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text:
Photographer's Note
Displayed here are the facades of 4 buildings.
-The silver building with the external walkway is the SVB building, designed by Abe Bonnema
-The building with the blue side faηade is the Hogeschool InHolland building by Erick van Egeraat.
-The temporary Portacabin building with the coloured corrugated plating is also used by the Hogeschool InHolland.
-The red brick clad building in the back is the Tax collectors and customs office by Cees Dam & Partners architects.
The buildings were erected between 1997 and 2004.
In a WS I posted a pic which offer a broader view of this piece of high density urban renewal which is part of the De Kop van Zuid. On this specific part of this quarter, the east side of the Rijnhaven (Rhine-docks) once three massive warehouses were located, an example of a still existing Rotterdam warehouse (veem), the St Jobs-veem in the Lloyd quarter, another former harbour area in reconstruction is here
The buildings line the Posthumalaan (formerly: Rijhaven Oost zijde) . The portacabin-building was an afterthought arisen from the need to accommodate the fast growing number of students of the Hogeschool InHolland, rather spoiling the lines of sight and the feeling of design quality of this quarter. I used it here as a framing device.
De Kop van Zuid is the city quarter where I live and is a very large urban restructuring project in progress which is well underway in converting the old harbor area on the South bank of the Maas (with its defunct ports, warehouses and railway heads) into a integrated living, shopping and commercial service quarter. Its comparable with the Docklands in London and the waterfront re-developments in Barcelona and New York.
This is my entry for the Fall Scav hunt 3.Building face. It was shot with the new Kodak DX6440 - it is a free replacement of my faithful Kodak DC-4800 that expired on one of the workbenches of the Kodak service centre. Kudos to this company, the DC-4800 was more then 3 years old & battle-scarred ;-)
PS-E2 tech: skewed, heavily cropped, saturation (master, reds, yellows) boosted/altered, USM-ed, frame added and saved for the web.
Critiques | Translate
Cool-ice (5289) 2004-11-11 11:10
Again a nice graphism, Bert !
The perspective is excellent, it looks like a construction game ;o)
And a good complementarity between the different colors.
Well done, and thank you for the complete note.
ydavid (294) 2004-11-11 13:02
This excellent composition shows modern architecture at it's best. The cropping and saturation are perfect.
jhm (194879) 2004-11-11 13:08
Hallo Bert, hier hebben wij het met wel een zeer progressieve bouwstijl te maken, de kleuren zijn wel zacht op zijn geheel, de lijnen zowel horizontaal als verticaal geven wel een goed perspectief, uw nota Bert is zeer verhelderen, alles zeer scherp en duidelijk.
Goed gedaan Bert!
SylFondacci (4087) 2004-11-11 13:14
Beautiful graphical shot, playing with geometrical shapes, lines and bright colours.
Well done.
Hanssie (11018) 2004-11-11 14:09
Ik zat naar beneden te scrollen en zag dit en jawel, daar is'ie weer !
Dit heb je heel erg leuk in elkaar gezet, Bert, een paar zeer moderne onderdelen van gebouwen, tevens moderne kleuren, mooi werk, ik had het niet gevonden !
sunny (3069) 2004-11-11 14:12
Lovely contrasting colours and strong graphical impact, Bert! Amazingly crowded composition, yet so neat, with every detail set in the right place and superbly lit up. Sharp and clean image. Wonderful capture!
ps: maybe next time it will be Hogeschool Rotterdam...?;-)
nwoehnl (122) 2004-11-11 14:22
It comes in your distinctive style again, Bert. You really have a good eye for spotting these angles and making interesting architectural compos from them. A very intriguing interplay of verticals and diagonals, colors and shapes. And of course a good add for your theme. Very well presented with the technical parameters handled to the best as usual.
pamastro (7290) 2004-11-12 0:12
Another excellently composed shot. The tight view is great and really shows your great eye for these things. The lines are excellent and the colors, too. Especially the orange. I really like how the most distant building is framed way in the back. The layers all add excellent depth. Very nice job.
carper (96) 2004-11-12 3:40
Een ware architect Hans,
Leuk weer eens wat van je te zien. Prachtig lijnenspel, met mooie vlakken, lijnen die extra diepte geven, Moet ik nog langer doorgaan Hans ik vind hem mooi. Goed werk
maphoto (8082) 2004-11-13 23:15
Very nice Bert, great graphic, lines and very modern, colorful, nicely cropped, this is good work, thanks for sharing.
ma-tibou (4568) 2004-11-15 7:37
Bonjour Bert, une composition trθs graphique beau rιsultat, bien vue.
Amitiι/ Martine.
veimann (1269) 2004-11-15 17:47
Hi Bert!
I have again come last and I have completely nothing to add to already told :) Unless that your geometrical compositions very much like me.
Rinie_Hoff (9340) 2004-11-18 19:32
Hi Bert, almost forgotten! Like a lot of other pictures. Too busy, and on the other hand, too unproductive. Either way, I hate it.
I like this strong composition of lines altogether, the zigzag of all directions, the color combinations used here, are very fresh, but also knowing you a little bit, you have boosted some. I myself have a picture of the temporary Port-a-cabin from the other side, and the colors are wonderful. I find some noise at the ceiling, though.
I thought the Kodak went to W.?
rudymalou (800) 2004-11-25 7:20
Hi Bert, welcome back. This picture is definitively in what I call now the Bertolucci Style. Norbert has perfectly commented this picture so I wont repeat
alvaraalto (39444) 2005-01-31 16:44
Three in one. You hide in this picture the awful colours InHolland is using. On the other hand..... Ugly colours can give interesting pictures. There are in that neighbourhood many subject to make great shot like this one, but you must have an eye for it. And you have.
Stepan (27210) 2005-10-02 14:20
Modern, abstract, graphic...
A little cold but I like the design here !
Very well composed in a good dof.
sergio1 (12152) 2006-04-13 5:20
Hi bert
You never let to me surprise. Its point of view is exquisite. The sharpness, contrasts and compo also is excellent. Thank you very much by everything. Greetings and happy day.
Photo Information
- Copyright: Bert Hoetmer (bertolucci) (14050)
- Genre: Τοποθεσίες
- Medium: Έγχρωμο
- Date Taken: 2004-11-07
- Categories: Αρχιτεκτονική
- Camera: Kodak DX6440, Schneider 33-132mm int, internal memory 16M
- Έκθεση: f/4.8, 1/180 δευτερόλεπτα
- Έκδοση φωτογραφίας: Πρωτότυπη έκδοση, Workshop
- Θέμα(τα): Outdoor Type, Rotterdam-Kop van Zuid [view contributor(s)]
- Date Submitted: 2004-11-11 9:29
- Αγαπημένα: 1 [view]
- To sunny: HRo (1)
by bertolucci, last updated 2004-11-11 02:32
- To Hanssie: dank dank (1)
by bertolucci, last updated 2004-11-11 02:48
- To nwoehnl: thanx Norbert (1)
by bertolucci, last updated 2004-11-11 02:53
- To jhm: bouwstijlen (3)
by bertolucci, last updated 2004-11-14 11:41
- To carper: namen ;-) (2)
by bertolucci, last updated 2004-11-15 06:39
- To Rinie_Hoff: Kodak (1)
by bertolucci, last updated 2004-11-19 08:09
- To rudymalou: merci Rudy (1)
by bertolucci, last updated 2004-11-26 09:25

subset: commoncrawl


text:
- The sequence of one heart beat ending to the beginning of another is known as the cardiac cycle
- The heart consists of two pumps that work together where both sides of the heart relax and contract with one another
- Systole is the repetition in contractions of the heart chambers
- Diastole is the relaxation of the heart chambers
- Blood moves through the circulatory system from regions of high pressure to low pressure
- There are three stages to the cardiac cycle; atrial systole, ventricular systole and diastole
- Atrial systole is where the atria within the heart contract and tops up the ventricles that are already filled with blood.
- Ventricular systole is where the ventricles chambers contract and forces the blood up and out of the heart and into the arteries
- Diastole is where all four chambers in the heart relax and re-fill with blood
- SAN causes the atria to contract and the AVN causes a delay before the ventricles contract
Stages of the cardiac cycle
All the events that come and play within the heart come together to become the cardiac cycle. The cardiac cycle is a series of contractions that ensure blood is flowing in the correct direction
The cycle is broken down into three stages:
• Cardiac Diastole – the entire heart is relaxed
• Atrial Systole, also known as Ventricular Diastole
• Ventricular Systole, also known as Atrial Diastole
Cardiac Diastole
In cardiac diastole the entire heart is relaxed. Both the atria and ventricles are relaxed and the blood enters at low pressure through the veins, the pulmonary vein and the vena cava into the atria. As the blood flows in the atria the blood pressure begins to increase causing the AV valves to become opened allowing for the blood to enter the ventricles.
Atrial Systole
This is where the atria contracts when they are around 50% empty. This ensures all the blood is emptied from the atria and enters the ventricles. This causes the pressure within the ventricles to slightly increase shutting the AV valves, to prevent the backflow of blood back to the atria.
Ventricular Systole
The next stage is ventricular systole, where the ventricles contract from the bottom of the heart also known as the apex of the heart and upwards. Now the pressure further increases in the ventricles above the pressure within the arteries (pulmonary arteries and aorta). Due to the pressure change, blood is able to flow out through the semilunar valves and allows for the blood to leave.
Actions of the valves aand pressure changes
Pressure changes – The pressure graph
Mammals have a double circulatory system; blood is kept within the blood vessels allowing the pressure within them to be maintained and regulated. Figure 2 illustrates the pressure changes that occur within the heart during one cardiac cycle.
- Pressure changes in the left side of the heart
- The atrial pressure shown in Figure 2 (yellow line) has the least changes in the pressure. The pressure is always relatively low due to the thin walls of the atrium and therefore, cannot create a force to increase the pressure. The atria fill up with blood that leads to a slight increase in blood however, there is drop again when the left AV valve opens allowing some of the blood to enter the ventricles.
- The ventricular pressure (green line) begins low at first, but then undergoes a huge pressure change during ventricular systole since the ventricles fill with blood as the atria contracts. This shuts the left AV valve and the pressure rises dramatically due to the ventricles thick muscular walls. As the pressure arises the aorta blood is passed through the semilunar valves and enters the aorta. Pressure then falls as the ventricles empty and the walls relax.
- The pressure in the aorta remain high to begin (shown with the red line)
- It is important to remember the key time when the valves open and when they close; the AV valve closes when the ventricular pressure exceeds the atrial pressure, indicated on Figure 2. The AV valve will only open again when the ventricular pressure is below the atrial pressure.
- Looking above on the top part of the graph shows when the semilunar valve opens and closes. The semilunar valve will open when the ventricular pressure exceeds above the aortic pressure and the semilunar valve will close when the ventricles pressure would be below the pressure within the aortic artery.
- The sounds of a heartbeat "lub" and "dub" are made by the sounds of the valves snapping shut (below).
Coordination and regulation of the cardiac cycle
For the heart to function effectively, there must be a fine control and balance of events that take place during the cardiac cycle described above.
Heart tissue is myogenic which means it would initiate its own contraction allowing the heart to contract without needing to be connected to the body. If cardiac muscle was left to contract by itself this could bring some problems to the heart. Since the atria contract faster than the ventricles this could lead to fibrillation hence there is a need for a way to control the cardiac cycle.
In the heart there are two nodes that are responsible to keep the cardiac cycle running correctly. A heart beat starts off at the region of tissue called the sinoatrial node (SAN) which is located above the right atrium. The SAN acts as the hearts pacemaker and ensures the heart is beating at a constant regular rate. This is achieved by the SAN giving out regular electrical signals spread throughout the heart and spreads throughout the atrial muscles that causes the atria to contract (atrial systole). This is what starts the contraction of the atria.
The second node is the known as the atrioventricular node (AVN) which is located near the AV valve. The role of the AVN is to pass the electrical signal to the middle of the heart also known as the septum. There is also a delay in the electrical pulse at the AVN (between the atria and ventricles contracting) which allows the atria to fully empty before the ventricles control. The electrical pulse next passes the signal into extremely insulated fibres called the Bundle of His. The insulation of these fibres ensures the electrical signal cannot escape. The Bundle of His carries the electrical signal to the apex, bottom of the heart. At the bottom of the heart the Bundle of His splits into two. The non-insulated fibres called Purkinje fibres spread up the walls of the ventricles and cause the ventricles to contract from bottom of the heart to cause the blood to be forced out from the ventricles, out of the semilunar valves and towards the aorta and pulmonary artery.
Electrocardiograms (ECG)
An electrocardiogram (ECG) is a medical device that measures the electrical pulse of the heart. Since the heart undergoes a series of electrical changes related to the waves of excitation, sensors that are connected to a monitor can detect these electrical signals. The ECG produces a trace of electrical activity shown in Figure 6.
Doctors can use the ECG trace to examine the patients electrical activity within the heart and understand better about the status of the patients health.
A normal healthy ECG trace shows what is known as the "PQRST shape" (Figure 5). Wave P is the stage that corresponds to atrial systole, excitation and contraction of the atria. Ventricular systole forms the "QRS" complex and the start of ventricular diastole corresponds to the "T" wave.
Below in Figure 6 shows the ECG from a healthy patient, patient suffering from a heart attack and one patient suffering from fibrillation. Fibrillation, already mentioned above is where the atria and ventricles contract and relax in an irregular manner causing strenuous stress to the heart that leads to death if not treated immediately (Figure 6c).
[1]. https://openstax.org/books/biology-2e/pages/40-3-mammalian-heart-and-blood-vessels
[2]. https://commons.wikimedia.org/wiki/File:Wiggers_Diagram_2.svg#/media/File:Wiggers_Diagram_2.svg
[3]. https://openstax.org/books/biology-2e/pages/40-3-mammalian-heart-and-blood-vessels
[4]. https://en.wikipedia.org/wiki/Electrocardiography


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The Neighborhood Council Board is made up of up to fifteen (15) volunteer Board Members elected by popular vote, who serve 2-year terms. The Board should include representation from the recognized neighborhood areas within the North End Neighborhood Council, and a majority of the Board Members shall be neighborhood residents.
Neighborhood residents are encouraged to take a leadership role in serving as board members and officers of the Neighborhood Council. Annual Board elections are held in November, and the board elects its officers in December. For more information view the NENC Bylaws.
2019 Board Members
Kyle Price, Chair
Jodi Cook, Vice Chair
Doug Crane, Treasurer
Michael Lafreniere, Secretary
Stephanie Breedlove
Jim Bush
Bea Christophersen
Cindy Feist
Gary Foreman
Alex Morganroth
Laura Pettit
Mitch Robinson
Susan Ryan
Joe Tieger


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© 2020


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Social Events
Monday, 9 September 2019
19.00-21.00 – Welcome Reception in the Cloister of Abbey of San Pietro
Thursday, 12 September 2019
Social Excursion to Todi and its surroundings and Gala Dinner
Small, charming piazzas and gardens tucked away within the city walls as well as a dense labyrinth of streets and alleyways where tourists can easily and pleasantly lose themselves whist discovering the city's architectural features, hidden away and exquisite, protected by the silence of a more than thousand-year old civilisation.
This is Todi, which rose up and developed thanks to its exceptional architecture, allowing it to rise to a position of pre-eminence over the surrounding areas. Todi is the mirror of its more than one-thousand years of history, retracing the steps of the Umbri (the ancient tribe that first settled in the area) and their Etruscan neighbours, the influence of the Roman Empire and the strength of the free township, up to the glory of the Renaissance and the few remaining signs of the Baroque era.
It is well worth the effort of walking up the hills and steps of one of the country's most popular tourist destinations to discover the breathtaking views of its forested countryside as well as the Tiber valley. Whether it be the soft blues of the morning or hazy pink sunsets, Todi's landscape changes with the seasons and always inspires a sense of wonder.
Guest Program
Guided tours will be organized in different locations of Umbria.
The program will be available soon.
Tuesday 10 September at 19:00 it will be possible to go to a concert by the Choir of the University of Perugia at the San Lorenzo cathedral.
Wednesday 11 September from 10:00 to 13:00 is planned a visit to Perugia for accompanying people. The visit is in English language and is free.
Interested people can send an e-mail by September, 5th at 13:00 to email@example.com. It will be possible to book also on 9th September evening and on 10th morning at the registration desk
Consulta Umbria is available for any need or request.


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Someone once asked me what was the best part of about running TrueHorror.net. Ever since then the answer has always been the same, the new friends I have made. Through it all I have made all kinds of new friendships and acquaintances. There is something very fulfilling about meeting a like minded individual and hitting it off from the start. Diandra was no exception. We had been wanting to work together for quite sometime, sadly the timing never worked out, until now.
Diandra just happens to be the biggest A Nightmare On Elm Street fan I have ever met. I do not say that lightly as I know quite a few great Freddy fans, but while Diandra adores Freddy Krueger, she loves Freddy's arch nemesis Nancy Thompson even more! From dressing up as her favorite heroine for fun and at conventions, to eventually getting to meet and become friends with Nancy herself, Heather Langenkamp. I could go on and on about it all, but to do it justice, I will let Diandra tell it.
"I didn't start out a horror fan. My life consisted of a wholesome, Midwestern family and catholic school education. I was terrified of practically everything, but I was always curious. I used to sneak viewings of bits and pieces of JAWS and the Universal monsters, but never saw my first actual horror film until 7th grade. I was traumatized. Freddy Krueger would also pop up throughout my life occasionally. It wasn't until a fateful night when a very realistic Freddy dream changed my life and I woke up a horror fan (ironic). It took me a while to gather up enough courage to actually watch the first film, but upon doing so I realized I had an instant connection with Nancy. She was the Midwestern girl next door with a spunky best friend. She displayed independence, loyalty and resourcefulness. She didn't really give a care about being fashion forward. Plus, she had poofy brown hair! Over the years, Nancy has become very much ingrained in who I am today. Her spirit and my fandom have shaped me. I love her more than words can say. The same goes for Heather Langenkamp, who has been a very kind spirit to me since the day I met her – 5 years ago today actually.
Nancy will always be with me and having the honor of being able to recreate this poster means a lot to me and I absolutely love it." -Diandra
After getting to know Diandra and her story it was evident this concept was truly exciting to her. I was honored to get the shot to help her bring to life an image that holds so much importance to her, and it was her enthusiasm that helped hype me up to get this done the way we both knew it could and should be done! So without further ado, here is our recreation of the original one sheet for A Nightmare On Elm Street.
Seeing this image complete was extremely satisfying. Diandra fits the role perfectly and it was an absolute pleasure working with her on this. She is a Nightmare/Nancy fan through and through and it showed throughout the entire process. I absolutely look forward to working with her on more projects in the near future. We hope you enjoyed learning a bit more about the person behind the photo and dig the final product as much as we do! Sweet Dreams everyone!
Diandra is also heavily involved in an upcoming Nightmare fan film entitled Don't Fall Asleep where she stars as who else? Nancy Thompson. You can find out more about that project at the link below as well as keep up with her on her social media accounts.
Instagram: https://www.instagram.com/sassysledgehammer/ (@sassysledgehammer)
Twitter: https://twitter.com/SassySledgehmmr (@SassySledghmmr)
Facebook: https://www.facebook.com/NancyTOfElmSt
Don't Fall Asleep Facebook: https://www.facebook.com/DontFallAsleepFanFilm


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The technician that helped me explained everything very well.
Posted by ANTHONY G. on 04-11-2019
The sonogram technician was very nice. I think her name was Pulwei.
Posted by SCOTT S. on 03-15-2019
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The 1965 Art Education Seminar at Penn State is a landmark in the field. Participants and attendees included leaders who shaped the field into the next century. Organized by art education faculty members Ed Mattil (Penn State), David Ecker and Jerome Hausman (Ohio State), and moderated by Harlan Hoffa (Penn State), the seminar was funded by the U.S. Office of Education and planned in conjunction with leading art educators such as Elliot Eisner (University of Chicago), Kenneth Beittel (Penn State), and Manuel Barkan (Ohio State). Participants at the 1965 Seminar argued that curriculum in visual arts education should draw on contemporary art practice, and introduce art history and criticism to school children. They carried these ideas back to their institutions, exploring them with succeeding generations of graduate students who then implemented new paradigms. Join us April 1–3, 2016, as we reflect on the 50th anniversary of this important event.
"The conference will serve as a critical reflection on the 1965 seminar held at Penn State, which served as a milestone event in the field," said Professor B. Stephen Carpenter, II, member of the conference planning committee. Current and emeritus Penn State Art Education faculty and students, as well as alums and scholars from across the globe, will present at the conference. President Barron is scheduled to make a few remarks on Friday afternoon. The conference format will include panels, roundtable sessions, a poster session, and discussions. Additional planned outcomes of the 2016 conference include published proceedings, online resources, and documentation.
The 1965 seminar was a ten-day gathering at Penn State, and became a benchmark in academic art education. The 1965 seminar influenced profoundly the study and practice of art education in the United States as it brought together artists, art historians, critics, art educators, curriculum experts, and psychologists. The seminar is a landmark in the field as it shifted the focus of art education from psychologically grounded, developmental approaches to teaching and researching to a more self-conscious stance as part of the humanities and interdisciplinary scholarship.
For complete details, and to register for the event, visit: http://sites.psu.edu/arteducationseminarfifty/. Attendees can pay for single day or full conference registration. PreK-12 teachers can earn ACT 48 hours by registering for and attending the conference Friday (5 hours), Saturday (6 hours), and Sunday (2.5 hours). For more information, contact firstname.lastname@example.com
The conference is made possible through a grant from the Institute for the Arts and Humanities at Penn State, and support from the College of Arts and Architecture, the Penn State School of Visual Arts, and the Art Education Program.


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LW Scientific Semen Evaluation LabScope with Heated Stage
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The Semen Evaluation LabScope
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No Longer Available


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At this site we maintain a list of the 5000 Largest Known Primes which is updated hourly. This list is the most important databases at The Prime Pages: a collection of research, records and results all about prime numbers. This page summarizes our information about one of these primes.
field (help)value
Description:405356632768 + 1
Verification status (*):Proven
Official Comment:Generalized Fermat
Proof-code(s): (*):p316 : Yama, GeneFer, AthGFNSieve, PrimeGrid, OpenPFGW
Decimal Digits:216526 (log10 is 216525.61096135)
Rank (*):22737 (digit rank is 1)
Entrance Rank (*):2863
Currently on list? (*):no
Submitted:9/7/2011 09:13:08 CDT
Last modified:9/7/2011 11:20:22 CDT
Removed (*):3/10/2012 04:08:51 CDT
Database id:101873
Status Flags:none
Score (*):41.9325 (normalized score 0.1072)
There are certain forms classed as
archivable: these prime may (at times)
remain on this list even if they do not make
the Top 5000 proper. Such primes are tracked with archival
Generalized Fermat (archivable *)
Prime on list: no, rank 1393
Subcategory: "Generalized Fermat"
(archival tag id 213433, tag last modified 2020-03-26 21:50:24)
The Top 5000 Primes is a list for proven primes only. In order to maintain the
integrity of this list, we seek to verify the primality of all submissions.
We are currently unable to check all proofs (ECPP, KP, ...), but we will at least trial
divide and PRP
check every entry before it is included in the list.
machineRedHat P4 P4
notesCommand: /home/caldwell/client/TrialDiv/TrialDiv -q 1 4053566 32768 1 2>&1[Elapsed time: 7.566 seconds]
modified2011-12-27 16:48:29
created2011-09-07 09:18:01
machineRedHat P4 P4
notesCommand: /home/caldwell/client/pfgw -t -q"4053566^32768+1" 2>&1PFGW Version 22.214.171.124BIT.20110215.x86_Dev [GWNUM 26.5]Primality testing 4053566^32768+1 [N-1, Brillhart-Lehmer-Selfridge]Running N-1 test using base 3Calling Brillhart-Lehmer-Selfridge with factored part 59.08%4053566^32768+1 is prime! (5422.8239s+0.0129s)[Elapsed time: 1.51 hours]
modified2011-12-27 16:48:29
created2011-09-07 09:36:57


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Mnk1 and Mnk2 are protein kinases that are directly phosphorylated and activated by extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein (MAP) kinases and implicated in the regulation of protein synthesis through their phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) at Ser209. To investigate their physiological functions, we generated mice lacking the Mnk1 or Mnk2 gene or both; the resulting KO mice were viable, fertile, and developed normally. In embryonic fibroblasts prepared from Mnk1-Mnk2 DKO mice, eIF4E was not detectably phosphorylated at Ser209, even when the ERK and/or p38 MAP kinases were activated. Analysis of embryonic fibroblasts from single KO mice revealed that Mnk1 is responsible for the inducible phosphorylation of eIF4E in response to MAP kinase activation, whereas Mnk2 mainly contributes to eIF4E's basal, constitutive phosphorylation. Lipopolysaccharide (LPS)- or insulin-induced upregulation of eIF4E phosphorylation in the spleen, liver, or skeletal muscle was abolished in Mnk1−/− mice, whereas the basal eIF4E phosphorylation levels were decreased in Mnk2−/− mice. In Mnk1-Mnk2 DKO mice, no phosphorylated eIF4E was detected in any tissue studied, even after LPS or insulin injection. However, neither general protein synthesis nor cap-dependent translation, as assayed by a bicistronic reporter assay system, was affected in Mnk-deficient embryonic fibroblasts, despite the absence of phosphorylated eIF4E. Thus, Mnk1 and Mnk2 are exclusive eIF4E kinases both in cultured fibroblasts and adult tissues, and they regulate inducible and constitutive eIF4E phosphorylation, respectively. These results strongly suggest that eIF4E phosphorylation at Ser209 is not essential for cell growth during development.
Mitogen-activated protein kinases (MAPKs) are activated by various extracellular signals, such as growth factors, stresses, and cytokines, and play crucial roles in the determination of cell fate through proliferation, differentiation, survival, and apoptosis (1, 5, 24, 38). Three classes of MAPK families, the ERK, Jun N-terminal kinase/stress-activated protein kinase, and p38 MAPK, are differentially activated depending on the signaling context and in turn phosphorylate target proteins, which include transcription factors and protein kinases. These proteins can be common targets for subsets of MAPK proteins or specific targets for individual MAPKs. The direct downstream protein kinases, comprehensively called the MAPK-activated protein kinase (MAPKAPK) family, can be categorized into four subclasses, the Rsk, MK, Mnk, and Msk families. The Rsks (Rsk1, Rsk2, and Rsk3) are activated specifically by ERKs, whereas the MKs (MK2/MAPKAPK2, MK3/MAPKAPK3/3pK, and MK5/PRAK) are activated mainly by p38 MAPK in vivo. In contrast, the Mnks (Mnk1 and Mnk2) and Msks (Msk1 and Msk2) are targeted in vivo by both the ERK and p38 MAPK pathways, resulting in the activation of Mnks and Msks by a broad spectrum of extracellular stimuli. Mnk1 (MAPK signal-integrating kinase 1/MAPK-interacting kinase 1) and Mnk2 were identified independently by phosphorylation screening for ERK substrates and in a two-hybrid screen for ERK-binding proteins (12, 60). Both Mnk1 and Mnk2 are directly activated in vitro by the ERK and p38 MAPK, which phosphorylate at least two threonine residues (e.g., Thr197 and Thr202 of the mouse Mnk1) located in the so-called activation loop (12, 46, 60, 61). Studies with cell lines overexpressing Mnk cDNAs have shown that Mnk1 is strongly activated by growth factors, cellular stresses, and inflammatory cytokines via ERK and/or the p38 MAPK, depending on the signaling context, whereas Mnk2 has a relatively high basal activity that is hardly affected by changes in MAPK activity (12, 46, 59, 60).
Although the physiological functions of Mnk1 and Mnk2 remain unknown, several studies have demonstrated that one of the Mnk substrates is eukaryotic translation initiation factor 4E (eIF4E), also known as cap-binding protein. eIF4E specifically binds the 5′ m7GpppN cap structure found in all eukaryotic mRNAs and plays a critical role in cap-dependent translation initiation as a central component of the eIF4F complex, which also contains eIF4A and eIF4G, and this complex facilitates the assembly of the translation initiation complex consisting of mRNA, eIF3, eIF4F itself, and the 40S ribosomal subunit (13). Formation of the eIF4F complex is negatively regulated by 4E-binding proteins (4E-BPs), which bind eIF4E and block its interaction with eIF4G and thus inhibit cap-dependent translation. Phosphorylation of 4E-BP through the mTOR-dependent pathway liberates eIF4E from this inhibition (14). Elevated levels of eIF4E protein are observed in various human cancers (7), and overexpression of eIF4E in cultured cells causes malignant transformation (6, 27). Furthermore, recent studies have shown that overexpression of eIF4E promotes tumor formation in vivo and cooperates with c-Myc in lymphomagenesis, indicating that cellular levels of eIF4E are involved in controlling cell growth, survival, and oncogenesis (44, 62). Previous studies showed that either Mnk1 or Mnk2 can phosphorylate eIF4E at serine 209 in vitro (40, 46, 60, 61) and that the phosphorylation of eIF4E is regulated in response to extracellular signals that activate ERK and/or p38 MAPKs in accordance with the activation spectrum of Mnk1 (12, 33, 59, 60). Furthermore, the overexpression of active Mnk1 or Mnk2 proteins increases the phosphorylation of eIF4E, whereas the expression of a dominant-negative Mnk1 mutant reduces the levels of eIF4E phosphorylation (46, 61). These results collectively established the notion that Mnk1 and Mnk2 are physiological eIF4E kinases that regulate cap-dependent translation initiation in response to extracellular signals (41, 47). This idea was also supported by the observation that Mnk1 and Mnk2 bind to eIF4G in vivo (40, 46, 61).
Serine 209, located near the C terminus, is the major eIF4E phosphorylation site in rabbit reticulocytes and in serum-stimulated hamster CHO or mouse NIH 3T3 cells (11, 18, 63). However, the functional significance of Ser209 phosphorylation in translation initiation remains unclear. Since phosphorylation of eIF4E is upregulated in response to growth factors and cytokines, it has been postulated that eIF4E phosphorylation plays a positive role in cell growth by stimulating translation efficiency. In accordance with this idea, in Drosophila, a point mutation of the eIF4E phosphorylation site (Ser251, corresponding to Ser209 in mammals) results in reduced viability, developmental delay, and a reduction in body size (25). Some other reports, however, do not necessarily support this idea. Increased eIF4E phosphorylation induced by overexpression of active Mnks is reported not to facilitate eIF4F complex formation or general protein synthesis (16, 46). In addition, one study reported that a specific Mnk1 inhibitor, CGP57380, suppresses eIF4E phosphorylation but has no effect on the de novo initiation of translation during the recovery of cells from hypertonic stress (34). In another report, the expression of active Mnk1 or Mnk2 decreased the level of cap-dependent translation relative to cap-independent translation, suggesting a negative role for Mnks in translational regulation (21).
To investigate the physiological functions of Mnk1 and Mnk2, we generated knockout (KO) mice by targeted disruption of their genes. Using these mice, we demonstrated that Mnk1 and Mnk2 are essential for the phosphorylation of eIF4E in embryonic fibroblasts and adult tissues and that they play distinct roles in the regulation of eIF4E phosphorylation. Surprisingly, however, not only the single KO mouse lines but also the Mnk1-Mnk2 double KO (DKO) line were viable and showed no apparent abnormalities or defects in translation. These results strongly suggest that the phosphorylation of eIF4E is not essential for cell growth during development.
Cells and mice.Mouse R1 embryonic stem (ES) cells were cultured as described previously (22). Primary embryonic fibroblasts were prepared from day 13.5 mouse embryos. After removing the head and internal organs, the remaining tissues were cut into small pieces, and single cells were liberated by trypsin digestion. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) that was supplemented with 10% fetal calf serum (FCS; Invitrogen). Embryonic fibroblasts at passage 3 or 4 were used throughout this study. Mice were purchased from Charles River Japan, Inc. (Yokohama, Japan) or Nippon SLC, Inc. (Shizuoka, Japan). All mice were housed in a specific-pathogen-free facility at Osaka University Medical School, and all animal experiments were carried out in accordance with protocols approved by the Osaka University Medical School Animal Care and Use Committee.
Construction of targeting vectors and generation of KO mice.Mnk1 and Mnk2 genomic DNA clones were isolated by plaque hybridization of a mouse 129/Sv genomic DNA library with full-length mouse Mnk1 and Mnk2 cDNA probes, respectively. To construct targeting vectors, a neomycin resistance gene (Neor cassette) was inserted between the MunI site in intron 4 and the AvrII site in intron 6 of the Mnk1 gene or between the NcoI site in intron 4 and the EcoT22I site in intron 9 of the Mnk2 gene. The diphtheria toxin A fragment gene (DTA cassette) for negative selection was ligated to the 3′ end of the 3′ homologous arm.
Mouse R1 ES cells were transfected with the Mnk1 or Mnk2 targeting vector by electroporation, and G418-resistant clones were screened for homologous recombination by PCR. ES clones carrying the single Mnk1- or Mnk2-targeted allele were injected into BDF1 blastocysts, which were then implanted into recipient ICR female mice. Chimeric mice with a high ES contribution were crossed to C57BL/6 females. Germ line transmission was identified by coat color and then confirmed by PCR. Single KO mice for either the Mnk1 or Mnk2 gene were generated by crossing the respective heterozygous mice. Mnk1−/− Mnk2−/− DKO mice were generated by crossing Mnk1−/− Mnk2+/− mice with Mnk1+/− Mnk2−/− mice.
PCR and Southern blot analyses.Genomic DNA for PCR was prepared from tail snips. The genotype of the Mnk1 and Mnk2 genes was determined by PCR with Taq polymerase (Amersham Biosciences) by using the GeneAmp PCR system 9700 (Applied Biosystems). For the wild-type (WT) and mutant alleles of the Mnk1 gene, a sense primer specific for the WT (5′-GACCCAGGAATGACACCTTC-3′) or mutant (5′-GATTCGCAGCGCATCGCCTTCTATCG-3′, a sequence in Neor) allele was used with the Mnk1-specific antisense primer (5′-GCGCAAACCACATGTGCTTT-3′). The WT and mutant alleles of the Mnk2 gene were detected by PCR with the WT (5′-ATTGAGAAGCAGCTGGGCCACATCCGCAGC-3′) or mutant (5′-GATTCGCAGCGCATCGCCTTCTATCG-3′) sense primer and the Mnk2-specific antisense primer (5′-GTCGCAGCGCTTGTCGTAGATGCTGGC-3′).
For Southern hybridization, genomic DNA from an adult liver was digested with XbaI (for Mnk1) or HindIII (for Mnk2), separated by agarose gel electrophoresis, and transferred to a BA85 nitrocellulose filter (Schleicher & Schuell). Hybridization of the Mnk1 gene was carried out by using the 0.68-kb XbaI-PmaCI DNA fragment containing exon 3 as a probe. For Mnk2-specific hybridization, the 2.5-kb HindIII-XbaI fragment including exon 1 was used as a probe.
Stimulation of embryonic fibroblasts and preparation of cell extracts.Mouse embryonic fibroblasts were seeded at 5 × 105 cells/well in a six-well plate and grown in DMEM with 10% FCS for 24 h. Cells were serum starved in DMEM containing 0.5% calf serum (CS) (starvation medium) for 20 h and then stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) (500 nM, 15 min), FCS (25%, 15 min), anisomycin (10 μg/ml, 15 min), UV-C irradiation, tumor necrosis factor alpha (TNF-α) (10 ng/ml, 30 min), interleukin-1β (IL-1β) (10 ng/ml, 15 min), or osmotic shock followed by isotonic recovery. In the experiment with UV-C irradiation, cells in a six-well plate were washed once with phosphate-buffered saline (PBS) prewarmed to 37°C, exposed to UV-C light at 40 J/m2 with a Stratalinker 2400 (Stratagene), and cultured in starvation medium for 20 min. For osmotic shock, serum-starved cells were incubated for 30 min at 37°C in starvation medium supplemented with an additional 0.2 M NaCl and then cultured in normal starvation medium for 30 min at 37°C. After stimulation, the cells were washed with ice-cold PBS and lysed in 1 ml of NP-40 lysis buffer (NLB; 50 mM HEPES-NaOH [pH 7.4], 150 mM NaCl, 1% NP-40, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, 20 mM NaF, 20 mM β-glycerophosphate, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 U of aprotinin/ml, and 10 μg of leupeptin/ml). The supernatant was recovered after centrifugation for 10 min at 4°C.
For the preparation of tissue extracts, the spleen, liver, femoral muscle, and brain were dissected from 8-week-old mice and a portion of each tissue (0.2 to 0.4 g) was homogenized in 1 ml of extraction buffer (20 mM HEPES-NaOH [pH 7.4], 10% glycerol, 1 mM EGTA, 20 mM NaF, 20 mM β-glycerophosphate, 1 mM PMSF, 10 U of aprotinin/ml, and 10 μg of leupeptin/ml) by using a plastic homogenizer pestle (Kontes) in a microcentrifuge tube. The supernatant was recovered after centrifugation for 30 min at 4°C.
Antibody preparation and immunoblot analyses.Anti-Mnk1 and anti-Mnk2 antisera were prepared by immunizing rabbits with recombinant mouse Mnk1 (amino acid residues 1 to 415) or Mnk2a (residues 48 to 459) protein that had been cleaved with thrombin from its respective glutathione S-transferase (GST) fusion protein produced in Escherichia coli (BL21). Both antibodies were affinity purified on glutathione-agarose beads that had been covalently coupled to GST-Mnk1 (residues 1 to 415) or GST-Mnk2a (residues 48 to 459) protein, respectively.
For immunoblot analysis, 10 μg of fibroblast extract or 18 μg of tissue extract were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane filter (Immobilon-P; Millipore). For the immunoblotting of Mnk proteins, we used affinity-purified anti-Mnk1 or anti-Mnk2 antibodies. Other antibodies used were an anti-phospho-Mnk1 (Thr197/202) antibody (Cell Signaling), anti-phospho-eIF4E (Ser209) antibody (Cell Signaling), anti-phospho-ERK antibody (anti-active-MAPK; Promega), anti-phospho-p38 antibody (anti-active-p38; Promega), anti-phospho-HSP27(Ser82) antibody (Cell Signaling), and anti-eIF4E monoclonal antibody (clone 87; Transduction Laboratories). Immunoblot detection was carried out with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin antibody (Dako) and the enhanced chemiluminescence detection system (PerkinElmer).
Metabolic labeling of eIF4E.For the 32P in vivo labeling of eIF4E, 5 × 105 embryonic fibroblasts were seeded into 35-mm-diameter dishes and grown in DMEM with 10% FCS for 24 h. Cells were serum starved in phosphate-free DMEM (Invitrogen) supplemented with 0.5% dialyzed CS and 0.6-mCi/ml [32P]orthophosphate (8,500 to 9,120 Ci/mmol; PerkinElmer) for 20 h and then stimulated with 500 nM TPA for 15 min. Cells were washed twice with ice-cold PBS and lysed in 0.9 ml of NLB, and the supernatant was recovered after centrifugation for 10 min at 4°C. For the immunopurification of eIF4E, 1 μg of an anti-eIF4E monoclonal antibody (clone P-2; Santa Cruz) and 20 μl of protein G-Sepharose were added to 0.4 ml of the cell extract and incubated for 1 h at 4°C. For affinity purification, 20 μl of m7GTP-Sepharose (Amersham Biosciences) was added to 0.4 ml of the cell extract and incubated as above. The protein G- or m7GTP-Sepharose resin was washed four times with 1 ml of NLB, suspended in 50 μl of Laemmli's sample loading buffer, and heated for 5 min at 95°C. After centrifugation, 10 μl of the eluted proteins was resolved by SDS-PAGE, electrotransferred to a PVDF membrane, and exposed to X-ray film for 18 h at −80°C with an intensifying screen. After autoradiography, the filter was subjected to immunoblotting with the anti-eIF4E antibody. For enhanced chemiluminescence detection, the filter was exposed to X-ray film for 2 s.
Protein synthesis measurements.Embryonic fibroblasts (3 × 105 cells) were seeded into 35-mm-diameter dishes, grown in DMEM with 10% FCS for 24 h, and then serum starved for 20 h. Cells were cultured in 0.8 ml of methionine/cysteine-free DMEM (Invitrogen) supplemented with 0.5% dialyzed CS for 30 min, and then 0.2 ml of dialyzed FCS and 20 μl (40 μCi) of a [35S]methionine/[35S]cysteine mixture (redivue Pro-mix l-[35S] in vitro cell labeling mix; Amersham Biosciences) was added. After 90-, 120-, and 240-min incubations, the cells were washed three times with ice-cold PBS and lysed in 0.4 ml of RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 50 U of aprotinin/ml, and 20 μg of leupeptin/ml). The supernatant was recovered after centrifugation for 10 min at 4°C. Cell extracts (20 μl) were diluted with 230 μl of water containing 10 μg of bovine serum albumin, mixed with 250 μl of 10% (wt/vol) trichloroacetic acid (TCA), and heated for 15 min at 90°C to hydrolyze the aminoacyl-tRNA. After incubation for 30 min on ice, each sample was passed through a glass fiber filter (GF/C; Whatman), and the filter, containing the labeled polypeptides, was washed three times with 5% TCA and once with ethanol and then air-dried. The radioactivity was measured by scintillation counting with Ultima Gold scintillation cocktail (PerkinElmer).
Translation reporter assay.The bicistronic translation reporter plasmids, pEF-FFL-IRES-SPL and pEF-SPL-IRES-FFL, were constructed by the sequential subcloning of firefly luciferase (FFL) cDNA from pGL3 (Promega), sea pansy luciferase (SPL, also called Renilla reniformis luciferase) cDNA from pRL-SV40 (Promega), and the internal ribosome entry site (IRES) sequence from pIRES2EGFP (BD Bioscience Clontech) into the pEF-BOS-EX plasmid, which carries the EF-1α enhancer/promoter as an expression driver.
Embryonic fibroblasts were seeded at 5 × 104 cells/well into a 24-well plate, grown for 24 h, and transfected with 1 μg of either pEF-FFL-IRES-SPL or pEF-SPL-IRES-FFL plasmid by the calcium phosphate coprecipitation method. At 5 h after transfection, the medium was changed to fresh DMEM with 10% FCS and further cultured for 36 h. Cell lysates were prepared and assayed for FFL and SPL activities by using the dual-luciferase reporter assay system (Promega). The error bars in the figures indicate standard errors of the mean of the results from two independent transfection experiments.
Generation of Mnk1 and Mnk2 KO mice.The targeting constructs for the mouse Mnk1 and Mnk2 genes are shown in Fig. 1A and B. By sequence analysis of the mouse Mnk2 gene locus and reverse transcription-PCR analysis of the RNA from embryonic fibroblasts, we found that the mouse Mnk2 gene encodes, in its exons 1 and 2, a 47-amino-acid extension (MVQKRTAELQGFHRSFKGQNPFELAFSLDLAQHRDSDFSPQCEARPD) to the N terminus of the previously published Mnk2 coding sequence (60). The N-terminal extension is highly conserved in the rat and human Mnk2a proteins (50), indicating that the entire coding sequence (459 amino acids), including this extension, represents a mouse Mnk2a homologue (data not shown) (DDBJ/EMBL/GenBank accession number AB164081).
In each targeting construct, exons 5 and 6 of the Mnk1 gene (chromosome 4) or exons 5 to 9 of the Mnk2 gene (chromosome 10) were replaced with a neomycin resistance gene cassette. Mouse ES cell clones containing either the Mnk1- or Mnk2-targeted allele were identified by PCR and Southern blot analyses and used to generate KO mice. Mating of either the Mnk1+/− or Mnk2+/− heterozygous mice yielded healthy littermates of three genotypes (+/+, +/−, and −/−) at Mendelian frequencies (Table 1). Both the Mnk1−/− and Mnk2−/− single KO mice were viable and fertile with no apparent abnormalities for at least 12 months after birth. We then generated mice heterozygous for both Mnk1- and Mnk2-disrupted alleles by mating the Mnk1 and Mnk2 single KOs, and the doubly heterozygous mice were further intercrossed to yield DKO mice. Like the single KO mice, Mnk1−/− Mnk2−/− DKO mice were born normally, were fertile, and did not show developmental abnormalities or morbidity for at least 8 months after birth. The mating of Mnk1+/− Mnk2−/− and Mnk1−/− Mnk2+/− mice yielded littermates of the four genotypes at nearly the same frequencies, indicating independent segregation of the KO alleles with Mendelian inheritance (Table 1). The chromosomal structure of the targeted genes in the KO mice was confirmed by Southern blot analysis of the genomic DNA prepared from WT, doubly heterozygous, and DKO mice (Fig. 1C).
A single Mnk1 protein and two isoforms of Mnk2, Mnk2a and Mnk2b, have been identified in human cells (12, 49, 50), but there has been no report of the characterization of the endogenous protein products of the mouse Mnk1 and Mnk2 genes. To examine the expression of the Mnk1 and Mnk2 proteins in mice, we prepared rabbit antibodies against the mouse proteins and analyzed spleen extracts from WT and KO mice by immunoblotting with these antibodies (Fig. 2). On the blot with the anti-Mnk1 antibody, a single Mnk1 protein of about 47 kDa was detected in the spleen extracts from WT and Mnk2 KO mice (lanes 1 and 3) but not in the extracts from Mnk1 KO or DKO mice (lanes 2 and 7). In contrast, the anti-Mnk2 antibody detected two protein products that were present in the WT and Mnk1 KO extracts (lanes 1 and 2) but absent in the extracts from Mnk2 KO and DKO mice (lanes 3 and 7). The large product (about 51 kDa) is thought to be an Mnk2a product whose calculated molecular size is 51.6 kDa. The small product is likely to be the counterpart of an Mnk2 splice variant, Mnk2b, identified in human cells (49, 50), although we have not been able to find mouse cDNA sequences that would encode the Mnk2b-specific C-terminal region in the DNA sequence databases. The anti-Mnk2 antibody cross-reacted with Mnk1, which was confirmed by the immunoblotting of bacterially produced recombinant Mnk1 and Mnk2 proteins (data not shown). Mice heterozygous for the KO alleles (lanes 4 to 6) showed reduced protein levels of Mnk products, indicating that expression of the Mnk proteins in the spleen is faithfully dependent on the gene dosage.
Mnks are essential for the phosphorylation of eIF4E in embryonic fibroblasts.We next investigated the phosphorylation of eIF4E in primary embryonic fibroblasts prepared from embryonic day 13.5 KO embryos in response to MAPK activation. Expression of the single Mnk1 protein and two Mnk2 proteins was detected in the WT mouse embryonic fibroblasts (Fig. 3B). To examine activation of the Mnk proteins, we utilized an anti-phospho-Mnk1 (Thr197/202) antibody, which recognizes the Mnk1 protein phosphorylated at Thr197 and Thr202 and cross-reacts with the phosphorylated Mnk2 protein. As shown in Fig. 3A, Mnk1 was barely phosphorylated in growing or serum-starved fibroblasts (lanes 1 and 2) but was inducibly phosphorylated upon the stimulation of cells with FCS, TPA, anisomycin, UV-C irradiation, TNF-α, IL-1β, and osmotic shock (NaCl) (lanes 3 to 9). In contrast, nearly constant levels of phosphorylated Mnk2 proteins were detected irrespective of stimulation. These bands were absent in the immunoblots of extracts from Mnk1-Mnk2 DKO embryonic fibroblasts (lanes 10 to 18), confirming the specificity of the anti-phospho-Mnk1 antibody. Immunoblotting with anti-phospho-ERK and anti-phospho-p38 antibodies revealed that FCS and TPA preferentially activated ERK, whereas anisomycin and UV strongly activated p38 MAPK and weakly activated ERK. TNF-α, IL-1β, and osmotic shock activated both ERK and p38 modestly. In accordance with results from earlier studies (12, 59, 60), these results demonstrate that, depending on the extracellular stimulus, the ERK and/or p38 MAPKs are differentially activated and in turn phosphorylate Mnk1 in embryonic fibroblasts. Immunoblotting of WT fibroblasts with anti-phospho-eIF4E (Ser209) revealed that eIF4E was weakly phosphorylated in growing and serum-starved cells and became highly phosphorylated in response to all extracellular stimuli tested (lanes 1 to 9). On the other hand, Ser209 phosphorylation was not detected in DKO fibroblasts under any conditions (lanes 10 to 18). Immunoblotting with an anti-eIF4E antibody showed the same expression levels of the eIF4E protein in WT and DKO fibroblasts. Furthermore, the activation spectrum of the ERK and p38 MAPKs in response to extracellular stimulation was similar, and HSP25, a direct target of MAPKAPK2, was properly phosphorylated in the DKO as well as the WT cells, suggesting that the absence of Mnk proteins in the DKO fibroblasts did not have a deleterious effect on MAPK signaling pathways other than on the Mnks themselves.
Mnk1 and Mnk2 play distinct roles in regulating eIF4E phosphorylation.Earlier studies showed that Mnk2 has a high basal activity but is relatively insensitive to the activation of ERK and p38 MAPKs (21, 46). Our anti-phospho-Mnk immunoblotting results also suggested that the activation state of Mnk1 is tightly regulated by these MAPKs, whereas Mnk2 is constitutive. To examine whether the two Mnks play distinct roles in phosphorylating eIF4E under various conditions, we analyzed the phosphorylation of eIF4E in embryonic fibroblasts that possessed different combinations of Mnk1 and Mnk2 KO alleles (Fig. 3B). Under the serum-starved condition, eIF4E was phosphorylated at comparable levels in WT and Mnk1−/− Mnk2+/+ fibroblasts (lanes 1 and 2) but at much lower levels in Mnk1+/+ Mnk2−/− fibroblasts (lane 3). In contrast, under conditions in which ERK and/or p38 MAPKs were rapidly activated, the phosphorylation of eIF4E was equally induced in WT, Mnk1+/+ Mnk2−/−, and Mnk1+/− Mnk2−/− fibroblasts (lanes 1, 3, and 4), but no increase in phosphorylation was observed in the Mnk1−/− Mnk2+/+ and Mnk1−/− Mnk2+/− fibroblasts (lanes 2 and 5). These results indicate that Mnk1 plays an essential role in the inducible phosphorylation of eIF4E in response to extracellular signals that are transduced through MAPKs, whereas Mnk2 is involved in the constitutive phosphorylation of eIF4E when MAPK activities are low. Interestingly, in Mnk1−/− Mnk2+/+ and Mnk1−/− Mnk2+/− fibroblasts, eIF4E phosphorylation decreased upon stimulation compared with its phosphorylation under the serum-starved condition (lane 2 and 5). This observation may indicate that the activation of MAPKs promotes the dissociation of Mnk2 from the eIF4G/eIF4E complex, which results in the subsequent dephosphorylation of eIF4E when Mnk1 is absent.
To confirm the results obtained by anti-phospho-eIF4E (Ser209) immunoblotting, we carried out metabolic labeling of eIF4E with [32P]orthophosphate and analyzed the incorporation of radioactivity into eIF4E after stimulation with TPA. As shown in Fig. 3C, we purified eIF4E from cell lysates either with m7GTP-Sepharose or by immunoprecipitation with an anti-eIF4E antibody, which resulted in similar patterns. In the WT fibroblasts, eIF4E was phosphorylated at a basal level after serum starvation and became further phosphorylated upon TPA stimulation. In Mnk1 KO cells, however, eIF4E phosphorylation remained at the basal level, even after stimulation. In clear contrast, Mnk2 KO cells showed a lower level of basal phosphorylation but TPA-dependent induction of eIF4E phosphorylation that was comparable to that in the WT cells. Neither basal nor induced phosphorylation was detected in the DKO fibroblasts. A control immunoblot with the anti-eIF4E antibody showed that the same amount of eIF4E was recovered from every cell extract. These results confirmed the distinct roles of Mnk2 and Mnk1 in the constitutive and inducible phosphorylation of eIF4E, respectively. Furthermore, the absence of 32P radioactivity in eIF4E in DKO cells indicated that Mnk1 and Mnk2 are exclusive eIF4E kinases, at least in embryonic fibroblasts, which is consistent with previous studies demonstrating that Ser209 is the major and perhaps the only phosphorylation site of eIF4E (11, 18, 63).
Mnks are essential for steady-state and inducible phosphorylation of eIF4E in vivo.We next examined the expression of Mnk1 and Mnk2 proteins and the phosphorylation of eIF4E in several tissues of the KO mice. As shown in Fig. 4A, immunoblot analysis revealed that both the Mnk1 and Mnk2 proteins were highly expressed in the spleen and modestly expressed in the liver and muscle, although the expression of Mnk2a in muscle could not be determined because of a nonspecific band of the same molecular size. In the brain, relatively low levels of Mnk1 and Mnk2 were expressed, as previously suggested by Northern analysis (60). In accordance with the ubiquitous expression of Mnks, basal levels of eIF4E phosphorylation were detected in all of the WT tissues studied. The phosphorylation levels of eIF4E were partially reduced in the Mnk1 and Mnk2 single KO mice and completely abolished in the DKO mice, indicating that both kinases contribute to the steady-state phosphorylation of eIF4E. Although a band comigrating with eIF4E was seen in the anti-phospho-eIF4E blot of the liver extract from DKO mice, by performing immunoblotting after affinity purification of the eIF4E with m7GTP-Sepharose, we confirmed that this was not phosphorylated eIF4E but rather a nonspecific signal because no eIF4E phosphorylation was detected under these conditions (Fig. 4A, bottom panels).
We next addressed whether the two Mnks play distinct roles in the regulation of eIF4E phosphorylation in adult tissues, as they do in embryonic fibroblasts. For this purpose, Mnk1+/− Mnk2−/− mice were mated with Mnk1−/− Mnk2+/− mice, and the resulting littermates at 8 weeks of age were administered lipopolysaccharide (LPS). LPS treatment induced the activation of ERK and p38 in the spleen 1 h after injection (Fig. 4B, lanes 1 to 8). Accordingly, phosphorylation of Mnk1 and eIF4E was induced in the Mnk1+/− Mnk2+/− and Mnk1+/− Mnk2−/− mice (lanes 1 to 4). In contrast, the basal levels of Mnk2a and eIF4E phosphorylation in Mnk1−/− Mnk2+/− mice did not change after LPS injection (lanes 5 and 6). Phosphorylated eIF4E was undetectable in the Mnk1−/− Mnk2−/− DKO mice, even after LPS treatment (lanes 7 and 8). Similar results were observed with immunoblots of liver extracts (lanes 9 to 16). We also tested the effects of insulin injection on eIF4E phosphorylation (Fig. 4C). Although we could not estimate activation of ERK or p38 in muscle and liver by immunoblotting, because there was a high background of nonspecific bands at around 40 kDa, insulin administration resulted in upregulation of both Mnk1 and eIF4E phosphorylation in these tissues from Mnk1+/− Mnk2+/− and Mnk1+/− Mnk2−/− mice (lanes 1 to 4 and 9 to 12). However, phosphorylation of eIF4E was constant in the Mnk1−/− Mnk2+/− mice and totally absent in the DKO mice (lanes 5 to 8 and 13 to 16). These results collectively indicate that the Mnks are indeed exclusive eIF4E (Ser209) kinases in adult tissues and that Mnk2 and Mnk1 are responsible for the constitutive and inducible phosphorylation, respectively.
Elimination of the Mnks does not affect global protein synthesis in embryonic fibroblasts.We next examined the effect of Mnk deficiency on protein synthesis. Primary embryonic fibroblasts were serum starved for 20 h and then stimulated with 20% FCS, and the rate of protein synthesis was determined by incorporation of [35S]methionine and [35S]cysteine into the TCA-insoluble polypeptide fraction. As shown in Fig. 5A, the time course of 35S incorporation revealed that the basal level of protein synthesis under the serum-starved condition was similar in the WT and DKO fibroblasts. Stimulation of cells with FCS enhanced the 35S incorporation into polypeptides to a similar extent in these fibroblasts. These data show that the absence of Mnk1 and Mnk2 does not significantly affect the basal or serum-stimulated global protein synthesis. SDS-PAGE analysis of proteins from 35S-labeled embryonic fibroblasts showed a general increase in the intensity of radiolabeled proteins after FCS stimulation and no detectable differences in the synthesis of any individual proteins between the WT and DKO fibroblasts (Fig. 5B).
Knauf et al. showed that expression of constitutively active mutants of Mnk1 and Mnk2 decreased the cap-dependent translation relative to cap-independent translation by using a bicistronic luciferase reporter system (21). To examine whether the absence of eIF4E phosphorylation affected the cap-dependent translation efficiency, we employed a similar reporter system, in which two reporter cistrons, FFL and SPL, are separated by an IRES (21, 39). In the pEF-FFL-IRES-SPL construct, translation of the FFL cistron is cap dependent, whereas the SPL cistron is translated by the cap-independent, IRES-directed initiation (Fig. 5C, left). Transfection of WT fibroblasts with this plasmid resulted in an average ratio (2.05) of relative luciferase light units of FFL and SPL. All of the other fibroblasts, Mnk1 KO, Mnk2 KO, and DKO cells, showed similar ratios of FFL and SPL activities. When the reporter assay was carried out with the reciprocal construct, pEF-SPL-IRES-FFL, the average ratio of SPL light units to FFL light units was 2.62 ± 0.13 in all fibroblasts tested (Fig. 5C, right). These results suggest that the elimination of eIF4E phosphorylation does not affect the cap-dependent translation relative to cap-independent translation in embryonic fibroblasts.
A previous study isolated mouse Mnk2 cDNA encoding a 412-amino-acid protein (60), but later, the identification of human Mnk2a cDNA suggested that the reported mouse Mnk2 clone might be a partial cDNA or a splice variant lacking the N-terminal region (49, 50). In this report, we have identified a cDNA sequence and chromosomal exons for mouse Mnk2a that include the N-terminal 47-amino-acid extension. By immunoblot analyses of tissues from WT and KO mice, we showed that, in addition to Mnk2a, a small Mnk2 product of about 44 kDa is expressed in various tissues and embryonic fibroblasts. Although the small product is likely to be a mouse counterpart of human Mnk2b that is produced by an alternative usage of the last exon (exon 13), encoding the C-terminal region (49, 50), we were not able to find a DNA sequence that would carry the Mnk2b-specific coding region in the Mnk2 gene locus we have sequenced or in databases for the mouse DNA sequence, and therefore, we cannot exclude the possibility that the smaller product is generated by some other splicing.
Previous studies have demonstrated that Mnk1 and Mnk2 phosphorylate eIF4E at Ser209 in vivo (21, 46, 49, 61). However, it has not been clear whether other protein kinases are also involved in the phosphorylation of eIF4E. It was previously reported that protein kinase C and MK3/MAPKAPK3/3pK, both of which are activated by extracellular stimuli, could phosphorylate eIF4E at least in vitro (33, 61, 63). Here, we unequivocally demonstrate that both Mnk1 and Mnk2 are physiological eIF4E kinases, not only in cultured fibroblasts but also in mouse tissues such as spleen, liver, muscle, and brain. We did not detect even a trace amount of phosphorylated eIF4E in embryonic fibroblasts or tissues from DKO mice by immunoblot analysis with an anti-phospho-4E (Ser209) antibody. The metabolic labeling of eIF4E with [32P]orthophosphate showed that the phosphorylation level of eIF4E in DKO fibroblasts was very low, if there was any, by comparison with WT and single KO cells. The latter result confirmed the findings from the immunoblot analysis and also supported the previous conclusion that Ser209 is the major and perhaps the only phosphorylation site of eIF4E in vivo (11, 18, 63). Therefore, it is likely that Mnks are the only physiological protein kinases that phosphorylate eIF4E.
Analyses of the single KO mice indicated that the protein kinase activities of Mnk1 and Mnk2 are differentially regulated by changes in MAPK signaling. In Mnk2 KO fibroblasts and tissues, the basal level of eIF4E phosphorylation was very low, but the induction of eIF4E phosphorylation was comparable to that of WT cells. In contrast, the inducible phosphorylation of eIF4E was completely lost, but the basal phosphorylation was not impaired in Mnk1 KO cells. These results indicated that the dynamic regulation of Mnk1 activity by MAPKs determines the magnitude of the changes in the phosphorylation state of eIF4E, whereas the basal phosphorylation of eIF4E is directed primarily by Mnk2, whose activity is relatively constant even when MAPK activities are perturbed. This is consistent with an earlier report that Mnk2 has a high level of basal activity and is less sensitive to changes in the MAPK signaling status than Mnk1 (46). Considering that phosphorylation of at least three threonine sites by MAPKs is likely to be essential for Mnk2 activity (46), these results suggest that Mnk2 is more susceptible than Mnk1 to low levels of MAPK activity and/or is less sensitive to the protein phosphatase(s) that inactivates Mnks.
The biological significance of eIF4E phosphorylation is not well understood. Recently, a positive function of eIF4E phosphorylation in cell growth was suggested by the finding that, in Drosophila melanogaster, a point mutation of the phosphorylation site (Ser251) of eIF4E resulted in reduced viability, developmental delay, and reduction in adult body size (25). In the present study, however, DKO mice, which showed no detectable eIF4E phosphorylation, were born normally and were fertile. We found no apparent differences in the morphology or size of the tissues between WT and DKO mice. Furthermore, primary fibroblasts from DKO embryos grew normally and showed no difference in general protein synthesis compared with WT fibroblasts. These facts strongly suggest that, at least in mammals, the phosphorylation of eIF4E is not required for cell growth and ontogenic development.
Previous studies have not reached a consensus on the physiological function of eIF4E phosphorylation in the regulation of protein synthesis. On one hand, it has been suggested that the phosphorylation of eIF4E is likely to play a role in enhancing the eIF4F assembly and subsequent translation initiation. The activation of cap-dependent mRNA translation in response to growth-stimulatory signals correlates with increased eIF4E phosphorylation, whereas the inhibition of protein synthesis resulting from heat shock, osmotic stress, or viral infection correlates with the dephosphorylation of eIF4E (3, 4, 9, 10, 13, 20, 32, 33, 59). On the other hand, recent studies have suggested that the phosphorylation of eIF4E is not sufficient or required for the upregulation of general protein synthesis (30). The expression of active Mnk1 or Mnk2 increased eIF4E phosphorylation but did not enhance the overall rate of protein synthesis (16, 21, 45); rather, it decreased the cap-dependent translation relative to the cap-independent translation (21). Furthermore, treatment of cells with an Mnk-specific inhibitor, CGP57380, blocked the eIF4E phosphorylation but did not impair the mitogen-stimulated translational activation or de novo protein synthesis following recovery from hypertonic stress (21, 34). An initial study reported that the phosphorylation of eIF4E increased its affinity for cap analogs (31), but more recent studies demonstrated that the phosphorylation of eIF4E markedly reduces its affinity for capped mRNA (48, 64). Based on these studies and the cocrystal structure of eIF4E-cap analog complexes (29, 53, 54), Scheper and Proud proposed two models for the physiological role of eIF4E phosphorylation, in which the phosphorylation of eIF4E by Mnks may stimulate either the rapid and sequential loading of multiple initiation complexes and ribosomes to the same mRNA or the rapid recycling of the initiation factors from one mRNA to another, depending on the time point at which the eIF4E phosphorylation occurs during the initiation process (47). According to these models, the phosphorylation of eIF4E may play a role in reprogramming the protein synthesis pattern by promoting polysome assembly on a limited group of mRNAs or the redistribution of translational machineries from preexisting mRNAs to newly synthesized mRNAs. In any case, it is likely that the mitogen-stimulated activation of general protein synthesis is regulated primarily by other mechanisms, such as mTOR-dependent phosphorylation of 4E-BP1 and S6 kinase, and that the phosphorylation of eIF4E may instead play a role in the fine control of limited aspects of mRNA translation, stabilization, or processing. A recent report showed that overexpression of Mnk1 in adult cardiocytes increased the translational efficiency of a reporter mRNA that has pronounced secondary structure in its 5′ untranslated region (57). Another report has indicated that phosphorylation of eIF4E by Mnk1 is critical for the viral protein synthesis and replication of herpes simplex virus type 1 in quiescent cells (58). These studies suggest that the eIF4E phosphorylation is involved in the translational control of specific groups of mRNAs.
The strong expression of both Mnk1 and Mnk2 in the spleen suggests that they play a role in the immune response, such as in the activation of lymphocytes or phagocytes, antibody production, or cytokine production. MK2/MAPKAPK2 has been shown to regulate the LPS-induced production of TNF-α and IL-6 in splenocytes by posttranscriptional mechanisms (23, 35). Although eIF4E is unlikely to be the target of MK2 in this process, Mnk-dependent eIF4E phosphorylation may regulate acute-phase cytokine production directly or indirectly at the translational level. In this regard, it has been suggested that Mnk1 regulates the production of the chemokine RANTES through translational control of RFLAT-1, a transcription factor for RANTES gene expression in T cells (36).
Most eIF4E is present in the cytoplasm, but a fraction of it localizes to the nucleus, especially to speckled structures called promyelocytic leukemia (PML) nuclear bodies (8, 26, 28). Recent studies have suggested that the nuclear eIF4E regulates the nucleocytoplasmic transport of cyclin D1 mRNA in conjunction with PML protein (2, 42, 43, 55, 56). Likewise, the Mnk1 and Mnk2a proteins are present mainly in the cytoplasm, but recent studies have shown that both Mnk1 and Mnk2 possess a potential nuclear localization signal and that a fraction of human Mnk2b localizes to the PML nuclear bodies, suggesting that the Mnks have some nuclear function (37, 49). Although we have not determined the subcellular distribution of the phosphorylated eIF4E, it would be intriguing to examine whether the nuclear function of eIF4E is regulated by Mnk-dependent phosphorylation at Ser209.
Accumulating evidence has shown that local protein synthesis at the synaptic area in dendrites plays a critical role in the control of neuronal plasticity, including the control of long-term potentiation and long-term depression (17, 52). A recent study showed that brain-derived neurotrophic factor induces the translocation of eIF4E to the so-called mRNA granules, cytoskeleton-associated granules for mRNA transport in dendrites (51). A more recent study showed that conditional inhibition of ERK activity in the postnatal murine forebrain results in a reduction in hippocampus-dependent memory retention, translation-dependent long-term potentiation, neuronal activity-induced translation, and phosphorylation of eIF4E, 4E-BP1, and S6 kinase in hippocampal neurons, suggesting that synaptic plasticity is regulated by ERK-dependent translational control (19). Analysis of the neural activities of Mnk KOs would elucidate whether eIF4E phosphorylation is involved in the local protein synthesis and synaptic plasticity.
Besides eIF4E, no physiological targets for Mnk1 or Mnk2 have been identified except for cytosolic phospholipase A2 (15), which catalyzes the first step of the arachidonate cascade for eicosanoid synthesis. Mnk1 and Mnk2 KO mice and their cultured cells would be useful systems for investigating the physiological significance of eIF4E phosphorylation in the fine control of mRNA translation, as speculated above, and to explore other Mnk functions that are executed through unidentified targets of these protein kinases.
We are grateful to Kohki Kawane for help in the generation and analysis of KO mice. Special thanks go to Tony Hunter for critical reading of the manuscript.
This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
- Received 12 March 2004.
- Returned for modification 24 April 2004.
- Accepted 1 May 2004.
- American Society for Microbiology


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120 West Exchange Ave
Fort Worth, TX 76164 Stockyards Rate and review
Fort Worth, TX 76164 Stockyards Rate and review
If you love live Texas music, then put this place on your must see destinations list while your in the DFW area. Recently reopened in the Historic Fort Worth Stockyards, the Thirsty Armadillo is a local spot for several of the hot Texas music artist of today. Live music nightly with drink specials. Come hear music how it is meant to be heard...LIVE.
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Planning + Design
Office Energy: What it Takes to Create a Great Space
Kristin Christensen on February 20, 2020
An impressive compensation package, stellar retirement and health benefits, and the opportunity to engage in meaningful and transformative work. These are just a few of the items that job seekers consider when evaluating employment opportunities. To compete effectively in today's marketplace, business executives need to utilize a range of resources to retain and attract talent, which also includes providing a great workspace for teams to thrive.Read More
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We believe office design and company culture truly work hand in hand to inspire and invigorate teams, and have the ability to dramatically impact businesses in many ways. Our Planning + Design team works to shape the perfect functioning workspace for clients' teams in order to enhance space and productivity, to ultimately reinforce a positive company culture. Follow our blog to read tips on how to enhance your own workspace!


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Take your family to the city of sandy beaches, parks and historic landmarks. Trivandrum, the capital of the state of Kerala is a mesmerizing city constructed on seven hills. The city gets its name from the word, Thiruvananthapuram, meaning the city of Anantha or the abode of the sacred Serpent Anantha on which Lord Vishnu, the preserver of the Hindu trinity, reclines.
The famous Sri. Padmanabha Swami Temple is dedicated to Vishnu and is a
landmark in the city. It is also an excellent base to explore the interiors
of Kerala, the southernmost region of India, and from Kochi, the Lakshadweep
Islands. The city offers all modern facilities needed by a visitor and is a
center for cultural activities.
Major Tourist Destination
Sree Anantha Padmanabha Swamy Temple
Dedicated to Lord Vishnu Sree Anantha, Padmanabha Swamy Temple possess
an excellent structure that has a 100 ft high 'gopuram', built in
Dravidian style. The temple has a magnificent array of gods, goddesses,
nymphs, sprites and demons which adorn the seven-storey 'gopuram' that
forms the temple tower. Only Hindus are allowed inside and dress
regulations are strictly followed.
Shankhumugham Beach
Just 8 km from the city, this is a favourite place of sunset watchers.
The beach is adjacent to the Trivandrum Airport and Veli Tourist
Village. An indoor recreation club, the matsya kanyaka, a gigantic, 35 m
long sculpture of a mermaid and a restaurant shaped like a starfish are
some of the attractions here.
Napier Museum
museum houses a rare collection of archaeological & historic
artifacts. The collections are as varied and electric as the building
and you will spend a pleasant few hours browsing through them. The
museum has a unique natural air-conditioning system.
Veli Tourist Village
This picnic spot, where the Veli Lake meets the Arabian Sea, offers
boating facilities. For a quick ride over the waters, speedboats are
available for hire. Children enjoy climbing over the huge sculptures
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Kovalam Beach
Kovalam beach is at a distance of 16 km that is 1/2 hour from
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southernmost, known as the Lighthouse Beach, is the most popular.
Vettukad Church
Situated at a distance of 7 km from Thiruvananthapuram, the 'Madre De
Deus' Church popularly known as the Vettukad Church attracts thousands
of devotees from all religions. The annual celebration here is called
the 'Feast Of Christ, The King'.
Peppara Wildlife Sanctuary
Situated 50 km from Thiruvananthapuram, Peppara is a great attraction
for wildlife lovers as it is rich in flora and fauna, also it is solid
with hillocks, forests and eucalyptus plantations.


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/ FABRICA FEATURES - New Collection
FABRICA FEATURES - New Collection
10 January, 2007
Contributors: Sam Baron, Valentina Carretta, Miren Marañon
presents a new "Express" collection of travel bags, inspired by the 'mobile' way of life of Fabrica community
Three different suitcases made of a resistant plastic structure, covered by industrial textiles in a variety of pastel colors, each related to different moments of the day.
The small version for working purposes.
The medium version for instant trips and for your sport activities.
The large version for the weekend, perfect for airplane cabin dimensions.
Pastel green, pastel pink, black and military green are the colors available for the moment in all the Fabrica Features shops.
Sam Baron, Valentina Carretta, Miren Marañon
Production and distribution:
Euromark (Italy)
© 1994-2009 Fabrica S.p.A. - P.I. 01926330265 |


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Enchanting zest and glowing bubbles
Soak yourself in spellbinding waters that glow like the light of the moon and seal the charm firm and good with bewitching lime and neroli bubbles.
You'll love this if you like:
– Banishing negative energy with uplifting lime and neroli
– To bestow your bathwater with a supernatural glow
– Citrusy scents like Calacas body spray, Calavera bath bomb and Key Lime Pie lip balm
– Fair Trade organic cocoa butter softens and soothes your skin – Lime oil banishes the spirit of tiredness
– Neroli oil lifts your mood to a higher plane
How to use: Crumble into running water to create your bubbling cauldron. Don't forget to charge this bar under the light of the full moon (actually any light will do) to get the best glow up!
How to store: Keep away from water and don't feed after midnight. Give it a good charge in direct light before using it in your bath.
Sodium Bicarbonate, Sodium Laureth Sulfate, Cream of Tartar (Potassium bitartrate), Luminescent Zinc Sulfide, Popping Candy, Sodium Lauryl Sulfate, Lauryl Betaine, Perfume, Lime Oil (Citrus aurantifolia), Neroli Oil (Citrus aurantium amara), Olibanum Oil (Boswellia carterii), Fair Trade Organic Cocoa Butter (Theobroma cacao), Guar Gum (Cyamopsis tetragonoloba), Alpha-Isomethyl Ionone, Benzyl Salicylate, *Citral, Hydroxycitronellal, *Limonene, *Linalool


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Year Walk 12+
Simogo AB
- #133 in Role-Playing
- £3.99
In the old days man tried to catch a glimpse of the future in the strangest of ways.
Experience the ancient Swedish phenomena of year walking through a different kind of first person adventure that blurs the line between two and three dimensions as well as reality and the supernatural.
Venture out into the dark woods where strange creatures roam, on a vision quest set in 19th century Sweden. Solve cryptic puzzles, touch and listen in your search to foresee the future and finally discover if your loved one will love you back.
Mysteries and clues await everywhere in Year Walk, but to fully understand the events that took place on that cold New Year's Eve, you will have to delve deeper than the adventure and lose yourself between fact and fiction.
For the complete Year Walk experience, or if you're curious about the folk lore on which Year Walk is based, we recommend downloading Year Walk Companion, available for free on the App Store.
"An iOS game like nothing else you've seen on the format to date.
This is a brave and truly original work"
Edge - 9/10
"Elegant and artful, Year Walk is an unmissable piece of work.
You can close the app and put down the phone, but the forest may spread beyond its glassy confines, its spindly, silver-skinned trees taking root in your own home, your own dreams."
Eurogamer - 9/10
"A game this distinctive, this different, this thrillingly new is the best possible demonstration of the format's versatility; indeed, of the narrative power of the interactive medium. Simogo has made something very special here."
The Telegraph - 5/5
"It's a gripping, somber, atmospheric, and elegantly-designed game, and everyone should play it.
I've never played anything quite like it."
Touch Arcade 5/5
"An incredible showcase for the potential of tablet games and will undoubtedly become influential over the next few years."
"Simogo has packed Year Walk with a hefty share of spine-tingling jump scares, but it's these external trappings that cause the game's lore to get under your skin and stick with you long after your phone or tablet has been turned off."
Gamezebo - 4,5/5
"Year Walk is smart, thrilling, and absolutely stunningly crafted. It'll throw you for a loop, leaving you transfixed to the screen until you shake out all of its dark secrets."
Mac|Life - 5/5
"As with any good page-turner, you won't want to put this game down until you get to the end. It's quite a journey, too. Year Walk is a beguiling adventure, and a truly memorable experience."
Pocketgamer - 8/10
What's New
Version 1.4
- Added support for new devices
Ratings and ReviewsSee All
Editors' Choice
The eclectic studio behind Bumpy Road stretches its creative wings once again in this haunting first-person adventure game, nominated for a 2014 British Academy Games Award for Game Innovation. Whether you're tracking a path in the snowy wilderness or piecing together the creepy storyline, the Year Walk experience overflows with style and atmospheric allure.
Not as good as Device 6
I loved Device 6, perhaps my favourite game. And the reviews seemed to indicate that this was even better. Well, disappointingly, it's not. The navigation for a start was so confusing that I had to draw a map. Then I was stuck, and resorted to look at a walkthrough to get further (which I never had to do with Device 6). But when I saw the answers to the puzzles I never thought "Oh, why didn't I see that!", instead I thought "I'd never have thought of doing that in a million years" - not intuitive solutions at all. In fact, I have now given up. I hate having to rely on walkthroughs. Shame. Still a great company.
Very interesting environment but engaging and puzzling, recommended for those who have tried device 6 although these puzzles are harder to solve
- Provider
- Simogo AB
- Size
- Category
- Games
- Compatibility
Requires iOS 8.0 or later. Compatible with iPhone, iPad and iPod touch.
- Languages
- Age Rating
- 12+
- Frequent/Intense Horror/Fear Themes
- Infrequent/Mild Cartoon or Fantasy Violence
- Infrequent/Mild Mature/Suggestive Themes
- Infrequent/Mild Realistic Violence
- Copyright
- © 2013 Simogo, All rights reserved.
- Price
- £3.99
Family Sharing
Up to six family members will be able to use this app with Family Sharing enabled.