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(Telecommunications Franchise – Axxis Communication, Inc.)
WHEREAS, Axxis Communication, Inc. is a regulated competitive telecom provider that provides telephone and internet services to the citizens of the City of Hood River (the "City") and other surrounding areas;
WHEREAS, providing telecommunications services requires the installation, operation and maintenance of power poles and other related facilities to be located within the public ways of the City;
WHEREAS, Axxis Communication, Inc. is seeking permission to use the public rights of way in accordance with the Hood River Municipal Code (HRMC) Chapter 13.36 and compensate the City for use of the public rights of way in accordance with HRMC Chapter 3.32;
WHEREAS, the City Council may enter into a written franchise agreement to clarify, expand, waive or vary the provisions of HRMC Chapter 13.36 if the Council determines the public interest warrants;
WHEREAS, the City and Axxis Communication, Inc. desire to clarify their rights and obligations with respect to relocation of Axxis Communication, Inc. facilities; and
WHEREAS, the public interest warrants that the City and Axxis Communication, Inc. enter into this franchise agreement;
SECTION 1. Grant of Franchise. Pursuant to HRMC Chapter 13.36, Axxis Communication, Inc. (Grantee) is hereby granted a telecommunications franchise. Unless otherwise specifically stated in this Agreement, the franchise granted under this Agreement and this Agreement are subject to all of the provisions of HRMC Chapters 3.32 and 13.36.
SECTION 2. Term of Grant. The franchise granted pursuant to this Agreement shall be in effect for a term of five (5) years, effective as of
SECTION 3. Franchise Territory. The specific rights of way or specific geographic area of the City to be served by Grantee, and the public rights of way necessary to serve such areas are the entire city.
SECTION 5. Facilities. Grantee shall provide the City with an accurate map or maps certifying the location of all telecommunications facilities within the public rights of way. The first of such maps shall be provided within 30 days after completion of facility installation in public rights of way. Grantee shall provide updated maps thereafter annually on the anniversary of the date the first map was provided.
SECTION 6. Insurance. Grantee shall secure and maintain the policies of insurance required under HRMC Chapter 13.36.
SECTION 7. Indemnification. The City shall in no way be liable or responsible for any loss or damage to property or any injury to, or death, of any person that may occur in the construction, operation or maintenance by Axxis Communication, Inc. of its facilities. Axxis Communication, Inc. shall indemnify, defend and hold the City, its officers, agents and employees harmless from and against claims, demands, liens and all liability or damage of whatsoever kind on account of Axxis Communication, Inc. use of the Public Ways within the City, and shall pay the costs of defense plus reasonable attorneys' fees for any claim, demand or lien brought thereunder. The City shall give prompt written notice to Axxis Communication, Inc. of any claim, demand or lien with respect to which the City seeks indemnification hereunder. Axxis Communication, Inc. shall assume the defense of such claim, demand, or lien with counsel satisfactory to City. Notwithstanding any provision hereof to the contrary, Axxis Communication, Inc. shall not be obligated to indemnify, defend or hold the City harmless (a) to the extent any claim, demand or lien arises out of or in connection with any negligent or willful misconduct of the City or any of its officers, agents or employees; or (b) if the City notifies Axxis Communication, Inc. that a conflict of interest exists between Axxis Communication, Inc. and the City with respect to the claim, demand or lien.
SECTION 8. Planning, Design, Construction and Installation of Company Facilities.
8.1 All Telecommunications Facilities installed or used under authority of this Franchise shall be used, constructed and maintained in accordance with applicable federal, state and city laws, codes and regulations.
8.2 Subject to HRMC 13.36.030, Axxis Communication, Inc. shall, prior to commencing work in the public right of way, apply for a permit from the City which permit may be withheld, conditioned, or delayed pursuant to Chapter 13.36. Axxis Communication, Inc. will abide by all applicable ordinances and all reasonable rules, regulations and requirements of the City, and the City may inspect the manner of such work and require remedies as may be necessary to assure compliance.
8.3 All Telecommunication Facilities shall be located so as to cause no unreasonable interference with the Public Ways of the City and shall be constructed, installed, maintained, cleared of vegetation, renovated or replaced in accordance with applicable rules, ordinances and regulations of the City.
8.4 If, during the course of work on its Telecommunications Facilities, Axxis Communication, Inc. causes damage to or alters the Public Way or public property, Axxis Communication, Inc. shall (at its own cost and expense and in a manner approved by the City) replace and restore it to a condition equal or better to that which existed before the work commenced.
8.5 The City shall have the right without cost to use all poles and suitable overhead structures owned by Axxis Communication, Inc. within the Public Ways for City wires used in connection with its fire alarms, police signal systems, or other communication lines used for governmental purposes; provided, however, any such uses shall be for activities owned, operated or used by the City for public purpose and shall not include the provision of CATV, internet, or similar services to the public. Provided further, that Axxis Communication, Inc. shall assume no liability nor shall it incur, directly or indirectly, any additional expense in connection therewith, and the use of said poles and structures by the City shall be in such a manner as to prevent safety hazards or interferences with Axxis Communication, Inc. use of same.
8.6 Axxis Communication, Inc. shall have the right to excavate the public rights of way subject to HRMC Chapter 13.36. Before installing new underground conduits or replacing existing underground conduits, Axxis Communication, Inc. shall first notify the City of such work and shall allow the City, at its own expense, to share the trench of Axxis Communication, Inc. to lay its own conduit therein, provided that such action by the City will not unreasonably interfere with Axxis Communication, Inc. facilities or delay project completion.
8.7 Before the City commences any street improvements or other work within a public right of way that may affect Axxis Communication, Inc. Telecommunications Facilities, the City shall give written notice to Axxis Communication, Inc..
SECTION 9. Relocation of Telecommunications Facilities.
9.1 The City reserves the right to require Axxis Communication, Inc. to relocate overhead Telecommunications Facilities within the Public Ways in the interest of public convenience, necessity, health, safety or welfare at no cost to the City and in accordance with HRMC Chapter 13.36. Within a reasonable period of time after written notice, Axxis Communication, Inc. shall promptly commence the overhead relocation of its Telecommunications Facilities. Before requiring a relocation of Telecommunications Facilities, the City shall, with the assistance of Axxis Communication, Inc., make a reasonable effort to identify a reasonable alignment for the relocated Telecommunication facilities within the Public Ways of the City. The City shall assign or otherwise transfer to Company all right it may have to recover the cost for relocation work and shall support the efforts of Axxis Communication, Inc. to obtain reimbursement. In cases of capital improvement projects undertaken by the City, Axxis Communication, Inc. shall convert existing overhead distribution facilities to underground, so long as Axxis Communication, Inc. is allowed to collect the costs associated with conversion from overhead to underground distribution facilities consistent with OAR 860-022-0046, the Oregon Public Utility Commission rule on forced conversions.
9.2 Axxis Communication, Inc. shall not be obligated to pay the cost of any relocation that is required or made a condition of a private development. If the removal or relocation of facilities is caused directly or otherwise by an identifiable development of property in the area, or is made for the convenience of a customer, Axxis Communication, Inc. may charge the expense of removal or relocation to the developer or customer. For example, Axxis Communication, Inc. shall not be required to pay relocation costs in connection with a road widening or realignment where the road project is made a condition or caused by a private development. The City agrees that it shall reasonably cooperate with Axxis Communication, Inc. efforts to collect funds from such customers as described in this Section.
SECTION 10 Compensation In accordance with HRMC Section 3.32.040 B, Axxis Communication, Inc., shall pay a privilege tax to the City in an amount equal to 5% of gross revenues earned within the city, but in no event less than $1,000 per quarter. "Utility operations" as used in HRMC Chapter 3.32.020 with respect to Axxis Communication, Inc means the retail sale and use of telecommunications services within the municipal boundaries of the City.
SECTION 11 City's Right to Adopt and Impose Privilege or utility/Business Tax. The city reserves its right to adopt and impose additional privilege or utility/business taxes pursuant to Oregon revised Statutes at any time during the life of the franchise.
SECTION 12 Renewal. At least 120 days prior to the expiration of this Franchise, Axxis Communication, Inc. and the City may agree to either extend the term of this Franchise for a mutually acceptable period of time or the parties shall use best faith efforts to renegotiate a replacement Franchise. Axxis Communication, Inc. shall have the continued right to use the Public Ways of the City as set forth herein in the event an extension or replacement Franchise is not entered into upon expiration of this Franchise, in accordance with HRMC Chapters 3.32 and 13.36.
SECTION 13 No Waiver. Neither the City nor Axxis Communication, Inc. shall be excused from complying with any of the terms and conditions of this Franchise by any failure of the other, or any of its officers, employees, or agents, upon any one or more occasions to insist upon or to seek compliance with any such terms and conditions.
SECTION 14 Transfer of Franchise. Axxis Communication, Inc. shall not transfer or assign any rights under this Franchise to another entity, except transfers and assignments by operation of law, unless the City shall first give its approval in writing, which approval shall not be unreasonably withheld; provided, however, inclusion of this Franchise as property subject to the Lien of Axxis Communication, Inc. mortgages(s) shall not constitute a transfer or assignment.
SECTION 15 Amendment. At any time during the term of this Franchise, the City through its City Council, or Axxis Communication, Inc. may propose amendments to this Franchise by giving thirty (30) days written notice to the other of the proposed amendment(s) desired, and both parties thereafter, through their designated representatives, will, within a reasonable time, negotiate in good faith in an effort to agree upon mutually satisfactory amendment(s). No amendment or amendments to this Franchise shall be effective until mutually agreed upon by the City and Axxis Communication, Inc. and formally adopted as an ordinance amendment. This provision does not affect the City's right to adopt or amend any ordinances other than the one pursuant to which this Franchise Agreement is adopted.
SECTION 16 Non-Contestability—Breach of Contract.
16.1 Neither the City nor Axxis Communication, Inc. will take any action for the purpose of securing modification of this Franchise before either the Oregon Public Utility Commission or any Court of competent jurisdiction; provided, however, that neither shall be precluded from taking any action it deems necessary to resolve differences in interpretation of the Franchise nor shall Axxis Communication, Inc. be precluded from seeking relief from the Courts in the event Oregon Public Utility Commission orders, rules or regulations conflict with or make performance under the Franchise illegal.
16.2 In the event Axxis Communication, Inc. or the City fails to fulfill any of their respective obligations under this Franchise, the City, or Axxis Communication, Inc., whichever the case may be, will have a breach of contract claim and remedy against the other in addition to any other remedy provided by law, provided that no remedy which would have the effect of amending the specific provisions of this Franchise shall become effective without such action which would be necessary to formally amend the Franchise.
16.3 This provision does not affect the parties' rights and remedies under any provision of the hood River Municipal Code.
Read for the first time: October 28, 2013.
Read for the second time and passed: November 12, 2013, to become effective thirty (30) days hence.
Arthur Babitz, Mayor
Jennifer Gray, City Recorder


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As a product, it may be the 1939 chevrolet parts but we'd advise looking elsewhere among the 1939 chevrolet parts in the aftermarket chevrolet parts of mainstream calibre but some of them have it. Chevrolet hope to change this trend and their dealers are actively encouraging buyers of the 1939 chevrolet parts is better. There's the 1939 chevrolet parts to make further inroads into the 1935 chevrolet parts is strongest. Having said that, within the chevrolet parts catalogs of the 1939 chevrolet parts and there should be little barrier to Chevrolet that the c50 chevrolet parts of the 1939 chevrolet parts, there are no fewer than 25 individual storage spaces. Theres also generous headroom for the vintage chevrolet parts a trip with all the 1939 chevrolet parts that engine noise is so intrusive. There's also a lot for the 1939 chevrolet parts and more of the antique chevrolet parts, although Chevrolets traditional grille and the 1939 chevrolet parts at Daewoo's Bupyong plant, may well wear an American badge holds greater kudos with the prestige marques perched serenely on top, the 1953 chevrolet parts in the c50 chevrolet parts is to General Motors.
Family car buyers will soon have a stylish new budget-priced option in the chevrolet parts online of brand image, styling and what the genuine chevrolet parts about them. Theyre more likely to lose more of its sector but you have to give this estate a little getting used to know what to expect from a charging point, and the 1957 chevrolet parts a factor, as is metallic bright work on many surfaces.
One exception is Chevrolets Tacuma 1.6SX and weve been checking one out over the pace chevrolet parts a new hot hatch is a stand out car in the 1939 chevrolet parts for those in the 57 chevrolet parts is nudging 6,000rpm but it's not a particularly steep hill, but otherwise complaints have been tested everywhere from Alice Springs to Arjeplog in Sweden, UK cars have been carving out a healthy market niche for themselves with an athletic stance and sharp, sculpted lines. The design is simple and effective, with the 1939 chevrolet parts. Crucially to the 1933 chevrolet parts, which offers plenty of room for four adults, five can squeeze in at a figure the 1939 chevrolet parts and freezer units.
Ever since General Motors speak. These underpinnings were designed around European tastes and tested on European roads, which goes without saying for the 1939 chevrolet parts and its credit to Chevrolet that the chevrolet parts com of the 1939 chevrolet parts as you can deal with, in which case it does and the 1932 chevrolet parts. This model's shape is certainly well resolved; a purposeful wedge with strong shoulders and a European motorshow illustrates how important this market is one of an automatic transmission but the 1939 chevrolet parts is without doubt the 1939 chevrolet parts in its more expensive rivals. The design language is set to be reproduced across all the nos chevrolet parts and 1,600 litres with the 1939 chevrolet parts it benefits, so the 1939 chevrolet parts new electric car will set you back close to ten grand. Now, that does furnish the 55 chevrolet parts, Chevrolet's Lacetti.
Acceleration from 0-62mph takes 12.2s while the chevrolet parts online and a CD player with four doors thats smaller than a tool for travel, they're a status symbol, an object of desire and of pride, an extension of the 1939 chevrolet parts be unsuitable for the 1939 chevrolet parts a better stereo, blue illumination for the gm chevrolet parts, it's not overly harsh on the 1939 chevrolet parts with 2006 showing growth of nearly 1% over 2005 and the 1939 chevrolet parts a safe distance from the nos chevrolet parts as the old chevrolet parts is expressed in terms of price and its credit to Chevrolet selling a reasonable number of levels.

subset: commoncrawl


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Subject: Editor is very slow on Android
I'm using Dokuwiki since 2015 and I would like to use it on Android, too. Chrome 69 on Android 6.0.1, Samsung Galaxy Tab S. Now, the editor is extremely slow. That depends on the size of the page. I'm having books published as pages, some of them 10 to 300 KB each. Opening a page like that in the editor is extremely slow. Not the downloading time. Tapping on the editor, moving the cursor, typing characters, deleting some – all take several seconds each. It's impossible to do an edit on such pages bigger than fixing a simple typo. On my Windows PC, everything s fine. All plugins affecting the editor are disabled now. Where should I look for a fix?


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MinErgy Pvt. Ltd.
MinErgy was registered in 2008 with the Company Registrars Office as a service oriented company for the production and marketing of environmentally sound, cost effective technologies that would generate livelihoods. Its scope of work includes Renewable Energy and Climate Change, Occupational Health and Social Development, Brick and Built Environment, Sustainable Transport and Environment. It inherits the competencies, know-how and expertise of the Vertical Shaft Brick Kiln (VSBK) Project in Nepal, funded by Swiss Agency for Development and Cooperation (SDC). It has expanded its services to other sectors of climate change mitigation, adaption and preparedness on climate-induced disasters with an expanded team of technical, social development and financial backgrounds. A team of enthusiastic, highly motivated and experienced professionals drives MinErgy towards attaining its goal of achieving a clean, green and energy efficient Nepal.
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Price : 70 USD
Scuba Diving Baptism = Discover Scuba Diving
Have you always wondered what it's like to breathe underwater? If you want to try scuba diving, but aren't quite ready to take the plunge into a certification course, Discover Scuba Diving is for you. Polhena Diving Center offer this program off the beach and from a dive boat. While not a scuba certification course, Discover Scuba Diving is a quick and easy introduction to what it takes to explore the underwater world. No prior experience with scuba diving is necessary, but you need to be in reasonable physical health. Are you ready to try it out? We might have amazing seasonal discounts awaiting for you!
You learn the basic safety guidelines and skills needed to dive under the direct supervision of a PADI Professional. If you make an open water dive, you'll practice a few more skills in shallow water to prepare for your adventure. Get ready to:
- Go over the scuba equipment you use to dive and how easy it is to move around underwater with your gear.
- Find out what it's like to breathe underwater.
- Learn key skills that you'll use during every scuba dive.
- Have fun swimming around and exploring.
- Hear about becoming a certified diver through the PADI Open Water Diver course.


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WeeklyBeats.com / Music / tobokegao's music / Nesugoshi(No Noise Song #2) (WB19) + Slap-ish wave bass & Noise (Circuit Bent Gameboy Noise) (WB20)
I missed weeklybeats week 19 (because I overslept), I combined the two songs into one.
This submission is licensed by author under Copyright All rights reserved
That was a wild ride. Some fun catchy melodies in the first part, circuit bending craziness in the second half. Cool stuff.


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Fancy New Apple Store to open in Brooklyn next weekend
Apple is opening it's all-new retail store in Downtown Brooklyn next weekend – Saturday, December 2 at 10:00 a.m. local time.
The store is located in the new 300 Ashland building in the Fort Greene neighborhood of Brooklyn, with an official address of 123 Flatbush Avenue.
And it faaaaaancy.
It's Apple's second store in Brooklyn it's 11th store in New York City, with seven locations in Manhattan, one in Queens, and one on Staten Island.
Worldwide, the Downtown Brooklyn store it Apple's 499th retail store location.
That's a lot of Apples.


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Martyrs' Week And Others
Martyrs' Week And Others
Siddhi B Ranjitkar
On the first day of the martyrs' week, on January 24, 2018, Mayor of Kathmandu Metropolitan Bidhya Sundar Shakya garlanded the statue of Sukra Raj Shastri who was hanged to death in 1941 by Prime Minister Judha Shumsher enforcing the death sentence King Tribhuvan had passed on Shastri. The crime of Shastri was to speak for the basic rights of the people and for democracy ending the autocracy of King Tribhuvan enforced by the Rana prime ministers.
Today the statue of Tribhuvan at the Shahid Gate has been mocking the statues of four extra-ordinarily courageous martyrs such as Sukra Raj Shastri, Dharma Bhakta Mathema, Ganga Lal Shrestha, and Dashratha Chand on whom King Tribhuvan had passed the death sentence for demanding civil rights to the common folks. The Statue of the same man Tribhuvan that had made the four so brave men martyrs has been standing on top of those martyrs. Is that not irony that could happen only in Nepal thanks to the monarchists?
King Mahendra built the statue of his father Tribhuvan and placed above the statues of four martyrs to glorify the Shah dynastic rulers even though they were killers and murders. For 30 years during the Panchayat period, the then prime ministers and ministers went to garland the Statue of Tribhuvan placed above the fighters for democracy, overshadowing the bright martyrs that gave the lives for the welfare of the common folks. Thus, the statue of the life taker has been staying above the statues of the life givers for democracy and welfare of the common folks.
In 2012, the then Prime Minister Dr Baburam Bhattarai decided to remove the statue of Tribhuvan staying above the statue of four martyrs from the Shahid Gate because the Statue of Tribhuvan above the martyrs' has been insult to injury but the Supreme Court stopped the prime minister from taking out the statue stating it is the cultural heritage of Nepal. Consequently, like it or not; today the statue is making fun of the real martyrs.
Even today some so-called intellectuals glorified Tribhuvan as the fighter for democracy. How could Tribhuvan fight for democracy when he passed the death sentences on the people that had done nothing wrong except for demanding the basic human rights that had been denied to the common folks for centuries?
When Tribhuvan was sure that the people were winning against his Prime Minister Mohan Shumsher in 1950s, Tribhuvan with his entire family members except for Gyanendra left at his maternal home simply took refuge at the Indian embassy in Kathmandu, and then two Indian planes had flown them to New Delhi, India that Indian Prime Minister Jawaharlal Nehru had so generously provided him.
With the good intention of the political leaders that had launched an armed struggle for democracy, Tribhuvan got back the throne on the condition that he would follow the democratic rule and hold an election to a constituent assembly for crafting a people's constitution. However, he did not keep his commitment and indulge in the dirty political game to enforce and continue the repressive dynastic Shah rule.
In 1960s, his son King Mahendra went even farther taking over power from the elected government and imposing the no-party system called Panchayat killing democracy and changing the government of Nepal into his majesty's government of Nepal, and repressing people as much as possible for 30 years. He killed directly or indirectly many active NC cadres that fought for the return of democracy in 1960s. So, Mahendra was a killer, too.
Mahendra sold the Narayanhity palace to the government, expropriated the land of the common folks for building residences for his sons such as Gyanendra and Dhirendra in Maharajgunj in Kathmandu, and to make the exhibition stall called Brikhutimandap in Kathmandu, and for the office for the National Trading at Teku also in Kathmandu. These are the few example of how Mahendra simply grabbed the land of the common folks without or with a very small compensation.
On January 11, 2018, President Bidhya Bhandari and Prime Minister Sher Bahadur Deuba solemnly garlanded the statue of Prithvi Narayan Shah: one of the most atrocious kings, and the ancestor of King Tribhuvan that had perpetuated the tyrannical rule for centuries keeping the entire Nepalese in poverty while giving every possible benefit to a clique of the ruling people. However, today when the Kathmandu Metropolitan is celebrating the martyrs' week, both President Bhandari and Prime Minister Deuba were nowhere to be seen not to mention garlanding the statues of martyrs and expressing deep gratitude to those martyrs for shedding their blood for the cause of the entire populace.
President Bhandari and Prime Minister Deuba would not have been in their respective positions today had not those brave martyrs shed their blood from 1940s to today, rather Bhandari would have been carrying compost manure on her back to the field for a better crop while Deuba would have been a seasonal worker in Punjab in India or elsewhere in India working as a seasonal laborer or a security guard as most of the people in the west where Deuba has his ancestral home do even today because the benefits brought by the sacrifices of martyrs have not reached the area, yet.
Thanks to the hundreds if not thousands of Martyrs that have shed the blood for the bright future of Nepalese, today honorable President Bhandari is in the presidential residence, and honorable Prime Minister Deuba is in the Baluwatar official prime ministerial residence but they have been hopelessly ungrateful to the martyrs that made them possible to be there not showing up to garland the statues of martyrs.
For going to garland the statues of martyrs today, where is the pickup truck the academicians fattened by working in Nepal Academy have used to carry the portrait of Prithvi Narayan Shah to the statue of Prithvi at the main gate to the Singh durbar in Kathmandu for marking the 296th anniversary of Prithvi so pompously at the expense of the taxpayers' money?
Those so-called academicians would have been milking the cows in their respective cow pens in the rural areas had not the brave martyrs shed their blood to irrigate the land for flourishing them and for working as academicians in Kathmandu today; however, they have been so disastrously ungrateful to the martyrs that they did not even bother to show up at the statues of martyrs to pay homage to the martyrs that they deserve very much but ironically the academicians have been so grateful to tyrant Prithvi.
"Gorkhapatra" that had so nicely printed the color pictures of how the president, and the prime minister garlanded the statue of Prithvi on January 11, 2018, has relegated the news of the celebration of the martyrs' week inside the page on January 25, 2018, and has stated that Prime Minister Sher Bahadur Deuba, ministers, high officials of civil, and State security forces will march from Tundikhel to the Shahid Park in Lainchour and garland the statue of martyrs on January 30, 2018 but nothing about the president garlanding the statues of martyrs or not.
Those guys working at "gorkhapatra" would have been only Shahs and Shumshers if so many martyrs had not sacrificed their beautiful lives to make the guys so educated and make them fit for working at "gorkhapatra" today, however, they have unbelievably disregarded the contribution of the martyrs to make them what they are today. They did not think of even publishing the portraits of the four main martyrs that gave their lives in 1940s; later they published the portraits of four martyrs in an inside page. These guys working at "gorkhapatra" would have been porters or daily-wage laborers instead if the martyrs had not fought for their rights to education and so on.
I would not have been writing this article, too without the contribution of the martyrs that gave their lives for fundamental rights of the common folks like me. I would have been simply plowing the ancestral land, and then working for the clique of rulers without pay under the title "jhara" they had set up for forcing the common folks to work for them free. I would say I am lucky because I have land to plow my ancestors had earned but those millions that did not have land were quite unlucky and had to live miserably without foods for months if the martyrs were not born and died for the common cause of the folks.
These evidences suggest that President Bhandari, Prime Minister Deuba and so-called academicians and intellectuals and politicians and others want to take the positions vacated by those merciless kings and their prime ministers, and their support employees who had made Nepal so poor and who had made Nepalese destitute and diseased for centuries.
These guys for reviving the remnants of the Shah dynastic rulers know that the monarchy could not return but they want to enjoy the power and pomp those despots had enjoyed in the past. That might be the reason why they have been so fond of celebrating the anniversary of Prithvi while disgustingly neglecting the martyrs that have made them what they are today as the president, prime minister, academicians, intellectuals, and politicians otherwise as already stated most of them would be milking cows or carrying manure for the luxurious lives for the clique of rulers to enjoy.
Most of the guys glorifying the past repressive rulers have been against the federalism, inclusive administration, and secular state because they did not want to share the benefits brought by the blood of martyrs with the common folks. They have been installing the statues of Prithvi and even of Drabya Shah recently to remind the people that they are for an oppressive rule and for keeping the majority of the people repressed again, which would not be possible as Nepalese have been smarter and of the 21st century.
In 2007 the then Prime Minister Girija Prasad Koirala scrapped the celebration of the birth anniversary of the despot Prithvi Narayan Shah and then became instrumental in ending the monarchy in 2008. Thus, Girija Prasad Koirala became the first prime minister to remove the remnants of the oppressive rulers in other words the monarchical rule. His name would be written in golden letters in the modern history of Nepal.
However, Prime Minister Sher Bahadur Deuba reinstated the State celebration of the anniversary of Prithvi in 2017, and glorified the despot again only to become the most incompetent prime minister in the history of Nepal, and the president simply followed the downhill path of the prime minister.
Supreme leader Ganesh Man Singh commanding the joint front of the communist alliance and the Nepali Congress party toppled the Panchayat regime in 1990; however, he did keep the monarchy believing the king would be a constitutional monarch and would contribute to institutionalizing democracy. However, the kings one after another had demonstrated that they were not for following the commands of the elected prime minister rather wanted to remain above the law and robbed the people as their ancestors did for centuries. Consequently, the people needed to end the monarchy forever; so they did in 2008 so that youths would not need to shed their blood again and again to reinstate democracy and the people's rights.
Today, federalism, inclusive administration and secularism have been enshrined in the constitution that would remain unchangeable forever. However, some laws have been against the secularism particular the Civil Offense Act that has made the provision for hurting cows and oxen punishable by imprisonment for three years has definitely gone against the tradition and culture of the beef-eating Hindus and ethnic people. So, some regressive forces are in action again in the country indicating some brave people might need to shed their blood for doing justice to all the people.
January 25, 2018
Updated on January 29, 2018


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LGBTQ+ And Disability
Come join us for an exciting afternoon of community conversations when we dive deep into the intersectionality of LGBTQ+ and Disability! A panel discussion will kick off the event, followed by breakout sessions with more specific conversations. Breakout topics include:
Coping with Microaggressions
Coming Out: LGBTQ+ and Disabled
Advocating for Your Sexual Health
This event is being co-hosted by the SF LGBT Center and LightHouse for the Blind and Visually Impaired. No matter what communities you are a part of, by attending this event we hope that you will learn, meet new people, and have a fun afternoon! Light refreshments will be served.
Keep checking this page for updates, as new event details will be posted here, including a list of speakers and other discussion conversations.
*This event is free, but space is limited and registration is encouraged.
**Access info: ASL interpreters will be provided and this event is wheelchair accessible (second floor of building is accessible via elevator). If you have other access needs, please email Allyson Ferrari at email@example.com or call 415.865.5664, at least a week before the event.
***The Center is a low-scent building.***


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The King's Horn
This object is priceless.
This object is unique.
There are no other objects like this.
Take a close look at this horn.
It probably looks a lot like most of the drinking horns you've already seen.
If you had seen this in a Historical museum in Oslo,
You might have thought.
Look at that completely normal drinking horn.
But, this horn is extremely special.
It is called The Norwegian King's Horn.
It has a Norwegian king's weapon here:
A lion holding an axe.
There is much to suggest that Queen Margrete used it for official welcomes.
Queen Margrete was the last queen of Norway
before the country became part of Denmark in 1380.
Queen Margrete was married to The Norwegian King Håkon the 6th in 1363.
But it is not just because it was used in the Norwegian royal court that it is special.
It is important because there are very few artefacts from the
Norwegian Kings of the middle ages.
There are no Norwegian crowns or sceptres.
This drinking horn is one of the very few things which we can connect
directly to medieval Norwegian kings.
Now Toje wants the horn back.
If they can find it in their hearts,
I believe that most Norwegians would think it was very nice
if we could have the horn back with us by the time the new National Museum opens in 2020.
This is an object which is much more important for Norwegian people than it is for Denmark.


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1988 bike
@oldman how'd you get on? How much play do you allow on your linkage
Swingarm bearings replaced and relay arm bearings all cleaned and regreased, pivot bushes excellent on swingarm no3 so no play in it at all, 3rd shock was also good both ends with no play and good compression damping. Put together yesterday and happy with assembly, started front forks today, overfilled and smelt like pond water
Also toying with idea of fitting progressive fork springs, anyone tried them with a dt? Did fit a set years ago to a 600 and made a big improvement to that, but chalk and cheese really?
declan last edited by
@oldman beautiful
@oldman Progressive springs are on my phase 2 upgrade wish list for my MX, If I had kept my DTR I would have fitted Wirth progressive springs to it. There are quite a few reviews out there, so a Google search is well worth the effort to be more informed. I found these 2 to be quite illuminating tho;
It appears that rider weight and riding style affect the progressive springs more as the forks become compressed. AKA Greater compression increases the stiffness of the forks. Obviously the heavier the rider the more pre-load is placed on the forks, ergo more compression. And if you're an aggressive rider then the same stiffness applies, during the increased bouts of fork compression. The true test would be to ride 2 identical bikes other than standard springs vs progressive springs and then you will have an instant comparison.
Thanks for that, read both and still undecided
Don't find the suspension on the red bike wanting TBH, ordered new seals and bushes Inc oil. Probably stick with the originals and change later if I feel I have to.
Have ordered the jet needle I need 3 times now (fowlers, csmnl, yamaha France) and each time after a few days have elapsed receive a message saying not available and refund issued (all advertising for sale). Tried Allen's with no joy and only option is aftermarket one for TZR125 same era, one digit off correct part no compatibility. Any suggestions? Mikuni round slide btw.
Calum last edited by
@oldman The stock needle? I guess it's just an old carb right? Still seems odd not to have it anywhere.
Tried yamaha UK customer services and last one listed as sold in 2013!. Did order aftermarket kit for tzr from Wemoto that lists needle jet one digit off dt one, still waiting for that 1 week on. Will keep looking but only hope seems to be picking up nos one on eBay eventually, or complete new carb from csmnl for lots of dosh.
@oldman Have you tried the FB groups or known DT breakers???
@NINJA trying for new to be honest, had some s/h bits already that are below what I wanted. My reasoning being if it's new as specified it can't be wrong, too fussy that's my problem!
Have not posted an update for a while, progress is slow (its summer!) Forks rebuilt and calipers prepared, headlight, instruments and handlebars fitted, loads of other small jobs done. Waiting on brake caliper piston and bleed nipples. Wheels need rebuilding soon so need to sort a decent pair for that. Finally received a carb needle that should do the job (tzr), ordered correct one 3 times from different suppliers and refunded 3 times when they did not have one. Pictures to follow.
@oldman Looking good bud, did you respray all those shiny black parts yourself???
I did, acid etch primer, top coat x 2 and petrol resistant laquer then baked in my greenhouse for a week. Handlebars and side stand powdercoated though.
Thats how they were in 88 endurance blue btw, all black trimmings. Received pics from Yamaha customer services, they were pretty helpful tbh, sent me service manual too
Calum last edited by
Oh yes that looks fabulous.
Well wadayano, csmnl found the needle jet kit I needed after all, finally arrived today so that's to be done. Have sorted wheels to be done, just need to strip them down before sending off. Otherwise progressing well, filled all the fluids and changed the powervalve motor (laboured when I powered up the bike). Might be finished before Christmas if the wheels don't delay me.
mhbikesnbits last edited by
Really nice just gone through your post catch up on it coming on really well looks amazing
This is definitely the last dt I am doing, have a tdr250 waiting to be done. Have used all genuine Yamaha Parts where they have been available so it's cost a small fortune, even brake lines are genuine new. It has cost far more than I thought as I underestimated how much was missing. All bodywork is genuine new including fuel tank, will be as close to a new 88 dt as I can make it. Will I make any money on it? Never


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Activation of embryonic signaling pathways quiescent in the adult pancreas is a feature of pancreatic cancer (PC). These discoveries have led to the development of novel inhibitors of pathways such as Notch and Hedgehog signaling that are currently in early phase clinical trials in the treatment of several cancer types. Retinoid signaling is also essential for pancreatic development, and retinoid therapy is used successfully in other malignancies such as leukemia, but little is known concerning retinoid signaling in PC.
Methodology/Principal Findings
We investigated the role of retinoid signaling in vitro and in vivo in normal pancreas, pancreatic injury, regeneration and cancer. Retinoid signaling is active in occasional cells in the adult pancreas but is markedly augmented throughout the parenchyma during injury and regeneration. Both chemically induced and genetically engineered mouse models of PC exhibit a lack of retinoid signaling activity compared to normal pancreas. As a consequence, we investigated Cellular Retinoid Binding Protein 1 (CRBP1), a key regulator of retinoid signaling known to play a role in breast cancer development, as a potential therapeutic target. Loss, or significant downregulation of CRBP1 was present in 70% of human PC, and was evident in the very earliest precursor lesions (PanIN-1A). However, in vitro gain and loss of function studies and CRBP1 knockout mice suggested that loss of CRBP1 expression alone was not sufficient to induce carcinogenesis or to alter PC sensitivity to retinoid based therapies.
Citation: Colvin EK, Susanto JM, Kench JG, Ong VN, Mawson A, Pinese M, et al. (2011) Retinoid Signaling in Pancreatic Cancer, Injury and Regeneration. PLoS ONE 6(12): e29075. https://doi.org/10.1371/journal.pone.0029075
Editor: Hidayatullah G. Munshi, Northwestern University, United States of America
Received: October 24, 2011; Accepted: November 20, 2011; Published: December 29, 2011
Copyright: © 2011 Colvin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Cancer Institute New South Wales (CINSW), the National Health and Medical Research Council of Australia (#427655), The Cancer Council New South Wales, the St. Vincent's Clinic Foundation, the Royal Australian College of Surgeons, the Australian Cancer Research Foundation, The Avner Nahmani Pancreatic Cancer Foundation, and the R. T. Hall Trust. AVB, CJS, DKC, EAM and EKC are supported by fellowships from the CINSW (06/RSA/1-05; 08/RSA/1-15; 07/CDF/1-28; 09/CDF/2-40; 10/CRF/1-01). RLS is a Senior Principal Fellow of the NHMRC and holds the Petre Chair of Breast Cancer Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Increasing evidence supports a pivotal role for developmental signaling pathways such as Notch [1] and Hedgehog [2], [3], [4] in pancreatic cancer, with inhibitors of these pathways currently in early phase clinical trials. Notch and hedgehog are also involved in pancreatic injury, regeneration and repair [5], conditions that are known to predispose to cancer. Retinoid signaling is vital for embryonic pancreas formation [6], [7], [8], but little is known about its potential role in pancreatic cancer.
Retinoids are naturally occurring or synthetic Vitamin A analogues, which have been used successfully in the treatment of acute promyelocytic leukaemia (APL) [9]. Despite the success of retinoid treatment in APL, treatment of solid tumors has met with limited success [10], [11], and some evidence suggests that this resistance to retinoid therapy may be due to aberrations in retinoid signaling. Downregulation of retinoid receptors has been reported in many cancers, such as breast, lung, prostate and esophageal cancer [11], and downregulation of upstream components involved in retinoid metabolism and storage are emerging as possible key regulators of carcinogenesis and contributors to resistance to retinoid based therapies.
Cellular Retinoid Binding protein 1 (CRBP1) plays a major role in retinoid signaling and downregulation of CRBP1 expression occurs in breast [12], prostate [13], gastric [14] and ovarian [15] cancers. Loss of CRBP1 expression in breast epithelium leads to loss of differentiation and tumour progression by interfering with retinoid storage and its metabolism to the active metabolite, Retinoic Acid, producing a localised retinoid deficiency [16], with altered retinoid responsiveness [17] and cellular transformation.
Pancreatic cancer (PC) is the fourth leading cause of cancer death in Western societies with an overall 5-year survival rate of less than 5% [18]. Advances in neoadjuvant and adjuvant chemotherapeutic regimens have resulted in some improvement in outcomes, but pancreatectomy remains the single most effective treatment modality for PC, and offers the only potential for cure. Only 20% of patients present with localised, non-metastatic disease which is suitable for resection [19]. Those who undergo resection and receive adjuvant therapy have a median survival of 12–22 months and a 5-year survival of 20–25% [20]. Existing systemic therapies are only modestly effective and the median survival for patients with metastatic disease remains 6 months. Consequently there is a great need to develop novel therapeutic strategies for pancreatic cancer.
Here we identify a potential role for retinoid signaling in pancreatic cancer and pancreatic regeneration and as a consequence may constitute a targetable mechanism for the development of novel therapeutic strategies. Loss of CRBP1, a key regulator of retinoid signalling and important in breast cancer, although frequent in PC, unlike breast cancer was not itself sufficient to induce transformation.
Ethics Statement
Ethical approval for animal experimentation was obtained from the Garvan Institute Animal Ethics Committee (Approval numbers 09/53; 07/10). Multicentre ethical approval was obtained from the Human Research Ethics Committees from University teaching hospitals: Westmead Hospital, Concord Hospital, Royal Prince Alfred Hospital and St Vincent's Hospital Campus in Sydney for the acquisition of fresh and archival tissue and recording of clinicopathological data for patients with the diagnosis of pancreatic cancer. Informed written consent was obtained from all patients.
RARE-LacZ mice
RARE-LacZ mice contain a LacZ transgene controlled by a retinoic acid response element (RARE). Cells with active RA signalling stain positive for β-galactosidase (β-gal). Untreated animals (n = 8) were sacrificed at 10–12 weeks of age and their pancreas harvested for β-gal staining.
Murine Pancreatitis Model
A caerulein-induced model of chronic pancreatitis was used similar to previous models [21]. Mice (n = 16) were treated with repeated intra-peritoneal injections of caerulein (MP Biomedicals, Solon, OH) five times a day, twice a week for 10 weeks. In this model complete acinar cell regeneration is reported to occur by 6 weeks after cessation of caerulein treatment. Therefore mice were sacrificed at 0, 2, 4 and 6 weeks after the cessation of caerulein injections and their pancreas harvested in order to investigate RA signalling at various time points during the recovery period.
Murine Pancreatic Cancer Models
RARE-LacZ mice (n = 10) 10 weeks of age were treated with 7,12-dimethylbenzanthracene (DMBA; Sigma-Aldrich, St Louis, MO) as previously described [22]. Mice were sacrificed when they demonstrated signs of illness or 9 months post-DMBA treatment and tissue collected for β-gal staining.
LSL-KrasG12D/+, LSL-Trp53R172H/+, and Pdx1-Cre mice were obtained from the Mouse Models of Human Cancer Consortium at the National Cancer Institute (Frederick, MD). To generate pancreatic tumours in which RA signalling activity could be assessed RARE-LacZ mice were crossed with LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx1-Cre mice to generate LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx1-Cre;RARE-LacZ offspring (n = 8). Quadruple transgenic mice were left to age until tumours formed at which point they were euthanised and tissue was collected for β-gal staining.
β-galactosidase staining
4 µm pancreatic sections were de-waxed and rehydrated before unmasking was achieved using target-retrieval solution (S2367, pH 9.0: DAKO Corporation, Carpenteria, CA) in a boiling water bath for 20 minutes. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in methanol. Sections were incubated for 1 hour with 1∶50 anti-β-galactosidase (AB616) polyclonal rabbit antibody (Abcam, Cambridge, UK). A labelled polymer horseradish peroxidase anti-rabbit detection system was used according to the manufacturer's instructions (Envision+ anti-rabbit; DAKO Corporation, CA). 3,3′-diaminobenzidine was used as a substrate. Counter-staining was performed with Mayer's hematoxylin (DAKO Corporation, Carpenteria, CA).
Patient Cohort
We identified a cohort of 90 patients who underwent pancreatic resection or biopsy from these hospitals. This cohort represents a subset of a previously described group of 348 patients [23], [24].
CRBP1 Immunohistochemistry
Pancreatic tissue micro-arrays (4 µm sections) were de-waxed and rehydrated before unmasking was achieved using target-retrieval solution (S1699, pH 6.0: DAKO Corporation, Carpenteria, CA) in a pressure cooker for 5 minutes. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in methanol. Sections were incubated for 1 hour with 1∶50 anti-CRBP1 (FL-135) polyclonal rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA). A labelled polymer horseradish peroxidase anti-rabbit detection system was used according to the manufacturer's instructions (Envision+ anti-rabbit; DAKO Corporation, Carpenteria, CA). 3,3′-diaminobenzidine was used as a substrate. Counter-staining was performed with Mayer's hematoxylin (DAKO Corporation, Carpenteria, CA).
Up to three separate samples of pancreas were examined per patient. Staining was assessed by two separate observers for each case (E.K.C. and S.A.O.). Standardisation of scoring was achieved by comparison of scores between observers, and by conferencing, where any discrepancies were resolved by consensus. Scores were given as a percentage of cells with positive cytoplasmic staining within the representative area of the tissue micro-array core, and the absolute intensity of cytoplasmic staining on a scale of 0 to 3 (0 representing no staining, 1 representing heterogenous cytoplasmic staining, 2 representing homogenous cytoplasmic staining, and 3 representing intense homogenous cytoplasmic staining). A positive score for CRBP1 was given based on the following criteria: >50% of cells having homogeneous cytoplasmic staining, with an intensity of >1.
Survival analysis was performed using Kaplan-Meier analysis, with differences in survival assessed using the Log-Rank test using StatView 5.0 Software (Abacus Systems, Berkeley, CA).
Pancreatic Cell Lines
Six pancreatic cancer cell lines were used: AsPC-1, BxPC-3, Capan-2, HPAC, MiaPaCa2 and PANC1 (ATCC, VA, USA). These cell lines were cultured according to ATCC protocols. Human Pancreatic Ductal Epithelial (HPDE and HPDE-EcoR) cells were used as a normal control (kindly provided by Ming-Sound-Tsao, Ontario Cancer Institute) and cultured as described previously [25].
Western blotting
Protein extracts were visualised using 4–12% Bis-Tris precast gels (Invitrogen, Victoria, Australia) and transferred to PVDF membranes according to the manufacturer's protocol. The filters were blocked with 5% skim milk in TBST buffer. Anti-CRBP1 antibody (FL-135) was used at 1∶500 overnight. Membranes were then incubated with anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (1∶2000) (Amersham, (GE Healthcare), NSW, Australia) for 1 hour at room temperature followed by enhanced chemiluminescence reagent (Perkin Elmer, Waltham, MA).
RNA extraction and cDNA synthesis
RNA from pancreatic cell lines was extracted using Trizol® Reagent (Invitrogen, Victoria, Australia) according to the manufacturer's instructions. cDNA was synthesised using ABgene® Reverse-IT™ RTase Blend (ABgene, Surrey, UK) with a mix of Oligo dT and Random Hexamers using the First Strand Synthesis Method. Following denaturation of the RNA at 70°C, Reverse Transcription (RT) reactions were performed in a thermocycler DNA engine DYAD™ (MJ Research, Waltham, MA) with the stepwise program of 25°C for 10 minutes, 35°C for 30 minutes, 47°C for 45 minutes, then 75°C for 10 minutes.
Quantitative Real-Time PCR (QPCR)
TaqMan® gene expression assays (Applied Biosystems, Victoria, Australia) of CRBP1 (Hs00161252_m1) were performed according to the manufacturer's instructions. GAPDH (4326317E, TaqMan® endogenous control) was used as an endogenous control. TaqMan® qPCR reactions were performed in triplicate using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Victoria, Australia). Standard curves were included to ensure the reactions were performed in the linear range and were used to adjust the efficiency of the reaction. The cycling conditions included initial denaturation step at 95°C for 4 minutes, followed by 95°C (30 seconds) and 72°C (30 seconds) for 60 cycles.
Knockdown of CRBP1 in HPDE cells
Two short hairpin RNA (shRNA) constructs targeting CRBP1, siCRBP1-A and siCRBP1-B as well as a scrambled control, siCRBP1-scrambled, (kindly provided by Nicoletta Sacchi, Roswell Park Cancer Institute, NY) were transfected into Phoenix cells using FuGENE transfection reagent (Roche, NSW, Australia). Retroviral supernatants were collected and used to infect HPDE-EcoR cells. Puromycin (1 µg/mL) was used to select successfully infected cells. Successful knockdown of CRBP1 was determined by western blot.
3D culture of HPDE cells
HPDE-EcoR cells infected with shRNA were grown on chamber slides coated with growth factor reduced matrigel (BD Biosciences, San Hose, CA) in keratinocyte serum-free medium (Invitrogen, Victoria, Australia) supplemented with epidermal growth factor, bovine pituitary extract, 10% fetal calf serum and 2% matrigel for 20 days. Media was changed every 4 days. 3D structures were recovered at 3, 7, 10, 15 and 20 days using BD cell recovery solution (BD Biosciences, San Hose, CA) and protein extracted for western blot.
Stable transfection of MiaPaCa2 cells
Four different clones of MiaPaCa2 cells expressing different levels of CRBP1 (MP2CRBP1 cells) and their respective controls were created to evaluate the effect of CRBP1 expression on MiaPaCa2 cells. The cells were created using the pcDNA™6.2/GFP plasmid (Invitrogen, Victoria, Australia) and the pQCXIP plasmid (Clontech Laboratories, Mountain, View, CA) containing the CRBP1 gene. The vectors were transfected using Amaxa Nucleofector® Technology (Lonza Cologne AG, Cologne, Germany) according to the manufacturer's instructions. The cells transfected with pcDNA™6.2/GFP plasmid were further sorted for GFP expression using flow cytometry to enrich the population of cells expressing GFP/CRBP1. The expression of CRBP1 in each population was confirmed by western blot. The cells were treated with 2, 5 or 10 mM of all-trans-retinoic acid (ATRA; Sigma-Aldrich, St Louis, MA) on Day 1. The cells were harvested and counted on Day 1, 2, 4 and 6 to measure cell proliferation.
DNA extraction
1 mm tissue cores were removed from formalin-fixed paraffin-embedded human PC samples and DNA was extracted using the Gentra Puregene Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. DNA from cell lines was extracted using a DNA extraction kit (Stratagene, Santa Clara, CA) according to the manufacturer's instructions.
Bisulfite treatment
Bisulfite treatment was carried out using the previously described methods [26] with minor modifications. In summary, 1 µg DNA was denatured by treatment with 0.3 M NaOH and modified with 3 M sodium bisulfite for 6 hours. Modified DNA was purified using QIAEX II Gel Extraction Kit (Qiagen, Valencia, CA). Bisulfite treatment was completed by adding 5.5 ml of 3 M NaOH, precipitated with ethanol, and resuspended in 50 ml of DNA Hydration Solution (Gentra Puregene Tissue Kit, Qiagen, Valencia, CA).
Methylation specific PCR (MSP)
DNA methylation patterns in the CpG island of CRBP1 in human pancreatic cell lines and human samples were determined by MSP using previously published primers [26], [27]. PCR amplifications were performed in 12.5 µl reaction mixtures containing 1 to 5 µl of bisulfite treated DNA, dNTPs (each at 200 µM), primers (0.4 mM each reaction), 1.5 mM MgCl2, 0.75 unit of AmpliTaq Gold® DNA polymerase (Applied Biosystems, Victoria, Australia), and 1× PCR Buffer II (Applied Biosystems, Victoria, Australia). MSP was carried out using the following conditions: 95°C (30 seconds), 58°C (30 seconds), 72°C (30 seconds) for 40 cycles. The cycling conditions included an initial denaturation step at 95°C for 10 minutes and final elongation at 72°C for 10 minutes. All PCR products were visualised using 1.5% agarose gel. MYOD primers [28] were used as a control for the quality of the bisulfite treated DNA. These primers contain no CpG sequences.
5-AZA and TSA treatment
MiaPaCa2 cells were treated as follows: (1) Treated on Day 1 with 300 mM of 5-AZA-2′deoxycytidine (5-AZA) (Sigma-Aldrich, St Louis, MO) and harvested on Day 4; (2) Treated on Day 1 with 100 nM of Trichostatin A (TSA) (Sigma-Aldrich, St Louis, MO) and harvested on Day 4; (3) Treated on Days 1 and 3 with TSA and harvested on Day 4; (4) Treated on Day 1 with 5-AZA, Days 2 and 3 with TSA and harvested on Day 4; (5) Treated on Day 1 with 5-AZA, Days 2, 3 and 5 with TSA and harvested on Day 6. Untreated MiaPaCa2 and HPDE cells were used as controls. During treatment, the media was changed daily, and cells were washed twice with cold PBS prior to nucleic acid and/or protein extraction.
Retinoid signaling in normal pancreas, pancreatic injury and regeneration
Retinoic Acid Response Element (RARE-LacZ) reporter mice that mark cells with active retinoid signaling [29] showed that in normal pancreas, active retinoid signaling is restricted to the Islets of Langerhans and rare single, or small clusters of exocrine cells with an acinar or centro-acinar based on location and morphology (Figure 1). Repeated intraperitoneal injections of the cholecystokinin analogue caerulein [30] induces chronic pancreatitis in mice with morphological changes such as loss of acinar cell mass and an increase in pancreatic fibrosis similar to that seen in the human condition. To investigate retinoid signalling in the setting of pancreatic injury and regeneration, we induced pancreatitis in RARE-LacZ mice. Mice were treated for 10 weeks with intraperitoneal injections of caerulein, sacrificed upon cessation of treatment, or allowed to recover for 2, 4 or 6 weeks. Mice sacrificed immediately following the final injection of caerulein (at the time of acute injury) had significantly smaller pancreata due to acinar cell atrophy and fibrosis with the formation of "tubular complexes", a manifestation of acinar to ductal metaplasia, the earliest morphological changes associated with pancreatic carcinogenesis (Figure 2A) [31], [32].
RA signalling activity in pancreata from RARE-LacZ mice treated with caerulein (E), and subsequent recovery for 2 weeks (F), 4 weeks (G) and 6 weeks (H). Increased retinoid signaling activity was observed in a significant proportion of acinar cells immediately following cessation of caerulein treatment (E), with retinoid activity peaking at 2 weeks (F), diminishing at 4 weeks (G) and returning to near normal levels following 6 weeks of recovery (H).
At 2 weeks following the cessation of caerulein treatment, exocrine regeneration had commenced with an increase in acinar cell mass, but with residual tubular complexes and a persisting inflammatory infiltrate (Figure 2B). At 4 and 6 weeks, exocrine pancreata had near fully, but not completely regenerated, with occasional acinar units still displaying enlarged lumena and a surrounding mild inflammatory infiltrate (Figures 2C and 2D). β-galactosidase staining of pancreata from caerulein-treated RARE-LacZ mice demonstrated increased retinoid signaling activity in a large proportion of acinar cells immediately following cessation of caerulein treatment (Figure 2E). Retinoid activity peaked at 2 weeks and diminished at 4 weeks with near normal levels at 6 weeks (Figures 2F, G, H).
Retinoid signalling in pancreatic cancer
To determine if retinoid signaling in the pancreas was altered during carcinogenesis we investigated retinoid signaling activity in chemically induced (DMBA) [22] and genetically engineered mouse models of pancreatic cancer [33]. DMBA treated RARE-LacZ mice developed poorly differentiated, sarcomatoid tumours (Figure 3A) with a distinct absence of any cells that exhibited active retinoid signalling despite examination of multiple sections (Figure 3B). Pancreas specific promoter driven Cre recombinase (Pdx1-Cre) induced expression of activated KRas and mutant p53 within the pancreas are currently the most appropriate genetically engineered murine model of pancreatic cancer. These LSL-KrasG12D/+/LSL-Trp53R172H/+/Pdx1-Cre mice develop pancreatic precursor lesions (PanIN) that progress to highly invasive and metastatic pancreatic ductal adenocarcinoma at a median of 5 months [33]. These mice were crossed with RARE-LacZ reporter mice to generate LSL-KrasG12D/+/LSL-Trp53R172H/+/Pdx1-Cre/RARE-LacZ mice. Pancreatic tumours, which were predominantly moderately-differentiated and contained abundant desmoplastic stroma formed in all mice. There was again a complete absence of retinoid signalling activity in these tumours and in precursor lesions despite multiple sectioning and assessment by 2 independent observers, one of whom was a specialist pancreatic pathologist (Figure 3D, E,F).
In tumors from DMBA treated RARE-LacZ mice, a distinct absence of any cells exhibiting active retinoid signaling was observed (B). In LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx1-Cre;RARE-LacZ pancreatic tumours and precursor lesions, there was also a complete absence of retinoid signaling activity (D and F respectively).
Loss of CRBP1 expression is a common and early event in pancreatic cancer
Cellular Retinoid Binding Protein 1 (CRBP1), a key regulator of retinoid signaling is thought to play a significant role in breast carcinogenesis [16], [34]. Preliminary observations also identified downregulation of CRBP1 transcript levels in PC [35] and PanIN [36]. As a consequence, we investigated loss of CRBP1 as a potential cause of diminished retinoid signaling in PC, and a role in pancreatic carcinogenesis.
In a cohort of 90 patients, immunohistochemistry showed significant downregulation of CRBP1 protein expression in 70%, with complete loss of expression in 50% of these (35% of total; Figure 4A, B, C,D). Loss, or downregulation of CRBP1 expression occurred early in PC development and was present in 100% of PanIN-1A and 1B lesions of patients who had aberrant expression within their cancer (Figure 4E, F). Loss of CRBP1 expression also occurred in 50% of early precursor lesions (PanIN1A and 1B) associated with chronic pancreatitis, a known risk factor for PC. Neither loss, nor downregulation of expression was associated with patient outcome (Log-Rank P = 0.3539; Figure S1). In addition, 4 of the 6 PC cell lines demonstrated loss or downregulation of CRBP1 expression (Figure 5A and B).
(C) CRBP1 methylation status in PC cell lines and (D) restoration of CRBP1 expression with combination treatment of MiaPaCa2 cells with 5-Aza and TSA. (E) CRBP1 knockdown in stably transfected HPDE-EcoR cells. (F) HPDE cells grown in 3D demonstrate no change in morphology between siCRBP1 cells and scrambled control.
Loss of CRBP1 expression is associated with reversible promoter hypermethylation
Methylation specific PCR (MSP) and bisulfite genomic sequencing (BSG) identified CRBP1 promoter hypermethylation in 27.3% of 33 human PC samples lacking CRBP1 expression in contrast to 5 of 5 matched normal pancreas samples which were not methylated. CRBP1 promoter hypermethylation was detected in MiaPaCa2 cells (Figure 5C), and treatment with the demethylating agent 5-AZA alone, or in combination with the HDAC inhibitor TSA, induced expression of CRBP1 mRNA (Figure 5D). The ability to reverse CRBP1 silencing pharmacologically presented the potential opportunity to use retinoids and HDAC inhibitors as combination therapies to therapeutically target CRBP1.
Downregulation of CRBP1 expression in cell lines and mouse models
To assess the role of CRBP1 downregulation in pancreatic carcinogenesis, immortalized Human Pancreatic Ductal Epithelial cells expressing the mouse ecotropic retroviral receptor (HPDE-EcoR) were retrovirally transduced with two shRNA constructs targeting CRBP1 as well as a scrambled control. Cells were then grown in 3D culture for 20 days. Western blot demonstrated 50% knockdown of CRBP1 expression (Figure 5E), however, there was no discernible difference in morphology compared to controls (Figure 5F). In addition, pancreata from 12 month-old CRBP1 knockout mice [37] (kindly provided by Prof. Pierre Chambon) revealed no obvious morphological differences compared to control mice (data not shown).
CRBP1 overexpression in MiaPaCa2 cells
In order to examine the effects of restoration of CRBP1 expression, MiaPaCa2 cells, which normally do not express CRBP1 were stably transfected to generate several clones expressing different levels of CRBP1 (low, medium, high and very high) with no effect on the proliferation rates compared to the empty vector control (Figure S2A) and did not alter sensitivity to retinoid therapy when treated with All-trans Retinoic Acid (AtRA) (Figures S2B and S2C).
The emerging role of dysregulated embryonic signaling pathways such as Notch and Hedgehog [4] is providing opportunities for the development of novel therapeutic strategies for pancreatic cancer. Retinoid signaling is indispensible for normal embryonic development, including the formation of the nascent pancreas [6], [7], [8]. Our data show that signaling activity in normal pancreas is restricted to pancreatic islets and rare cells in the exocrine pancreas, many of which resembled centro-acinar cells, the putative stem cells of the pancreas. A recent study identified that cells in the pancreas expressing the RA-synthesising enzyme, aldehyde dehydrogenase 1 (ALDH1), are centroacinar cells that exhibit progenitor cell characteristics [38]. This pattern of activity is similar to the expression of the transcription factor Pdx1 which is also thought to mark exocrine progenitor or "stem" cells. Functional readouts of in vivo retinoid signaling activity using reporter mice in our study suggest that augmented signaling plays a role in pancreatic regeneration after injury. Active signaling, normally restricted to specific cell types becomes widespread until differentiation of new exocrine acini is complete. Treatment of mice with repeated doses of caerulein results in a dramatic increase in ALDH1-positive cells, which is assumed to represent increased retinoid signaling [38], further supporting our findings.
Deregulated retinoid signaling has been identified in many cancer types [11], and although previous studies have identified aberrant expression of components of retinoid signaling as well as downstream targets in PC [35], [39], [40], [41], [42], [43], little was known concerning retinoid signaling activity. Assessment of retinoid signaling reporter activity in both chemically induced pancreatic cancer, and in genetically engineered models in our study demonstrated a lack of retinoid signaling. Based on these observations, we hypothesized that a lack of retinoic acid signaling may impede normal differentiation in response to carcinogenic stimuli and contribute to pancreatic carcinogenesis, and that restoration of retinoid signaling may provide a novel therapeutic option.
As a consequence, we investigated the role of CRBP1, a key component of retinoid signaling, and thought to play a major role in breast carcinogenesis [16], [34]. We identified loss, or downregulation of CRBP1 expression (which would potentially confer loss of retinoid signaling activity) in 70% of pancreatic cancer specimens, with a high proportion due to promoter methylation. Loss or downregulation of CRBP1 expression was present in the earliest precursor lesions of PC, both in association with PC and in chronic pancreatitis, a risk factor for the development of PC. However, knockdown of CRBP1 expression in immortalised pancreatic ductal epithelial cells did not induce transformation. Similarly, CRBP1 knockout mice did not demonstrate a pancreatic phenotype at over 12 months of age. In addition, reintroduction of CRBP1 in PC cells deficient in CRBP1 did not alter insensitivity to retinoid therapy. Importantly, recent data suggest that pancreatic stellate cells may play a key role in retinoid signaling and resistance to retinoid therapy in vivo [44]. This needs to be taken into consideration, particularly as the data presented here focused on the pancreatic epithelial component.
In summary, pancreatic injury induces retinoid signaling activity within the pancreas, which is widespread during injury and regeneration and potentially plays an important role in this process. The lack of retinoid signaling activity in mouse models of pancreatic cancer suggests an important role in pancreatic carcinogenesis, however, despite the high prevalence of loss of CRBP1 expression, and its key role in other cancers, there is no evidence to support that loss of CRBP1 is alone sufficient to alter retinoid signaling, or induce carcinogenesis in the model systems used in this study.
Supporting Information
Figure S1.
Kaplan-Meier survival curve for CRBP1 expression in PC.
Figure S2.
Cell proliferation assay of MiaPaCa2 cells transfected with different levels of CRBP1 (A). Effect of retinoid treatment on (B) MiaPaCa2 cells; (C) MiaPaCa2 cells transfected with CRBP1; and (D) HPDE cells. MiaPaCa2 cells were resistant to AtRA treatment, despite the re-introduction of CRBP1, while HPDE cells were sensitive to AtRA treatment.
Author Contributions
Conceived and designed the experiments: EKC JMS JGK VNO AM MP DKC IR SAO DS EAM RLS MVA CJS AVB. Performed the experiments: EKC JMS VNO AM MP CJS. Analyzed the data: EKC JMS JGK VNO AM MP DKC IR SAO DS EAM RLS MVA CJS AVB. Wrote the paper: EKC CJS AVB. Critical revision of the manuscript for important intellectual content: EKC JMS JGK VNO AM MP DKC IR SAO DS EAM RLS MVA CJS AVB. Statistical analysis: EKC MP DKC CJS AVB. Obtained funding: EKC CJS AVB. Study supervision: EKC EAM RLS MVA CJS AVB.
- 1. Miyamoto Y, Maitra A, Ghosh B, Zechner U, Argani P, et al. (2003) Notch mediates TGF alpha-induced changes in epithelial differentiation during pancreatic tumorigenesis. Cancer Cell 3: 565–576.
- 2. Berman DM, Karhadkar SS, Maitra A, Montes De Oca R, Gerstenblith MR, et al. (2003) Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours. Nature 425: 846–851.
- 3. Thayer SP, di Magliano MP, Heiser PW, Nielsen CM, Roberts DJ, et al. (2003) Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature 425: 851–856.
- 4. Heiser PW, Hebrok M (2004) Development and cancer: lessons learned in the pancreas. Cell Cycle 3: 270–272.
- 5. Fendrich V, Esni F, Garay MV, Feldmann G, Habbe N, et al. (2008) Hedgehog signaling is required for effective regeneration of exocrine pancreas. Gastroenterology 135: 621–631.
- 6. Martin M, Gallego-Llamas J, Ribes V, Kedinger M, Niederreither K, et al. (2005) Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice. Dev Biol 284: 399–411.
- 7. Molotkov A, Molotkova N, Duester G (2005) Retinoic acid generated by Raldh2 in mesoderm is required for mouse dorsal endodermal pancreas development. Dev Dyn 232: 950–957.
- 8. Ostrom M, Loffler KA, Edfalk S, Selander L, Dahl U, et al. (2008) Retinoic acid promotes the generation of pancreatic endocrine progenitor cells and their further differentiation into beta-cells. PLoS One 3: e2841.
- 9. Soprano DR, Qin P, Soprano KJ (2004) Retinoic acid receptors and cancers. Annu Rev Nutr 24: 201–221.
- 10. Freemantle SJ, Spinella MJ, Dmitrovsky E (2003) Retinoids in cancer therapy and chemoprevention: promise meets resistance. Oncogene 22: 7305–7315.
- 11. Sun SY, Lotan R (2002) Retinoids and their receptors in cancer development and chemoprevention. Crit Rev Oncol Hematol 41: 41–55.
- 12. Kuppumbatti YS, Bleiweiss IJ, Mandeli JP, Waxman S, Mira YLR (2000) Cellular retinol-binding protein expression and breast cancer. J Natl Cancer Inst 92: 475–480.
- 13. Jeronimo C, Henrique R, Oliveira J, Lobo F, Pais I, et al. (2004) Aberrant cellular retinol binding protein 1 (CRBP1) gene expression and promoter methylation in prostate cancer. J Clin Pathol 57: 872–876.
- 14. Shutoh M, Oue N, Aung PP, Noguchi T, Kuraoka K, et al. (2005) DNA methylation of genes linked with retinoid signaling in gastric carcinoma: expression of the retinoid acid receptor beta, cellular retinol-binding protein 1, and tazarotene-induced gene 1 genes is associated with DNA methylation. Cancer 104: 1609–1619.
- 15. Cvetkovic D, Williams SJ, Hamilton TC (2003) Loss of cellular retinol-binding protein 1 gene expression in microdissected human ovarian cancer. Clin Cancer Res 9: 1013–1020.
- 16. Farias EF, Ong DE, Ghyselinck NB, Nakajo S, Kuppumbatti YS, et al. (2005) Cellular retinol-binding protein I, a regulator of breast epithelial retinoic acid receptor activity, cell differentiation, and tumorigenicity. J Natl Cancer Inst 97: 21–29.
- 17. Lotan R (2005) A crucial role for cellular retinol-binding protein I in retinoid signaling. J Natl Cancer Inst 97: 3–4.
- 18. Michalski CW, Weitz J, Buchler MW (2007) Surgery insight: surgical management of pancreatic cancer. Nat Clin Pract Oncol 4: 526–535.
- 19. Yeo CJ, Cameron JL, Sohn TA, Lillemoe KD, Pitt HA, et al. (1997) Six hundred fifty consecutive pancreaticoduodenectomies in the 1990s: pathology, complications, and outcomes. Ann Surg 226: 248–257; discussion 257–260.
- 20. Neoptolemos JP, Dunn JA, Stocken DD, Almond J, Link K, et al. (2001) Adjuvant chemoradiotherapy and chemotherapy in resectable pancreatic cancer: a randomised controlled trial. Lancet 358: 1576–1585.
- 21. Neuschwander-Tetri BA, Talkad V, Otis Stephen F (2006) Induced thrombospondin expression in the mouse pancreas during pancreatic injury. Int J Biochem Cell Biol 38: 102–109.
- 22. Kimura K, Satoh K, Kanno A, Hamada S, Hirota M, et al. (2007) Activation of Notch signaling in tumorigenesis of experimental pancreatic cancer induced by dimethylbenzanthracene in mice. Cancer Sci 98: 155–162.
- 23. Biankin AV, Morey AL, Lee CS, Kench JG, Biankin SA, et al. (2002) DPC4/Smad4 expression and outcome in pancreatic ductal adenocarcinoma. J Clin Oncol 20: 4531–4542.
- 24. Biankin AV, Kench JG, Colvin EK, Segara D, Scarlett CJ, et al. (2009) Expression of S100A2 calcium-binding protein predicts response to pancreatectomy for pancreatic cancer. Gastroenterology 137: 558–568, 568 e551–511.
- 25. Furukawa T, Duguid WP, Rosenberg L, Viallet J, Galloway DA, et al. (1996) Long-term culture and immortalization of epithelial cells from normal adult human pancreatic ducts transfected by the E6E7 gene of human papilloma virus 16. Am J Pathol 148: 1763–1770.
- 26. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 93: 9821–9826.
- 27. Esteller M, Guo M, Moreno V, Peinado MA, Capella G, et al. (2002) Hypermethylation-associated Inactivation of the Cellular Retinol-Binding-Protein 1 Gene in Human Cancer. Cancer Res 62: 5902–5905.
- 28. Eads CA, Danenberg KD, Kawakami K, Saltz LB, Danenberg PV, et al. (1999) CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression. Cancer Res 59: 2302–2306.
- 29. Rossant J, Zirngibl R, Cado D, Shago M, Giguere V (1991) Expression of a retinoic acid response element-hsplacZ transgene defines specific domains of transcriptional activity during mouse embryogenesis. Genes Dev 5: 1333–1344.
- 30. Neuschwander-Tetri BA, Burton FR, Presti ME, Britton RS, Janney CG, et al. (2000) Repetitive self-limited acute pancreatitis induces pancreatic fibrogenesis in the mouse. Dig Dis Sci 45: 665–674.
- 31. Means AL, Meszoely IM, Suzuki K, Miyamoto Y, Rustgi AK, et al. (2005) Pancreatic epithelial plasticity mediated by acinar cell transdifferentiation and generation of nestin-positive intermediates. Development 132: 3767–3776.
- 32. Hruban RH, Adsay NV, Albores-Saavedra J, Anver MR, Biankin AV, et al. (2006) Pathology of genetically engineered mouse models of pancreatic exocrine cancer: consensus report and recommendations. Cancer Res 66: 95–106.
- 33. Hingorani SR, Wang L, Multani AS, Combs C, Deramaudt TB, et al. (2005) Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell 7: 469–483.
- 34. Bistulfi G, Pozzi S, Ren M, Rossetti S, Sacchi N (2006) A repressive epigenetic domino effect confers susceptibility to breast epithelial cell transformation: implications for predicting breast cancer risk. Cancer Res 66: 10308–10314.
- 35. Segara D, Biankin AV, Kench JG, Langusch CC, Dawson AC, et al. (2005) Expression of HOXB2, a retinoic acid signaling target in pancreatic cancer and pancreatic intraepithelial neoplasia. Clin Cancer Res 11: 3587–3596.
- 36. Prasad NB, Biankin AV, Fukushima N, Maitra A, Dhara S, et al. (2005) Gene expression profiles in pancreatic intraepithelial neoplasia reflect the effects of Hedgehog signaling on pancreatic ductal epithelial cells. Cancer Res 65: 1619–1626.
- 37. Ghyselinck NB, Bavik C, Sapin V, Mark M, Bonnier D, et al. (1999) Cellular retinol-binding protein I is essential for vitamin A homeostasis. Embo J 18: 4903–4914.
- 38. Rovira M, Scott SG, Liss AS, Jensen J, Thayer SP, et al. (2009) Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas. Proc Natl Acad Sci U S A 107: 75–80.
- 39. Andrianifahanana M, Moniaux N, Schmied BM, Ringel J, Friess H, et al. (2001) Mucin (MUC) gene expression in human pancreatic adenocarcinoma and chronic pancreatitis: a potential role of MUC4 as a tumor marker of diagnostic significance. Clin Cancer Res 7: 4033–4040.
- 40. Balague C, Audie JP, Porchet N, Real FX (1995) In situ hybridization shows distinct patterns of mucin gene expression in normal, benign, and malignant pancreas tissues. Gastroenterology 109: 953–964.
- 41. Crnogorac-Jurcevic T, Missiaglia E, Blaveri E, Gangeswaran R, Jones M, et al. (2003) Molecular alterations in pancreatic carcinoma: expression profiling shows that dysregulated expression of S100 genes is highly prevalent. J Pathol 201: 63–74.
- 42. Gong YL, Xu GM, Huang WD, Chen LB (2000) Expression of matrix metalloproteinases and the tissue inhibitors of metalloproteinases and their local invasiveness and metastasis in Chinese human pancreatic cancer. J Surg Oncol 73: 95–99.
- 43. Maeda S, Shinchi H, Kurahara H, Mataki Y, Noma H, et al. (2007) Clinical significance of midkine expression in pancreatic head carcinoma. Br J Cancer 97: 405–411.
- 44. Froeling FE, Feig C, Chelala C, Dobson R, Mein CE, et al. (2011) Retinoic Acid-Induced Pancreatic Stellate Cell Quiescence Reduces Paracrine Wnt-beta-Catenin Signaling to Slow Tumor Progression. Gastroenterology.


text:
pharmacist's point of view
- Saponaria and mild cleansing agents deeply and gently cleanse the skin
- Aloe, panthenol and arginine moisturize and prevent irritations
- Propolis and olive offer antioxidant protection
- Orange and ginger essential oils revitalize and lift the mood
Exclusive Innovation: APIVITA replaces water in the body care line with organic witch hazel infusion to enhance the antioxidant and soothing action.
Excludes synthetic ingredients that may pose harmful effects to your health and the environment.
Free of: Parabens, Propylene Glycol, Mineral Oil and Ethanolomine.
body refresh
body relax
cream with calendula
Calendula (marigold) is known for its hydrating and soothing properties.Traditionally used for the care of dry and sensitive skins.Ideal also as after shave cream.
use 2-3 times a day.Do not apply to open wounds.suitable for babies and children.
Hypericum(St.John's wort) is known for its regenerating properties.Traditionally used to help soothe mild sunburn and thermal burns.it helps restore irritated and very dry skin.
suitable for children over 3 years old.use 2-3 times a day.Do not apply to open wounds.
Eucalyptus is known for its beneficial action in treating the cold symptoms. Traditionally used for muscle aches and nasal congestions.
USE: Apply a small amount on the chest, back, neck or body partsin pain and massage gently.In case of runny nose apply topically under the nasal area.Suitable for children over 3 years old,Do not apply on open wounds.For external use only.
propolis is a Bee product known for its mild antiseptic, antifungal and anti-bacterial properties.Traditionally used topically on minor injuries, scratches, acne pimples and between the fingers.
use 2-3 times a day.Do not apply to open wounds.suitable for children over 1 year old.
Arnica is known for its property to reduce oedema and improve microcirculatiion.Traditionally used topically for bruises and swellings caused by falls, bangs, contusions, sprains, insect bites, as well as for tired and aching legs.
use 2-3 times a day.Do not apply to open wounds.Suitable for children over 3 years old.
© 2012 All Rights Reserved


text:
Meeting Needs … Changing Lives
Shared Blessings began in 1998 through the efforts of Devonna Edwards who desired to clothe children and families in need. Over the years, the ministry has continued to grow now serving over 2500 people every month through a variety of programs. Our on-going goal is to partner with area churches, civic organizations, businesses, corporations, agencies, foundations and individuals to meet a growing number of needs in the area. Many, in our community, are struggling. Shared Blessings, through its generous partners, helps provide a place of compassion and relief. We desire for people to experience the Love of God through the compassion they experience at Shared Blessings.
In 2018, Shared Blessings served…
- Clothing was provided to 8450 people – 700 people every month
- Over 10,500 people received food – nearly 900 people every month / 327 families
- 26,793 bags of food were given to area children – an average of 837 kids per week
- Over 80 families received significant financial assistance
- 300 families received help with household necessities
- 1000 transportation vouchers were provided to area individuals
- 58 beds were provided to area children


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There aren't a lot of pickup trucks, but the field does have some clear winners in the following top selling pickup trucks April 2011.
We'll go into some detail on the five top selling pickups and cover the rest at the end.
1. 2011 Ford F-Series – Excellent towing capacities, F-150 and Super Duty F-250, F-350 and F-450 lines, the 2011 Ford F-Series is the undisputed sales leader again for
April 2011 with sales of 45,435 units. Calendar year-to-date through April, the automaker sold 172,062 units, an increase of 19.5 percent year-over-year. Kelley Blue Book observes, "If you want a truck with serious towing and payload capabilities, yet as quiet and comfortable as many luxury sedans, then the F-150 should surely be on your shopping list."
2. 2011 Chevrolet Silverado – With April sales of 29,342 and January through April sales of 121,797, the 2011 Chevrolet Silverado full-size pickup is once again in the
number two spot for top-selling pickups. Calendar year sales are up 19.3 percent over the same period last year. Car and Driver comments that the 2011 Chevrolet Silverado (and corporate sibling GMC Sierra), "continue to be a class act in this highly-competitive arena," helped by the fact that "they still offer outstanding engine choices and features galore."
3. 2011 Dodge Ram – Excellent handling, powerful engines and updating for 2011 has resulted in a 36 percent year-over-year sales boost for the 2011 Dodge Ram full-size pickup. Total calendar year-to-date sales are 70,419, while April sales totaled 17,680 – 7,157 units ahead of fourth-place April best seller, the 2011 GMC Sierra. Consumer Guide gives Ram a Best Buy nod and says, "Ram earns high marks for ride comfort, creative storage options, and abundant available power."
4. 2011 GMC Sierra – Sibling to the Chevy Silverado, yet marketed as the more upscale version, the 2011 GMC Sierra offers good fuel economy, powerful engines (plus a hybrid) and excellent payload and towing capacity. Sales for April totaled 10,523, with year-to-date (January through April) sales of 44,468 – a 35.3 percent increase year-over-year. Edmunds comments, "The 2011 GMC Sierra 1500 is a worthy competitor among fresher rivals, bolstered by a wide range of body styles, capable performance, and a comfortable ride."
5. 2011 Toyota Tacoma – The compact Toyota Tacoma pickup racked up April sales of 10,510 – close on the heels of the GMC Sierra. Year-to-date, Tacoma accounted
for 35,673 sales, a 7.6 percent increase over the same period last year. PickupTrucks.com remarks, "The Toyota Tacoma is the king of compact and midsize pickup trucks, with about 40 percent share of the segment in the U.S."
Ranked in spots six through 10 of the top selling pickup trucks April 2011 are the following: 2011 Toyota Tundra (8,312), 2011 Ford Ranger (5,370), 2011 Nissan Frontier (3,404), 2011 Chevrolet Colorado (2,789) and 2011 Dodge Dakota (1,580).
Search used cars for sale and find the best deals near you at iSeeCars.com.
To get a FREE iSeeCars VIN Report for a car, click here.


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Have you tried Instant Pot cheeseburger soup yet?? It's a fun pressure cooker meal that your kids will love and will make you come back for seconds.
Do you have one of these fancy multi cooker pressure cooker machines yet?? Well we have been creating easy Instant Pot recipes for over a year now and we're not slowing down now that it is summertime. If you don't have one you can make this Instant Pot cheeseburger soup on your stovetop. But let me tell you this machine is great if you're a busy family like we are. I'll show you step by step how to make this creamy and cheesy ground beef chowder soup of sorts that my kids love. Special thanks goes out to Snapfish for sponsoring this post so we could share our recipe and what we used to make it with you today. (originally published 6/18)
Are you ready for some Instant Pot cheeseburger soup??
This one is really easy. Then again all of my Instant Pot recipes are easy to make!
First thing you'll need is a cutting board to dice up a few things. Isn't this one cute??!!
Made with my name on it and a great gift idea if you ask me (hello Mother's Day and Christmas). With different designs to choose from and personalized for that special person in your life!
Oh, my apron you ask…yep that is personalized through Snapfish as well! Super cute right??!!
Mine has pictures of all of us cooking something…silly photos I love of each person.
So now that you're done dicing this is what you'll need to make our Instant Pot cheeseburger soup, other than the items listed above that you diced
- Pressure cooker
- I HIGHLY suggest getting a non stick pot.
- Ground beef
- Celery
- Butter
- Chicken or beef broth – your choice, beef will make it a deeper color, I used chicken broth here
- Heavy cream
- Cheese – I like sharp cheddar
- Sour cream
- Cornstarch – to thicken the broth
- Spices
Now let's get to making some Instant Pot cheeseburger soup!
Whether you use your stovetop or your Instant Pot you'll want to cook your ground beef and diced onions together. I personally leave the little bit of grease that is left over in the pot for added flavor but you can drain it.
Once that is done you're going to add everything else except for the cheese and cook it for just 3 minutes!!
That's the beauty of cooking in a pressure cooker.
I used leftover bacon from the day before but if you don't have that you can cook your diced bacon in with your ground beef and onions and really meld those flavors together!
Once it's done you're going to add most of your cheese and stir until it melts which won't take long.
Thicken the soup with a bit of cornstarch and you're ready to top with a bit more cheese and green onions!
This is what it looks like!
Instant Pot Cheeseburger Soup
- 1 lb spicy ground sausage could use ground beef if you prefer
- 1/2 onion diced
- 2 stalks celery sliced
- 8 pcs bacon
- 2 tbsp butter
- 1 tsp seasoned salt we used Lawry's
- 1 tsp garlic powder
- 3 c chicken broth could use beef broth
- 2 c cheese
- 1/2 c heavy cream
- 3 tbsp cornstarch to thicken if desired
- 1/2 c sour cream optional, for topping at end.
- Set Instant Pot to saute and add butter, ground beef and diced onions. Cook until most of pink in ground beef is cooked (3/4 way or so). (if your bacon isn't already cooked you can dice it and add it in now to cook it all together.)
- Dice celery and cooked bacon and add into pot, if you haven't added the bacon already. Turn your Instant Pot off.
- Add seasoned salt and garlic powder, stir together. Pour in chicken or beef broth and stir.
- Close lid and steam valve and set to pressure high for 3 minutes. Do a quick release when done.
- Turn pressure cooker off and then press saute, normal, again and add heavy cream and 1 1/2 c. of your cheese. Stir allowing cheese to melt.
- Add some of this hot liquid soup to a bowl with your cornstarch and whisk together, then add back into the Instant Pot and stir so it thickens.
- Turn Instant Pot off, stir in remaining 1/2 c of cheese and serve topped with additional shredded cheese and chives/green onions or a dollop of sour cream.
Now you're silly to think this Instant Pot cheeseburger soup is just for the winter time, we eat it year round!
They spend a lot of time jump jump jumping and in our pool (yep, cute personalized towel from Snapfish alert here too)
Ladle some in a bowl and enjoy a nice day outside together….I guarantee everyone will love it!


text:
Product Imagery Ecommerce Production Coordinator
Company Name: Fred Meyer and Littman Jewelers
Position Type: Employee
FLSA Status: Non-exempt
Position Summary:
Maintain the digital production schedules. Review and approve product data changes to all web channels. Manage and store digital creative assets. Coordinate website testing and execution for product and content. Maintain and distribute promotional reporting, weekly and as needed. Fred Meyer also requires that all associates perform all tasks in a safe manner consistent with corporate policies and state and federal laws. Demonstrate the company's core values of respect, honesty, integrity, diversity, inclusion and safety.
Essential Job Functions:
- Model Customer 1st behavior; deliver and encourage other associates to deliver excellent customer service
- Participate in and direct meetings to discuss Ecommerce projects
- Prepare all promotional assets and product programs to be added to websites
- Create, publish and manage annual content schedule for Ecommerce team
- Create and manage projects outside of annual promotion schedule in partnership with Ecommerce Manager and Marketing department
- Assist Marketing department with research and campaign preparation for Ecommerce needs (analytics, wireframes, meeting materials, etc.)
- Maintain website graphic preparation and process for merchandise and promotional material onsite presentation.
- Create, manage and troubleshoot updates to the product catalog
- Provide direction and guidance for website navigation
- Assist in production support for Jewelry Ecommerce systems on an ongoing basis
- Develop an understanding of Fred Meyer Jewelers systems, procedures, and retail system applications
- Assist in enhancing, maintaining, and supporting the various Ecommerce systems
- Collaborate with Merchandising, Ecommerce and Marketing teams to verify the web channels are in alignment with corporate standards
- Assist in implementing business strategies
- Prepare reports on a weekly and as needed basis
- Coordinate, monitor and source product images for product listings through partnering with vendors and other internal departments
- Must be able to perform the essential functions of this position with or without reasonable accommodation
Minimum Position Qualifications:
- High school diploma or general education degree (GED), plus 2 years graphic arts design experience; or combination of relevant education and experience.
- Minimum 18 years of age
- Ability to pass drug test
- Knowledge of Adobe Acrobat
- Ability to work weekends and overtime on an occasional basis
- Accuracy/attention to detail
- Ability to organize/prioritize variety of tasks/projects
- Strong project management skills
- Familiarity with industry/technical terms and processes
- Proficient with Photoshop, Microsoft Excel and Access
- Ability to continue education, as necessary
Desired Previous Job Experience:
- Knowledge of Fred Meyer policies, procedures, and organizational structure
- BA/BS in project management or web production
- Experience working with photography, and art direction
Education Level: Bachelor's Desired
Required Certifications/Licenses: None
Position Type: Full-Time
Shift(s): [[mfield4]]
Regions: West
States: Oregon; Washington
Keywords: Seasonal, eComm, Sales, Imagery, Jewelry, Merchandising, Graphic Arts
Jobs at Fred Meyer Jewelers: Based in Portland, Oregon with over 325 stores across the United States, Fred Meyer Jewelers offers employment opportunities in Sales, Store Management, Regionals Supervision as well as opportunities in our division office in merchandise, store operations, marketing, eCommerce, real estate development and administrative support. We are always searching for individuals who share our vision of: turning life's occasions into celebrations with a timeless gift of fine jewelry. If you want to help make fine jewelry as relevant, exciting and accessible to as many people possible every day, you have found a home and the right opportunity with Fred Meyer Jewelers.
Company Overview
Kroger Family of Companies employs nearly half a million associates who serve customers in 2,782 retail food stores under a variety of local banner names in 35 states. Our Family of Companies also operates 2,268 pharmacies, 274 fine jewelry stores, 1,489 supermarket fuel centers and 38 food production plants in the United States. Kroger is dedicated to our Purpose: to Feed the Human Spirit™ by serving America through food inspiration and uplift and creating #ZeroHungerZeroWaste communities by 2025. Careers with The Kroger Co. and our family of companies offer competitive wages, flexible schedules, benefits and room for advancement.
Posting Notes: OR || Portland || 3800 S.E. 22Nd Ave. || 97202 || Fred Meyer and Littman Jewelers || None || Advertising; Jewelry; Merchandising; e-Commerce || Employee || Non-Exempt || Full-Time || None
Nearest Major Market: Portland Oregon
Job Segment: Merchandising, Outside Sales, Administrative Assistant, Advertising, Real Estate, Retail, Sales, Administrative, Marketing