text:
Friday Fallback Story: HyQube Installed at Teesside Airport
Fuel Cell Systems has installed a refuelling station at Teesside International Airport which will serve hydrogen-powered vehicles based in the Tees Valley as part of a £2.5million region-wide trial.
As part of the Tees Valley Hydrogen Transport Hub, the airport, along with other key organisations, is testing out commercial and support vehicles fitted with 100% zero-emission hydrogen-fuelled commercial and support vehicles.
Element 2 contracted FCSL to build, install and commission the HyQube 500 refuelling syste to provide fuel for two Toyota Mirai hydrogen fuel cell vehicles and a forklift which have recently arrived at the site. This is alongside a Kangoo hydrogen-powered light van and a hydrogen tug due to arrive by May.
Toyota is deploying a number of hydrogen vehicles across the region's rapid response services, such as emergency response units for the Cleveland Police and NHS patient support, which can also take advantage of the new refuelling infrastructure. Northern Gas Networks, which is testing hydrogen as an alternative to natural gas in households in the region, also relies on a hydrogen vehicle
Until the launch of the new refuelling station at Teesside Airport the nearest place hydrogen vehicles could refuel was nearly 100 miles away in Rotherham.
In September 2020 it was announced that Tees Valley would be home to the UK's first Hydrogen Transport Hub. The hub, in partnership with Teesside University, will form an innovation campus focused on clean energy research and development. It will lead research, development, and testing of new hydrogen transport technologies including for cars, buses, trains, lorries, boats and planes.
Previous PostPlan France 2030: 800 Million Euros to Develop the Aircraft of the Future
Next PostRegistrations First H2Benelux Real Life Trials Are Open


text:
Q: Number of nodes of given height 'h' in an n element heap inconsistency The formula for the number of nodes of height 'h' in an 'n' element heap is given as ceil(n/2^(h+1)). For this given problem the number of nodes of height 2 as shown in figure is 2.

But from the formula, I get ceil(19/8) = 3.
Could someone help explain my mistake?

A:
The formula for the number of nodes of height 'h' in an 'n' element heap is given as ceil(n/2^(h+1))

Can you provide your source?
If this is regarding this problem (which I assume from your picture). It says:
A heap of size n has at most ceil(n/2^(h+1)) nodes with height h

redpajama_set_name: RedPajamaStackExchange


Site visitors: Over K in the past monthFollowers: Over M on Twitter. Shop GTM Sportswear for your team apparel, featuring the Champion brand! Warm-ups, uniforms, tees and accessories for cheer, dance, track, ball sports and more. Great selection of athleisure fanwear, shoes, and bags. Stock, blank, custom, and personalized merchandise available. Before you hit the gym you need to get the right gear on and here at BANG+STRIKE we have a wide range of sportswear. So pick up your jersey sport shorts, extreme racer tanks and sports mesh hoodies from designer brands like Rufskin, Diesel and Armani.. For out of the gym we have running tights and windbreakers for when you are pounding the street.


text:
Saint Jude-on-the-Hill
Archive of the Parish Church of Hampstead Garden Suburb London NW11 7AH twitter: @stjudeonthehill
Farewell to Benedict
on Friday, August 14, 2020 No comments:
Friday 8 May marks the 75thanniversary of VE Day and we had planned to host a deanery service at St Jude's this Sunday afternoon. The Corona pandemic of course makes that impossible.
Victory in Europe is only briefly mentioned in the St Jude's Gazette for 1945. The parish magazine, which had once been a substantial weekly, had by then been reduced to a slender monthly. It seems that wartime censorship extended even into such local publications, and very little is said about the war other than brief reports of lost or missing parishioners serving with the forces. The invasion of Europe following D Day in June 1944 receives no direct mention until September when it is announced that "the King has bidden his people to join in corporate prayer for the welfare of the Allied Nations and ultimate victory . . . [as] the great news of our victories sweeps in . . . ".
That same month a new book by the Vicar, the Rev W. H. Maxwell Rennie, and Mr L. D. Gibbin, "a valued member of the St Jude's congregation", entitled Our Men Returning is announced (Evans Brothers 1/6). "Detailed suggestions are given for contacting men and women returning from the Forces and from Industry, and for using their individual talents in the service of the Church." The cover ("in two colours") evidently carried a picture of Fr Rennie (we have no copy in our archive),as the reviewer (in the same edition of the Gazette) described it as "a mistake; it should have been a symbolical, or at least impersonal design. The incumbent, say, of Nether Wallop, to take a basic example, will think of his own flock returning and not of them being welcomed by the Vicar of St Jude's".
Some things could not be censored. The summer of 1944 saw the beginning of the 'doodle bug' (V1 flying bomb) attacks. These put an abrupt end to the recently reinvigorated children's activities inaugurated by a new curate, Father John Knight, who had arrived only that June. 'Doodling' had caused not inconsiderable damage on the Suburb, and the church had served as a refuge for local residents and for furniture from their bombed-out houses. "Censorship will not allow us to say more [but] the St John's Chapel is a very safe sleeping space . . .". We read also (in July) "that by the time this appears in print our scientists, one of whom is well known to St Jude's, who deal with Nazi machinations, will have gone a long way to solving the problem of the 'Buzz-Bomb'".
In September 1944 the St Jude's Director of Music, Charles Proctor, under the heading 'Wohin? (Wither?) The purpose of music is to teach (Wagner), offered a lengthy essay, a "psychological study of German obsessions', in which he suggested "the age in which we live can best be explained as a projection of the myth-laden mind of the rulers of Germany. If one considers for a moment the condition of Germany under Nazi rule, as but a projection of the [Wagnerian version of the] Nibelungen Saga upon its generation, one can perhaps attune oneself to the inconsistencies of the regime and even perhaps foretell its doom. . . What is the end of the story? 'Gotterdammerung'. The Twilight of the Gods . . . Had the leaders of Germany learnedfrom their great dramatic poet, Wagner, instead of glorifying him, they might have hesitated before setting out to emulate these primary legendary figures, without first considering the end of the story".
In a more mundane note in November, thanks are offered to "those who responded to our request for knitted garments for Liberated Europe".
Early 1945 editions of the Gazette make little mention of the war. In April a short series of Sunday evening addresses by distinguished speakers is announced. These include Sir Stafford Cripps, the Minister of Aircraft Production, and C. S. Lewis.
Victory in Europe is finally announced in the Gazette of June 1945. A Thanksgiving Mass had been held on VE Day itself when the choir and orchestra had performed Schubert's Mass in B Flat and the church was filled from end to end.
The vicar announced that he was under doctor's orders to take a month's rest. "One of the more tiring elements in my daily duties – the constant cycling uphill back to the Vicarage, for I could not have managed my job walking about the parish – will in future be abolished through the kindness of a parishioner who has presented me with a New-Hudson Autocycle."
In July we learn that the altar ornaments (gold and rock crystal crucifix and candlesticks by Lutyens) and the stained-glass window (by Robert Anning Bell) from the St John's (Harmsworth Memorial) Chapel have been stored "somewhere in England" and are about to be returned.
There was a strong recognition that the 'Japanese War' had not yet ended and that the time for the resumption of normal duties and family reunions was 'not yet'.
on Thursday, May 07, 2020 No comments:
Notes: Maxwell Rennie, Second World War
Mothering Sunday 2020
Mass was celebrated today for the parish and the world
on Sunday, March 22, 2020 No comments:
Notes: Mothering Sunday, StayathomeStayalive
StayathomeStayalive
Notes: StayathomeStayalive
UCL/Bartlett Visit 2020
Architecture Students from UCL Bartlett School with Professor Murray Fraser on their annual visit to the church.
on Monday, February 24, 2020 No comments:
Notes: Adrian Forty UCL, Murray Fraser
Visitors from Queen Mary University
London Architecture students from Queen Mary visited the church in February
on Saturday, February 15, 2020 No comments:
Notes: QMU
Photos by Cesar Rodriguez-Duran
on Sunday, January 05, 2020 No comments:
Notes: New Year, res, Residents Association
Older posts Aug 14 (1) May 07 (1) Mar 22 (2) Feb 24 (1) Feb 15 (1) Jan 05 (1) Dec 29 (2) Nov 11 (1) Oct 27 (1) Sep 28 (1) Sep 22 (1) Sep 08 (2) Jun 09 (2) May 19 (1) May 12 (1) Apr 22 (1) Apr 21 (1) Apr 20 (1) Apr 12 (1) Apr 09 (2) Mar 31 (1) Mar 03 (1) Feb 18 (1) Feb 17 (1) Feb 05 (1) Jan 06 (1) Jan 02 (1) Dec 17 (2) Dec 15 (1) Dec 09 (1) Nov 13 (1) Nov 11 (2) Nov 08 (1) Nov 05 (1) Oct 29 (1) Oct 26 (1) Sep 09 (1) Jul 28 (1) Jul 18 (1) Jul 09 (1) Jul 08 (1) Jul 05 (1) Jul 02 (1) Jun 30 (1) May 25 (1) May 23 (2) May 22 (1) May 20 (1) May 18 (1) May 16 (1) Apr 30 (2) Apr 02 (1) Apr 01 (2) Mar 30 (2) Mar 11 (1) Feb 28 (2) Feb 20 (1) Feb 06 (1) Jan 28 (1) Jan 21 (1) Jan 14 (2) Jan 06 (1) Jan 02 (1) Dec 26 (2) Dec 24 (2) Dec 17 (1) Dec 10 (1) Dec 03 (1) Nov 12 (1) Oct 29 (1) Oct 05 (5) Oct 02 (1) Oct 01 (1) Sep 24 (1) Sep 19 (1) Sep 09 (1) Sep 01 (1) Aug 11 (1) Aug 05 (1) Jul 04 (1) Jun 16 (1) Jun 13 (2) May 22 (1) May 19 (2) May 16 (2) May 14 (1) May 12 (2) May 07 (1) May 02 (1) Apr 28 (1) Apr 16 (1) Apr 15 (2) Apr 14 (3) Apr 09 (4) Apr 04 (1) Apr 02 (1) Mar 26 (2) Mar 19 (1) Feb 26 (1) Feb 21 (1) Feb 20 (1) Feb 12 (2) Feb 06 (1) Feb 05 (2) Jan 30 (1) Jan 15 (1) Jan 01 (2) Dec 29 (1) Dec 26 (1) Dec 25 (3) Dec 10 (1) Nov 21 (1) Nov 17 (1) Nov 07 (2) Nov 01 (1) Oct 30 (1) Oct 19 (1) Oct 11 (1) Oct 08 (1) Oct 02 (2) Oct 01 (1) Sep 25 (3) Sep 17 (2) Sep 06 (1) Sep 02 (2) Jul 28 (2) Jul 19 (1) Jul 17 (1) Jul 16 (1) Jul 15 (2) Jun 28 (1) Jun 20 (1) Jun 16 (1) Jun 12 (1) May 22 (1) May 19 (1) May 15 (2) May 12 (2) May 06 (1) May 03 (1) Apr 29 (1) Apr 23 (1) Apr 22 (1) Apr 20 (1) Apr 10 (1) Mar 29 (1) Mar 27 (1) Mar 20 (2) Mar 19 (1) Mar 15 (1) Mar 13 (1) Mar 12 (1) Mar 06 (1) Feb 29 (1) Feb 22 (1) Feb 12 (2) Feb 07 (1) Feb 01 (1) Jan 31 (1) Jan 26 (1) Jan 22 (1) Jan 19 (1) Jan 17 (2) Jan 08 (1) Jan 03 (2) Jan 02 (1) Jan 01 (2) Dec 28 (1) Dec 26 (1) Dec 25 (3) Dec 24 (1) Dec 23 (1) Dec 20 (1) Dec 19 (2) Dec 13 (2) Dec 12 (1) Dec 07 (1) Dec 06 (1) Dec 05 (1) Dec 01 (2) Nov 30 (1) Nov 28 (1) Nov 27 (1) Nov 23 (2) Nov 22 (1) Nov 21 (1) Nov 17 (1) Nov 13 (1) Nov 11 (2) Nov 06 (2) Nov 02 (2) Oct 25 (1) Oct 12 (1) Oct 11 (2) Oct 10 (1) Oct 08 (1) Oct 06 (1) Oct 04 (1) Sep 26 (2) Sep 20 (1) Sep 17 (1) Sep 15 (2) Sep 13 (1) Sep 08 (1) Sep 07 (2) Aug 30 (1) Aug 27 (1) Aug 01 (1) Jul 26 (2) Jul 22 (1) Jun 16 (1) Jun 10 (1) Jun 08 (1) Jun 05 (1) Jun 03 (1) Jun 02 (1) May 31 (1) May 30 (1) May 28 (1) May 24 (1) May 23 (1) May 18 (2) May 09 (1) May 08 (1) May 06 (1) May 04 (1) May 02 (1) Apr 30 (1) Apr 26 (3) Apr 15 (1) Apr 11 (1) Apr 08 (1) Apr 01 (3) Mar 22 (1) Mar 20 (1) Mar 19 (1) Mar 13 (2) Mar 08 (1) Feb 27 (1) Feb 09 (1) Feb 03 (1) Jan 26 (1) Jan 17 (1) Jan 05 (2) Jan 01 (2) Dec 31 (1) Dec 30 (1) Dec 24 (1) Dec 21 (1) Dec 17 (1) Dec 15 (1) Dec 14 (1) Dec 06 (1) Dec 01 (1) Nov 23 (2) Nov 14 (1) Oct 29 (1) Oct 26 (1) Oct 25 (1) Oct 24 (1) Oct 22 (1) Oct 15 (2) Sep 27 (1) Sep 17 (1) Sep 15 (1) Sep 02 (1) Jul 24 (1) Jul 23 (1) Jul 20 (1) Jul 18 (1) Jul 17 (1) Jul 07 (1) Jun 22 (1) Jun 14 (1) Jun 08 (1) Jun 07 (1) Jun 05 (1) May 23 (1) May 07 (1) May 04 (1) Apr 30 (1) Apr 24 (1) Apr 20 (1) Apr 13 (1) Apr 12 (1) Apr 10 (1) Mar 31 (1) Mar 28 (1) Mar 24 (1) Mar 22 (2) Mar 11 (1) Mar 06 (1) Feb 27 (1) Feb 24 (1) Feb 23 (2) Jan 27 (1) Jan 20 (1) Jan 12 (1) Jan 09 (1) Jan 04 (1) Jan 03 (1) Jan 01 (1) Dec 26 (1) Dec 16 (1) Dec 01 (1) Nov 25 (1) Nov 18 (1) Nov 15 (2) Nov 04 (1) Sep 04 (2) Aug 22 (1) Aug 02 (1) Jul 27 (1) Jul 19 (1) Jul 18 (1) Jul 14 (1) Jul 06 (1) Jul 01 (2) Jun 21 (1) Jun 20 (1) Jun 17 (1) Jun 10 (1) Jun 05 (2) Jun 03 (1) May 31 (1) May 23 (1) May 21 (1) May 17 (1) May 01 (2) Apr 15 (1) Feb 10 (1) Feb 04 (3) Jan 01 (1) Dec 27 (1) Dec 22 (1) Dec 17 (1) Dec 11 (1) Dec 08 (1) Dec 03 (1) Nov 26 (1) Nov 09 (2) Oct 14 (1) Oct 07 (1) Sep 27 (2) Sep 23 (1) Sep 16 (1) Sep 03 (1) Jul 27 (1) Jul 02 (2) Jul 01 (4) Jun 29 (1) Jun 26 (1) Jun 25 (1) Jun 23 (1) Jun 13 (1) Jun 09 (1) May 14 (1) May 05 (1) May 02 (2) Apr 30 (1) Apr 10 (1) Apr 07 (1) Apr 06 (1) Apr 05 (2) Apr 04 (2) Apr 02 (1) Apr 01 (2) Mar 24 (1) Mar 23 (2) Mar 18 (1) Mar 17 (1) Mar 12 (1) Mar 10 (1) Mar 03 (1) Feb 27 (2) Feb 25 (1) Feb 22 (2) Feb 19 (1) Feb 13 (1) Feb 06 (1) Feb 05 (1) Feb 03 (1) Jan 30 (2) Jan 21 (2) Jan 17 (1) Jan 01 (2) Dec 25 (1) Dec 24 (1) Dec 23 (1) Dec 14 (1) Dec 11 (2) Dec 10 (1) Dec 03 (1) Nov 24 (1) Nov 15 (1) Nov 13 (1) Nov 10 (1) Nov 07 (2) Oct 22 (1) Oct 02 (1) Sep 28 (1) Sep 25 (1) Sep 14 (1) Sep 07 (1) Aug 16 (1) Aug 15 (1) Aug 14 (1) Aug 12 (1) Aug 10 (1) Aug 07 (1) Jul 24 (1) Jul 10 (1) Jul 08 (2) Jul 07 (1) Jun 28 (1) Jun 26 (1) Jun 19 (2) Jun 15 (1) May 29 (2) May 26 (1) May 22 (1) May 21 (1) May 17 (1) May 16 (2) May 13 (1) May 10 (3) May 08 (2) May 04 (1) May 01 (1) Apr 29 (1) Apr 27 (1) Apr 26 (1) Apr 24 (1) Apr 22 (1) Apr 21 (1) Apr 20 (2) Apr 19 (1) Apr 17 (1) Apr 13 (1) Apr 12 (2) Apr 11 (1) Apr 09 (1) Apr 06 (2) Apr 03 (1) Apr 02 (2) Mar 30 (1) Mar 29 (2) Mar 28 (1) Mar 27 (1) Mar 26 (2) Mar 21 (1) Mar 19 (1) Mar 18 (1) Mar 07 (1) Mar 06 (1) Mar 01 (1) Feb 14 (1) Feb 11 (1) Feb 10 (1) Feb 08 (1) Feb 06 (1) Feb 04 (1) Feb 02 (1) Jan 31 (2) Jan 30 (1) Jan 16 (1) Jan 14 (1) Jan 10 (1) Jan 07 (1) Jan 01 (1) Dec 31 (1) Dec 30 (1) Dec 26 (3) Dec 24 (1) Dec 23 (1) Dec 18 (1) Dec 15 (1) Dec 14 (1) Dec 13 (1) Dec 11 (1) Dec 08 (1) Dec 05 (1) Dec 04 (1) Nov 24 (1) Nov 17 (2) Nov 15 (1) Nov 14 (1) Nov 13 (1) Nov 10 (1) Nov 06 (2) Nov 03 (2) Oct 31 (1) Oct 29 (1) Oct 24 (1) Oct 21 (1) Oct 20 (2) Oct 19 (1) Oct 18 (1) Oct 17 (2) Oct 16 (2) Oct 14 (2) Oct 10 (1) Oct 04 (1) Oct 03 (1) Oct 02 (1) Sep 19 (1) Sep 12 (2) Sep 11 (1) Sep 03 (1) Sep 01 (1) Aug 19 (1) Aug 08 (1) Jul 25 (1) Jul 18 (1) Jul 01 (1) Jun 30 (3) Jun 28 (1) Jun 27 (2) Jun 24 (1) Jun 23 (1) Jun 17 (1) Jun 16 (1) Jun 11 (1) Jun 06 (1) Jun 04 (1) May 27 (1) Apr 29 (1) Apr 26 (1) Apr 25 (1) Apr 23 (1) Apr 22 (1) Apr 16 (1) Apr 11 (2) Apr 09 (1) Apr 06 (1) Apr 05 (1) Apr 04 (1) Apr 03 (2) Apr 02 (1) Mar 28 (2) Mar 26 (1) Mar 21 (1) Mar 19 (2) Mar 14 (1) Mar 04 (1) Mar 03 (1) Feb 16 (1) Feb 14 (1) Feb 12 (1) Feb 11 (1) Feb 08 (2) Feb 01 (1) Jan 10 (1) Jan 09 (1) Jan 06 (2) Jan 03 (1) Jan 01 (2) Dec 29 (1) Dec 25 (1) Dec 24 (1) Dec 22 (1) Dec 21 (1) Dec 20 (2) Dec 19 (1) Dec 15 (1) Dec 12 (1) Dec 10 (1) Dec 07 (1) Dec 06 (2) Dec 05 (1) Dec 04 (1) Nov 28 (1) Nov 27 (3) Nov 26 (1) Nov 25 (1) Nov 24 (1) Nov 23 (1) Nov 21 (1) Nov 12 (1) Oct 23 (1) Oct 22 (1) Oct 10 (1) Sep 28 (1) Sep 12 (1) Jul 22 (1) Jun 25 (1) Jun 01 (1) Apr 06 (1) Dec 14 (1)
Search previous posts 2007-18

redpajama_set_name: RedPajamaCommonCrawl


text:
You are here: Home / Schools / Bay Path University
Long Meadow, MA
Log in or Upgrade
To make use of this feature, you need to be a member.
Photo: Bay Path University. Retrieved from: www.baypath.edu
A historically all women's undergraduate institution, Bay Path University is located in Long Meadow, Massachusetts in Hampden County. The school also offers online and graduate programs that both men and women can participate in. There are two additional Bay Path campuses in Burlington and Sturbridge, Massachusetts.
Along with academics, it is the goal of the administrators at Bay Path University to encourage students to focus on leadership, communication, and technology in hopes that students will become, "confident and resourceful contributors to our increasingly interdependent world."
Bay Path has over 20 majors to choose from including Child Psychology, Forensics, and Marketing. Unlike many colleges who make students take multiple courses in areas other than their major, students at Bay Path University take a majority of their classes in their specific majors. This allows the students to get the most out of their education. Along with Baccalaureate degrees, students can also obtain associates degrees, Graduate degrees, and certificates.
Along with seven other colleges such as Springfield College, Western New England University, and Westfield State University, Bay Path University is part of the Cooperating Colleges of Greater Springfield. This consortium allows students from the eight different colleges to take advantage of the classes, libraries, and opportunities of the other seven colleges.
Bay Path University was founded in Springfield, Massachusetts in 1897 as a coeducational school. When it moved to Long Meadow in 1945 it became an all women's college. It wasn't until 1988 that Bay Path University became a four-year institution.
Just 5 minutes from Springfield, the 46-acre campus gives students a campus-feel but is close enough to a city to take advantage of the many opportunities available. Students can obtain part-time jobs in Springfield, apply for internships or simply take in the rich culture that Springfield has to offer.
There are three resident halls on campus that come equipped with at least one lounge and computer room. Freshmen and Sophomores are required to live on campus as the faculty feel this is an important part of the college experience. While upperclassmen can live on campus, many rent apartments near by. Currently, only 25% of students live on campus.
There are numerous activities for students to participate in on their down time. They can join the choir, or student government, or they can play Division III athletics in sports such as basketball, field hockey, and softball.
Throughout the year, Bay Path University offers events on campus that students and the community can attend. For instance every year since 1996, Bay Path University hosts the Women's Professional Development Conferences. It has featured the likes of PBS/ABC news correspondent Cokie Roberts, former U.S. Secretary of State Madeleine Albright, and writer Maya Angelou.
Bay Path University calls itself one of the most affordable, private colleges in New England. Costing around $43,000 a year to attend, Bay Path strives to make their school both "financially feasible" and an "investment in your life plan."
Around 95% of students receive some form of financial aid from Bay Path University but only 6% have their needs fully met. The average package given out to a first-year student is just shy of $27,000. Upon graduation, students can expect to be $38,000 in debt.
Location: Long Meadow, MA
Number of Students: 1001 to 2000
Avg Net Price: $ 22,000
Retention Rate (%): 80
Reported Test Type: SAT
Test Scores (75% percentile): 1170
Test Scores (25% percentile): 840
Freshman in Top 10% of HS Class (%): 1
Admit Rate (%): 77
School Selectivity: Moderately Selective
4 Year Graduation Rate (%): 50
Note: Facts compiled from school website as well as other data sources including: US Department of Education, IPEDS, College Board and NCES.


text:
Dysfunction of Kidney Endothelium after Ischemia/Reperfusion and Its Prevention by Mitochondria-Targeted Antioxidant
S. S. Jankauskas1, N. V. Andrianova1, I. B. Alieva1, A. N. Prusov1, D. D. Matsievsky2, L. D. Zorova1,3, I. B. Pevzner1, E. S. Savchenko4, Y. A. Pirogov5, D. N. Silachev1, E. Y. Plotnikov1*, and D. B. Zorov1*
1Lomonosov Moscow State University, Belozersky Institute of Physico-Chemical Biology, 119991 Moscow, Russia; E-mail: zorov@genebee.msu.ru, plotnikov@genebee.msu.ru
2Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences, 125315 Moscow, Russia
5Lomonosov Moscow State University, Faculty of Physics, 119991 Moscow, Russia
Received July 1, 2016; Revision received August 1, 2016
One of the most important pathological consequences of renal ischemia/reperfusion (I/R) is kidney malfunctioning. I/R leads to oxidative stress, which affects not only nephron cells but also cells of the vascular wall, especially endothelium, resulting in its damage. Assessment of endothelial damage, its role in pathological changes in organ functioning, and approaches to normalization of endothelial and renal functions are vital problems that need to be resolved. The goal of this study was to examine functional and morphological impairments occurring in the endothelium of renal vessels after I/R and to explore the possibility of alleviation of the severity of these changes using mitochondria-targeted antioxidant 10-(6ʹ-plastoquinonyl)decylrhodamine 19 (SkQR1). Here we demonstrate that 40-min ischemia with 10-min reperfusion results in a profound change in the structure of endothelial cells mitochondria, accompanied by vasoconstriction of renal blood vessels, reduced renal blood flow, and increased number of endothelial cells circulating in the blood. Permeability of the kidney vascular wall increased 48 h after I/R. Injection of SkQR1 improves recovery of renal blood flow and reduces vascular resistance of the kidney in the first minutes of reperfusion; it also reduces the severity of renal insufficiency and normalizes permeability of renal endothelium 48 h after I/R. In in vitro experiments, SkQR1 provided protection of endothelial cells from death provoked by oxygen–glucose deprivation. On the other hand, an inhibitor of NO-synthases, L-nitroarginine, abolished the positive effects of SkQR1 on hemodynamics and protection from renal failure. Thus, dysfunction and death of endothelial cells play an important role in the development of reperfusion injury of renal tissues. Our results indicate that the major pathogenic factors in the endothelial damage are oxidative stress and mitochondrial damage within endothelial cells, while mitochondria-targeted antioxidants could be an effective tool for the protection of tissue from negative effects of ischemia.
KEY WORDS: ischemia, kidney, Doppler, blood vessels, oxidative stress, mitochondria
Abbreviations: AKI, acute kidney injury; DCF, 2,7-dichlorodihydrofluorescein; I/R, ischemia/reperfusion; IREC, isolated renal epithelial cells; OGD, oxygen–glucose deprivation; ROS, reactive oxygen species; SkQR1, 10-(6ʹ-plastoquinonyl)decylrhodamine 19.
The problem of acute kidney injury (AKI) treatment remains relevant despite decades of intense experimental work aimed at deciphering the mechanisms of organ damage and approaches to affect them. Many data on AKI pathogenesis have been accumulated; however, effective ways to treat this pathology have not been found. AKI often occurs as a complication of various severe clinical conditions such as sepsis, severe trauma, cardiac arrest, and dehydration; it contributes significantly to mortality among patients in intensive care units.
Disorders in blood supply is the most common cause of AKI; it results from the drastic reduction of blood pressure in systemic vasodilatation or heart disorders or in case of excessive vasoconstriction of renal vasculature. In all cases, the renal blood flow autoregulation system is unable to maintain perfusion of renal parenchyma at the level corresponding to the energy requirement of the nephron. If such a period of ischemia is long enough, then even after removal of the factors that caused the ischemia, the restoration of blood supply does not lead to complete restoration of renal blood flow – it remains somewhat decreased [1, 2]. Such secondary hypoperfusion, which develops after the initial ischemic episode, is caused by pathological changes in the functioning of the renal vascular bed. The latter leads to the loss of renal blood flow autoregulation [3, 4], enhancement of constrictor response to stimulation of the sympathetic nervous system, disappearance of reaction to vasodilating compounds [4], attraction and adhesion of immunoinflammatory cells in microvascular renal bed [5, 6], and blood stasis in capillaries [2].
These functional changes are associated with destructive processes in cells of the vascular wall, initiated by ischemia followed by reperfusion (I/R). In both in vitro and in vivo experiments, I/R was shown to damage cytoskeletal structure and organization of tight junctions in endothelial and smooth muscle vascular cells [7-10]. Similar changes in renal vascular cells were also found in patients with AKI [11]. An increase in the concentration of endothelial activation markers in blood of experimental animals [9] and the reduction of the number of endothelial cells lining the renal capillaries (visualized by immunohistochemical methods) also indicated endothelial damage after I/R [12, 13].
Mitochondria deserve special attention among cell organelles subjected to the damaging effect of I/R [14]. Increased production of reactive oxygen species (ROS) by mitochondria is one of the main consequences of I/R [15]. ROS are known to play an important role in the regulation of renal vascular tone. For example, administration of antioxidants increases renal blood flow [16], and inhibition of intracellular antioxidant systems results in its decrease [17]. In addition, ROS regulate the tone of afferent arteriole in the tubuloglomerular feedback system [18], which ultimately regulates nephron blood supply. ROS also play the key role in signaling cascades triggered by angiotensin II [19, 20].
We have previously shown that mitochondria-targeted antioxidant SkQR1 (10-(6ʹ-plastoquinonyl)decylrhodamine 19) has a pronounced nephroprotective effect in a number of renal pathologies, including experimental cold and warm kidney ischemia, rhabdomyolysis, gentamicin nephrotoxicity, and acute pyelonephritis [21-26]. SkQR1 effects in kidneys are primarily associated with the reduction of oxidative stress associated with these pathologies and increased tolerance of kidney cells to damaging factors. However, the effects of this antioxidant on renal hemodynamics and the functional state of renal endothelium have not been previously studied. The aim of this study was to test the hypothesis that the protective effect of SkQR1 on renal I/R is linked to the prevention of the renal vascular dysfunction.
Experiments on cell cultures. Cells of endothelial line EA.hy926 were studied (courtesy of Prof. C. J. Edgel, North Carolina State University, USA). Cell culture EA.hy926 was obtained by hybridization of the primary endothelial line HUVEC with A549 lung carcinoma cells; it reproduces all the main morphological, phenotypic, and functional characteristics inherent in microvessel endothelial cells. EA.hy926 line cells were cultured in complete growth medium consisting of DMEM/F-12 (1 : 1), 10% fetal calf serum (PAA, USA), 100 U/ml penicillin-streptomycin, 2 mM L-glutamine, 0.1 mM hypoxanthine, and 16 µM thymidine (all from PanEco, Russia). For the experiments, cultures were seeded in 96-well plastic plates. Oxygen–glucose deprivation (OGD) was carried out by a conventional procedure; cultured cells were washed twice with balanced salt solution containing (in mM) NaCl 116, KCl 5.4, CaCl2 1.8, MgSO4 0.8, NaH2PO4 1, pH 7.3, and then incubated for 5 h in the same solution in a humidified chamber filled with nitrogen at 37°C. Culturing conditions remained the same for control cultures. Immediately after completion of OGD, saline solution was replaced by cultivation medium. The number of surviving cells was assessed after 24 h based on their ability to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan as measured by spectral properties (standard MTT-test [27]).
Induction of renal I/R. Experiments were performed on outbred male albino rats (350-400 g) kept in a vivarium on a standard diet. Work with laboratory animals was carried out in accordance with the requirements of the Commission of Bioethics of Belozersky Institute of Physico-Chemical Biology (protocol #1, April 8, 2013).
Left kidney ischemia was performed as previously described [21]. The animal was anesthetized with chloral hydrate intraperitoneally (i.p., 300 mg/kg), abdominal incision was performed, and the vascular bundle of the left kidney was clamped with an atraumatic microvascular clamp for 40 min. In acute experiments, the right kidney stayed intact. To assess the level of damage in the kidney subjected to I/R, we conducted nephrectomy of the contralateral (intact) kidney, because no renal failure is observed when at least one kidney remains intact. Ligation of the right ureter was performed to prevent retrograde urine release into the abdominal cavity. During anesthesia, the animal's temperature was maintained at 37°C. Control animals were subjected to similar procedures except the clamping of the vascular bundle of the left kidney (sham-operated animals). Since elevation of the creatinine concentration in blood is an indicator of the degree of deterioration of renal filtration function, creatinine concentration in blood served as an indicator of renal failure in animals. Creatinine was measured in serum taken from the tail vein using an AU480 Chemistry System (Beckman Coulter, USA).
Magnetic resonance imaging. Renal vessels were studied using magnetic resonance imaging with a BioSpec 70/30 device (Bruker, Germany) with induction of magnetic field 7 T and gradient system 105 mT/m. To obtain T2-weighted imaging, a pulse sequence based on the spin echo RARE (Rapid Acquisition with Relaxation Enhancement) was used with the following parameters: TR = 2800 ms, TE = 90 ms, slice thickness 1.5 mm, field of view 8 × 4 cm, matrix size 256 × 384. Before conducting magnetic resonance imaging, the animals were anesthetized and placed in a positioning device equipped with a system of stereotaxis and thermoregulation.
Study of blood flow using high-frequency Doppler technology. High-frequency ultrasound Doppler operating at a frequency of 27 MHz was used in the experiments on hemodynamics (developed in the Laboratory of Bioengineering, Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences). Blood flow was measured using bandage-type transducer with internal diameter of 1.5 to 2 mm, calibrated in units of volumetric flow rate. The transducer was applied to the renal artery. Blood flow velocity was registered 10-15 min after fixation of transducers (this time was needed for blood flow stabilization).
Blood flow velocity was recorded for short time periods (up to 30 s) at regular intervals: after blood flow stabilization, immediately after the clamping of the vascular bundle (clamps were placed distal to the transducer), 40 min after ischemia, during the first 60 s of reperfusion, and then every 5 min until the 30th min. For experiments with Nω-L-nitroarginine (L-NA) administration, blood flow velocity was recorded also immediately after the administration of this compound and 5 and 10 min after its administration.
Linear (V) and volumetric (Q) blood flow velocity were recorded during the experiment. Data from the ultrasonic device was digitized using the Graph2Digit software. Renal hemodynamics was analyzed based on the following parameters: heart rate (HR, beats/min), volumetric blood flow velocity (Q, ml/min), peak systolic blood flow velocity (Vmax, cm/s), end diastolic flow velocity (Vmin, cm/s), resistivity index (Pourcelot index): (Vmax – Vmin)/Vmax.
Evaluation of permeability of the renal vascular endothelium. In vivo administration of Evans Blue dye was used to evaluate permeability of the renal vascular bed. Anesthetized animals were injected intravenously with 2 ml 0.5% Evans Blue. After 70 min, the animals were transcardially perfused with 200 ml PBS to wash Evans Blue out of blood vessels. After that, the left kidney was excised, dried, weighed, cut, and photographed. Then the kidney was homogenized, centrifuged for 10 min at 14,000g, and the supernatant was collected. For protein precipitation, the supernatant was mixed with 50% trichloroacetic acid (1 : 2 v/v) and centrifuged for 20 min at 14,000g. Then 90 µl of ethanol was added to 30 µl of the supernatant, and fluorescence intensity was measured (excitation – 535 nm, emission – 625 nm). Solution with known Evans Blue concentration was used for calibration.
Electron microscopy. The renal cortical section was cut into pieces of approximately 1 mm3 and fixed with 2.5% glutaraldehyde (Sigma, USA) in phosphate buffer (pH 7.4), then the samples were additionally fixed with 1% OsO4 solution, dehydrated (70% ethanol containing 2% uranyl acetate), and embedded in Epon 812 (Fluka, USA). After polymerization in Epon, ultrathin sample sections were made on an LKB Ultratome III microtome. Sections were contrasted with citrate and examined with a JEM-1400 electron microscope (JEOL, Japan) at accelerating voltage 100 kV.
Detection of endothelial cells circulating in blood using polymerase chain reaction with reverse transcriptase (RT-PCR). A 1.5-ml blood samples were taken from the rat jugular vein with a syringe containing EDTA as anticoagulant. RNA was isolated from the whole blood using a QIAamp RNA Blood Mini kit (Qiagen, USA) following the manufacturer's instructions. The resulting total RNA was used as a template for reverse transcription and PCR. RT-PCR was performed in single vial format on a Bio-Rad CFX96 device using a One-Step RT-PCR kit (Alpha-enzyme, Russia) according to the manufacturer's instructions. vcam-1 relative expression was evaluated from the curves of fluorescent signal accumulation, crossing the threshold, and normalized to the expression of housekeeping gene Gapdh. Amplification products were detected in real time using TaqMan probes according to the principle of 5′-terminal tag excision. The temperature parameters of RT-PCR reaction were: reverse transcription cycle, 50°C for 15 min; Taq-AT enzyme activation cycle, 95°C for 5 min; then 45 cycles of successive denaturation for 15 s at 95°C, annealing for 15 s at 50°C, and elongation for 10 s at 72°C with fluorescent signal registration. PCR efficiency for all oligonucleotides was tested by obtaining standard curves using RNA serial dilutions. As efficiency was similar and close to 100% (data not shown) for all the cases, we calculated normalized expression assuming efficiency to be 100%.
Primers and fluorochromes used for vcam-1 and Gapdh amplification are given in Table 1.
Table 1. Oligonucleotides used in the study
Statistics. At least five animals were used in each group during in vivo experiments. For in vitro studies, the experiments were repeated at least three times. Statistical significance was evaluated using Student's test. Data are presented as mean ± SEM.
Effects of ischemia on endothelial cell culture. To study endothelial vulnerability to the damaging effects of I/R, we used the model that most accurately reproduces I/R conditions in vitro – oxygen–glucose deprivation (OGD). Over 50% of endothelial cells in EA.hy926 culture died 24 h after 5-h OGD. Mitochondria-targeted antioxidant increased endothelial cells survival after ischemia: survival of EA.hy926 cells increased to 64% after incubation for 1 h with 25 nM SkQR1 prior to OGD (Fig. 1a).
Fig. 1. SkQR1 reduces ischemia-induced endothelial damage. a) Death of EA.hy926 cells after oxygen–glucose deprivation (OGD) was reduced after 1-h preincubation with 25 nM SkQR1; * p < 0.05. b, c) Evans Blue dye accumulation in kidneys 70 min after i.v. injection of 2 ml 0.5% Evans Blue. Visual analysis of the dye distribution in the kidney (b) and quantification of the amount of dye extracted from kidney tissue homogenates (c). SkQR1 was administered i.p. 3 h prior to the incidence of ischemia; 1, 18, 30, 42 h after the beginning of reperfusion (each injection – 100 nmol/kg). d) Changes in the level of vcam-1 mRNA in blood after 40 min of ischemia and 10 min of reperfusion; # p < 0.1.
Effects of ischemia on endothelial barrier function. The degree of vascular endothelial damage after renal ischemia was also evaluated in vivo. In 48 h after 40-min renal ischemia, the permeability of the renal vascular bed was studied, which reflects the state of one of the main endothelial functions – barrier function. Comparing the accumulation of Evans Blue in the kidneys of control rats and rats subjected to I/R, we found a small increase in its concentration in postischemic kidneys (Fig. 1, b and c), suggesting increased permeability of renal vessels to albumin (99% of the dye is bound to this protein). Visual analysis of Evans Blue localization in different kidney portions showed that the main accumulation of the dye is observed in the renal pelvis and inner medulla, whereas it is hardly detectable in the cortex and outer medulla (Fig. 1b). I/R results in the increase in Evans Blue content in all kidney parts (Fig. 1b). We observed the dye accumulation in the cortex and outer medulla, i.e. in the kidney sections most sensitive to ischemic damage. We measured the amount of Evans Blue extracted from the total kidney homogenates and found the difference between the control animals and rats subjected to I/R (Fig. 1c). After the animals were treated with mitochondria-targeted antioxidant (i.p. administration of 100 nmol/kg SkQR1 3 h prior to ischemia; 1, 18, 30, and 42 h after the beginning of reperfusion), we observed a tendency toward normalization of the renal vascular bed permeability. In SkQR1-treated animals, Evans Blue concentration after I/R in the total kidney homogenate did not differ from that of intact animals (Fig. 1c). Visual analysis also showed extremely low dye content in the cortex and outer medulla (Fig. 1b).
Assessment of vascular endothelium damage by levels of endothelial cells markers in circulating blood. Appearance in the bloodstream of freely circulating endothelial cells is one of the conventional markers of vascular endothelium damage. In this study, we evaluated the content of mRNA of VCAM-1 in peripheral rat blood (this protein is a specific marker of endothelial cells). Ten minutes after the beginning of reperfusion, the amount of VCAM-1 mRNA was significantly increased compared to sham-operated animals, indicating a significant increase in the number of severely damaged endothelial cells desquamated into the vessel lumen and/or the level of endothelium activation, as VCAM-1 expression increases in the inflammatory response of endothelial cells (Fig. 1d).
Ultrastructural changes in mitochondria of endotheliocytes. Since mitochondria play the key role in the fate of cells subjected to I/R, and mitochondria-targeted antioxidant SkQR1 had a positive effect on the survival and maintenance of the normal functioning of endothelium after I/R, we evaluated the effect of I/R on the ultrastructure of the mitochondrial reticulum of endothelial cells. Electron microscopic study of the renal cortical zone showed significant damage in endothelial mitochondria already in the early stages of reperfusion after 40 min of ischemia. Endothelial cells in the cortex of an intact kidney with mitochondria in orthodox configuration were considered as normal (Fig. 2, a and b). In a similar zone of the kidney fixed 10 min after the beginning of reperfusion we detected both endotheliocytes with mitochondria maintaining orthodox structure and endotheliocytes with altered mitochondrial morphology. In particular, we observed a significant increase in intercristae and intermembrane space accompanied by a local enlightenment of the mitochondrial matrix and their swelling (Fig. 2, c and d). In some mitochondria, the number of visually determined cristae was decreased up to their complete disappearance (Fig. 2, e and f); cells with such mitochondria showed signs of degradation. This data indicated that mitochondrial damage in renal endothelial cells is an early event observed in the first minutes after the beginning of reperfusion.
Fig. 2. Ultrastructural changes in renal endothelial mitochondria after ischemia. a, b) Intact kidney, endothelial cells with mitochondria (M) in the orthodox state; BM, basal membrane, V, vessel lumen. c-f) Kidney after 40 min of ischemia and 10 min of reperfusion. Alterations in mitochondrial morphology (M) starting from minor (c, d: swelling of inter-cristae (large arrows in (c)) and intermembrane space (arrow in (d)) and matrix (small arrow in (c)) to significant (local, shown by arrow in (e) or spacial in (f)) mitochondrial swelling. N, nucleus; E, part of erythrocyte in vessel lumen.
Changes in renal hemodynamics parameters. Renal vascular response to I/R was studied at the same time frames, in which mitochondria of endothelial cells are damaged. We visualized segmental and partially interlobar renal arteries using magnetic resonance imaging. Ten minutes after the beginning of reperfusion, the number of visualized segmental and interlobar renal arteries was significantly reduced, indicating a strong decrease in the diameter of their lumen. Ten minutes after the beginning of reperfusion, the number of visualized vessels was higher in the kidney of animals injected with SkQR1 (100 nmol/kg, i.p.) 3 h before I/R, compared to animals which did not receive the treatment, but lower than in intact kidneys (Fig. 3a).
Fig. 3. Changes in renal blood flow after 40 min ischemia. a) Representative kidney images obtained using magnetic resonance imaging after 40 min ischemia and 10 min reperfusion. SkQR1 increases the number of blood-filled renal vessels after 40 min ischemia. b-e) Representative recording of the linear (b, d) and volumetric (c, e) blood flow velocity in the left renal artery using a high frequency ultrasound (27 MHz) bondage-type Doppler transducer. Rat after I/R (b, c) and rat after I/R with SkQR1 (100 nmol/kg) administered i.p. 3 h prior to I/R (d, e).
The ultrasound Doppler was used for quantitative assessment of renal blood flow after I/R. We observed severe changes in linear and volumetric blood flow velocity in the renal artery after 40 min of ischemia, which were largely prevented by preliminary SkQR1 administration (Fig. 3, b-e).
Measurement of volumetric blood flow rate in the renal artery, reflecting the total renal blood flow, showed significant renal hypoperfusion after 40 min of ischemia (Fig. 4a). One minute after the beginning of reperfusion, the volumetric blood flow rate was only about 30% of the level prior to ischemia. Measurements of this parameter at the 5th, 10th, 15th, and 20th min of reperfusion showed that renal blood supply remains low. Some recovery in blood flow was found only at the 25th min, but hypoperfusion persisted until the end of the observation period (30 min) (Fig. 4a).
Fig. 4. SkQR1 reduces the negative effect of 40 min ischemia on renal hemodynamics. Volumetric blood flow rate (a) and resistivity index (b) of the left renal artery. Administration of 100 nmol/kg SkQR1 3 h prior to I/R increased renal blood flow and reduced vasoconstriction of renal vessels as evidenced by a decrease in resistivity index. Intravenous administration of 1 mg/kg L-NA abolished the positive effect of SkQR1 on renal hemodynamics; * p < 0.05, ** p < 0.1 compared to I/R.
We calculated the resistivity index using the maximal peak systolic and end diastolic blood flow velocity during the cardiac cycle (the parameter indicates the degree of resistance to blood flow in the areas distal to the spot of blood flow registration). Since in our experiments the transducer was applied to the renal artery, changes in the resistivity index reflected the changes in renal vascular resistance. After 40 min of ischemia, resistivity index significantly increased, and within 1 min after the beginning of reperfusion, it was about 20% higher than before ischemia (Fig. 4b). Resistivity index reached its maximum value in the 10th min and remained increased until the 30th min of reperfusion (Fig. 4b), when the experiment was terminated. The data was consistent with the results obtained with MRI scanning of the kidney and indicated pronounced vasoconstriction of renal vessels after I/R.
Administration of SkQR1 (i.p. 100 nmol/kg) 3 h prior to the beginning of ischemia resulted in a more rapid recovery of renal blood flow. For example, at the 1st min of reperfusion, the volumetric blood flow rate in rats treated with the mitochondrial antioxidant increased to 49% (compared to the values prior to ischemia), while in rats received no treatment this parameter was only 30%. Volumetric rate continued to grow and reached 67% at the 15th min, while in animals received no treatment the blood flow was restored only to 36% of the pre-ischemic value (Fig. 4a).
Resistivity index before ischemia did not differ significantly in animals with or without administered SkQR1. After 40 min of ischemia, we observed no positive effect of mitochondria-targeted antioxidant on the increase in resistivity index caused by ischemia in the 1st min of reperfusion. However, resistivity index decreased already in the 5th and 10th min of reperfusion, returning to the values observed before ischemia. Starting from the 15th min, vascular resistance again began to increase (Fig. 4b). Thus, the mitochondria-targeted antioxidant reduced renal vascular resistance after ischemia at the very time when mitochondrial damage occurred in vascular endothelium.
We previously showed that systemic hemodynamics did not undergo significant changes after I/R [28]. In this study, heart rate measurements also showed no statistically significant differences, both with and without SkQR1 (Table 2).
Table 2. Changes in heart rate (HR) during the experiment
Note: Animals were subjected to 40-min renal ischemia. SkQR1 was administered 3 h prior to the beginning of ischemia (i.p. 100 nmol/kg).
Since NO is the main vasodilatory factor, we studied the effect of SkQR1 on renal hemodynamics against the background of inhibited NO synthesis. Treatment with nonselective inhibitor of NO-synthases L-NA led to the disappearance of the positive effect of SkQR1 on renal hemodynamics. Dynamics of renal blood flow restoration under coadministration of SkQR1 (i.p. 100 nmol/kg, 3 h prior to ischemia) and L-NA (i.v. 1 mg/kg, 10 min prior to ischemia) was not significantly different from the animals that received no treatment (Fig. 4a). Decrease in the resistivity index was not observed after coadministration of L-NA and SkQR1, in contrast with animals treated only with SkQR1 (Fig. 4b).
We previously showed that SkQR1 preserved the function of kidney subjected to I/R [22]. To assess the role of this effect on the renal vascular bed for the nephroprotective SkQR1 impact, we evaluated renal function 48 h after I/R based on serum creatinine concentration. SkQR1 treatment resulted in almost twofold reduction of creatinine concentration compared to animals that received no treatment, indicating a decrease in the severity of renal failure. However, when this SkQR1 treatment protocol was combined with intravenous L-NA administration 10 min before I/R, we observed no decrease in creatinine concentration (Fig. 5).
Fig. 5. Effect of L-NA on the protective effect of SkQR1 on renal function after 40-min ischemia. Renal function was assessed by creatinine concentration in blood serum 48 h after ischemia. L-NA (1 mg/kg) was administered intravenously 10 min prior to the beginning of ischemia. SkQR1 (100 nmol/kg) was injected i.p. 3 h before the beginning of ischemia and 1, 18, 30, and 42 h after the beginning of reperfusion; * p < 0.001; n/d, no significant differences; SO, sham-operated animals.
In this study, we show that damage of endothelial mitochondria plays an important role in the mechanisms of impairment of renal hemodynamics resulting from I/R. This is indicated by the fact that pronounced impairments in renal vascular bed functioning (vasoconstriction of blood vessels, increase in vascular resistance, reduction of renal blood flow) occur within the same timeframe as the impairments of mitochondrial ultrastructure. Electron microscopic study showed that already 10 min after the beginning of reperfusion, destructive processes are triggered in mitochondria of endothelial cells, leading to the impairment of their ultrastructure. In addition, 10 min after the beginning of reperfusion, we observed the reduction of the renal blood flow, which obviously leads to insufficient oxygen supply to cells. Renal blood flow does not return to the pre-ischemic values after the cessation of ischemia; it remains considerably reduced for a long time due to a persistent increase of renal vascular resistance. Administration of SkQR1, a compound selectively accumulated in mitochondria that protects endothelial cells in vitro, had a beneficial effect on the observed parameters.
It is known that I/R increases ROS production in mitochondria [14]. Since I/R results in the damage to endothelial cells of glomerular and peritubular capillaries and tubular epithelial cells, ROS hyperproduction may be within all these types of cells. It has been shown in a number of studies that ROS can regulate renal blood flow [16-18]. In these studies, the effect of ROS on blood flow was similar to the post-I/R changes that we observe; i.e. a decreased renal perfusion. We previously showed that I/R leads to considerable oxidative stress in renal parenchyma, and mitochondria are the main source of ROS [29]. It is important to note that the peak of ROS production in the kidney was observed 10 min after the beginning of reperfusion – the time when endothelial mitochondria were damaged. Administration of SkQR1 3 h before ischemia prevented the development of oxidative stress in the kidney 10 min after the beginning of reperfusion [22]. Although in that study the ROS production in renal tubules only was studied, we assumed that similar changes also take place in endothelium. This was confirmed by our observation that mitochondria-targeted antioxidant improves renal blood flow, which emphasizes the possible role of mitochondrial ROS in changes of renal blood flow. This conclusion is confirmed by other studies. For example, it has been shown that mitochondrial ROS production plays an important role in the development of experimental hypertension [20, 30].
Noteworthy that disruptions in mitochondrial functioning occurring in the first minutes of reperfusion, apparently have a long-term effect on the processes taking place in the vascular wall, because hemodynamic parameters remain abnormal for a long time after I/R – at least for 30 min. Moreover, in the preliminary experiments we observed that hemodynamic parameters reached by the 10th min of reperfusion remained basically unchanged even at the 60th min of reperfusion. We consider that the primary oxidative stress of mitochondrial nature induced in the early phase of I/R is sustained for a long time due to a positive feedback between ROS-producing systems [14, 31]. For example, it has been shown that mitochondrial ROS can activate NADPH-oxidase (a ROS-producing enzyme), and ROS generated by this enzyme can, in turn, cause oxidative stress in mitochondria [32].
Since SkQR1 is accumulated in mitochondria of all renal cells, it raises a question: Is the positive effect of SkQR1 specifically related to the protection of endothelial cells? (Earlier it has been shown that SkQR1 normalizes mitochondrial functions in tubular cells [22].) Therefore, in this study we assessed the effect of SkQR1 on the consequences of I/R in endothelial cells in the absence of any intermediates. I/R simulation in vitro showed that preincubation of EA.hy926 endothelial cells with SkQR1 decreased OGD-induced cell death, indicating the possibility of the primary protection of endothelial cells.
In our work, we demonstrate an increase in the number of endothelial cells circulating in blood 10 min after the beginning of reperfusion, indicating that I/R may cause the destruction of endothelium in vivo. The phenomenon is a marker of pronounced damage of the vascular bed [33]. Apparently, it happens due to the desquamation of the critically damaged cells into the vessel lumen. Loss of endothelial lining may be another cause of the long-term disorders in renal hemodynamics. Thus, the density of the renal capillary bed has been shown to be reduced even one week after renal ischemia [12]. In our study, we observed significant impairment of endothelial barrier function 2 days after I/R. SkQR1 treatment reduced the permeability of renal blood vessels to normal values.
Decrease in NO availability or reduction of its production by endothelial cells due to disorders in their functioning and their destruction are the most probable mechanism of vasoconstriction after ischemia. We can increase NO production in post-ischemic kidney by protecting endothelial cells with SkQR1; it allows to avoid (at least partially) the harmful effects of the secondary ischemia due to vasoconstriction. In our study, we used the nonselective inhibitor of NO-synthases L-NA to test whether the positive effect of SkQR1 on blood flow is associated with an increase in NO production. Indeed, when L-NA was administered 10 min before ischemia to rats who had previously received SkQR1, we observed disappearance of the positive effect of the mitochondria-targeted antioxidant on the renal blood flow.
Positive effect of SkQR1 on restoration of renal blood flow was limited in time (improvement of these parameters became statistically unreliable already by the 30th min of reperfusion), and L-NA inhibition of this short positive effect at the early stages of reperfusion also resulted in the reduction of the protective effect of the mitochondria-targeted antioxidant on the renal functions 2 days after I/R. Our data suggest the fundamental importance of inhibiting the very first events of the pathological cascade of vascular dysfunction, which are apparently associated also with the dysfunction of endothelial mitochondria.
Early studies have shown the positive effect of the mitochondria-targeted antioxidant SkQ1 on endothelial cell communication impaired by tumor necrosis factor. It led to general conclusions on the involvement of ROS in the inflammatory response and SkQ1 having antiinflammatory activities [34, 35].
This study was supported by the Russian Science Foundation (project No. 14-15-00147).
1.Ajis, A., Bagnall, N. M., Collis, M. G., and Johns, E. J. (2003) Effect of endothelin antagonists on the renal hemodynamic and tubular responses to ischemia-reperfusion injury in anesthetized rats, Exp. Physiol., 88, 483-490.
2.Olof, P., Hellberg, A., Kallskog, O., and Wolgast, M. (1991) Red cell trapping and postischemic renal blood flow. Differences between the cortex, outer and inner medulla, Kidney Int., 40, 625-631.
3.Adams, P. L., Adams, F. F., Bell, P. D., and Navar, L. G. (1980) Impaired renal blood flow autoregulation in ischemic acute renal failure, Kidney Int., 18, 68-76.
4.Kelleher, S. P., Robinette, J. B., and Conger, J. D. (1984) Sympathetic nervous system in the loss of autoregulation in acute renal failure, Am. J. Physiol., 246, F379-F386.
5.De Greef, K. E., Ysebaert, D. K., Dauwe, S., Persy, V., Vercauteren, S. R., Mey, D., and De Broe, M. E. (2001) Anti-B7-1 blocks mononuclear cell adherence in vasa recta after ischemia, Kidney Int., 60, 1415-1427.
6.Takada, M., Nadeau, K. C., Shaw, G. D., Marquette, K. A., and Tilney, N. L. (1997) The cytokine-adhesion molecule cascade in ischemia/reperfusion injury of the rat kidney. Inhibition by a soluble P-selectin ligand, J. Clin. Invest., 99, 2682-2690.
7.Kwon, O., Phillips, C. L., and Molitoris, B. A. (2002) Ischemia induces alterations in actin filaments in renal vascular smooth muscle cells, Am. J. Physiol. Renal Physiol., 282, F1012-F1019.
8.Kuhne, W., Besselmann, M., Noll, T., Muhs, A., Watanabe, H., and Piper, H. M. (1993) Disintegration of cytoskeletal in energy-depleted endothelial structure cells of actin filaments in energy-depleted endothelial cells, Am. J. Physiol. Heart Circ. Physiol., 264, H1599-1608.
9.Sutton, T. A., Mang, H. E., Campos, S. B., Sandoval, R. M., Yoder, M. C., and Molitoris, B. A. (2003) Injury of the renal microvascular endothelium alters barrier function after ischemia, Am. J. Physiol. Renal Physiol., 285, F191-198.
10.Kevil, C. G., Oshima, T., Alexander, B., Coe, L. L., and Alexander, J. S. (2000) H(2)O(2)-mediated permeability: role of MAPK and occludin, Am. J. Physiol. Cell Physiol., 279, C21-C30.
11.Kwon, O., Hong, S.-M., Sutton, T. A., and Temm, C. J. (2008) Preservation of peritubular capillary endothelial integrity and increasing pericytes may be critical to recovery from postischemic acute kidney injury, Am. J. Physiol. Renal Physiol., 295, F351-F359.
12.Cantaluppi, V., Gatti, S., Medica, D., Figliolini, F., Bruno, S., Deregibus, M. C., Sordi, A., Biancone, L., Tetta, C., and Camussi, G. (2012) Microvesicles derived from endothelial progenitor cells protect the kidney from ischemia-reperfusion injury by microRNA-dependent reprogramming of resident renal cells, Kidney Int., 82, 412-427.
13.Basile, D. P., Friedrich, J. L., Spahic, J., Knipe, N., Mang, H., Leonard, E. C., Changizi-Ashtiyani, S., Bacallao, R. L., Molitoris, B. A., and Sutton, T. A. (2011) Impaired endothelial proliferation and mesenchymal transition contribute to vascular rarefaction following acute kidney injury, Am. J. Physiol. Renal Physiol., 300, F721-F733.
14.Zorov, D. B., Juhaszova, M., and Sollott, S. J. (2014) Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release, Physiol. Rev., 94, 909-950.
15.Silachev, D. N., Plotnikov, E. Y., Pevzner, I. B., Zorova, L. D., Babenko, V. A., Zorov, S. D., Popkov, V. A., Jankauskas, S. S., Zinchenko, V. P., Sukhikh, G. T., and Zorov, D. B. (2014) The mitochondrion as a key regulator of ischaemic tolerance and injury, Heart Lung Circ., 10, 897-904.
16.Ahmeda, A. F., and Johns, E. J. (2012) The regulation of blood perfusion in the renal cortex and medulla by reactive oxygen species and nitric oxide in the anesthetized rat, Acta Physiol., 204, 443-450.
17.Majid, D. S. A., and Nishiyama, A. (2002) Nitric oxide blockade enhances renal responses to superoxide dismutase inhibition in dogs, Hypertension, 39, 293-297.
18.Liu, R., Ren, Y., Garvin, J. L., and Carretero, O. A. (2004) Superoxide enhances tubuloglomerular feedback by constricting the afferent arteriole, Kidney Int., 66, 268-274.
19.Zhang, H., Schmeisser, A., Garlichs, C. D., Plotze, K., Damme, U., Mugge, A., and Daniel, W. G. (1999) Angiotensin II-induced superoxide anion generation in human vascular endothelial cells: role of membrane-bound NADH-/NADPH-oxidases, Cardiovasc. Res., 44, 215-222.
20.Dikalova, A. E., Bikineyeva, A. T., Budzyn, K., Nazarewicz, R. R., McCann, L., Lewis, W., Harrison, D. G., and Dikalov, S. I. (2010) Therapeutic targeting of mitochondrial superoxide in hypertension, Circ. Res., 107, 106-116.
21.Plotnikov, E. Y., Silachev, D. N., Chupyrkina, A. A., Danshina, M. I., Jankauskas, S. S., Morosanova, M. A., Stelmashook, E. V., Vasileva, A. K., Goryacheva, E. S., Pirogov, Y. A., Isaev, N. K., and Zorov, D. B. (2010) New-generation Skulachev ions exhibiting nephroprotective and neuroprotective properties, Biochemistry (Moscow), 75, 145-150.
22.Plotnikov, E. Y., Chupyrkina, A. A., Jankauskas, S. S., Pevzner, I. B., Silachev, D. N., Skulachev, V. P., and Zorov, D. B. (2011) Mechanisms of nephroprotective effect of mitochondria-targeted antioxidants under rhabdomyolysis and ischemia/reperfusion, Biochim. Biophys. Acta, 1812, 77-86.
23.Plotnikov, E. Y., Silachev, D. N., Jankauskas, S. S., Rokitskaya, T. I., Chupyrkina, A. A., Pevzner, I. B., Zorova, L. D., Isaev, N. K., Antonenko, Y. N., Skulachev, V. P., and Zorov, D. B. (2012) Mild uncoupling of respiration and phosphorylation as a mechanism providing nephro- and neuroprotective effects of penetrating cations of the SkQ family, Biochemistry (Moscow), 77, 1029-1037.
24.Jankauskas, S. S., Plotnikov, E. Y., Morosanova, M. A., Pevzner, I. B., Zorova, L. D., Skulachev, V. P., and Zorov, D. B. (2012) Mitochondria-targeted antioxidant SkQR1 ameliorates gentamycin-induced renal failure and hearing loss, Biochemistry (Moscow), 77, 666-670.
25.Plotnikov, E. Y., Morosanova, M. A., Pevzner, I. B., Zorova, L. D., Manskikh, V. N., Pulkova, N. V., Galkina, S. I., Skulachev, V. P., and Zorov, D. B. (2013) Protective effect of mitochondria-targeted antioxidants in an acute bacterial infection, Proc. Natl. Acad. Sci. USA, 110, E3100-E3108.
26.Bakeeva, L. E., Barskov, I. V., Egorov, M. V., Isaev, N. K., Kapelko, V. I., Kazachenko, A. V., Kirpatovsky, V. I., Kozlovsky, S. V., Lakomkin, V. L., Levina, S. B., Pisarenko, O. I., Plotnikov, E. Y., Saprunova, V. B., Serebryakova, L. I., Skulachev, M. V., Stelmashook, E. V., Studneva, I. M., Tskitishvili, O. V., Vasilyeva, A. K., Victorov, I. V., Zorov, D. B., and Skulachev, V. P. (2008) Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 2. Treatment of some ROS- and age-related diseases (heart arrhythmia, heart infarctions, kidney ischemia, and stroke), Biochemistry (Moscow), 73, 1288-1299.
27.Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods, 65, 55-63.
28.Jankauskas, S. S., Matsievsky, D. D., Plotnikov, E. Y., and Zorov, D. B. (2014) High-frequency ultrasound Doppler system for study of renal blood flow during ischemia/reperfusion of kidney, Nephrol. Dial., 16, 169-175.
29.Plotnikov, E. Y., Kazachenko, A. V., Vyssokikh, M. Y., Vasileva, A. K., Tcvirkun, D. V., Isaev, N. K., Kirpatovsky, V. I., and Zorov, D. B. (2007) The role of mitochondria in oxidative and nitrosative stress during ischemia/reperfusion in the rat kidney, Kidney Int., 72, 1493-1502.
30.Nazarewicz, R. R., Dikalova, A. E., Bikineyeva, A., and Dikalov, S. I. (2013) Nox2 as a potential target of mitochondrial superoxide and its role in endothelial oxidative stress, Am. J. Physiol. Heart Circ. Physiol., 305, H1131-1140.
31.Zorov, D. B., Filburn, C. R., Klotz, L. O., Zweier, J. L., and Sollott, S. J. (2000) Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes, J. Exp. Med., 192, 1001-1014.
32.Dikalov, S. (2011) Cross talk between mitochondria and NADPH oxidases, Free Radic. Biol. Med., 51, 1289-1301.
33.Woywodt, A., Kirsch, T., and Haubitz, M. (2008) Circulating endothelial cells in renal disease: markers and mediators of vascular damage, Nephrol. Dial. Transplant., 23, 7-10.
34.Zinovkin, R. A., Romaschenko, V. P., Galkin, I. I., Zakharova, V. V., Pletjushkina, O. Y., Chernyak, B. V., and Popova, E. N. (2014) Role of mitochondrial reactive oxygen species in age-related inflammatory activation of endothelium, Aging (Albany N. Y.), 6, 661-674.
35.Demyanenko, I. A., Popova, E. N., Zakharova, V. V., Ilyinskaya, O. P., Vasilieva, T. V., Romashchenko, V. P., Fedorov, A. V., Manskikh, V. N., Skulachev, M. V., Zinovkin, R. A., Pletjushkina, O. Y., Skulachev, V. P., and Chernyak, B. V. (2015) Mitochondria-targeted antioxidant SkQ1 improves impaired dermal wound healing in old mice, Aging (Albany N. Y.), 7, 475-485.


In the latest Mega64 video, it seems Derrick has become a bit too enamored with the game Papers, Please. He's set up a border patrol booth at the entrance to the Mega64 offices, which has now been dubbed the country of Acosta (after his own last name). Rocco only wants to go inside to get his iPad that he left behind, but Derrick is getting a little too into the role. Hilarity naturally ensues.
If only bypassing the patrol in the game was as easy as it is to get past Derrick. But at what cost? WHAT COST?!


Dress: Topshop. Bag: Chanel Vintage. Shoes: Chockers. Necklace: Claires. Sunglasses: Bershka.
OK, so I am channelling a bit of last-season-style here with the Clogs and Chanel tattoo but it's a look I still love even though I wore it to death last summer! This cute dress is from Topshop (£34.00) and has this amazing cut-out detail at the waist acting as a waistband. Sitting on the grass I had a feeling of Audrey Hepburn in Breakfast At Tiffanys with a modern day twist.


It's often not easy. Most of the time it is very difficult. There will be obstacles, but the final success will make you forget them. It's a life lesson you need to learn. Sometimes a motivational quote is just the push you need. Here we will discuss 12.
Learning life lessons is essential if you are to grow as a person. In a continuation of my I've Learned Series, today's post offers motivation for you to keep pursuing your dreams.
Some life lessons will help you cope with tragedy and discouragement. Many will help you live peaceably while being more empathic and compassionate. Still others, the ones you will read about today, will give you the push you need to get started and keep going.
Whether you are dreaming of being a published author, a renowned stage actor or just a few pounds lighter, these quotes will help get you moving.
It is easy to put off a dream or goal because we are not where we think we should be. Have you ever said any of these things?
· I will do it when I get a little older and have more time.
· I will start when my kids are grown.
· I will start exercising on January 1st.
· I will start when the weather gets warmer.
· I will start when I'm in a better position financially.
There are lots of reasons to put off your dreams. The truth is, there will never be the perfect time or circumstances. Start here. Start now.
Success never happens overnight. Start today in the direction you want to go. Tomorrow, you will wake up one step closer to your goal.
Disclosure: This site is a participant in the Amazon Services, LLC. Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to Amazon.com. This post also contains affiliate links. Any purchase made through such links will award me a small commission or referral fee, at no extra cost for you.
A leader with motivational quotes, Tony Robbins gives one of the keys to success.
You already have your dream. The final prize is clearly in your mind. Setting goals is about more than just setting the ultimate destination. Success is achieved through reaching many smaller goals. These small goals serve as footholds and milestones that are an important part of your journey.
Even small goals are important.
Suppose your ultimate goal is to write a novel. Most novels are in the range of 80,000-100,000 words. Very few people could pump out that many words in a day. Most can't even do it in a week.
That's why you start with smaller goals.
If you want to write a 100,000-word novel in a year, that means you need to write 275 words per day. Writing 275 words isn't nearly as daunting as 100,000. Up to this point in this post you have already read 350 words. It's not that much to commit to writing each day. It's a realistic and achievable goal.
The same is true for every goal. Use this life lesson and break your goal down into its smallest parts. Then set each of those parts as a goal that you can knock off one by one.
A few lucky people are born into wealth and privilege. Due to their family legacy, they have all the tools they need to work towards virtually any goal. Or at least the means to buy the tools.
The rest of us usually like to wait for opportunity to find us. Stop waiting and start actively looking for opportunities. That truth makes this one of the most popular motivational quotes.
I got started in the financial services field completely by chance. One day, while visiting one of my neighbors at his office, I noticed an abundance of files scattered around in piles on the floor.
Often not the most tactful person, I made a comment about them and how unprofessional they looked.
My friend explained that he desperately needed a new filing system, but his entire staff was so busy that no one had time to work on it. I had some free time in my schedule, so I offered to spend a few hours a week putting things in order.
That simple act led to a twenty-year career in financial services.
The point here is that opportunity is all around us. Rarely does it come and knock on our door, but if we keep our eyes and minds open, we will see opportunity all around us.
Look around your life for the opportunities you need that will help you achieve your dreams.
A lie that people like to tell themselves, especially as they get older, is that they aren't the right age to pursue their dream. That lie is complete garbage.
In my life I have had the pleasure of watching friends achieve their goals, even starting late in life. One friend started taking piano lessons when she was nearly 60. She is a very accomplished pianist today. Another friend about the same age started taking art classes. His paintings now garner more attention than those from painters with decades of experience.
Browse the internet and you will find stories of many octogenarians that have started running marathons, taken up sky diving or gone back to school. They learned the life lesson and apply it in their lives.
You are not too young or too old to start a new goal. Get that thought out of your head.
Forget your age. It's only a number. Keep this message with you favorite motivational quotes.
Another obstacle people put in the way of their dreams is the past. Maybe you tried to lose weight before but then gained it all back. Perhaps you started a business and it didn't succeed. You might have started writing a book that you never finished.
All those things are okay. There is nothing you can do to change them. Stop letting them halt your future progress.
Tomorrow is a blank page. Nothing from yesterday needs to color it. Choose for tomorrow to be the day that gets you closer to your goal.
Now that the past is behind you, and you have your goal broken down into more manageable parts, it's time for action.
Do at least one thing every day to move you forward. It doesn't have to be a huge task. But do something every day.
Sometimes it might be just making a decision.
For instance, if you are starting a blog, one of the things most bloggers struggle with is picking a domain name. Your goal for today could be to make a list of possible names. If you already have a list, today's assignment could be to pick one and sign up with a hosting company.
Reaching a goal isn't about making big strides every day. Doing so might get you there faster, but any forward movement is just that – forward movement.
Do one thing today that moves you forward and count today as a win.
It's a crazy truth, but many people never achieve their dreams because they are afraid of success. Singer song-writers may fear that fame will change their lives in negative ways. Actors may fear that they won't be able to keep the momentum after one big success. Authors fear that they might not have another good book in them.
Fear also rears its ugly head from the other direction. An author might fear publishing a book that no one reads or likes. I venture that every author feels that way, at least with their very first book.
An artist may fear that no one will understand or like their art. A musician might fear that no one will connect with their songs.
Don't let fear stop you. Most things we fear will never happen. Learn and believe that life lesson.
There are more than 7-billion people on the planet. 7 billion! Many of them are going to like what you do no matter how good or bad it is.
Think of some of the worst films you have ever seen. Probably they all have some sort of cult following that love the film.
And if you don't succeed? That's not even an option. If you reach your goal, whatever that goal is, you have already succeeded. That success will change everything else in a good way.
Imagine that your dream is to build a boat and sail across the ocean. You work hard for months or years in crafting an excellent vessel. If you never put that boat in the water and actually set sail, your dream will never reach its true fulfillment.
Many writers do this with books. They write books, sometimes dozens of them, but then they never pursue publication. One writer share on Twitter recently that she has a whole wall of books she wrote and never pursued.
Most likely fear comes back into play here, but I cannot say that for sure. I myself have written at least five books, but only one is published. Getting that one published with a struggle that I stopped many time. You can read my experience here.
Reaching a milestone is not the place to stop. Keep your goal in front of you always. Then keep moving forward.
Life lesson? If you already have the boat finished, now is the time to get in the water.
The brutal truth is that failure is an integral part of success. But failure never has to be the end. Just because something doesn't succeed the first time, or even the second or hundredth time, doesn't mean that it never will.
Nearly all the biggest achievements in the world arrived with a past littered with numerous failures. Thomas Edison is a famous example. For each of his world-changing successes were thousands of ideas that had not worked.
To read more about Thomas Edison and perseverance, read my post 7 Reasons to Never Give Up.
What's necessary is to change your mindset. Expect you will have at least some failures during your journey. Then, when they happen, meditate on what you can learn from the failure and move on.
Your dream is still achievable.
Perhaps the biggest barrier to success is procrastination. Continually putting off the steps that need to be completed to reach your goal keeps your prize forever out of arm's reach.
The good news is procrastination is another thing that is in your control. There is a great post about how to beat procrastination. 4 Ways to Beat Procrastination – With Frogs is another life lesson that gives you four easy things you can do to change your procrastinating habit .
Resolve that you will keep moving forward. Set deadlines for yourself and do everything in your power to achieve them.
Only you can do what needs to be done. No matter how many online courses you pay for, how many classes your take or how many personal trainers you see, at the end of the day you are the only one that can do what needs to be done. You are the one that needs to write, exercise or eat better.
You have the ability to reach your goal. Remind yourself of that daily. Hourly if you have to. It is possible. You can do it.
No matter what happens along the pathway to achieving your dream, don't give up. There will be bad days. There might be bad weeks and months. I've even had bad years. But don't let anything stop you from moving forward.
Even if circumstances cause your project to stall for a while, as soon as possible, get moving again.
Remember that the small things count. That's another life lesson.
If your goal was to fill a bucket with rocks, big rocks would get the job done quicker. But even the smallest rocks tossed into the bucket add to the whole.
Acknowledge the small successes as well as the big ones. Each one is moving you toward reaching your dream.
This post contains just a few of the quotes that have helped to reaffirm this life lesson in my mind. Are there quotes that keep you motivated? Please share them in the comments below.
If you enjoyed this post, please share it with friends and on social media.
Thank you for sharing my link!


The Sorting question type is an interactive tool used to re-arrange answers in the correct order (for example, smallest to largest).
Student may receive partial credit for having a partially correct response.
Under Choose Question Type, select Sorting.
The default is set to three items. If you would like more than three items, click Add Item.
In the correct order, enter the information you wish to have sorted in the text boxes provided.
(Optional) If you wish to re-order the text boxes, use the move ( ) icon.
(Optional) Width (px): specifies the size (original width) of the item being sorted.
(Optional) Height (px): specifies the size (original height) of the item being sorted.
(Optional) Background color: specifies the color of the item being sorted.
(Optional) To add, view or delete Algorithms, Custom CSS, and/or Feedback, see Adding and Editing Algorithms and Adding and Editing Feedback for more details.
Click Save & Close to save the question.
Enter the following for Question Text: Order these fractions in increasing order (from least to greatest).
Click Add Item twice. (This means you have a total of five text boxes).
Enter the following information in the text boxes. Important: Ensure that the responses are in the correct order.
Width (px): specify the width of the item to 50.
Height (px): specify the height of the item to 100.
Click Save and then click Preview to see the question, as shown in Figure 5.56 below.
(Optional) To add, view or delete Algorithms, Custom CSS, and/or Feedback, see Adding and Editing Algorithms and Adding and Editing Feedback for more details. Click Save.


Learn how we spend $5 and turn it into $94 in just 24 hours using Bing and Clickbank.
My exact blueprint of setting up highly profitable campaigns literally in minutes.
Copy my step by step proven and reliable method to generate thousands of keywords in minutes using my free secret resources.
Get instant access to my own personal bidding formula that drives me faster traffic and results.


The Dellatera Z-Glide Standard is Dean Zelinsky's personal take on making a traditional 2-single-coil pickup guitar with something extra special. The unique body carve and narrowed waist cut creates a style that sets it apart from the sea of dual-single guitars. The body is made from select Alder creating a light, yet better sustaining guitar…and yields the best overall tone. This guitar is very comfortable with weight averaging 7.5 lbs.


WHEN a delinquent is arrested by any of the means mentioned in the preceding chapter, he ought regularly to be carried before a justice of the peace. And how he is there to be treated, I shall next show, under the second head, of commitment and bail.
THE justice, before whom such prisoner is brought, is bound immediately to examine the circumstances of the crime alleged: and to this end by statute 2 & 3 Ph. & M. c. 10. he is to take in writing the examination of such prisoner, and the information of those who bring him: which, Mr. Lambard observes,1 was the first warrant given for the examination of a felon in the English law. For, at the common law, nemo tenebatur prodere seipsum [no one was obliged to betray himself]; and his fault was not to be wrung out of himself, but rather to be discovered by other means, and other men. If upon this inquiry it manifestly appears, either that no such crime was committed, or that the suspicion entertained of the prisoner was wholly groundless, in such cases only it is lawful totally to discharge him. Otherwise he must either be committed to prison, or give bail; that is, put in securities for his appearance, to answer the charge against him. This commitment therefore being only for safe custody, wherever bail will answer the same intention, it ought to be taken; as in most of the inferior crimes: but in felonies, and other offenses of a capital nature, no bail can be a security equivalent to the actual custody of the person. For what is there that a man may not be induced to forfeit, to save his own life? and what satisfaction or indemnity is it to the public, to seize the effects of them who have bailed a murderer, if the murderer himself be suffered to escape with impunity? Upon a principle similar to which, the Athenian magistrates, when they took a solemn oath, never to keep a citizen in bonds that could give three sureties of the same quality with himself, did it with an exception to such as had embezzled the public money, or been guilty of treasonable practices.2 What the nature of bail is, has been shown in the preceding book;3 viz. a delivery, or bailment, of a person to his sureties, upon their giving (together with himself) sufficient security for his appearance: he being supposed to continue in their friendly custody, instead of going to jail. In civil cases we have seen that every defendant is bailable; but in criminal matters it is otherwise. Let us therefore inquire, in what cases the party accused ought, or ought not, to be admitted to bail.
AND, first, to refuse or delay to bail any person bailable, is an offense against the liberty of the subject, in any magistrate, by the common law;4 as well as by the statute Westm. 1. 3 Edw. I. c. 15. and the habeas corpus act, 31 Car. II. c. 2. And lest the intention of the law should be frustrated by the justices requiring bail to a greater amount than the nature of the case demands, it is expressly declared by statute 1 W. & M. St. 2. c. 1. that excessive bail ought not to be required: though what bail shall be called excessive, must be left to the courts, on considering the circumstances of the case, to determine. And on the other hand, if the magistrate takes insufficient bail, he is liable to be fined, if the criminal does not appear.5 Bail may be taken either in court, or in some particular cases by the sheriff, coroner, or other magistrate; but most usually by the justices of the peace. Regularly, in all offenses either against the common law or act of parliament, that are below felony, the offender ought to be admitted to bail, unless it be prohibited by some special act of parliament.6 In order therefore more precisely to ascertain what offenses are bailable.
LET us next see, who may not be admitted to bail, or, what offenses are not bailable. And here I shall not consider any one of those cases in which bail is ousted by statute, from prisoners convicted of particular offenses; for then such imprisonment without bail is part of their sentence and punishment. But, where the imprisonment is only for safe custody before the conviction, and not for punishment afterwards, in such cases bail is ousted or taken away, wherever the offense is of a very enormous nature: for then the public is entitled to demand nothing less than the highest security that can be given; viz. the body of the accused, in order to ensure that justice shall be done upon him, if guilty. Such persons therefore, as the author of the mirror observes,7 have no other sureties but the four walls of the prison. By the ancient common law, before8 and since9 the conquest, all felonies were bailable, till murder was excepted by statute: so that persons might be admitted to bail before conviction almost in every case. But the statute Westm. 1. 3 Edw. I. c. 15. takes away the power of bailing in treason, and in diverse instances of felony. The statute 1 & 2 Ph. & Mar. c. 13. gives farther regulations in this matter: and upon the whole we may collect,10 that no justices of the peace can bail, 1. Upon an accusation of treason: nor, 2. Of murder: nor, 3. In case of manslaughter, if the prisoner be clearly the slayer, and not barely suspected to be so; or if any indictment be found against him: nor, 4. Such as, being committed for felony, have broken prison; because it not only carries a presumption of guilt, but is also superadding one felony to another: 5. Persons outlawed: 6. Such as have abjured the realm: 7. Approvers, of whom we shall speak in a subsequent chapter, and persons by them accused: 8. Persons taken with the mainour, or in the fact of felony: 9. Persons charged with arson: 10. Excommunicated persons, taken by writ de excommunicato capiendo [for taking an excommunicated person]: all which are clearly not admissible to bail. Others are of a dubious nature, as, 11. Thieves openly defamed and known: 12. Persons charged with other felonies, or manifest and enormous offenses, not being of good fame: and 13. Accessories to felony, that labor under the same want of reputation. These seem to be in the discretion of the justices, whether bailable or not. The last class are such as must be bailed upon offering sufficient surety; as, 14. Persons of good fame, charged with a bare suspicion of manslaughter, or other inferior homicide: 15. Such persons, being charged with petit larceny or any felony, not before specified: or, 16. With being accessory to any felony. Lastly, it is agreed that the court of king's bench (or any judge thereof in time of vacation) may bail for any crime whatsoever, be it treason,11 murder, or any other offense, according to the circumstances of the case. And herein the wisdom of the law is very manifest. To allow bail to be taken commonly for such enormous crimes, would greatly tend to elude the public justice: and yet there are cases, though they rarely happen, in which it would be hard and unjust to confine a man in prison, though accused even of the greatest offense. The law has therefore provided one court, and only one, which has a discretionary power of bailing in any case: except only, even to this high jurisdiction, and of course to all inferior ones, such persons as are committed by either house of parliament, so long as the session lasts; or such as are committed for contempts by any of the king's superior courts of justice.
1. Eirenarch. b. 2. c. 7.
2. Pott. Antiq. b. 1. c. 18.
3. See Vol. III. pag. 290.
4. 2 Hawk. P. C. 90.
5. 2 Hawk. P. C. 89.
6. 2 Hal. P. C. 127.
7. c. 2. § 24.
10. 2 Inst. 186. 2 Hal. P. C. 129.
12. 2 Hal. P. C. 122.
13. 2 Inst. 381. 3 Inst. 34.
14. Flet. l. 1 . c. 26.


The Budget retractors provide easy banner installation & changeover. The units all come with a padded carry bag and come finished in a choice of black powder coat or anodised aluminum.
Conect Retractors are available in two sized 850mm and 900mm.


Seeking an experienced hands on Production Supervisor to join a small and supportive team in a fast paced environment.
A passion for and knowledge of animal welfare.
A unique opportunity with responsibility for development and implementation of RWWA's animal welfare strategies and retirement programs.
Laconik is a start-up with technology that improves fertiliser decisions Australian farmers make to maximise food production and environmental care.
Metro Farm Hand - N.O.R.
Farm Hand - Turf Harvesting Crew. This physical role, operating agricultural machinery, driving tractors and general farm duties.


Using a vegetable peeler or knife, thinly slice the comté into strips. Place the slices on top of the crackers. Trim the comté to fit size of cracker if necessary.
In a pan toast hazelnuts, continuously shaking the pan. Toast until hazelnuts are golden and they begin to give off an aroma.
Using a pestle and mortar or blender, roughly crush the hazelnuts into small pieces.
On the plate arrange the comté crackers. Drizzle lightly with truffle honey and sprinkle with the hazelnuts.
To finish, scatter with micro parsley.


Insurance discussion ruled the evening in an NSWCA meeting hosted by Garden City Park Water District Commissioners Kenneth Borchers, Peter Chimenti and Chris Engel. Guest speaker Mr. Ray Florio, representing Allstate Benefits, educated the assembly on the advancements in voluntary benefits and disability benefits. Mr. Florio also discussed the latest changes and features in other topics typically of interest to Districts such as accident coverage, group critical illness and universal life coverage.


text:
NOFX Brings Beer and Punk Rock Together With Punk in Drublic (Again)
Josh Chesler | Posted on September 29, 2017
When Stone Brewing announced their partnership with NOFX to create an official Punk in Drublic beer, it seemed to make a lot of sense. Much like the recent creation of Pennywiser, NOFX and their fans like to partake in a good brew as much as anyone. But while most bands only dream of someday reaching the level of success where breweries are naming beers after your iconic albums, the guys in NOFX are just excited to finally have a beer company take them seriously rather than just using the catchy name.
"I think it's cool because a couple of breweries have tried to work with us before, but it's always been janky," says NOFX frontman and bassist "Fat Mike" Burkett. "But when Stone wanted to work with us, they were taking it seriously. I was like 'This is the kind of beer I want to do' and they listened to me. It's a hoppy lager, and they don't do lagers here very often. They had to bring in a certain malt that they don't usually use, but it's a really drinkable beer."
But along with the limited edition beer (which is in stores now), the punks and brewers also teamed up for a traveling festival. With six weekend dates on and near the West Coast — concluding with October 28 in Huntington Beach — the Punk in Drublic Festival is bringing a handful of punk bands to each stop. Why only a handful instead of a few dozen on multiple stages? Because more than that is too much for an adults-only (21+) punk rock and beer festival, and the point isn't to cram as many acts as possible into a day.
"I did [Ye Scallywag] in San Diego last year with Stone as a sponsor, and it was so fun," Burkett says. "It was five or six bands — which is about how many bands I want to see — it was on a weekend, and it had free beer tasting. The promoter was talking about doing one in LA, and I was like 'Let's just do it everywhere!' So this year we have a few of them, and hopefully next year we'll have a bunch of them."
Of course, even if Punk in Drublic is among the first festivals Fat Mike's been primarily responsible for, a quarter-century of playing massive stages has taught him enough lessons to know what he's doing this fall. In some ways, Burkett says he's actually more comfortable onstage a lot of the time than he is in real life (except when he has to do "Cokie the Clown," but that's another story), and it always helps when the band can add friends like Bad Religion and Goldfinger to the bill — like they did for Huntington Beach.
"No one knows this, but one of the deals we had with Warped Tour the last three times we did it was that we'll only do it if Bad Religion does it too," Burkett says. "We wouldn't do the Warped tour without Bad Religion, and now we're doing our own festival with Bad Religion. They're a little older than us, but we're from the same time and we like the same things. We're adults — or we're old."
But whether they're playing alongside Bad Religion, Pennywise, Goldfinger, Less Than Jake, or any of the other various bands NOFX likes sharing a festival stage with, it's the brotherhood mentality of punk rock that's allowed these types of events to continue on for so long. While other genres often get stuck with infighting and drama tearing apart their scene (or at least preventing it from growing), most of the established punk bands get along swimmingly — with a few exceptions of course.
"Punk rock's not like metal or rap," Burkett says. "We're all bros. There are no beefs — well maybe some bands have them. I had one with Underoath, but that's different because they're Christian."
Punk 'n Brew Craft Beer & Music Festival will be in Huntington Beach on October 28. Tickets start at $59 and are available through the festival's website.
Josh Chesler
Josh Chesler used to play baseball for some pretty cool teams, but now he just writes about awesome stuff like tattoos, music, MMA and sneakers. He enjoys injuring himself by skateboarding, training for fights, and playing musical instruments in his off time.
CategoriesMusic


text:
Russia's Novatek Allies With French-American TechnipFMC on New Arctic LNG 2 Project
By: Rauf Mammadov
June 3, 2019 05:29 PM Age: 2 months
Yamal LNG (Source: Novatek)
A joint venture led by Russia's Novatek has chosen the French-American company TechnipFMC to spearhead its new Arctic LNG 2 liquefied natural gas project on the Gydan Peninsula, in northern Siberia. The same Western firm previously oversaw the work on Novatek's first LNG facility on the Yamal Peninsula (see below). According to the newly signed deal, TechnipFMC will be in charge of the engineering, procurement, supply, construction and commissioning of the planned Arctic LNG 2 complex, which will have an annual liquefaction capacity of 19.8 million tons of gas (1prime.ru, May 20). Work on the four-year project will begin in the third quarter of 2019.
Novatek owns 70 percent of the Arctic LNG 2 venture. France's Total owns 10 percent and two subsidiaries of China National Petroleum Corporation control 10 percent each. Novatek plans to sell the other 10 percent to Saudi Aramco or Japan's Mitsui by the end of June. Last December, the joint venture chose Italy's Saipem and Turkey's Renaissance for the on-shore engineering and construction part of the work—a contract worth $2.2 billion (Saipem.com, accessed May 31).
Novatek chose a non-Russian project overseer once more because Russian companies lack the technology to operate in the Arctic's severe weather and permafrost soil. TechnipFMC oversaw the work on Novatek's Yamal LNG project in 2017. That facility has an annual capacity of 17.5 million metric tons, and Novatek plans to add an additional million tons by the end of 2019 (Novatek.ru, accessed May 31).
The choice of TechnipFMC as Arctic LNG 2's overseer is the latest example of Russian gas-exporting partnerships with European companies. Total, which owns 19 percent of Novatek, has stakes in both Yamal LNG and Arctic LNG 2. Meanwhile, Russian state-owned natural gas giant Gazprom has partnered with French companies in its own LNG projects in the Arctic region as well as with German companies on the Nord Stream Two gas pipeline, which will deliver additional volumes of Russian gas to Europe across the Baltic seafloor.
One reason Russian companies may have chosen German and French companies as their Arctic LNG partners was to preemptively minimize the risk of United States and European Union sanctions against them. The US has already sanctioned the oligarchs who own a combined 51 percent of Novatek—Leonid Michelson and Gennady Timchenko—but not the company itself. Washington has also been threatening to sanction companies working on Nord Stream Two, which it opposes on the grounds that this pipeline will make Europe more dependent on Russian gas.
Novatek's Arctic projects have become the linchpin for a recent Russian LNG export offensive. In early May, President Vladimir Putin signed a new national energy security doctrine that makes expansion of Russia's LNG capacity a priority (Minenergo.gov.ru, May 13). While the previous version of the doctrine saw other countries' LNG as the main threat to Russia's energy export markets, the new version sees it as a way to increase gas revenue while avoiding Western sanctions. Putin has said that Russia plans to expand its LNG capacity to 100 million metric tons a year by 2035 (The Barents Observer, April 3).
Novatek is using dozens of Arc7 ice-breaking vessels to transport its Arctic-sourced LNG. South Korean companies built the medium-sized tankers specifically for extreme Polar conditions. Recently, Novatek awarded shipbuilding contracts to the Russian companies Sovcomflot and Zvezda. Igor Sechin, a close associate of President Putin, runs two of the companies that own Zvezda—Rosneft and Gazprombank (Sovcomflot.ru, January 11).
Thanks to its ice-breaking tankers, Novatek became the largest LNG supplier to the EU in the first quarter of 2019—a development that reduced Gazprom's gas market share in northwest Europe (see EDM, May 6). Gazprom complained about this to the Kremlin, but Novatek has shown no signs of backing away from its determination to expand the company's European market share. Indeed, it has already signed contracts to sell some Arctic LNG 2 output to Spain's Repsol and Switzerland's Vitol (LNG World News, April 2).
Russia has been flexing its muscles in the Arctic to try to dominate the area's energy landscape as well as the Russian High North's main maritime transport corridor, the Northern Sea Route (NSR), which climate change is making increasingly navigable. In addition to establishing new military bases in the Arctic, Russia is requiring foreign military and commercial vessels to notify it before using the NSR (Maritime Executive, March 8; see EDM, May 14, 30).
The United States and Europe have yet to apply sanctions to Russian natural gas projects to, for instance, match the preexisting US sectoral sanctions on Russia's oil exploration operations in the Arctic. And the personal sanctions against Michelson and Timchenko have not prevented Novatek from expanding its business. When Arctic LNG 2 is commissioned in 2023, Novatek's overall LNG capacity will jump to 37 million metric tons a year.
Russian Intrigues in the Middle EastNext
Political Crisis Underlines Need for Con..
Militant Leadership Monitor – December 2018
The December 2018 issue of the Militant Leadership Monitor features a brief on the jihadist rivalry taking place between Abu Mus'ab al-Barnawi and Abubakar Shekau in Nigeria; an update on Somali founder of al-Shabaab turned repentant jihadist Shaykh Mukhtar Robow Ali, who now sees himself once again as an... MORE


Wynn's work is a "a smack of Picasso, a smattering of Dali, a soupcon of Blake"
The following are links to news articles and press releases about my artwork and exhibitions. Some articles require Adobe Acrobat Reader to view them.
© Daniel Wynn, Wynn Design Group. All Rights Reserved.