buy out an ex-spouse. In some cases, divorcing parties agree that both will retain ownership of the home and sell at a later date, splitting the proceeds at the time (perhaps with an offset for the resident who continues to build equity by paying the mortgage).
If spouses cannot agree to such an arrangement, they may simply have to sell the home, split any profit, and each move into new residences in the aftermath of divorce. This could be hard on kids, but perhaps easier on parents, especially if one parent cannot support the existing family home without the aid of a second income, or even with the help of spousal and child support.
2. Divvying Debts
Assets are only one side of the coin when it comes to division of assets. Spouses are also legally liable for marital debts, including mortgages, auto loans, credit card debt, and in some cases, even medical debt (although generally, student loan debt is assigned to the person who accrued it).
Michigan is not a community property state, which means divorcing couples are subject to equitable division of assets. In other words, assets and debts are divided fairly, but not necessarily equally. This may affect who ends up being responsible for specific debts.
3. Planning for Future Tax Returns
Just because you are divorced doesn’t mean you are totally done with joint financial obligations, especially if you have children. For years to come, you may have to contend with issues like who will claim children as dependents. This is something you’ll want to decide before the divorce is final.
If you’re struggling with how to divide assets and debts during divorce, you want the qualified experts at The Gucciardo Law Firm on your side. Call today at 248-723-5190 for legal guidance and support.
Too much information?
We focus exclusively on family law matters so we are always available to answer your questions and help.


text:
Where to buy a Pet Rock? Right Here. The alternative pet that is clean, never hugry, and never bites. Unlike cats or dogs, they are very easy ...
Pet Rocks for Sale
Summer Acceleration Pet Rock Sale
Funny Pet Rocks for Sale 9.99
Disclaimer: All content from the website users to share, if violated your rights, please email: email@example.com , we will promptly remove the infringing content, thank you!

domain: pet


Schubert Quartettsatz in C minor | Baermann – Adagio for clarinet and strings | Beethoven – Septet
Tickets: £22 stalls, £20 gallery, £15 online
In celebration of the 250th anniversary year of Beethoven’s birth, our traditional annual NCO Soloists concert gives our principal players the chance to enjoy some of the limelight and features one of his most celebrated chamber works. The Septet, scored for clarinet, horn, bassoon, violin, viola, cello and double bass, is one of Beethoven’s most successful and popular works – an uplifting piece of music that is rewarding and enjoyable for the musicians and audience alike. The concert also includes Baermann’s Adagio for clarinet and strings, the second movement of his Septet op 23, which is often performed as a stand alone piece, and Schubert’s Quartettsatz, the opening movement of a string quartet that he never finished but nonetheless feels complete and whole.
Due to social distancing guidelines, this concert is being sold as unreserved seating; we will allocate you a seat after your purchase that will allow for enough space around you. If you have specific access needs, please forward your confirmation email to email@example.com with the access information after purchase.
There is also the option to purchase a ‘ticket’ for the online stream – this will be available to watch from the following day for a week and the link will be sent directly to you.
If another lockdown is put in place, tickets will be refunded.
If you would like to purchase a season ticket for all three 2020 concerts at a discounted price (3 October, 31 October, 5 December), please email firstname.lastname@example.org to arrange. You can purchase a season ticket to attend in person or online.
Macclesfield Heritage Centre


text:
Dwarf hamsters eat hamster food, like all hamsters, but there are some things they CAN'T eat.
Dwarf hamsters can't eat too many sunflower seeds because they will get Diabetes. Also, they can't eat citrus fruits (lemon, orange, grapefruit) and tomatoes. Other veggies are okay.
dwarf hamster eat sunflower seeds Snake eats dwarf hamster hawk eats snake
yes other dwarf hamsters not that much with bigger hamster because the bigger hamster might eat it (the dwarf hamster)
No. Dwarf hamsters and all hamsters cannot eat tomatoes!
You shouldn't let them live together because it's regular for the regular sized hamster to eat the dwarf hamster.
yes but it is unlikely. hamsters can be cannibals. my hamster had a sister but she killed her and ate her. most hamsters are nice though. but if a dwarf hamster was dead then the syrian hamster would likely eat it
A dwarf hamster only has to eat once a day if you fill the bowl all the way... If you fill it half way dwarf hamsters should eat twice a day
yes the hamster will not get hurt if it eats a green bean
of course, any rodent can
no they cant they will die and choke
The Dwarf Hamster would be smaller than a regular hamster because of the "dwarf" part in it. Unless the regular hamster were to be a runt.
A robo dwarf hamster is a roborovski hamster which is a dwarf hamster so yes.
There are many breeds of hamster, but the ones kept as pets are: Syrian hamster, Winter White Dwarf hamster, Campbell's Dwarf hamster, Roborovski Dwarf hamster, and Chinese hamster.
There are nineteen types of hamsters in the world: The Mongolian Hamster Eversmann's Hamster Gansu Hamster Tibetan Dwarf Hamster Chinese Striped Hamster Chinese Hamster Kam Dwarf Hamster Long-tailed Dwarf Hamster Gray Dwarf Hamster Sokolov's Dwarf Hamster European Hamster Golden or Syrian hamster Turkish Hamster Romanian Hamster Ciscaucasian Hamster Campbell's Russian dwarf hamster Desert hamster Winter White Russian hamsters Greater Long-tailed Hamster
There is lots like, Syrian, Chinese dwarf hamster, Campbells dwarf hamster, Robo dwarf hamster, Russian hamster. My favorite, is the blueberry campbells dwarf hamster and the Syrian hamster. They are the sweetest and easy to hold. NOT THE ROBO"S!!!
about big will a dwarf hamster grow about big will a dwarf hamster grow about big will a dwarf hamster grow
A dwarf hamster is about 5 inches.
* * * == == * * * == == == ==
there is no such thing but if you mean a roborovski hamster yes its a dwarf
Dwarf hamsters can eat seeds one week after they are able to eat solid foods. This is when they are about 4 weeks old.
yes....in some of the hamster formula food they have peanuts in it
It is good and healthy for your hamster to eat fruits and vegetables and they enjoy it as well :)
Yes, it's a good source of Vitamin A.
yes,but theymay turn yellow
no, a teddy bear hamster is not a dwarf. a teddy bear hamster is a syrian.

domain: biology


text: Figure 5. The biological function and signalling pathway of the key genes affect the prognosis of breast cancer. (A) The bubble chart shows BP and KEGG enriched by key genes. (B) GSEA of genes expressed by breast cancer patients with survival times greater than 5 years. (C) The same BP as GSEA was clustered into three types of biological functions.

domain: medical


text:
The nutritional values given are approximate and should be taken as a guide.
1. Wash and shake dry rocket. Peel avocado, pit and cut into cubes.
2. Rinse and drain chick peas in a sieve. Heat oil in a pan and pan the chick peas until they are warm.
3. Heat the tortillas shortly in a pan or microwave and coat them with cream cheese. Spread rocket, Kühne Sliced Beetroots, Kühne Pumpkin Cubes, avocado and chickpeas on the wraps and season with salt and pepper.
4. Roll up the wraps, smash the ends, cut in half and serve.

domain: food


text:
NEWS RELEASE 17-238 - NOVEMBER 15, 2017
The Sarasota County Sheriff’s Office is pleased to begin offering front desk services at their new headquarters located at 6010 Cattleridge Boulevard, Sarasota, beginning Monday, November 20.
The front desk will be staffed every day from 7:00 a.m. to 7:00 p.m. Citizens can walk in to report a crime, commend a deputy, drop-off or receive property. As always, citizens can also dial 941.861.5800 to speak with a deputy by phone or email the agency’s general inbox at firstname.lastname@example.org. Also moving to the headquarters is the agency’s permanent prescription drop box which gives citizens a safe place to dispose of unused or unwanted medication. With the placement of front desk deputies at the agency’s headquarters, the front desk in downtown Sarasota will be permanently closed. The only remaining sheriff’s office services offered at 2071 Ringling Blvd. will be civil processing and public records requests.
As the agency continues to adapt to new technology and the changing needs of the public, personnel will remain staffed at the south county front desk located at 4531 S.R. 776 in Venice. Eventually, both the Siesta Key and Englewood training offices will be consolidated to the south county facility where services to the public remain available from 8:00 a.m. to 8:00 p.m.
The sheriff’s office will host a formal ribbon cutting and grand opening event on Saturday, December 9 from 10:00 a.m. to 2:00 p.m. Members of the public are invited to enjoy special demonstrations, meet sheriff’s office personnel, and visit the new front desk. For more information about the event, visit www.SarasotaSheriff.org or call the agency’s Community Affairs Office at 941.861.4005.


text:
Jump to navigation
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Please note: This content may be out of date and is currently under review.
View recent regional and seasonal content by using the filters on the right.
This page provides seasonally relevant information on conditions and management options for June, July and August 2019 in the agricultural areas of Western Australia (WA).
The whole farm nutrient mapping procedure described here applies to pastures in the greater than 600mm rainfall zone of south-west Western Australia.
In May 2019, Agriculture and Food Minister Alannah MacTiernan announced the independent panel that would oversee a public review of the operation and effectiveness of the Animal Welfare Act 200
The Western Australian Government recognises the value of animal welfare to the community and strives to ensure that all animals receive appropriate standards of care.
Calling a vet to investigate disease protects our markets: The WA Livestock Disease Outlook provides information about recent livestock disease cases in Western Australia and diseases...
Livestock disease investigations protect our markets: Australia’s ability to sell livestock and livestock products depends on evidence from our surveillance systems that we are free of particular...
Fifteen projects will share in $582,800 in funding through the eConnected Grainbelt Project’s new WA Internet of Things (IoT) DecisionAg Grant Program.
The Pastures from Space service generates maps to estimate pasture feed on offer (FOO) as kilograms of dry matter per hectare, and plant (pasture and crop) growth rates (PGR) as kilograms of dry ma
Comprehensive survey data is collected on sheep enterprises and producer intentions every four years by DPIRD.
Pastures from Space estimates green feed-on-offer (FOO) and the pasture growth rate (PGR).
Feral cats are the same species as domestic cats, but survive in the wild without human reliance or contact.
The full biomass mapping data is available in more detail on the Australian Renewable Energy Mapping Interface (AREMI) – see the links below the dashboard. The AREMI map also allows you to overlay
Testing for herbicide susceptibility allows you to determine what herbicide options are still available to control weeds on your farm.
Herbicides can be applied by a variety of means including boom sprayers, aerial spraying, misters, blanket wipers, rope wick applicators, weed seekers and back-pack sprayers.
A Quarantine Area is in place to direct the movement and treatment of host plants to contain and control the Tomato potato psyllid (TPP) in Western Australia.
The Final Tranche of recipients has been announced, as part of Round One of the State Government's successful Digital Farm Grants Program.
The release of the nation’s first Digital Infrastructure Atlas will provide WA enterprises with increased opportunities to improve their telecommunications services.
The Western Australian Government, through the Department of Primary Industries and Regional Development, is focusing resources on supporting the growth of premium and value-add food and beverage b
The Lambing Planner is a simple tool that allows you to change a lambing date or a joining date to see the impacts of that on other key times in the reproductive year.
The Department of Primary Industries and Regional Development’s (DPIRD) Statistical Seasonal Forecast (SSF) outlook for winter, June to August 2019 and June to October 2019 is indicating less than...

domain: agronomy


NTN 4R3039 bearing description
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As Hamas launches rockets from the Gaza Strip into Israel, casualties are kept at a minimum thanks to Israel’s superior air defense system. In the current conflict, up to 90 percent of the rockets engaged by the “Iron Dome” are intercepted as they approach high-value Israeli targets. The Rafael-Raytheon air defense system has held a high rate of success since it first intercepted a Gaza rocket in April 2011. What is the Iron Dome and how does it operate? Heritage policy analyst Michaela Dodge answers these questions and more.
What is the Iron Dome?
The Iron Dome is an Israeli air defense and a short-range defense system designed to destroy enemy rockets, mortars, and artillery shells.
How does it work?
Each Iron Dome battery is comprised of interceptors (Tamir), radars, and command and control systems. The system shoots down incoming rockets mid-air before they hit the target. The system only intercepts rockets that would hit targets of value (population centers, military facilities, etc.). This allows the system to preserve interceptors and save more resources than if it were shooting down every single rocket. In April 2011, the Israelis found out that they needed to launch an interceptor against only about 20 percent of the rockets launched by Hamas.
How often is it activated?
This depends on the threat level. The Iron Dome is deployed close to population centers. It is mobile so it can be deployed to different parts of the country depending on where the threat is coming from. Once radars get information about incoming rockets, the interceptors are fired in a manner that increases the probability of the intercept.
Since it’s a short-range air defense system, what type of capabilities does the IDF have for countering long- and medium-range threats?
Israel is building a layered missile defense system, which means that it is developing a range of systems for different ballistic missile threats. Arrow 3 is designed to intercept short- and medium-range ballistic missiles. David’s Sling, currently under development, will be designed to intercept short-range ballistic missiles.
What role did the US play in developing the system?
The U.S. is contributing funds toward developing the Iron Dome system. In the fiscal year 2014, the Department of Defense requested more than $220 million to fund the system. Raytheon, U.S. defense contractor, is under contract for co-producing the system’s components and interceptors.
How effective is it? What percentage/number of incoming rockets has it intercepted?
In the current conflict, the Iron Dome has intercepted up to 90 percent of rockets that it engages. Note that it does not engage every single rocket, but only the ones that would actually target valued areas.
What other countries have the Iron Dome technology?
Other countries (for example, Russia, the U.S., Japan, South Korea) have air defense systems and while they are conceptually similar, other air defense systems are based on different technologies that the Iron Dome uses. No other country in the world has the Iron Dome system today.
Can you briefly address the criticisms of the system?
Critics have argued that the Iron Dome is too expensive, since limited numbers of interceptors (about $60 000 apiece) are fielded against large numbers of cheaper offensive rockets (about $500 to $1,000 apiece). These critics miss the point. What matters is the value of defended area and what would costs of a successful attack. The damage in blood and treasure caused by a successful attack could be much larger than a cost of an interceptor.
Critics also argue that the system does not work based on a “visual analysis” of rockets’ contrails. This argument simply does not stand. Blurry videos from cameras and smartphones are not sufficient to make a judgment whether a threat was destroyed or not. The IDF uses different types of radars to confirm intercepts.
There are other significant benefits that the critics of the system often omit from their analysis. Until the deployment of the Iron Dome system, the Israelis faced a stark choice: They could absorb the attacks or escalate the conflict by conducting retaliatory offensive operations against their enemies. Escalation, for justifiable reasons, is what the Israelis chose to do both against Hezbollah in 2006 and against Hamas periodically. To the extent the Israelis used these retaliatory strikes in attempts to destroy enemy rocket launchers—itself a fundamentally defensive purpose—they found the offensive counterstrikes themselves to have limited effectiveness and cost effectiveness. The Iron Dome system provides the Israeli leadership with the political, policy, and military options as well as the space and the time to make the right decision in order to prevent an escalation.
The use of the Iron Dome system permits Israel to draw an even clearer distinction between itself and its enemies as to which side is the aggressor (not that there should be much doubt by anyone making an honest assessment). Nevertheless, there is considerable, albeit intangible, value in depriving an enemy of plausible arguments against an effective defensive military system. For Hamas and Hezbollah to argue against Iron Dome, they will have to assert that Israeli use of this defensive system is an aggressive measure. The argument is implausible.


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This week, 90 degree temperatures are expected to deliver the warmest and driest conditions for summer. Even for many that have been “wet” this season, but are already seeing dry areas, expect more drying to occur. As Labor Day weekend approached, USDA top soil moisture shortages continue to grow.
“Normally we would recommend renovation programs, including seeding and sodding to commence at this time with favorable temperatures and rainfall normal expected. However, unless your turf area looks like the damaged turf in the picture, it might be best not to stress your existing turf at this time. Furthermore, if you are relying on rainfall to get new turf established, consider your existing soil moisture levels and the ability to keep the turf moist over the next few weeks with expected high temperatures. This is likely to lead to more problems with seedling damping off issues. “
- Frank S. Rossi, Ph.D.
Read the full article here at Cornell Turfgrass ShortCutt.
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This work has been selected by scholars as being culturally important, and is part of the knowledge base of civilization as we know it. This work was reproduced from the original artifact, and remains as true to the original work as possible. Therefore, you will see the original copyright references, library stamps (as most of these works have been housed in our most important libraries around the...
Hardcover: 282 pages
Publisher: Palala Press (May 7, 2016)
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Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.
21 Related JoVE Articles!
Development of an IFN-γ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation
Institutions: Université de Montréal, Université de Montréal, Université de Montréal.
Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-γ production by T lymphocytes in response to in vitro
stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens.
Immunology, Issue 89, Varicella zoster virus, cell-mediated immunity, T cells, interferon gamma, ELISpot, umbilical cord blood transplantation
Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production
Institutions: Keck School of Medicine, University of Southern California.
In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine γHV68 is a natural pathogen of murine rodents. γHV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo
virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that γHV68 hijacks MAVS and IKKβ, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NFΚB activation and antiviral cytokine production. Specifically, γHV68 infection activates IKKβ and that activated IKKβ phosphorylates RelA to accelerate RelA degradation. As such, γHV68 efficiently uncouples NFΚB activation from its upstream activated IKKβ, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.
Immunology, Issue 85, Herpesviridae, Cytokines, Antiviral Agents, Innate, gamma-HV68, mice infection, MEF, antiviral cytokine
4D Imaging of Protein Aggregation in Live Cells
Institutions: Hebrew University of Jerusalem .
One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1
. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2
, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1
. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4
. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1
, target them for degradation by the ubiquitin-proteasome system5
, or direct them to protective aggregation inclusions6-9
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10
: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1
- model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12
) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts
is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 °C, or when the cell faces misfolding stress, Ubc9ts
misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.
Cellular Biology, Issue 74, Molecular Biology, Genetics, Proteins, Aggregation quality control, protein folding quality control, GFP, JUNQ (juxtanuclear quality control compartment), IPOD (insoluble protein deposit), proteostasis sensor, 4D live cell imaging, live cells, laser, cell biology, protein folding, Ubc9ts, yeast, assay, cell, imaging
Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells
Institutions: Tel Aviv University.
Attachment of the Glc3
precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3
H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8
. In contrast, unstable ERAD substrates are trimmed to Man6-5
and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.
Cellular Biology, Issue 38, N-linked oligosaccharide, mannose-labeling, endoplasmic reticulum associated degradation, calnexin, glycosylation, mannosidase
Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors
Institutions: Twincore, Centre for Experimental and Clinical Infection Research, The Rockefeller University, NY.
Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5
. HCV in vitro
research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8
. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans
-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans
-complementation, which is determined by measuring de novo
production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected naïve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9
) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10
. Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11
) and subsequently challenged with infectious HCV particles (HCVcc), de novo
production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans
-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.
Virology, Issue 65, Immunology, Molecular Biology, Genetics, cell fusion, HCV, restriction factor, heterokaryon, mouse, species-specificity
Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy
Institutions: University of Texas Medical Branch.
Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1
, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus
in the family Bunyaviridae
and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3
as well as wild-type RVFV strains 4-6
, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3
, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8
and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10
IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11
, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14
. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.
Immunology, Issue 57, Rift Valley fever virus, reverse genetics, NSs, MP-12, vaccine development
Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples
Institutions: Institute for Hepatitis and Virus Research, Thomas Jefferson University , Drexel University College of Medicine, Van Andel Research Institute, Serome Biosciences Inc..
In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6
. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12
. In fact, most current cancer biomarkers, such as the L3 fraction of α-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15
, and CA199 for pancreatic cancer 16, 17
are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al.
has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18
. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis
-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4
in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20
. This method has been used successfully in multiple different labs 1, 7, 13, 19-31
. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.
Molecular Biology, Issue 63, Glycoproteins, glycan-binding protein, specific protein glycosylation, multiplexed high-throughput glycan blocked antibody microarray
The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice
Institutions: Goethe University Hospital Frankfurt.
Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2
. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5
. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5
. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6
. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8
. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9
for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFNγ-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.
Medicine, Issue 60, autoimmunity, liver, autoantigen, fibrosis, perfusion
Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro
using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro
. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo
. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo
tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro
and in vivo
and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
Assaying Proteasomal Degradation in a Cell-free System in Plants
Institutions: Stony Brook University, State University of New York.
The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium
, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via
its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.
Biochemistry, Issue 85, Ubiquitin/proteasome system, 26S proteasome, protein degradation, proteasome inhibitor, Western blotting, plant genetic transformation
Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance
Institutions: University of Central Florida.
AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1
. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4
. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5
. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12
To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15
. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.
Immunology, Issue 59, Surface plasmon resonance, AB toxin, translocation, endoplasmic reticulum, cell culture, cholera toxin, pertussis toxin
Reconstitution Of β-catenin Degradation In Xenopus Egg Extract
Institutions: Vanderbilt University Medical Center, Cincinnati Children's Hospital Medical Center, Vanderbilt University School of Medicine.
egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus
egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3
. Herein, a method is described for isolating Xenopus
egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, β-catenin. Two different methods are described to assess β-catenin protein degradation in Xenopus
egg extract. One method is visually informative ([35
S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess β-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus
egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of β-catenin degradation.
Molecular Biology, Issue 88, Xenopus laevis, Xenopus egg extracts, protein degradation, radiolabel, luciferase, autoradiography, high-throughput screening
A Protocol for Analyzing Hepatitis C Virus Replication
Institutions: Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA.
Hepatitis C Virus (HCV) affects 3% of the world’s population and causes serious liver ailments including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae
. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla
luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro
transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.
Infectious Diseases, Issue 88, Hepatitis C Virus, HCV, Tumor-virus, Hepatitis C, Cirrhosis, Liver Cancer, Hepatocellular Carcinoma
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein
Institutions: Institute for Biomedical Aging Research, Leiden University Medical Center.
Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2
. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3
, cell cycle regulation and cellular differentiation 4
or in immune system response 5
. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6
. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10
. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)–dgn fusion protein (GFP–dgn, unstable) and a variant carrying a frameshift mutation (GFP–dgnFS, stable 11
) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP–dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.
Cellular Biology, Issue 69, Molecular Biology, Medicine, Biomedical Engineering, Virology, proteasome activity, lentiviral particles, GFP-dgn, GFP-dgnFS, GFP, human diploid fibroblasts, flow cytometry, plasmid, vector
A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments
Institutions: Delft University of Technology, Delft University of Technology.
This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli
and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9
addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli
to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native β-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2
) of the alkane hydroxylase system from Gordonia
were transformed into E. coli
. For the conversion of long-chain alkanes (C15-C36), theladA
gene from Geobacillus thermodenitrificans
was implemented. For the required further steps of the degradation process, ADH
and ALDH (
originating from G. thermodenitrificans
) were introduced10,11
. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli
K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFDα and β, both from Pyrococcus horikoshii
OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and β improve the protein folding process e.g.
under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n
-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli
to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.
Bioengineering, Issue 68, Microbiology, Biochemistry, Chemistry, Chemical Engineering, Oil remediation, alkane metabolism, alkane hydroxylase system, resting cell assay, prefoldin, Escherichia coli, synthetic biology, homologous interaction mapping, mathematical model, BioBrick, iGEM
Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism
Institutions: Ben-Gurion University of the Negev.
The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro
, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans
. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo
, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.
Biochemistry, Issue 82, aging, Caenorhabditis elegans, heat shock response, neurodegenerative diseases, protein folding homeostasis, proteostasis, stress, temperature-sensitive
Institutions: UVP, LLC, Keck Graduate Institute of Applied Life Sciences.
Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.
Basic Protocols, Issue 16, Current Protocols Wiley, Immunoblotting, Biochemistry, Western Blotting, chromogenic substrates, chemiluminescent substrates, protein detection.
Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation
Institutions: La Jolla Institute for Allergy and Immunology.
Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens. Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells. The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells. Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy. Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.
Immunology, Issue 10, Natural Killer T cells, NKT cells, CD1 Tetramers, antigen presentation, glycolipid antigens, CD1d, Mucosal Immunity, Translational Research
Detection of Protein Ubiquitination
Institutions: The Sanford Burnham Institute for Medical Research.
Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo
and test tubes in vitro
. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.
Cell Biology, Biochemistry, Issue 30, ubiquitination, cultured cell, in vitro system, immunoprecipitation, immunoblotting, ubiquitin, posttranslational modification


text:
Maritime Day Celebration!
219 Pilottown Road
Lewes, Delaware 19958
About Maritime Day Celebration!
The Lewes Historical Society is proud to be a co-sponsor of the 2022 Maritime Day Celebration.
Experience and celebrate maritime history and its impact on the region at the annual celebration at the Lightship Overfalls on Saturday, May 21, from 10:00 a.m. to 3:00 p.m.
Co-sponsored by the Lewes Historical Society, Cape May – Lewes Ferry, Zwaanendael Museum, and Overfalls Lightship, this celebration – complete with events, displays, vessel tours and activities – is free to all.
A brief remembrance ceremony to honor sailors who perished on the Lewes beaches, recognize departed members of the Overfalls Foundation, and to celebrate the opening of the Lightship Overfalls (LV-118) for the 2022 season will start the day at 10:00 a.m.
Other Maritime Day activities include a life raft demonstration by the Ferry, a water cannon display by the Lewes Fire Boat, and a U.S. Coast Guard rescue helicopter fly-by. The day’s finale will be "The Wreck of the DeBraak" Program presented by the Zwaanendael Museum, starting at 2:30 p.m. The program explores the history of the British warship DeBraak which sank off the coast of Lewes in May of 1798 only to be recovered by treasure hunters nearly 200 years later.
In 1933, the United States Congress declared May 22 as National Maritime Day. The designation honors the contributions of the American merchant marine – civilians who have defended the freedom of the United States since 1775 and who executed the largest sealift the world has ever known during World War II.


text:
The purpose of parts 6125.8000 to 6125.8700 is to set forth procedures and standards to be followed by the commissioner of natural resources when leasing state-owned lands for the exploring for, mining, and removal of selected industrial minerals. Each lease issued under parts 6125.8000 to 6125.8700 will identify which of the industrial minerals are covered by that particular lease.
Due to the variable nature of mining, selling, and processing various industrial minerals, several of the provisions of each lease will need to be developed within the framework of the model lease form contained in part 6125.8700. Parts 6125.8100 to 6125.8700 prescribe minimum standards as to the term of the lease, rental rates, and royalty rates.
MS s 93.25
19 SR 2433
August 7, 2009
Official Publication of the State of Minnesota
Revisor of Statutes

domain: law