text: MWC 2014 Preview: MIMOtech launches high capacity small-cell radio link20 Feb 2014 03:10 PM StarLink 60G mm-wave link is optimised for small cells and mobile backhaul in urban network deploymentsMIMOtech, a specialist in ultra high capacity wireless point-to-point backhaul, has announced the launch of StarLink 60G, a fully-integrated millimeter-wave radio link optimised for short-range small-cell backhaul in urban network deployments.The StarLink 60G operates in the 57-66GHz frequency band (V-band), providing cost-effective high-capacity wireless point-to-point Gigabit Ethernet connectivity for network operators and service providers."StarLink 60G has been designed to meet the stringent requirements of mobile network operators and service providers, offering an easily-installed carrier-grade backhaul solution that provides high capacity combined with low total cost of ownership," said Buks Fouche, managing director of MIMOtech."Its unique electronically-stabilized lens antenna system is designed to overcome pole sway, twist and tilt, and it has a near-zero footprint, making it ideal for street-level installations."StarLink 60G features a patented automatic antenna alignment technique that makes deployment fast, simple and cost-effective. Channel bandwidth is scalable from 56MHz up to 250MHz with flexible modulation from QPSK to 64QAM and hitless Adaptive Modulation (HAACM).This allows for management of spectral bandwidth versus capacity requirements—and in some cases annual spectrum costs—depending on local 60GHz regulations. The small aperture size of 0.2m and low mass make the StarLink 60G ideal for pole mounting in urban environments, minimizing site leasing costs.The underlying millimeter-wave technology is an advanced silicon germanium (SiGe) chipset with a highly integrated transceiver design, allowing both time-division (TDD) and frequency-division duplexing (FDD) operation. This millimeter-wave technology has several advantages—most importantly the use of standard manufacturing techniques and the capability to cover higher millimeter-wave frequencies, including the 70/80GHz ultra high capacity backhaul band.MIMOtech plans to release a 70/80GHz product later this year to complement the StarLink 60G, expanding its product offering to address higher capacity, longer range backhaul requirements in the 70/80GHz band in addition to the short-range 60GHz requirements for small cell backhaul that are covered by the StarLink 60G.MIMOtech will be exhibiting on Stand H07 in Hall 7 at the Mobile World Congress in Barcelona, 24-27 February 2014.James Atkinson EditorLinkedIn |Google + |Email Leave a CommentPlease write code to prove you're humanMore on... Small cellsParallel Wireless expands indoor vRAN small cell solution MWC 17: ip.access to unveil latest Viper small cell platform Italian service provider Fidoka Srl to deploy DragonWave Harmony radiosLatest in Mobile infrastructureQuortus and Expeto announce PaaS virtual LTE networks for enterprisesMWC17: Zyxel to debut new LTE and Wi-Fi indoor gatewayMWC17: Ericsson expands 5G access and transport portfolio


text:
Pay-Per-Minute Viewing
You can now enjoy this movie on your iPhone®, iPad®, or compatible Smart TV & Android devices.
Purchase Options
Pay-Per-MinutePurchase Minutes
Streaming Rentals
Available MP4 sizes:SD 480p (1.77 GB)
DRM-free downloads offer content that is not limited by standard Digital Rights Management (DRM) access technologies. These technologies allow hardware manufacturers, publishers, copyright holders and individuals to limit the usage of digital content.
When you purchase DRM-free downloadable content, that content is available for you to download on any computer and save to a disk whenever you like. You can also download to multiple computers. There is no timeframe associated with your purchase and the content will not expire.
Running Time: 113 minutes
Released: 08/2003
Studio Name: Diabolic Digital
Series: Gangbang Auditions
Director: Christopher Alexander
Categories: Amateur GangBang Anal Cumshot Interracial Gonzo Double Penetration
Description: Young and eager sluts step up to the plate. They will show us if they have what it takes to be a Gangbang girl. They fuck, suck and eat cum in a frenzy! Anal, double anal, double penetrations, standing anal, ass-to-mouth, ass-to-mouth merry-go-rounds and more! Enjoy!
3 Scenes from Gangbang Auditions 9
Gangbang Auditions 9: Scene 1 Free Preview
Starring: Vivienne La Roche ... More Details
Stars: Vivienne La Roche
Sex Acts: Ass To Mouth, Anal Sex, Rim Job, Handjob, Masturbation, GangBang, Gapes, Double Penetration, Vaginal Sex, Choking, Cum Swallowing, Oral Sex, Blowjob
Positions: Standing Side Saddle, Standing Doggy Style, Standing, Side Saddle, Sitting, Missionary, Laying, Reverse Cowgirl, Cowgirl, Kneeling
Settings: Generic Room
Gangbang Auditions 9: Scene 2 Free Preview
Starring: Lanny Barbie ... More Details
Stars: Lanny Barbie
Sex Acts: Ass To Mouth, Anal Sex, Handjob, Jousting, Masturbation, GangBang, Double Penetration, Vaginal Sex, Oral Sex, Blowjob
Positions: Standing Doggy Style, Crouching/Squatting, Standing, Side Saddle, Laying, Reverse Cowgirl, Cowgirl, Doggy Style
Settings: Living Room
Gangbang Auditions 9: Scene 3 Free Preview
Starring: Melanie Jagger ... More Details
Stars: Melanie Jagger
Sex Acts: Anal Sex, Rim Job, Handjob, Jousting, Masturbation, GangBang, Double Penetration, Vaginal Sex, Cum Swallowing, Oral Sex, Blowjob
Positions: Standing Doggy Style, Standing, Standing Reverse Cowgirl, Spoons position, Missionary, Laying, Reverse Cowgirl, Cowgirl, Kneeling, Doggy Style
Settings: Generic Room
Recommended Movies
AEBN Cams

subset: commoncrawl


text:
can't make head nor tail of sthUS also can't make heads or tails of sth
› to not be able to understand something:
I can't make head nor tail of these instructions on the packet.
(Definition of "can't make head nor tail of sth" from the Cambridge Advanced Learner's Dictionary & Thesaurus © Cambridge University Press)
Get a quick, free translation!
Create and share your own word lists and quizzes for free!
Word of the Day
lend a helping hand
to help someone


text:
for Art + Innovation
Located in a historic neo-Gothic church in Uptown Charlotte, McColl Center for Art + Innovation offers more than 5,000 square feet of Gallery space. We welcome the public to explore our exhibitions and attend our various events, including open studios, community engagement initiatives, workshops and more.
NovellerNew Frequencies at McColl Center / Music
Thursday, March 2, 2017
Doors open at 7 PM
$10 McColl Center Member ▶ BUY TICKETS
$14 Non-member ▶ BUY TICKETS
$60 Become an Emerging McColl Center Member ▶ BUY TICKETS
($50 membership + $10 ticket; Become a member of McColl Center now and qualify for the reduced member price. You'll also receive McColl Center Member pricing on upcoming/future New Frequencies at McColl Center events, artist-led workshops, special events, and other membership perks.)
Noveller is the solo electric guitar project of Los Angeles-based composer and filmmaker Sarah Lipstate. Handling the guitar as her muse, Lipstate summons a sonic palette so rich as to challenge the listener to conceive of how it's housed in a single instrument manipulated by a solitary performer. She toured Europe with Iggy Pop in 2016, and has also participated in Rhys Chatham's Guitar Army, Ben Frost's "Music for 6 Guitars" Ensemble, and Glenn Branca's 100 Guitar Ensemble.
▶ Noveller "No Dreams":
New Frequencies at McColl Center showcases innovative and exciting performances from today's most adventurous creators and acclaimed artists. Selected "Best New Arts Programming" by Charlotte Magazine in 2016: "Here, the future of art is on display.
Image: Courtesy of the artist


text:
Vic & Anthony's Steakhouse Map
129 E. Fremont St. (First St.)
702-385-7111 | Make Restaurant Reservations | Menu
Find Vic & Anthony's Steakhouse with this handy map to the restaurant. If you don't see a pop-up, click on the map's green arrow to get driving directions to and from Vic & Anthony's Steakhouse.
Find out which restaurants and chefs top the list of GAYOT's 2016 Annual Restaurant Awards.


text:
1960 Fiat 2100
The saga began in the winter of 2004, when Editor Martin purchased a 1960 Fiat 2100 from SCMer Howard Jacobs of Ohio. He was very thorough in his description, including work he'd had done to the car, adding that it "can be driven and enjoyed as-is."
SCM could have shipped the Fiat back to World Headquarters in Portland, OR, but Martin had an idea: He instead asked readers to volunteer to drive this thing in stages from Cleveland to Portland. In a momentary lapse of sanity, our managing editor at the time, Jeff Sabatini, took the first leg, bringing it from Cleveland to Ann Arbor, MI, where it spent the winter.
Offers and professions of Fiat love flooded our inboxes. Our first subscriber/driver was Neil Johnson, who took it from Ann Arbor, MI, where it stained his garage floor, to Fort Wayne, IN (May 2004, 151 miles).
The next driver, Hugh Baldus, has the distinction of being the only pilot of the SCM Fiat to reach his destination without incident: Elkhart, IN (June 2004, 109 miles). Granted, he only took the car 109 miles. Baldus also procured the front- and rear-window "SCM America by Fiat" vinyl signs.
Najeeb Khan and his daughter Sarah (who loved the car) drove it to a Chicago suburb (July 2004, 120 miles), where they handed it over to Fred Panici, a task we had planned on accomplishing the previous February. The car did not see the 2004 Concorso Italiano, but its day would come.
While the car was in Panici's care (July to September 2004), SCM had seatbelts installed along with numerous other repairs, including "windshield gasket ineffectively caulked and duct-taped." It may not be pretty, but it is resourceful. Panici's last words: "I have no doubt that it can now make Portland, OR, without much more trouble."
Mike and Ann Griese picked the car up in Wilmette, IL, and drove it to their home in Byron, MN (October 2004, 390 miles). They even managed to get it to the Wheels and Wings car show in Osceola, WI, with the help of Lotus Owners, Oftha North. The rejoicing was short-lived, though, as the carb cracked and leaked gas once back in Minnesota, sidelining it for several weeks.
Charlie Gaetze of Sioux Falls, SD, not only offered to pick the car up and drive it home (December 2004, 235 miles), but also to get the Fiat to Scottsdale in time for the Barrett-Jackson auction in January 2005.
He and co-driver Rick Barrett made only one mistake: stopping at the Corn Palace in South Dakota. The Fiat refused to start up, necessitating a trailer ride to Scottsdale (January 2005, 1,593 miles—few of which were under its own power). Of course, once in the relative warmth of the Southwest, it started right up. Or maybe it was just happy to see Editor Martin and the SCM staff, our first glimpse of the car in person.
Airline pilot Matt Packard of Phoenix had the car when it suffered what he called a "catastrophic failure. The distributor started eating itself and parts started dropping out of the engine bay onto the road." Packard's accomplice Matt Hoza fortunately carries the title of "Fiat guru," and got it running again.
Packard and his wife Gina Cuciniello finally were able to deliver the Fiat to the Petersen Museum in Los Angeles (July 2005, 375 miles). It is worth noting that they also used the SCM fix-it of choice, duct-tape, to repair the cooling system overflow cap – a smart move before driving across the desert in 108-degree heat.
Petersen Museum Director, Dick Messer, delivered on his promise to have the car in Monterey for Concorso Italiano, the event the little Fiat had missed the previous year. He did not, however, drive it himself. He roped AutoWeek writer Mark Vaughn into making the trip, along with Museum intern Brian Martin (no relation to SCM's editor). With a case of oil in the trunk, the Fiat 2100 made its spectacular concours debut on the stage at Concorso, where Editor Martin was emcee (August 2005, 375 miles).
SCM contributor Martin Swig drove the final leg of the journey to Portland (October 2005, 735 miles), where Jim Feldman was kind enough to let the car rest – and drip – in his garage.
The 1960 Fiat 2100 is home now, in the official SCM garage (November 2005, 9 miles) after being pushed downhill by a Honda Element driven by General Manager David Slama. "I couldn't understand why Stefan [Lombard, Auction Editor] had the brake on going downhill," Slama said. Of course, the brake wasn't on at all, nor was the car out of gas when it conked out at the top of the hill. It was just the Fiat, doing what it does best. Nothing.
Months of ownership: 22
Miles driven since purchase: 4,421
Recent investments: Starter Fluid, $4.99; lunch with repairman, $25
Total investment: $3,828.65
Cost per month: $174.03
Cost per mile: $.87
Cost per day: $5.80
New car equivalent: 2006 Chevrolet Aveo, $173/ month, 60-month loan (That's right, we could buy instead of lease.)
– Kristen Hall-GeislerSports Car Market magazinewww.sportscarmarket.com
It began as a trip for two. But Joy Rainey's coast-to-coast drive across the United States will ultimately benefit thousands more — and perhaps ease her emotion … More
Editor's Note: Yoav Gilad is Editor in Chief at [keep it] Wide Open, a website dedicated to bringing the thrill and romance of cars to the enthusiast. Yoav rece … More
In 1961 Mom and Dad loaded my brother and me into our old '55 Plymouth Wagon and drove from New Jersey to Michigan to visit my aunt, uncle and cousins in Michig … More
Value data provided by Hagerty Valuation Tools®. Learn More
Get a quote
For Collector Vehicle Insurance.
Does your vehicle qualify | Retrieve your quote


text:
In my previous article, I have discussed the matters one should consider in deciding the childbirth in Saudi Arabia. Now, we will be talking about the steps you need to take after child's birth in Saudi Arabia. Paperwork starts as soon as a child gets birth. This article discusses the procedure you need to adopt if a child is born inside Kingdom of Saudi Arabia. However, if the child is born outside the Kingdom of Saudi Arabia, you need to follow a separate procedure to add him under your Iqama. This procedure is explained in this article.
Procedure to Apply for Iqama of Newly Born Baby – Born inside Saudi Arabia
- First of all you will have to apply for the Birth Certificate of the newly born child in Saudi Arabia. The procedure to get birth certificate for the newly born child has been explained in this link. Recommended: Procedure to get Birth Certificate in Saudi Arabia
- Next step is to get passport from your home country's embassy. In most of the countries, you will have to apply for national identity card to get the passport.
- Before visiting Embassy, make sure that Birth Certificate issued by Civil Affairs is attested by the Ministry of Foreign Affairs of Saudi Arabia. Recommended: Procedure to Attest Documents from MOFA, Saudi Arabia
- You may also need to get it translated and attested by your Embassy (the requirement varies from Embassy to Embassy) Recommended: Procedure to Apply for the Pakistani Passport of Newly Born Child in Saudi Arabia
- You need to take online appointment with Jawazat. Right now, online appointment is not needed for Makkah region. But I would recommend you to try to apply for online appointment of Jawazat for your region. You never know when they change the procedure. Recommended: Procedure to Book an Appointment with Jawazat for the issuance of Iqama of Newly Born Baby
- You need to fill a form to add your child as your dependent. I have attached the form below. You can download it from the below link. If you don't want to fill it yourself, agents sitting in front of Jawazat building will fill it for you for only SR 10. Recommended: Download "Iqama Form" for Saudi Jawazat (Passport Office)
- You need to pay the dependent's fee to process the Iqama. If dependent's fee for your existing family members is not yet paid, you would not be able to apply for the Iqama of your newly born child. Recommended: Procedure to pay Dependent's Fee for Iqama Renewal
- Original passport of your newly born baby
- 2 photos of your baby make sure at least one picture of your baby must be attached on baby's filled iqama form.
- Copy of your Passport
- Copy of your wife's passport
- Copy of your Iqama
- Copy of your wife's Iqama
- Copy of Birth Certificate
- If you are applying for Iqama after 12 months of the birth of your child, you will have to pay a fine of SR 1,000 through SADAD.
- Jeddah Jawazat: In Jeddah, you will have to go to the passport office located in Kandara. I have attached the map location of this office below. Firstly, the correct entrance to the first floor is located by taking the first left immediately after entering through the main entrance. You should continue straight ahead until you see an open courtyard to your right where you will then see a single door entrance at the end which will lead you to the correct staircase. You go straight to the 1st floor and enter through the main offices to the right. You'll find multiple booths numbering from 1 through to 18. For those born outside KSA, they should go to the last booths between 15 & 18. For those born in KSA, they should go to the first booths between 1 & 4.
- Riyadh Jawazat: In Jawazat normally you need to go to first floor, your targeted section is 'Azadafa Molod Jaded' – mean 'Addition of newly born baby'. If you come to first floor by elevator then it's in front of your just behind the stairs. Normally you need to go on window 1 0r 2 there your need to submit your document and get your baby's iqama in 5 to 10 minutes. Once you get your baby's iqama, before you leave the counter, make sure you take back your baby's Arabic birth certificate original one and also double check the baby's name in Arabic and English. At least make sure that in English, spellings of the baby's name should be correct and name is same in English and Arabic both. If you find any mistake on baby's iqama then inform the Jawazat officer on counter who gave you baby's iqama, If everything is ok on Iqama then congratulations it's end of your journey. Once your baby's iqama is in your hands it means your baby is added under your Iqama/Passport and you can check it in your MOI online account in the dependents tab.
Iqama for Newly Born


text:
langauge:Find link is a tool written by Edward Betts.
searching for Daniel W. Thompson 1 found (3 total)
alternate case: daniel W. Thompson
Marianopolis College
(1,089 words)
[view diff]
exact match in snippet
view article
find links to article
their stay at Marianopolis. Notable Marianopolis alumni include: Daniel W. Thompson ('81), President at Jones, Gable & Company Ltd. Dr. Avi Wallerstein


text:
That's about 20 pounds per leg by my estimation. I'm trying to hold off posting cakes until after lunchtime going forward otherwise this octopus isn't the only thing that's going to weigh over 200 pounds.
via make
200 Pound Octopus Cake
via make
Related Gift Ideas and Products
Stealthy Vulcan Salute Sweatshirt « Previous
» Next Neff Knitted Headphones
Other cool gadgets to check out:
1 comment to 200 Pound Octopus Cake
Leave a Reply
Recent Posts
Gadget Categories


text:
Nathan's Books at Wikia
Welcome to the Nathan's Books mini wiki at Scratchpad!
You can use the box below to create new pages for this mini-wiki. Make sure you type
[[Category:Nathan's Books]] on the page before you save it to make it part of the Nathan's Books wiki (preload can be enabled to automate this task, by clicking this link and saving that page. Afterwards, you may need to purge this page, if you still see this message).
This wiki has been created so I can test how wiki's are made. I am currently using it to create a catalog of my books. Feel free to edit if you like, by proving more information on the books.


text:
Doctor insights on:
How Long Does It Take To Heal A Broken Patella
How long does it take to heal a broken patella?
Fracture: It generally takes 6 to 10 weeks to heal a patella fracture. Certainly the severity of the injury and how it is treated will influence healing time. Some patella fractures are treated with surgery and some are not. ...Read moreSee 1 more doctor answer
Patella (Definition)
Patella is a thick piece of bone, more commonly known ...Read more
How long for a broken patella to heal
Healing is different: Healing is different in petella , is sesmoid bone buried in a tendon , unlike normal bone heals by fibrosis which takes longer time , some need surgical removal of smaller fragment . Speak to your orthopedic surgeon, also possible you may have other associated injuries to the ligaments of knee ...Read more
For a broken patella, how long would an elderly patient be expected to be on pain killers? Patient has been on pain killers for a month now.
Pain killers: This really depends on the healing - elderly folks take longer to heal - however the requirement for the medication should decrease as the healing proceeds,. ...Read more
How long does it take for a fractured patella to heal?
Varies: It varies somewhat on the nature of the fracture, but usually about 6-8weeks. Also, x-rays will show when it heals as well. ...Read moreSee 1 more doctor answer
How long will a hairline fractured patella(knee cap) normally take to heal and what do I do to speed up the process?
6-8 weeks: A fracture usually takes 6-8 weeks to heal, but there are many factors that can slow this down. Smoking, use of certain medications and excessive motion can all slow down the process. Nothing you can eat or srink will speed the process, but quitting smoking, avoiding Ibuprofen or Naproxen and proper immobility will help maximize healing. ...Read more
I have a broken patella. is surgery necessary?
Yes: yes surgery is require the patella is like the fulcrum where the knee is able to bend.Good luck ...Read more
What do I expect when my physio says that i need hard work on my recent broken patella?
Depends on what your: Orthopod advises, so check with him. Is it healed or is it recently broken? One loses the thigh muscles very quickly if knee joint is immobilized for any length of time, so you'll definitely need intensive pt to exercise the weakened muscles back to normal strength. It can 2-3 months. Good luck. ...Read more
What can I expect when my physio says that I need hard work on my recent broken patella?
Motion once healing: Once the patella is stable or healed enough to allow for active motion, your surgeon will want you to begin moving your knee and mobilizing the patella to alleviate the development of knee stiffness. You may be able to walk with the knee locked straight (in a brace), but your surgeon will be the one to make theis determination. ...Read more
Elder man had surgery for broken patella. Transferred to a care/rehab facility. What will they do for him, length of time, and purpose of stay?
Rehab: They will help him get his strength back and make sure his balance good and give him correct techniques for mobilization. Length of stay depends on his situation but could be week or so. ...Read more


text:
Greed, Envy, At Risk
All I wanted was...
to be wanted
a best friend
to help
to have land
to be important
But I didn't want...
to be handles
to feel alone
to be "extra weight"
to have nothing to my name
to be unknown
In the beginning Candy had a shepherd dog but Carlson kills it and then candy gets depressed because his dog has been shot but after spending time with George and Lennie he is happy once more for a little while.
Candy does not interact very well with Curley's wife. He is afraid that he will be kicked off the ranch just by talking to her. Candy acts well with Lennie and treats him with respect. Candy treats George as if he was a boss and him candy was a employ.
Candy helps develop the theme by being one of the nice guys and he also creates a early sadness in the beginning of the book by allowing his dogs death.


text:
|Home | About | Journals | Submit | Contact Us | Français
The unfolded protein response (UPR) controls the protein folding capacity of the endoplasmic reticulum (ER). Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase) domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis.
This analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥ 7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL) of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase.
Our data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L.
The unfolded protein response (UPR) is an intracellular signaling pathway that provides homeostatic feedback regulation between the endoplasmic reticulum (ER) and the gene expression program in the nucleus. To this end, the UPR senses the conditions inside the ER, detecting an imbalance between newly made proteins and the protein folding capacity in the ER, and activates a corrective response. For signaling, the UPR uses a transmembrane sensor of ER-lumenal unfolded proteins, Ire1.
Ire1 is an ER membrane-resident receptor that serves as a primary signal transduction device in the UPR conserved from yeast to mammalian cells [1-4]. Oligomerization of Ire1-lumenal domains is thought to be a key event in initiating signal propagation across the ER membrane that enables the cooperative assembly of Ire1's cytosolic kinase and RNase modules into an ordered oligomer with a defined three-dimensional structure [5]. The oligomer is stabilized by phosphates resulting from autophosphorylation of the kinase domain and allows juxtaposition of Ire1's RNase domains, which presumably activate the RNase [5,6]. Once activated, Ire1 initiates the non-conventional splicing of HAC1 mRNA (yeast) or XBP1 mRNA (metazoan) by cleaving the mRNA at two conserved sites to excise an intron [5,7]. An RNA ligase (tRNA ligase in yeast and a still unknown enzyme in metazoan cells) rejoins the severed exons to complete the reaction. Intron removal allows for the production of the UPR transcription activators Hac1 and XBP1, respectively, which upregulate UPR target genes.
Ire1's RNase domain thereby serves the primary role in signal transmission. The mechanism underlying activation of Ire1 RNase by oligomerization and the mechanism of mRNA recognition and cleavage have remained elusive. To date, there are no known structural homologues of the Ire1 RNase domain that could help answering these questions. The best attempt at defining the mechanism of RNA cleavage has been made based on sequence conservation arguments and on a crystal structure of Ire1 dimer with a ligand-free RNase domain, which also lacked a fragment of the RNase active site [6]. In this work, the RNase dimer was proposed to contain two independent catalytic centers, one per Ire1 monomer, which would simultaneously accommodate the two RNA stem-loops conserved in all known mRNA substrates of Ire1 [6]. According to the proposed model, two stem-loops would form a kissing interaction for docking and recognition by Ire1. Subsequent studies proposed [5] that such a kissing interaction is not likely because Ire1 exhibits no preference for RNA substrates with dual stem-loops over substrates with a single stem-loop, indicating that a single stem-loop structure is the cognate folded substrate of Ire1 RNase.
It has been also suggested [6] that putative active site residues are positioned similarly in Ire1 RNase and in pre-tRNA splicing endonuclease (SEN), although these two endoribonucleases share no apparent sequence or structural homology and cleave dissimilar RNA substrates (stem-loop versus bulge-helix-bulge motif). The authors point out [6] that the putative catalytic residues in Ire1 do not align completely with those in SEN and would require a 5-Å displacement of Y1043 (in yeast Ire1) to bring it into a position corresponding to that of Y249 in SEN. A 5-Å conformational change in Ire1 RNase upon substrate binding has been proposed based on these arguments [6]. A new cocrystal structure of Ire1 RNase with an oligonucleotide bound, which we provide in this work, does not support considerable conformational changes in the position of Y1043. Therefore, Ire1 and SEN apparently cleave RNA using different arrangements of active site residues. Underscoring this difference, substitution of a single catalytic histidine residue H1061 produced orders-of-magnitude greater effect on the catalytic activity of Ire1 (this work) compared to only a 28-fold rate reduction in SEN [8].
Conservation of residue H1061, the only invariant histidine in the RNase domain of Ire1, was used to propose that H1061 marks the catalytic center and serves for general acid-general base catalysis [6]. In agreement with this model, mutation H1061A reduced RNA cleavage [6]. However, this mutation was analyzed using a qualitative approach that had a narrow dynamic range and did not distinguish between effects on binding and catalysis. Furthermore, mutation H1061A not only removed the ability of histidine to transfer protons but also the ability to form hydrogen bonds, which alone could explain the observed effect on RNA cleavage, without invoking H1061 in general acid-general base catalysis. Considering that ribonucleases can cleave RNA stem-loops by using for proton transfer basic amino acids instead of conventional ionizable residues (histidine, glutamate and aspartate) [9], the very involvement of H1061 in general acid-general base catalysis could not be deduced convincingly from any of the available data.
To understand the mechanism of RNA cleavage by Ire1, we combined quantitative analysis of rationally designed Ire1 mutants and X-ray crystallography. We show that proton transfer by histidine H1061 contributes greater than five orders of magnitude (≥ 7.6 kcal/mol) to catalysis of RNA cleavage, which experimentally defines a key catalytic functionality in the active center of Ire1 RNase. Using the non-disruptive Y1043F mutation we experimentally demonstrate a catalytic role of the OH group of Y1043 and also show an important role in RNA binding of a helix-loop element (HLE) residue, R1039. Our findings suggest parallels between catalytic centers of Ire1 and the well characterized RNases A and T1 and propose an unexpected mechanism of oligomerization-induced in trans RNA recognition by Ire1.
To glean insights into Ire1 interactions with HAC1/XBP1 mRNA, we analyzed the RNA binding properties of the entire surface of the Ire1 oligomer [5]. Surface electrostatics calculations [10] are commonly used for such analyses, but they produce ambiguous results because electrostatics is not the sole determinant of RNA binding. Here we used a modified optimal protein-RNA area (OPRA) algorithm that previously demonstrated high prediction accuracy [11]. We refined this method by adding phylogenetic conservation to the scores and termed the resulting algorithm 'conservation-weighted OPRA' (cwOPRA; to be described elsewhere). The cwOPRA analysis of RNA binding properties of the Ire1 oligomer produced a high-contrast image of the surface RNA binding propensity. A single RNA binding site was strongly predicted in the center of every back-to-back RNase dimer [6]. The RNA binding site is proximal to the dynamic helix-loop element HLE, which becomes more ordered upon formation of Ire1/Ire1 interfaces in the high-order oligomer and has been proposed to serve as a part of the active site [5] (Figure 1a, b). The rest of the Ire1 surface was predicted not to interact with RNA (we note, however, that the crystal structure is lacking a highly basic N-terminus, which is disordered and could serve as a secondary RNA binding site). We next validated this computational prediction using a cocrystal structure of Ire1 with an oligonucleotide.
For crystallization trials, we tested a panel of RNase-resistant RNA analogs with 2'-deoxy or 2'-methoxy substitutions throughout and a variety of Ire1 constructs. Only a single combination led to formation of diffracting crystals as discussed below. Attempts to soak mononucleotides, dinucleotides, trinucleotides or oligonucleotides (0.5-20 mM) into previously characterized Ire1 crystals [5] were unsuccessful. No additional difference density was observed, which could correspond to the soaked-in substrate analogs.
By contrast, cocrystallization of Ire1KR32 with a 6-mer 2'-deoxy-substituted oligonucleotide bearing the consensus sequence of an Ire1 splice site CCGCAG (the use of 2'-deoxy substitutions prevents degradation by Ire1 RNase) resulted in crystals of the Ire1KR32·dCdCdGdCdAdG complex. Ire1KR32 construct contains the Ire1's kinase domain, the RNase domain, and a 32-amino-acid linker previously shown to be required for optimal RNase activity [5]. The dCdCdGdCdAdG oligonucleotide bound to Ire1KR32 with Kiapp = 200 μM determined from inhibition of the Ire1KR32-catalyzed cleavage of HP21, a small stem/loop structure containing the 3' splice site of XBP1 mRNA [5]. This value is approximately 40-fold weaker than that for the cognate stem-loop (Km = 5 μM, Figure Figure1c1c and Additional file 1, Figure S1) possibly due to a more flexible structure of the single-stranded oligonucleotide dCdCdGdCdAdG compared to HP21 RNA stem-loop and/or due to the lack of 2'OH groups. Unlike the oligonucleotide-free Ire1, which crystallizes in space group P21212 [5], the Ire1KR32·dCdCdGdCdAdG oligomer crystallized in space group C222 and diffracted to 6.6 Å (Additional file 1, Table S1). The presence of the oligonucleotide allowed Ire1KR32 to crystallize without a ligand occupying the kinase ATP binding pocket, providing an important insight into the mechanism of cofactor-dependent activation of Ire1 [12].
We used the 3.2-Å structure of Ire1 crystallized in the absence of a nucleic acid substrate [5] for molecular replacement and subsequent rigid-body refinement, as described in Methods and in the accompanying manuscript [12]. At 6-7 Å resolution, the secondary structure of proteins and the sugar-phosphate backbone, which is the most electron-dense part of nucleic acids, can be visualized in ribonucleoprotein complexes [13,14]. Accordingly, we observed a strong Fo-Fc difference electron density at the core of the RNase domain, which coincided with the cwOPRA-predicted RNA binding site (Figure (Figure1b).1b). The electron density was delineated by two separate contours related by twofold symmetry (Figure 1b, d). Each contour (at level 7σ) defines an elongated volume of approximately 20 Å in length, consistent with the length of one dCdCdGdCdAdG molecule bound per RNase monomer. We placed Ire1 side chains according to the 3.2-Å-resolution search model used for the molecular replacement (Figure (Figure1d).1d). Such an approximation is valid because the protein backbone traces and the quaternary structure of Ire1 oligomers superimpose in the 6.6Å and the 3.2Å structures, even though they crystallized in different space groups (P21212 vs C222). Based on the complete superposition of the polypeptide backbones in Ire1KR32·APY29 [5] and in Ire1·dCdCdGdCdAdG complexes, we can rule out the possibility of a large-scale movement in the catalytic core of Ire1 upon binding of the substrate analog.
The electron density for dCdCdGdCdAdG is located in the cleft formed by the HLEs [5]. The position of the electron density suggests that eight protonatable residues of Ire1 located proximal to the substrate mimic could, in principle, contribute to general acid-general base catalysis leading to the formation of the 2', 3'-cyclic phosphate [15]. Because Ire1 does not require divalent metal ions for RNase activity [5], it must rely exclusively on amino acid side chains to carry out catalysis. In the identified group of amino acids, only H1061 and D1064 have pKa values near neutrality, analogous to the catalytic residues involved in proton transfer in well characterized ribonucleases, such as RNase A [16] and RNase T1 [17]. The H1061 is invariant in all known Ire1 sequences and the only Ire1 homologue RNase L [18]. By contrast, D1064 is mutated in several organisms to residues incapable of proton transfer (Additional file 1, Table S2), indicating that it is dispensable for catalysis.
Mutation of the catalytic histidine to alanine, asparagine or glutamine diminishes catalysis by ribonucleases T1 and A by 102 to 104-fold [17,19,20]. In a previous study, an H1061A mutation in Ire1 was reported to inhibit the RNase activity; however, the quantitative effect of this mutation on catalysis has not been measured [6]. To determine the quantitative contribution of H1061 to catalysis, we expressed and purified a mutant of Ire1KR32 in which H1061 was replaced by asparagine. We chose mutation to asparagine because it best preserves the space filling and hydrogen bonding properties of histidine, while removing histidine's ability to transfer protons and to support catalysis. We observed that Ire1KR32(H1061N) displays a ≥ 3 × 105-fold rate reduction compared to wild-type Ire1KR32 (Figure (Figure2a),2a), strongly indicating that H1061 plays a central part in catalysis.
Next, we ruled out a possibility that the large effect of the H1061N mutation arose from a perturbation of oligomerization. To this end, we monitored the optical density of Ire1KR32(H1061N) solutions at 500 nm, which provides a quantitative measure of Ire1KR32 oligomerization (Methods and [12]). Ire1KR32(H1061N) mutant exhibited an indistinguishable oligomerization profile from that of wild-type Ire1KR32 (Figure (Figure2b),2b), suggesting that the H1061N mutation does not interfere with oligomer formation.
To further ascertain that the dramatic rate reduction does not arise from more local conformational perturbations of Ire1 RNase, uncoupled from oligomerization, we crystallized Ire1KR32(H1061N) in complex with the synthetic ligand APY29, which binds in Ire1's kinase ATP binding pocket [5]. We determined a 3.65-Å-resolution crystal structure of this mutant (Additional file 1, Table S3), which proved identical to that of wild-type Ire1KR32 (Figure (Figure2c;2c; Additional file 1, Figure S2). Our functional and structural results pertaining to H1061N mutant therefore indicate that the greater than 3 × 105-fold catalytic impairment of Ire1KR32(H1061N) arises from a direct role of H1061 in proton transfer during RNA cleavage.
Due to the twofold symmetry of the Ire1 RNase dimer [6], a single H1061N mutation removes two H1061 residues in the vicinity of the RNase active site (Figure (Figure1b).1b). We thus wondered whether the catalytic impairment of the H1061N mutant might arise from this symmetry: if Ire1 required both H1061 residues together to catalyze phosphodiester cleavage, we would de facto have measured activity of a double mutant. To explore this possibility, we built Ire1 oligomers in which each catalytic H1061 would be paired with a non-catalytic H1061N in chimeric RNase dimers. To this end, we titrated catalytically inactive Ire1KR32(H1061N) into a solution of wild-type Ire1KR32 held at a constant concentration just below its oligomerization threshold. For enzymes that follow Michaelis-Menten kinetics, addition of a catalytically inactive mutant to a wild-type reaction inhibits the rate, as inactive enzyme sequesters the available substrate. By contrast, we observed approximately tenfold activation of Ire1KR32 before onset of inhibition at excessive concentrations of Ire1KR32(H1061N) (Figure (Figure2d).2d). Stimulation occurs because wild-type Ire1KR32 becomes trapped in a wild-type/H1061N hetero-oligomer as enzyme concentration increases and causes Ire1 to oligomerize. Kinetic modeling of this experiment shows that such a response is expected only if wild-type:H1061N heterodimers in the Ire1KR32 oligomer were active RNases (Additional file 1, Figure S3 and Additional file 2). We conclude that only one of the two H1061 residues in the twofold symmetric RNase dimer is required for RNA cleavage.
Proximity to the electron density of the substrate, mutagenesis analysis, evolutionary conservation and the neutral pKa value of histidine all converge on the suggestion that H1061 serves a catalytic function. Cleavage of an RNA backbone by other non-metal RNases involves pairs of conserved residues that carry out general acid-general base catalysis and lie at a fixed mutual separation of 6-8 Å (for example, PDB ID 1r5c, 1rga; Figure Figure3a).3a). In Ire1, only two invariant amino acids, R1056 and Y1043, are located 5.5 Å and 8 Å from H1061, respectively (Additional file 1, Table S2), and hence emerge as candidates to be a catalytic partner of H1061. Steric considerations discussed below and high pKa value of arginine argue that R1056 is unlikely to fulfill this role, suggesting Y1043 as the most plausible partner of H1061 (Figure 3b, c; see Discussion). We note that the R1056 side chain is deeply buried in the active site, which would make it difficult to interpret results of mutational analyses. We therefore did not pursue analysis of R1056 at this time.
Previously, it was suggested that if Ire1 RNase resembled SEN endoribonuclease, then tyrosine Y1043 may serve as the general base [6]. The fact that Y1043 resides approximately 5 Å from the analogous tyrosine in SEN was used to propose that Ire1 RNase should undergo an obligate conformational change upon binding of substrate to position Y1043 relative to H1061 as in SEN. To test this possibility, we examined the electron density for the Y1043-bearing loop in the Ire1KR32·dCdCdGdCdAdG complex (Figure (Figure3b;3b; Additional file 1, Figure S4). We found that backbones of the HLE and of the adjacent Y1043-bearing loop occupy the same position as in the 3.2-Å crystal structure obtained without bound oligonucleotide (PDB ID 3fbv). These findings support a model that tyrosine Y1043 does not move over a significant distance upon substrate binding.
To assess whether Y1043 contributes to catalysis, we prepared and quantitatively characterized the mutant Ire1KR32(Y1043F), which is predicted to disable any involvement of Y1043 in proton transfer while ensuring minimal structural perturbation to the protein compared to a potentially far more disruptive Y1043A mutation tested previously [6]. We obtained an activation profile with Ire1KR32(Y1043F) (Figure (Figure3d,3d, open circles). At enzyme concentrations exceeding 3 μM, the observed rate constant for cleavage of HP21 RNA plateaued at a first-order single-turnover rate constant k2 = 0.022 s-1. This value is approximately tenfold smaller than the corresponding value for wild-type Ire1KR32 (k2 = 0.25 s-1), demonstrating that the OH group of Y1043 is important for catalysis. Similar to the H1061N mutation, the Y1043F mutation had no effect on the oligomerization equilibrium of Ire1KR32 (Figure (Figure2b).2b). The deleterious effect of the Y1043F mutation was greater at subsaturating k2/K1/2 conditions encountered below approximately 3 μM enzyme concentrations, indicating that Y1043 also contributes to binding of the RNA substrate (Figure (Figure3d3d).
Location of the dCdCdGdCdAdG electron density in proximity to the HLE (Figure 1b, d) confirms the previously posed hypothesis [5,6] that this element serves as an important part of the active site. It was proposed that residues from HLEs could serve for binding and recognition of RNA substrates, rather than for catalysis. Mutations in the HLE are therefore expected to provide a valuable control to contrast the catalytically important mutations of H1061 and Y1043 described above. To test the role of the HLE in RNA cleavage, we prepared Ire1KR32 with a mutation of an arginine residue within the HLE, Ire1KR32(R1039A) and measured the titration rate profile for the Ire1KR32(R1039A) mutant (Figure (Figure3d,3d, diamonds). At saturating enzyme concentrations Ire1KR32(R1039A) achieved the same first-order single-turnover rate constant k2 as did wild-type Ire1KR32 (Figure (Figure3d,3d, closed circles), indicating that R1039 makes no contribution to stabilization of the transition state. Under subsaturating conditions, however, the R1039A mutant became deleterious, revealing a role of R1039 in substrate binding. Analogous to other mutations tested here, Ire1KR32(R1039A) exhibited an unperturbed oligomerization profile (Figure (Figure2b),2b), indicating that this mutation too does not affect the enzyme's oligomerization equilibrium. Both structural (Figure (Figure1d)1d) and functional data with Ire1KR32(R1039A) therefore show that the HLE plays an important role in recognition of RNA substrates.
Electron density-guided docking of an RNA stem-loop into Ire1 RNase (below) suggests that the HLE of one RNase monomer may contribute to RNA cleavage in the active site of an adjacent RNase monomer, acting in trans. To test this possibility, we performed in trans activation experiments as in Figure Figure2d,2d, in which we analyzed the activity of Ire1KR32(wild-type)/Ire1KR32(R1039A) heterodimers. To ensure that effect of the R1039A mutation is indeed measured in trans, we additionally disabled the catalytic site of the R1039A mutant by the H1061N mutation, as outlined in Figure Figure4a.4a. The experiment was conducted as in Figure Figure2d,2d, but contained lower concentration of wild-type Ire1KR32 (0.3 μM or approximately 0.06 Km) to ascertain a regime in which subsaturating conditions (k2/K1/2) were maintained during the titration. This was an important consideration because if the reactions were allowed to approach the first-order k2 regime, the deleterious effect of the R1039A mutation on binding would have been masked (note loss of effect from the R1039A mutation in Figure Figure3d3d when the reaction plateaued at E0 > 5 μM). Using this experiment, we found that the RNase activity of wild-type Ire1KR32 stimulated in trans by the double mutant Ire1KR32(R1039A, H1061N) was an order-of-magnitude lower than with the H1061N single mutant (Figure (Figure4b).4b). The chimeric dimer with the double mutation (G in Figure Figure4a)4a) therefore binds RNA more weakly than the chimeric dimer with the single mutation (E in Figure Figure4a),4a), whereas the only difference between these two cases is an intact HLE (E) versus a HLE bearing a single R1039A mutation (G). It follows that the RNA cleavage reaction catalyzed by one RNase monomer is sensitive to a mutation in the HLE of the paired monomer. These observations strongly support a model of a composite RNA-binding surface in Ire1 RNase, which also emerges from the electron density-guided RNA stem-loop docking described below.
Ire1 recognizes and site-specifically cleaves conserved sites in HAC1 and XBP1 mRNA, which are characterized by a stem containing approximately five Watson-Crick base pairs and a seven-residue loop with a consensus sequence CNGNNGN [21]. To understand how Ire1 might recognize such stem-loops, we searched bioinformatically for RNA heptaloops with similar features. This search showed that the anticodon stem-loop of tRNAPhe closely resembles the Ire1 consensus substrate (Figure (Figure5a).5a). To verify that this sequence similarity meaningfully identifies an Ire1 substrate, we conducted RNA cleavage assays and compared the kinetics of cleavage by Ire1 of tRNAPhe anticodon stem-loop and cleavage of the stem-loop derived from XBP1 mRNA, HP21. Ire1 cleaved both stem-loops in a single site and with similar rates (Figure (Figure5b),5b), indicating that the tRNAPhe anticodon stem-loop indeed provides a good model of the cognate Ire1 stem-loops from XBP1 and HAC1 mRNA. As multiple crystal structures of tRNAPhe have been already solved, this finding enabled us to model the docking of an RNA stem-loop to the Ire1 RNase active site (Figure 5c-e).
Our conclusion that each Ire1 monomer contains a complete catalytic center in the context of the Ire1 oligomer agrees with the model proposed previously based on experiments in which several Ire1 variants with dissimilar RNase-inactivating mutations were mixed [6]. The previous observation showed that none of the mutant mixtures have RNase activity and was interpreted in favor of individual active sites over a composite catalytic center (created by association of two RNase monomers) [6]. Importantly, however, this lack of RNase activity can be reasonably reconciled with both, per monomer and composite active site architectures, as well as with a third possibility of indirect defects in Ire1 RNase due to misfolding or conformational perturbation of the mutants. By contrast, the observation of electron density for a substrate mimic in each RNase monomer (Figure 1b, d) and in trans activation of wild-type Ire1KR32 by Ire1KR32(H1061N) (Figure (Figure2d)2d) provide strong experimental support for an individual catalytic center in each RNase monomer.
We deduce the position of the scissile phosphate of a bona fide Ire1 RNA substrate in this catalytic center using the structure of the Ire1KR32 complexed with a DNA hexanucleotide, dCdCdGdCdAdG, which mimics the cognate Ire1 substrate. This substrate analog was chosen due to present experimental limitations imposed by the inability of Ire1 to cocrystallize with other oligonucleotides. While we recognize that the deoxy-substituted single-stranded substrate may be structurally distinct from RNA stem-loops, we surmise that its binding site on Ire1 represents that of the HP21 RNA because (i) dCdCdGdCdAdG efficiently inhibits cleavage of HP21 by Ire1KR32, and (ii) the electron density in the RNase active site directly contacts the critical catalytic residue H1061, which is involved in proton transfer and contributes ≥ 3 × 105-fold to catalysis. At high contour levels, we could identify three near-spherical peaks in a Fo-Fc Fourier map. The central peak was strongest and was visible in most Ire1 monomers in the asymmetric unit, while the other two peaks could be observed in several but not all Ire1 monomers. Because the central peak most likely represents the most-ordered phosphate group of the oligonucleotide and is located between the functionally validated catalytic residues H1061 and Y1043 (Figures (Figures2a2a and and3d),3d), we suggest that it corresponds to the position of the scissile phosphate of the substrate.
Placement of this phosphate and comparison of the active site of Ire1 with the active sites of RNase T1 and A suggest putative roles of Ire1 residues in catalysis (Figure 3a, b). Similar to Ire1, ribonucleases T1 and A do not use metal ions [5] and produce 2',3'-cyclic phosphate [21]. Importantly, the mechanisms of RNA cleavage have been established in detail for both RNase T1 and RNase A [20,22-24].
Structural comparison shows that Ire1 and RNases T1 and A have a single positively charged residue contacting the phosphate: R1056 (Ire1), R77 (RNase T1) and K41 (RNase A). These positively charged residues serve for catalysis by stabilizing the negative charge buildup on non-bridging oxygens in the transition state, as has been shown for RNase A [20]. By analogy, R1056 of Ire1 may also participate in transition state stabilization and is unlikely to directly participate in proton transfer. The last notion is supported by steric considerations: R1056-phosphate-H1061 connectivity would form an atypically acute angle for a general acid-phosphate-general base triad, placing phosphate entirely off the line connecting the two protein residues implicated in proton transfer (compare Figure Figure3a3a and and3b).3b). This arrangement would not be compatible with in-line geometry required for RNA cleavage to form a 2',3'-cyclic product.
In RNase T1 and RNase A, a single uncharged hydrophilic residue is positioned off the line connecting general acid, scissile phosphate and general base, and at a hydrogen-bonding distance from the phosphate oxygens: Y38 in RNase T1 and N11 in RNase A, respectively. In RNases T1 and A, these residues play a catalytic role by stabilizing the transition state via coordination of non-bridging oxygen [25]. Position of the electron density in Ire1 (Figure (Figure3b,3b, right panel) predicts an analogous catalytic function for N1057.
Finally, all three RNases contain two residues that act as a general acid/general base pair. For RNases T1 and A, general acids are H92 and H119 at a distance of 3.7 and 3.8 Å from the scissile phosphate, respectively. General bases are H40 and H12 at a distance 5.6 Å and 3.8 Å, respectively. The position of the electron density for the phosphate between the H1061 and Y1043 (Figure (Figure3b)3b) agrees with the role of these residues in Ire1 as the general acid/general base pair (Figure (Figure3c).3c). Whereas the role of H1061 parallels that of the histidines in RNase T1 and A, a catalytic tyrosine is unusual because of the high pKa of approximately 10. Experimental testing of the catalytic role of Y1043 using an Y1043F mutation revealed that the hydroxy group of Y1043 contributes a rate enhancement of approximately tenfold (or approximately 1.4 kcal/mol) to catalysis. Our data therefore are in agreement with the catalytic role of Y1043 and suggest two kinetically equivalent catalytic mechanisms shown in Figure Figure3c.3c. We note that the tenfold transition-state effect of the Y1043F mutation is modest and thus it could also be consistent with a third possibility that the OH group of Y1043 plays an indirect role in catalysis and forms an important hydrogen-bonding network in the active site rather than participates in proton transfer directly. This alternative possibility cannot be ruled out until an atomic resolution cocrystal structure of an Ire1-RNA complex becomes available. If Y1043 serves for proton transfer directly, then its relatively small catalytic contribution compared to the ≥ 3 × 105-fold contribution of the partnering H1061 residue or the 103 to 104-fold contributions of the catalytic histidines in RNase A or T1 [17,20] could be accounted for quantitatively by the 103 to 104 higher pKa of tyrosine compared to histidine.
While our data support the roles of the H1061 and Y1043 as a general acid/general base catalytic pair, they do not unambiguously define which residue serves as the general acid or the general base. Such an assignment has proven challenging for RNases and ribozymes, even when high-resolution cocrystal structures with substrate analogs or products were available [20,26]. At present, we cannot formally distinguish between the two plausible mechanisms for RNA cleavage by Ire1 shown in Figure Figure3c.3c. As we argue below, the scheme shown in the left panel appears more compatible with steric considerations upon docking an RNA stem-loop in Ire1 RNase.
Our model suggests that the Ire1 RNase active site is unique and does not superimpose with other structurally characterized RNases. However, the arrangement of the catalytic residues in Ire1 and their interactions with the scissile phosphate resemble those in the active sites of ribonucleases T1 and A. By extension, we predict that the same mechanism applies to mammalian Ire1 and RNase L, the Ire1 homologue involved in the interferon response pathway, in which all relevant residues discussed here for Ire1 are strictly conserved.
The crystal structure of tRNAPhe (PDB ID 1ehz) shows the stem-loop in a conformation that would preclude cleavage by Ire1 (Figure (Figure5c,5c, yellow trace). The RNA cleavage reaction imposes strict stereochemical requirements such that an incoming nucleophile can attack the phosphate in line with the leaving group. A similar stereochemical problem was encountered in studies of sarcin/ricin GAGA tetraloop cleavage by ribonucleases α-sarcin and restrictocin. A cocrystal structure of restrictocin with the sarcin/ricin stem-loop RNA suggested that scissile bases should undergo base flipping to position the incoming 2'OH in the 2'-endo conformation and to allow for in-line attack during catalysis [27]. Guided by this concept, we searched the PDB database for a tRNAPhe structure, in which the loop guanosine at position 3 is flipped out such that its ribose ring adopts the 2'-endo conformation. We found that the crystal structure of tRNAPhe complexed with the tRNA modifying enzyme MiaA [28] serves as a suitable model (Figure (Figure5c,5c, blue trace).
Accordingly, we used the stem-loop from the tRNAPhe·MiaA structure for rigid-body docking into Ire1 active site. As a restraint, we placed the scissile phosphate into the strongest electron density peak (Figure (Figure5d)5d) and the rest of the loop to allow sterically permissible interactions expected from the cleavage mechanism (I) in Figure Figure3c.3c. The docked model has no backbone intersections or clashes of backbone atoms. By contrast, cleavage mechanism (II) could not be satisfied using this docking approach due to seemingly irresolvable interchain clashes. The resulting model predicts that only one stem-loop can bind to the Ire1 RNase dimer and that one RNase domain in the dimer cleaves RNA, whereas the other RNase monomer completes the RNA-contacting surface in trans, suggesting that both RNase monomers and both HLEs participate in recognition of the RNA stem-loop structure (Figure (Figure5e5e).
Our model that the HLE of one RNase monomer contributes to RNA cleavage by the catalytic center of a different RNase monomer is functionally supported by the in trans complementation assay described above (Figure (Figure4).4). This experiment demonstrates that cleavage by the active site of an RNase monomer is impaired upon removal of an RNA binding residue from an adjacent, non-catalytic monomer. Therefore, although Ire1 RNase has individual per-monomer catalytic centers, two RNase monomers are involved in RNA cleavage and apparently form a composite RNA binding surface created via dimerization. The high-order oligomerization of Ire1 completes the active site by stabilizing the highly dynamic HLE [5] in a conformation suitable for recognition of cognate RNA stem-loops. This mechanism could explain how self-assembly of Ire1 into an oligomer activates the RNase function.
We found that, strikingly, Ire1 can recognize and cleave the anticodon stem/loop structure of tRNAPhe in addition to the cognate splice site stem-loops of HAC1 and XBP1 mRNA. This observation redefines the known substrate specificity of Ire1 and suggests that additional stem-loops besides consensus sites in HAC1 and XBP1 mRNA could be cleaved by Ire1 RNase. Whether tRNAPhe is an in vivo substrate of Ire1 remains to be investigated. In principle, tRNA destruction could serve to reduce the cell's translational capacity and thereby reduce protein influx into the ER, akin to protein kinase-like ER kinase (PERK) activation in metazoan cells. However, the scissile guanine in the anticodon of tRNAPhe carries a post-transcriptional 2'-methoxy modification [29], which precludes cleavage of the modified tRNAPhe pool by any RNase producing 2',3'-cyclic phosphate, such as Ire1, leaving only the unmodified pool as a possible substrate. It is tempting to speculate that the tRNA modification evolved to protect tRNAPhe from cleavage by Ire1 and that regulation of the methylated pool of tRNAPhe could provide a control point by which Ire1 may affect the translational capacity of cells.
We investigated the mechanism of RNA cleavage by Ire1 RNase by combining computational, structural and quantitative biochemical approaches. This work experimentally determined the location of the catalytic center in Ire1 RNase and converged on a model of docking and cleavage of cognate RNA stem-loops. Histidine H1061 was defined as a key catalytic residue that is required in single copy in the twofold symmetric active site of the Ire1 RNase. Presented here in trans mutagenesis studies using (R1039A) and (R1039A, H1061N) Ire1 mutants, as well as electron density-guided in silico docking of an RNA hairpin with the scissile bond correctly aligned for catalysis suggest that the HLEs of two Ire1 monomers participate in stem-loop binding. This model intuitively predicts that only one RNA stem-loop binds to the dimer of Ire1 at a time (Figure (Figure5e)5e) whereas the high-order oligomerization of Ire1 enhances RNA recognition via allosteric stabilization of the HLE [5]. These findings identify the link between oligomerization of Ire1 and recognition of cognate RNA stem-loops, providing a glimpse of the central event in Ire1 signaling.
Individual rate constants were determined from single-exponential fitting of time courses of RNA cleavage. Kinetic parameters were reproduced two or more times and were consistent between different days. Rate variations were typically within twofold; this uncertainty is small compared to the effects we describe as significant. Experimental errors are provided in the text and figure legends, when appropriate.
Ire1KR32 and its mutants were expressed as GST fusion proteins using pGEX-6P-2 plasmid (GE Healthcare, Waukesha, WI) and codon-compensated Escherichia coli (BL21-CodonPlus RIPL) (Stratagene, Santa Clara, CA). Expression was performed at room temperature for 4 h after IPTG induction. Cells were lysed using an Emulsiflex C-3 (Avestin Inc. Ottawa, Ontario, Canada) and proteins purified by affinity purification with subsequent cleavage of the GST tag by Prescission protease (GE Healthcare, Waukesha, WI). All protein mutants were fractionated by gel filtration on an S200 column to approximately 99% purity. Proteins stocks (10-20 mg/ml) were stored at -80°C in the presence of 5% glycerol.
RNA oligonucleotides were purchased from Dharmacon (Lafayette, CO), labeled at the 5'-terminus using T4 polynucleotide kinase and 32P-ATP (PerkinElmer, Waltham MA) and purified by 20% polyacrylamide gel electrophoresis (PAGE) that allowed a single-nucleotide resolution, as described previously [5].
Kinetics of RNA cleavage was conducted as described previously [5]. Typically, reactions were carried out in 10 μl volume at 30°C. Reactions were started by adding 1 μl of 32P-labeled RNA to 9 μl of premixture containing 20 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) pH 7.4, 70 mM NaCl, 2 mM MgCl2, 4 mM dithiothreitol (DTT) and 5% glycerol. The reactions contained ≤ 1 pM radioactively (32P) labeled RNA and were conducted under single-turnover conditions (except for measurements of kcat and Km on Additional file 1, Figure S1, which were conducted under multiple-turnover conditions). Unless noted otherwise, Ire1 concentration was 3 μM. The enzyme concentration was determined from Ire1KR32 sequence using absorbance at 280 nm (e280 = 40.8 × 103 M/cm was calculated with BiochemLabSolutions ELN software, Princeton, NJ). Reactions were quenched at time intervals with 6 μl stop solution containing 10 M urea, 0.1% SDS, 0.1 mM ethylenediaminetetra-acetic acid (EDTA), 0.05% xylene cyanol and 0.05% bromophenol blue. Samples were analyzed by 10% to 20% PAGE, gels were scanned using Typhoon (Molecular Dynamics-GE Healthcare, Waukesha, WI) and quantified using ImageQuant (Molecular Dynamics-GE Healthcare, Waukesha, WI) and GelQuant.NET (BiochemLabSolutions, Princeton, NJ) programs. The data were plotted and fit in SigmaPlot to exponential curves to determine observed rate constants and to hyperbolic curves to determine binding constants.
Ire1 was in the reaction buffer containing 20 mM HEPES (pH 7.4), 70 mM NaCl, 2 mM MgCl2, 2 mM ADP, 4 mM DTT and 5% glycerol. Transparency of the samples containing higher concentrations of Ire1 visibly changed immediately upon adding the enzyme. Samples were allowed to sit for 15 min to allow for a complete Ire1 oligomerization. The optical density of the samples was subsequently measured at room temperature (22°C), at 500 nm on a UV-visible spectrophotometer Ultrospec 3300 Pro (Amersham-GE Healthcare, Waukesha, WI). OD500 was obtained after subtraction of baseline absorbance from the buffer free of Ire1. Sample absorbance did not change upon repeated scans of the same sample, indicating that the oligomerization reaches equilibrium under the conditions of the experiments.
Ire1·(dCdCdGdCdAdG) complex was prepared by mixing Ire1KR32 and dCdCdGdCdAdG (IDT). When performed at relatively low NaCl concentration (300 mM or less) addition of the oligonucleotide causes profound precipitation of the oligomeric complex, as observed upon addition of ADP·Mg [5]. The oligonucleotide thus apparently promoted formation of Ire1KR32 oligomers analogous to ADP·Mg and to APY29. The precipitate readily redissolved upon slight increase in NaCl concentration, indicating that the oligomerization reaction was salt dependent and readily reversible. Crystallization was conducted in hanging drops using stock solution of premade complex containing Ire1KR32 (12 mg/ml) and 0.6 mM 2'-deoxy-CCGCAG. The well solution contained 0.12 M sodium citrate (pH 6.5), 7% polyethylene glycol (PEG) 3350 and 4% glucose. Single crystals grew overnight and were cryoprotected in well solution containing 25% ethylene glycol. The crystals belong to orthorhombic space group C222 distinct from the orthorhombic space group P21212 previously reported for Ire1KR32 oligomer with bound APY29. Crystallization of a variety of other nucleotides and protein constructs was also tested but produced either inferior crystals or no crystals.
Diffraction from Ire1·(2'-deoxy-CCGCAG) crystals was collected on Beam Line BL 8.3.1 at the Advanced Light Source (Lawrence Berkeley National Laboratory, Berkeley, CA), at an X-ray wavelength of 1.115872 Å and an oscillation angle of 1°. The data were indexed, integrated and scaled using the XDS package [30] (Additional file 1, Table S1, Figure S5). A total of 5% of the reflections were marked as a test set (Rfree). A molecular replacement solution was found using PHASER [31] starting from monomer C of PDB ID 3fbv as a search model. Seven copies of monomer C were found in the asymmetric unit and the resulting 7-mer of Ire1KR32 was used for rigid body refinement in Phenix [32]. Simulated annealing was attempted and, expectedly, resulted in deterioration of statistics and excessive separation between Rwork and Rfree (Additional file 1, Table S1). Simulated annealing was therefore used only for calculation of omit maps (2000°C). Fourier σA-weighted [33] Fobs-Fcalc difference maps were used for interpretation of the parts of the model missing from the starting structure. Electron density and structure were analyzed and graphed in Coot [34] and PyMol (Schrödinger, San-Diego, CA). The sevenfold non-crystallographic symmetry (NCS) of the model was used to considerably increase quality of the electron density maps by averaging. However, all elements of Ire1 secondary structure were clearly visible without NCS. Coordinates have been deposited with the Protein Data Bank http://www.rcsb.org/ under accession number 3SDM.
The H1061N mutant was crystallized and analyzed as described for the 3.2-Å structure of Ire1 [5]. Diffraction data were collected on the beamline 8.3.1 at the Advanced Light Source (ALS, Berkeley, CA) using wavelength 1.115879 Å. A total of 5% of reflections were marked as test set. The 3.2-Å structure [5] was used for molecular replacement. The H1061N mutation was introduced in Coot [34] prior to refinement. Rigid-body refinement and subsequent simulated annealing refinement (starting temperature 3200 K) using 14-fold non-crystallographic symmetry were performed in Phenix [32] (Additional file 1, Table S3). The resulting crystal structure had a good stereochemistry and no residues in disallowed regions on the Ramachandran plot. In order to reduce model bias, we also calculated an omit difference map from a model refined at 3200 K without the use of NCS restraints, from which a whole α-helix containing the H1061N residue was omitted prior to refinement. The resulting electron density clearly shows that the conformation of the H1061N mutant is identical to that of wild-type Ire1. Coordinates have been deposited with the Protein Data Bank http://www.rcsb.org/ under accession number 3SDJ.
AVK designed and conducted the biochemical experiments and prepared protein and RNA constructs. AVK and PFE carried out Ire1 crystallization and diffraction data collection. JFM determined space groups and conducted initial diffraction data analyses. AVK and AAK carried out model refinement and building. CZ prepared synthetic small molecule modulators of Ire1. PW, RMS and KMS supervised the work. AVK and PW wrote the manuscript. All authors read and approved the final manuscript.
Gepasi modelling files. Compressed archive with Gepasi kinetics modeling files used in Figure S3.
Supplementary information. File with supplementary figures and tables.
We thank the staff of beamline 8.3.1 and Chris Waddling at UCSF molecular structure group for help in data collection and structure analysis. We are grateful to David Morgan and Natalia Jura for critical reading and commenting on the manuscript. We thank Niels Bradshaw for valuable discussions and suggestions during the work. AVK was supported by Jane Coffin Childs fellowship and the Howard Hughes Medical Institute, CZ was supported by a grant from the National Parkinson Foundation and the Howard Hughes Medical Institute. RMS, JFM and PFE were supported by an NIH grant P50 GM073210. PW and KMS are Investigators of the Howard Hughes Medical Institute.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
Now that I am going to start a thesis with my group in college, one thing is on my mind, what to do afterwards?
I really don't have a passion to look for a job like being a QC because you'll just do the same thing over and over again. Plus, I really don't like to wake up a specific time to work.
I really like to draw manga, like food-related stuff, and tried my best to create a game that I want to publish eventually. I want to do all these stuff after I graduate. What must I do?
What I planned is to write my story early for my future manga and try to publish one, finish my TD game and try to publish it. I will not waste my studies for nothing, so I'll try to use my thesis to create new food products or try to use other local ingredients to create new products.
My final say is, I'll just do my best...


text:
About the Book
This is a photo album with over 600 images from Macau, Hong Kong, and Japan. Although many of the images are typical snapshots of family or friends, most of the photographs are views of Macau (and Hong Kong and Tokyo) that would be of general interest.
Features & Details
- Category Arts & Photography
Standard Portrait, 7.75×9.75 in, 20×25 cm
200 Pages
- Publish Date Aug 12, 2007
- Tags Hong Kong, Asia, Culture, Macau, Photography, Tokyo, Travel
See More
Log in or create an account to post a comment.
0 Comments
Delete this comment?
See More


text:
With First Tutors: Music you can find a Keswick cornet tutor. Cornet lessons are a brilliant method to start a stimulating new hobby, pick up a new talent and gain confidence in cornet. Please use our service to find Keswick cornet teachers and choose which suits your requirements so you can start your cornet lessons in Keswick immediately!
This is the fastest way to locate Keswick cornet tutors in the UK, helping you find Keswick cornet tuition in your area and Keswick tutors to assist with any instrument, for example, piano, violin or guitar. We can help you find Keswick cornet lessons or a Keswick cornet tutor to prepare for cornet grades and develop your cornet skills! Our search will order Keswick cornet teachers local to you by their hourly prices and distance from you.
If you are a cornet tutor and would like to offer cornet tuition services in Keswick or throughout the UK, please register with First Tutors: Music or learn about us here. Becoming a Keswick cornet tutor is a brilliant way to re-affirm your own knowledge through giving cornet tuition while assisting someone else, all in the Keswick area!


text:
Predictors of mortality among elderly dependent home care patients
Healthcare needs and demand
The purpose of this study is to identify which variables –among those commonly available and used in the primary care setting– best predict mortality in a cohort of elderly dependent patients living at home (EDPLH) that were included in a home care program provided by Primary Care Teams (PCT). Additionally, we explored the risk of death among a sub-group of these patients that were admitted to hospital the year before they entered the home care program.
A one-year longitudinal cohort study of a sample of EDPLH patients included in a home care programme provided by 72 PCTs. Variables collected from each individual patient included health and social status, carer's characteristics, carer's burden of care, health and social services received.
1,001 patients completed the study (91.5%), 226 were admitted to hospital the year before inclusion. 290 (28.9%) died during the one-year follow-up period. In the logistic regression analysis women show a lower risk of death [OR= 0.67 (0.50-0.91)]. The risk of death increases with comorbidity [Charlson index OR= 1.14 (1,06-1.23)], the number of previous hospital admissions [OR= 1,16 (1.03-1.33)], and with the degree of pressure ulcers [ulcers degree 1–2 OR = 2.94 (1.92-4.52); ulcers degree 3–4 OR = 4.45 (1.90-10.92)]. The logistic predictive model of mortality for patients previously admitted to hospital identified male sex, comorbidity, degree of pressure ulcers, and having received home care rehabilitation as independent variables that predict death.
Comorbidity, hospital admissions and pressure ulcers predict mortality in the following year in EDPLH patients. The subgroup of patients that entered home care programs with a previous record of hospital admission and a high score in our predictive model might be considered as candidates for palliative care.
A major challenge for family doctors today is to provide appropriate care for the growing elderly dependent population living at home (EDPLH) that are unable to pay a visit to existing health centre facilities. EDPLH patients represent approximately 10% of the population over 65 years of age
There is a reported increased risk of death following hospital admission in these patients, and primary care interventions that provide integrated social and health care services to them have demonstrated a reduction in hospital admission rates
Comorbidity, dependency and poor self-perceived physical and mental health status have also been identified as independent predictors of mortality among the elderly population
The purpose of this study is to identify which variables –among those commonly available and used in the primary care setting- best predict mortality in a cohort of EDPLH patients benefitting from home care programmes in primary care. Our interest is therefore to explore how patient's characteristics, carer's and services dimensions relate to the probability of dying, both for all EDPLH patients included in the study and for a subgroup of patients that were admitted into hospital care the year before inclusion in the home care program.
This is a longitudinal one-year follow-up study of a cohort of elderly patients living at home (EDPLH). The methodology of the study has been published elsewhere
The research protocol of this study was approved by the Ethics and Research Committee in the Jordi Gol i Gurina Primary Care Research Institute.
Criteria for inclusion required patients to be non-institutionalised, chronically ill
Patient exclusion criteria included refusal to participate, transitory patients (followed by the primary care team for less than 9 months/year), patients with a life expectancy below 4 months, patients receiving transitory post-surgical care, and when both the patient and the carer had been diagnosed with dementia.
Informal carers are defined in the study as those looking after the basic needs of the patient, receiving no specific remuneration for that task and having a close relationship (next-to-kin or friend) with the patient.
Regarding patient recruitment, each of the 378 primary health care professionals who agreed to cooperate in the study selected, on average, 3 patients fulfilling the inclusion criteria. Participating professionals work throughout the Catalan territory, including rural and urban areas. They collaborated in the study on a voluntary basis and no randomisation was performed. They were asked to include in the study at least the first three patients that were found eligible under the agreed inclusion and exclusion criteria. Each of these professionals acted as collaborative researcher and a member of the research team trained them in their tasks. Health professionals were responsible for the administration of the questionnaires used in this study and the gathering of the necessary patient data from clinical visits, clinical records and reports from other health care providers.
Data collection
The following data were collected from each patient at baseline and after one year follow-up: patient clinical characteristics, comorbidity level (Charlson test)
In addition we looked into variables that measured the utilisation of health and social services including hospital admissions (defined as the patient remaining in acute hospital care for over 24 h), emergency room visits, home emergency visits data, primary care and community services activity data. The utilisation of social services included home help, tele-assistance, meals on wheels, volunteer care, social work visits, day centre and nursing homes, among others. These variables were collected for all patients one year before the baseline assessment and during the one year follow up period.
The utilisation of health and social services the year before the baseline assessment was extracted from patient's clinical records. Collaborative researchers in the study also collected the same utilisation variables prospectively during follow up.
Researchers participating in the study received training for data collection standardisation and collected data were analysed centrally. A continuous data quality assurance process was in place in order to minimize errors and information losses. A research team member audited and validated 10% of the data gathered by collaborative researchers from patients' original clinical records. The Kappa index and interclass correlation index were used to test the concordance among registers
Statistical analysis
A descriptive analysis is provided including average and standard deviation for all continuous variables. We analysed baseline differences between individuals who died and those who did not die during the study period by means of the Mann–Whitney-Wilcoxon non-parametric test. Categorical variables were analysed using the chi-square test and Fisher's exact test. We agreed on a p-value significance level of p < 0.05.
Following a bivariate analysis we included significant variables in a logistic regression model, in which the dichotomous dependent variable was vital status. We used stepwise techniques for inclusion and exclusion of variables in the model, testing the significance of each individual variable by means of a deviance analysis. We ran two separate analyses, one including all patients in the sample, and a second analyses including only patients with an informal carer. Accordingly, carer-related variables were excluded from the first analysis since not all patients had an informal carer. The statistical results are expressed in terms of odds ratios (OR) and 95% confidence intervals (CI). The statistical package used was R
We fitted a second logistic regression model in order to identify those variables that best explained the event of death among those patients that were hospitalised the year before entering the home care program. The resulting predictive model shows the probability of death among home care patients that were hospitalised before entering the program, in terms of patient's characteristics, carer's and service variables. We used the Hosmer-Lemeshow test to report the goodness of fit and ROC curve analysis to assess the discriminatory power of the model.
A total of 1,093 EDPLH patients were included in the study. Patients that completed the study (1,001) had a mean age of 83.7 years (±6.8 sd.), 66.8% were female (729). 290 patients died during the one year follow-up (28.9%).
As reported in Table
Regarding personal characteristics, health status and life-styles of patients included in the study, Table
Furthermore, patients who died during follow-up received a higher proportion of informal care, largely provided by women carers, and these informal carers reported a higher burden of care as measured by the Zarit test (see Table
With respect to health and social services utilisation (Tables
Variables found to be independently associated to the risk of dying during follow-up in this EDPLH group (N=1,001) were male gender, comorbidity (as measured by the Charlson test), the number of hospital admissions the year before, and both the existence of and degree of pressure ulcers (see Table
Variables that predict the risk of dying among those patients that were hospitalised before entering the home care program were male gender, comorbidity (as measured by the Charlson test), having received home care rehabilitation, and both the existence and degree of pressure ulcers. The resulting predictive model is represented by the formula below. The Homer-Lemeshow goodness of fit test is p=0.4837. The probability of dying during the next year can be calculated by the following equation:
The area below the ROC curve is 0.754 (95%CI=0.689-0.820) (see Figure
Figure 1
Predictive model of death in EDPLH patients admitted to hospital the year before entering the home care program: ROC curve
Predictive model of death in EDPLH patients admitted to hospital the year before entering the home care program: ROC curve.
EDPLH patients constitute a frail population with a high risk of dying. Our study shows that approximately a quarter of these patients died during the one-year follow-up period. Variables that independently predict death are found to be male gender, comorbidity, the existence of pressure ulcers and the number of hospital admissions the year before the baseline assessment. Among patients with an informal carer, those EDPLH patients with worse perceived and objective health status who are looked after by heavily burdened informal carers have a greater probability of dying during the following year. Since hospital admission was identified as a risk factor for death, we went further and built a predictive model of death for that sub-group of patients. This model yielded a formula of an acceptable discriminative power that includes sex (male), comorbidity (as measured by the Charlson index), degree of pressure ulcers, and having received home care rehabilitation. The formula also accounts for the fact that the Charlson index predicts a higher risk of death among women that men. This is a simple formula, that can be easily calculated by the doctor at the bed-side when deciding which would be the best therapeutical path to follow.
The main limitation of this research is that the sample of patients included in the study is not representative of all patients visited by primary care teams, since it was not a randomised sample. However, the sample was large enough to fulfil the objectives of the study. In addition, as patients who were lost to follow-up were probably sicker, this might imply that some of the odds ratios obtained in the study are under-estimates.
Variables found as significant predictors of mortality in EDPLH patients might be associated to other variables such as malnutrition, frailty syndrome and severity of comorbidity, that were not collected in this study.
Contrary to other published research on very elderly populations
Previous research with the same cohort has found that hospital admissions are related to lack of coordination among services as well as demand induced by family relatives and patients themselves
Published studies have found that admission to hospital is not always to the benefit of these EDPLH patients because admission could aggravate the pre-existing pathological status of these patients
We have contributed to this literature by arguing that the risk of dying does not disappear after hospitalization but prevails thereafter. It is therefore of utmost importance to take the patient's record of previous hospital utilisation into account when entering a given home care program. Our predictive formula allows for the identification of those patients with a greater probability of a reduced life expectancy (below one year). It could therefore be of assistance in selecting those patients who would benefit from palliative care.
It is worth noticing that none of the variables related to the utilisation of primary health care services during the previous year are related to better survival. On the contrary, the number of hospital admissions increases the risk of death in this EDPLH population. Further, primary care services are bound to have no differential effect on the risk of death since they are available to all patients in largely the same regime. The few published studies looking at the connection between home care services and mortality, have found no relationship
In the light of the above findings we would recommend future research into management policies and practices addressing this group of patients. We encourage research on the effectiveness of preventive measures among EDPLH patients in three areas. First, to assess whether a more adequate detection, assessment and treatment of comorbidity would improve survival. Second, to investigate the effectiveness of pressure ulcer prevention actions such as informing carers and family relatives about adequate diet and postural management strategies, and counselling health care services on the public provision of specific mattresses for these patients. Finally, we suggest further research on management strategies for a subgroup of patients that could benefit from a more end-of-life oriented care in their home setting without hospitalisation. This might avoid unnecessary direct and indirect costs to the health care system and to the family. Information systems already in place could serve the purpose of identifying these high-risk patients, allowing for appropriate and timely professional action.
Family doctors and community nurses could play an active and central role in the management of EDPLH patients using mortality risks assessment in the management of each individual patient. The predictive model presented here could help doctors make decisions on therapeutic alternatives for these EDPLH. This would improve the quality of existing home care programs. High-risk patients could benefit greatly from a more end-of-life oriented care in their home.
EDPLH: Elderly dependent patients living at home; PCT: Primary care teams; SF-12: The short form (12) Health Survey is a survey of patient health; OR: Odds ratio; CI: Confidence interval.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JGB, ABS and EL participated in the study design, data acquisition, analysis and interpretation, and drafted the manuscript. JCCS, CAT, LCG and PGP were involved in the study design, analysis and interpretation, and drafted the manuscript. The HC>65 Research Team participated in data acquisition. All authors read and approved the final version of the manuscript.
The authors would like to thank all researchers who participated in the data collection process in the study. A full list of team members has been published elsewhere. This research project has been financed by a research grant from the Catalan Agency for Health Technology Assessment and Research (CAHTA) in 2004.
HC>65 Research Team
The full list of participants has been published elsewhere: Gené J et al. Los problemas de salud también explican la utilización de servicios sociales en atención domiciliaria Aten Primaria. 2009 Feb; 41(2):91–101.
Prevalencia de fragilidad en una población urbana de mayores de 65 años y su relación con comorbilidad y discapacidad
Toward a conceptual definition of frail community dwelling older people
Do Geriatric Interventions Reduce Emergency Department Visits? A Systematic Review
Effectiveness of a nurse-led case management home care model in Primary Health Care. A quasi-experimental, controlled, multi-centre study
Comorbidity and social factors predicted hospitalisation in frail elderly patients
Factores asociados a ingreso hospitalario en una cohorte de pacientes ancianos incorporados a un programa de atención domiciliaria
Pressure ulcer and mortality in frail elderly people living in community
Six –month mortality risks in long-term care residents with chronic ulcers
A new method of classifying prognostic comorbidity in longitudinal studies: development and validation
Geriatrics index of comorbidity was the most accurate predictor of death in geriatric hospital among six comorbidity scores
Predictors of Mortality in Community-Living Frail Elderly People with Long-Term Care Needs
Predictors of mortality in outpatient geriatric evaluation and management clinic patients
Self-Rated Health: A Predictor of Mortality Among the Elderly
Health, cognitive, and psychosocial factors as predictors of mortality in an elderly community sample
The predictive value of self assessed general, physical, and mental health on functional decline and mortality in older adults
Comorbid chronic diseases, discordant impact on mortality in older people: a 14-year longitudinal population study
Development of a new predictive model for polypathological patients. The PROFUND index
Seguimiento de una cohorte de Atención Domiciliaria
Chronic comorbidity and outcomes of hospital care: length of stay, mortality, and readmission at 30 and 365 days
Validación del Índice de Barthel
Adaptación y Validación al castellano del cuestionario de Pfeiffer (SPMSQ) para detectar la existencia de deterioro cognitivo en personas mayores de 65 años
Clinical utility of the Braden scale for Predicting Pressure Sore Risk
Interpretación de los cuestioanrios SF-36 y SF-12 en España: compronente físico y mental
La escala sociofamiliar de Gijón, instrumento útil en el hospital general
Escala Zarit reducida para la sobrecarga del cuidador en Atención Primaria
Prognostication in acutely admitted older patients by nurses and physicians
Prognosis of primary care patients aged 80 years and older with lower respiratory tract infection
Predicting mortality in older patients. The VELCA Study
Predictors of in-hospital mortality among hospitalised nursing home residents: an analysis of the National Hospital Discharge Surveys 2005–2006
Advanced Illness Index: Predictive Modeling to Stratify Elders Using Self-Report Data
Rehospitalizations among Patients in the Medicare Fee-for-Service Program
Impactibility Models: Identifying the Subgroup of High-Risk Patients Most Amenable to Hospital-Avoidance Programs
Outcomes from a program of home care attendance in very frail eldery subjects
Medicaid Home care Services and Survival in New York City
Day care Services Use is Associated with Lower Mortality in Community-Dwelling Frail Older People
Los problemas de salud también explican la utilización de servicios sociales en atención domiciliaria
Pre-publication history
The pre-publication history for this paper can be accessed here:


text:
6 posts 0 hypeJoined Sep 2012
- Last login Nov 12, 14 @ 6:25 am
[Supreme] 2012 Discussion Thread (Flaming Users = Ban / Selling Items = Ban)
snagged the orange painted camo the other day2 Weeks ago in Supreme
Official 5 Panels/ Camp Caps Thread
[Quote] I'd be willing to make some sort of arrangement.2 Weeks ago in Fashion
Official Blogger Thread*
lets get some traffic up in here2 Weeks ago in Visual Arts
Official Blogger Thread*
Bringing it back because I tried tumblr and I still dig blogspot better. Check mine out: [URL]. I want some people i can follow because so far on the seach bar i've only found a couple worth following. I know most of you dudes got interesting stories and shit to share2 Weeks ago in Visual Arts
Official Blog Thread
[URL] Music, Clothes, Movies, etc, Cool.2 Weeks ago in Visual Arts
Is this for real?
I got a check in the mail for 100 bucks from google for my blog. Is this shit real?2 Weeks ago in Fashion


text: june, 2014 This is a repeating eventJune 12, 2014 09:15:am 12jun9:15 am- 10:15 amLadies Golf Clinic Time(Thursday) 9:15 am - 10:15 am Event DetailsJune 12th - August 28th No Event Details June 12th – August 28th No clinic on July 3rd, July 24th or August 7th 9:15 – 10:15 a.m. at the Practice Field $10 per person Fun golf instruction for ladies takes place in the months of June, July and August – for everyone new to the game and those with years of experience. This is the place to both learn the basics and brush up on your current skills. The golf professional staff will focus on one aspect of the golf game for the first half-hour with open instruction for the final half-hour. Tee times are reserved for those wishing to enjoy play together following the clinic. Calendar GoogleCal No Comments Sorry, the comment form is closed at this time.


text:
Do you know any horse shows that are walk, trot and are in Ohio near Akron or Chagrin falls? If you could help that would be awesome. :)
Click here to post comments
Return to Share Your Horse Show Story!.
Are you horse crazy? Join our free horse club! Fun horse games and lots more!
By Sydney, Copyright 2007-2017 - All Rights Reserved


text:
Brisbane medical centre promotes staff wellbeing
Published: Monday 14th November 2016
The ACU Medical Centre, located in M Block on the Brisbane campus, is open Monday to Friday, providing convenient and affordable medical care for ACU staff and their families.
Dr Tania Burgess and Dr Rebecca Dunn are highly experienced General Practitioners and can assist you with all your medical needs including acute medical care, injury management, chronic disease management, travel advice and vaccinations, insurance and license medical examinations, women's and men's health issues, mental health care, return to work programs and much more.
Psychology appointments are also available with clinical psychologist Margaret Rebgetz.
The clinic is operating normal hours through to 9 December, so this is a great time to make sure that your health care needs are up to date before the holiday break. Please call (07) 3623 7740 for an appointment.


text:
Aspire – News & Magazine Clean Ghost Theme Free Download
Aspire is a modern, clean, and content and image focus theme for Ghost blogging platform. Aspire is created for those who want to create a beautiful online magazine, or news website. Aspire it is 100% responsive, that means it fits with all different kind of devices in different sizes. Aspire is compatible with Ghost version 0.7.x.
Aspire Features
- Clean and minimal design
- Ready for Ghost latest version – 0.7.3
- Instant Search with GhostHunter
- MailChimp newsletter subscription form
- Custom CSS (Sass Source Files)
- Clean well commented code, easy to customize
- HTML5 / CSS3 valid code
- Mobile first & 100% Responsive layout
- Sidebar with 6 types of widgets
- Instagram feed
- Twitter feed
- Multi author support
- Author page
- Google fonts (Merriweather)
- Evil Icons
- Tag page support
- Disqus comment system
- Static page support
- Post sharing
- 404 custom page
- SEO Friendly with Structured Data
- Next & Previous Post Navigation
- Easy setup with easy to follow instructions
Aspire have used the following scripts, fonts or other files as listed.
- Preview Images form &
----- 1.1 (28/1/2016) ----- Fix un-styled content when page load issue ## Files assets/css/main.min.css assets/js/app.min.js assets/js/scripts/script.js assets/sass/base/_base.scss assets/sass/sections/_search.scss default.hbs
----- 1.0 (12/24/2015) ----- Initial release


text:
Simplicity 6915; ©1985; Misses' Set of Easy-To-Sew Wrap Skirts: Skirt has panelled front and back wrap, with unpressed pleats into tie waistband. V. 1 has patch pockets. V. 2 has bias cut front panel.
Links to reviews/blog postsEdit
Add a photo to the gallery by clicking the "modify" button below.
[insert your username, and make sure your preferences allow for people to contact you via e-mail. Do not list your email address here!]