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Q: How to make div open/close slow? once again in a pickle. I have this js that I would like to when open and close it does it slow. As it is, it just snaps open and close. I tried SlideUp, slideDown, slow but it's always the wrong placement. Much appreciated. Ty.
The button:

js:
      $("document").ready(function () {
        $(".manualclose").click(function(){
        $(".child_box").toggle();
          });

      ls = localStorage.getItem('on')
      if(ls){
        $(".child_box").show()
      }

      $(".open_child").click(function(){
          localStorage.setItem('on',true)
          toggled = $(".child_box").toggle();
          if(toggled.is(":hidden")){
            localStorage.clear();
          }
      });
      $(".manualclose").click(function(){
        localStorage.clear();
        $(".child_box").hide()

      });
   })

A: Both .show() and .hide() will take a duration parameter that will fade in or out the target over that number of milliseconds.
E.g.
$( ".target" ).show(500);

There are lots of other things you can do, too. Take a look at the jQuery reference manual for details.


Why go for a rental office?
Offpro have the answer. Businesses searching for offices often choose a rental office as a home for their business due to their flexibility, ease of use and many other different features. The main reason why businesses often prefer to rent over purchasing properties is due to the uncertain economic changes that can occur. Small businesses often have aspirations of growing and expanding their business's and therefore just require small offices. It's also more convenient for small businesses to rent, in terms of cost, it wouldn't make much sense to purchase an office that can only accommodate 20 employees, when your business plan indicates that within the next 3 years you will require a work force of 100 employees. Renting also reduces the risk of your business going into liquidation as well as the pressure to perform every month, in order to cover overheads.
All the boring details to do with the building are completely taken care of, most of the time the bills are included within the rent that you are required to pay each month. This means you can just focus on what you want to – your business! This gives you the clarity and focus you need to achieve success. You get a flexible service, giving you exactly what you need. Small or medium sized rental offices are at your fingertips, with included telephone and broadband lines. Some (like ours) even include high speed lines if you need them, to ensure maximum productivity.
To get you going as soon as possible, we ensure any hassle involved in moving your team into your new serviced office is removed – productivity is, therefore, maintained when compared to setting up your own office. Our offices come fully furnished to your requirements and have telephone and internet connectivity ready and waiting for you to simply 'plug and play'.
Often, temporary rental office solutions will be used by businesses seeking a simple office for staff. One that can change far more quickly than a leased office. Business requirements may change, your continued growth might mean it's worth waiting for the building of your dreams rather than settling for less. Our offices mean you can get all you need with little commitment, whilst enjoying affordability and flexibility.
Here at Offpro we offer two stellar rental offices. One is based in Coventry and the other is in Leamington Spa. They're both reachable by all main transport links, and are chosen to be the ideal location for you to get the most out of your business. Parking is provided, as well as anything else you could ask for. We have catering, high quality furnishings and equipment, and you will be dealing directly with the directors to ensure all of your needs are met.


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Burton Bradstock C E VC Primary School
Church Street, Burton Bradstock, DT6 4QS, UK Bridport
www: http://www.burtonbradstock.dorset.sch.uk e-mail: office@burtonbradstock.dorset.sch.uk
Burton Bradstock is a Voluntary Controlled, Church of England school. The school is owned and funded by Dorset County Council and administered and controlled by the Dorset Education Partnership - the Local Education Authority, and managed by the Governing Body in conjunction with the Headteacher and staff.
Burton Bradstock is a rural primary school taking children from the parishes of Burton Bradstock, Shipton Gorge and Chilcombe. It covers the age range of 4 - 11 years and most children progress to Sir John Colfox School in Bridport. Our school is a member of the Bridport Cluster of schools, that work closely together to give opportunities to children to broaden their education through co-operation.
At Burton Bradstock School we believe that….
All children are individuals, to be treated equally in terms of access to all school activities and opportunities at school.
We provide a framework wherein each individual, whether a child or a staff member, is encouraged to achieve his/her potential, socially, intellectually, physically, spiritually and emotionally in a happy and challenging environment.
Education is a partnership between the parent, child, school and wider community and that this partnership needs to be nurtured and encouraged.
All children live a full life within the school and the wider community, demonstrating the values of friendship, patience, consideration for others and self control.
A successful learner is a child who is able to access, organise, select and apply resources and materials to enable them to further their learning. The true learner develops skills and strategies to control and direct their own learning, to move confidently through the various worlds they inhabit.
Children need to acquire knowledge, skills and practical abilities relevant to adult life and employment in a changing world. From the start of their life at Burton to the end we aim to gain a full picture of each child's development and progress to inform us of the future learning requirements.
We deliver a broad, balanced curriculum where varied learning experiences enable children to become independent, confident and responsible learners.
Our community, local, national and world-wide offers rich source of learning for our children and that we strive to blur the boundaries between school and the wider world, reaching out to it and drawing it into us.
Our school is like a caring family, building on the love and values that a good home provides. As a Christian school we place Christian values at the very heart of all we do.
CLASS ORGANISATION
To make best use of the financial resources at our disposal and to provide a balance and continuity the school is divided into two departments, Key Stage 1 and Key Stage 2 and four classes.
Infants Department
Class I and 2 - Years R, 1 and 2
Junior Department
Class 3 - Years 3 and
Class 4 - Years 5 and 6
The two Infant classes are located on the ground floor and have folding interconnecting doors, which allow the two rooms to open up into one large room. Due to the nature of the small school the children are split, with reception and some Yr. I children in Class I and the other Yr. 1 and all Yr.2 children in Class 2. Staff plan together to ensure that in Yr. 1 children receive the same curriculum.
Curriculum is delivered through a thematic or topic approach. This means that the class teacher, following an agreed programme that ensures that all National Curriculum subjects are taught, selects a topic, such as OURSELVES. Using this title the teacher devises a scheme of work that will last for part, or all of a term. The skill of the good teacher is then to develop the topic in such a way as to engage the interest of all the children and to make the learning purposeful and relevant.
Children, like adults, learn best when they are stimulated, excited and challenged by the activity they are engaged in.
The School Curriculum is composed of 9 subjects – 5 Core Subjects and 6 Foundation subjects.

redpajama_set_name: RedPajamaCommonCrawl


Some of these people are my heroes.
This is the first post in a little while,One has been procrastinating in part and otherwise occupied in other matters.
So the new University year has started and for what is the 3rd year of academic study in my journey to attaining a degree in the arts, I think it would be hard to ask for anything better, I now have several thick books with lots of writing and laden with images of beautiful art too, I could not be happier.
Studying aside, after 25+ years experience in the retail industry the time came to call an end to it all and have since moved into fundraising and the interesting thing is when I first moved to this city 23 years ago, I saw a notice for the people I know work for and something inside me said I would be working for them one day, well to be honest they have changed their name (once) in that time but their core mission and principles are the same. It beats stacking things on shelves, stock takes, positioning FSDU's and the like, the mileage in the car has shrunk immeasurably, have more time to myself and am doing something I thoroughly enjoy.
When you bear in mind my last employers were Crooks (literally) in name and practice. It is so refreshing not to be dealing with those people anymore, I do not like people who cannot maintain professional distance and the administrator was just like that, a real nosey so and so, from Leeds which for those, who do not know is in West Yorkshire, their local Association Football club ( the game played with a round ball and eleven players on each team) is leeds Utd.
Football and Yorkshire aside, relationship wise we are still together she has gone home back to the States for the time being after being over here for 5 months.


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Website Design and Branding by Godzspeed Communications
Release: Michael A. Johnston publishes in Criminal Law Quarterly – "Whelan Still Waiting"
Michael A. Johnston's article "Whelan Still Waiting" has been published in the Criminal Law Quarterly (at (2018), 66 C.L.Q. 19): Patrick Whelan received a "short drop" and died slowly. His procedurally flawed execution was the tragic punctuation to a death sentence passed pursuant to a procedurally flawed
in Blog, Michael A. Johnston, Release
Release: Michael A. Johnston submits memorandum to Parliament about Bill C-75
On 1 September 2018, Michael A. Johnston submitted a memorandum to Parliament about Bill C-75, An Act to amend the Criminal Code, the Youth Criminal Justice Act and other Acts and to make consequential amendments to other Acts, in particular on the proposed changes to jury selection procedures: Representativeness is,
Release: Michael A. Johnston publishes article in Law Times
Michael A. Johnston published an article on the Government of Canada's proposed Bill C-75, titled "Challenging trial by jury without cause," in the 4 June 2018 edition of Law Times: Trial by jury is constitutionally entrenched in our criminal justice system, but more subtle is its
Release: Michael A. Johnston interviewed on 1310 News
Earlier today, Michael A. Johnston was interviewed on Ottawa Today with Mark Sutcliffe at 1310 News. Michael discussed the Supreme Court of Canada's recent decision in R. v. Jordan, which changed the rules governing a defendant's right to a trial within a "reasonable time" under subsection 11(b) of
In the news: Publication Ban and the Presumption of Innocence
by Michael A. Johnston Being accused of a criminal offence has many indelible and irreversible aspects. Often, a criminal defendant is tried and convicted in the courts of public opinion before he or she has even made a first appearance, let alone made full answer and
in Blog, In the news, Michael A. Johnston
Release: A Criminal Record in Ontario = Taxation Without Representation
Following the publication of his article in the Criminal Reports last week, Michael A. Johnston has been cited by the Toronto Star: "Ottawa lawyer Michael A. Johnston, in a paper published last week, argues that up 10 per cent of Canadians are being unfairly barred from jury
Release: Michael A. Johnston publishes in The Last Line – "Spare the Rod, Spoil the Code"
Michael A. Johnston publishes his paper "Spare the Rod, Spoil the Code - The Unconstitutional Actions of the Supreme Court of Canada in Canadian Foundations for Children, Youth and the Law v Canada (Attorney General)" in the latest edition of the Defence Counsel Association of Ottawa's magazine, The
In the news: Aboriginal Youth Are More Likely To Face Pre-Trial Detention
by Michael A. Johnston Data from Statistics Canada shows that although the overall number of youth (including Aboriginal Youth) being held in pre-trial detention has decreased in the past few years, the percentage of aboriginal youth being held has increased dramatically.
In the news: The Rule of Law?
by Michael A. Johnston We have a system of justice that purports to treat all people equally; Lady Justice is most often blind-folded, for that reason. However, even as an ideal, the concept of equality can have differing effects on differing people. A philosopher once quipped: "the
Release: Michael A. Johnston publishes in For the Defence – "the Queen and Her Counsel"
Michael A. Johnston published his article "Should All the King's Horses and All the King's Men Put the Queen and Her Counsel Back Together Again" in the Criminal Lawyers' Association's magazine, For the Defence (at (2014) 35:3, 21): "In the Superior Court most of us wear wool
in Blog, Michael A. Johnston, Release, Uncategorised
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Market Size For Mobile Crusher In India 2012 Grinding Market Size For Mobile Crusher In India 2012 2013 It's ... ket size of mobile impact crushers in india Research .
size of mobile impact crushers in india - ddmsuncraft.
Apr 4, 2015 Stone crusher plant cost in india crushing plant design and layout . size of mobile impact crushers in india,coal mobile crusher supplier Y.


text:
U.K. Liability Markets Show Improvement Following Study
Lower increases in premiums, better communication between insurers and policyholders and a reduction in the number of businesses denied cover are among the key improvements identified by the OFT in its follow-up study of the UK liability insurance markets.
In a study published in June 2003, the OFT identified a number of concerns in the UK liability insurance markets, and undertook to review the markets in order to assess whether further action was necessary.
In its follow-up study, the OFT found that the average premium rises in 2004, while still ahead of general inflation, had fallen to seven per cent for employer's liability (compared to rises of 50 per cent observed in 2002), and four per cent for public liability and professional indemnity insurance (compared to rises of between 30 and 60 per cent in 2002).
Further, significantly fewer businesses than identified by the previous study were being denied renewal of cover by their insurer or denied cover by other insurers. In the case of employer's liability insurance, for example, the number of businesses denied cover fell by two thirds to three per cent. Those sectors which were identified in the 2003 report as experiencing significant problems in getting cover showed signs of improvement in both availability and lower than expected price increases this time around.
Insurers and policyholders should also be benefiting from initiatives, led by government departments and the industry itself, aimed at making the markets work better. Longer renewal notice periods and efforts to improve information and risk management, so that premiums more accurately reflect risk (for example, by taking better account of good health and safety practice), seem to be providing benefits for all concerned.
Graham Winton, a branch director in the OFT's Markets and Policy Initiatives Division, said:
'We have seen significant changes in these markets over the past two years aimed at making them work better for all parties. There will, however, continue to be cost pressures, and all sides must remain committed to pushing forward positive changes.'
The follow-up study also included some initial work on after the event insurance. The OFT will contribute to the work which the Civil Justice Council, in conjunction with the Department for Constitutional Affairs, will be undertaking in this area later in the year.
FBI Probes Russian-Linked Postcard Sent to FireEye CEO After Hack
Categories: International News


Spider-Man: Homecoming is a magnificent triumph that will obliterate fans expectations. Sony Pictures and Marvel Studios have achieved greatness here. They have not only made the best Spider-Man by far; they have created a funny, heartfelt, coming of age story that deftly captures the awkwardness of being a teenager. Every aspect of this film has been touched by brilliance. This review is completely spoiler free, but I will tease you with these two golden nuggets. The last scene of the film, and the very end post-credits scene, are...utterly...ingenious.
When last we saw Peter Parker (Tom Holland), he was helping Tony Stark (Robert Downey Jr.) fight Captain America (Chris Evans) in Civil War. Peter did his bit. But instead of being ushered into the esteemed ranks of The Avengers, he was unceremoniously dropped home. Go back to school kid, get your skills in order, here's Happy Hogan's (Jon Favreau) number. He'll call you, if and when Mr. Stark decrees it.
Months later, Peter Parker is struggling with just being a teen again. He moonlights as Spider-Man, fights local crime, or at least attempts to. Every call and text to Happy Hogan remains unanswered. He pines away. Daydreaming about the beautiful Liz (Laura Harrier), constantly bullied by Flash Thompson (Tony Revolori), with only the nerdy Ned (Jacob Batalon), and his doting Aunt May (Marisa Tomei), to wonder what's bothering him.
Peter's boredom is interrupted by the deadly Adrian Toomes (Michael Keaton). A hot-tempered tough guy, Toomes and his crew have spent years looting alien technology. His hybrid weaponry has hit the New York City streets. Peter's mission is clear. Stop the bad guys and prove to Tony Stark that he has what it takes. Unfortunately for Peter and those around him, he may not be ready to fight in the big leagues just yet.
Spider-Man: Homecoming can best be described as a John Hughes take on superheroes. The film, though loaded with stupefying action scenes, is in the vein of Sixteen Candles and The Breakfast Club. Peter Parker is a diminutive high school geek. He's not cool, cannot talk to girls, and only has one friend, who's goofier than he is. It's difficult for any teen to navigate high school, imagine hiding the fact that you have superpowers. Peter struggles mightily, but is not unprepared or unqualified for the deadly fight to come. He has courage, strength, and willpower; every characteristic needed to be the hero. Homecoming is his journey to realization.
The film has several relationships that define Peter, but none more important than his mentoring from Tony Stark. Peter idolizes Tony Stark. He is the big brother, father figure, towering hero that Peter wants to emulate more than anything. Tony Stark sees greatness in Peter, but doesn't have the warm, tender personality to foster a fawning acolyte. The pair, on the surface, seems different, but in truth are cut from the same superhero cloth. Tony Stark never connected with his father as a son, a loss that defines his character. He gets another chance with Peter. It is fantastic to see. Robert Downey Jr. and Tom Holland absolutely nail this vital bond.
The multi-cultural, ensemble cast reflects the true diversity of New York City. They are integral to this film's success. For the first time in a comic adaptation, Peter Parker's story is truly indicative of where and how he lives. Jacob Batalon stands out as Ned. He almost steals the show with his warmth and humor. Michael Keaton embraces villainy with gusto as Toomes/Vulture. Die-hard Spidey fans are going to love how authentic his character is. The same is true for Marisa Tomei's Aunt May. She is markedly different from what we've seen previously on film and in the comics, but is just perfect for the character. Aunt May has so much love for Peter. It's evident in every scene with Tom Holland.
It's rare in a review that I give credit to the Hollywood suits, but it has to be done. I can't imagine the army of lawyers and accountants it took to get this film made. Amy Pascal, the former head of Sony Pictures, and Kevin Feige, President of Marvel Studios, are the real heroes of Spider-Man: Homecoming. They brought Spider-Man into the Marvel Cinematic Universe. Then got the best filmmaking team possible to make this amazing film. Director Jon Watts, who had only made the indie feature Cop Car to this point, was allowed to pursue his vision of Spider-Man. So many tentpole films of this scope and magnitude are bungled because the studios can't get their act together. Spider-Man: Homecoming is what it looks like when everyone is on the same page. They have delivered a terrific film that will knock the socks off audiences.


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Browne's Confidence At 'An All Time High'
@pnefc
Alan Browne admits his confidence is at all all time high as he aims to cement a place in Simon Grayson's first team.
The 20-year-old, who has started eight games so far this season, featuring in a total of 13, has adapted well to life in the Championship, and the Irishman is hoping to keep that going throughout the season.
"I think I'm at all an time high at the moment confidence wise, I'm just enjoying playing and the gaffer has given me a chance and I'm looking to repay him.
"Every game I look to do that, it's a good experience for me, it was a bit up and down last season but hopefully this season I can stay on top of my game and keep playing."
The Republic of Ireland U21 midfielder has also impressed for his national side, and is enjoying the experience and hoping to bring the confidence from that into his game at PNE.
"I think anyone who goes away with their country wants to do well and thankfully I have been doing that with the U21s. We have been getting good results, I've been putting in good performances and getting a goal or two as well.
"Anytime I come back to the Club I try and do the same and my focus then changes to the Club and focus on the next performance so hopefully I can just keep it up."
To hear more from Alan Browne, go to PNE PlayerHD by clicking on the video below.


Obtain free marketing strategy templates and discover assist and advice on tips on how to write your marketing strategy. Figuring out early that a business idea is not going to work saves time, money and heartache later. Note also that every enterprise rule may be expressed in a number of formal rule statements, although each formal rule assertion must be an expression of just one (atomic) business rule. It's good to know what it is going to cost you to open your doorways the first day of what you are promoting. Securing monetary assistance to start your new business is instantly related to the energy of your marketing strategy.
Once the owner and the guide agree on a plan, the marketing consultant should enter the third section of consulting, which is the restructuring section, or the implementation of the plan. Initially, the analyst's project is to decompose these composite ramblings into 'atomic' business guidelines, where every atomic business rule is a specific, formal assertion of a single term, fact, derivation, or constraint on the business.
Your preferrred location will rely upon your enterprise wants, zoning restrictions, and the place your prospects and rivals are. Once you begin to match one model with one other, you're coming into the realms of strategy, with which business fashions are sometimes confused. The primary and doubtless probably the most-used function of a price range is to help you gather information for a marketing strategy , together with all the gadgets and expenses needed for start-up. The proliferation and increasing complexity of the legal guidelines governing enterprise have pressured growing specialization in company regulation. Throughout this process, the enterprise guide will uncover the main points of an organization's mission and what operations are in place.
The body of your online business letter ought to include a couple of concise paragraphs that convey your message clearly. In Why Business Fashions Matter," Magretta goes back to first ideas to make a simple and helpful distinction, pointing out that a enterprise model is an outline of how your online business runs, but a aggressive technique explains how you'll do higher than your rivals. Those enterprise processes which might be affected by the development and integration of IT programs are considered. In addition, a business that wishes to lift cash on a inventory market or to be owned by a variety of individuals will usually be required to undertake a specific authorized type to take action. Thus, the enterprise administrative which means of business system does not differ very a lot from our use of the time period enterprise system. This article offers a step-by-step course of for making a business startup price range.
Learn how international value chains can enhance competitiveness, profitability and lengthy-term sustainability for your corporation. Native jurisdictions can also require particular licenses and taxes simply to function a enterprise. Description of the Enterprise The business description often begins with a short rationalization of the trade. Within the model diagram, we see a policy – a general statement of path for an enterprise.


Gatis Flinters heads the Corporate & Commercial Law practice group at COBALT Latvia, advises on M&A transactions and is recognized for his particular expertise in energy and insolvency law. He brings over 15 years of field experience and has a proven record of success in large-scale investment and reconstruction projects while advising local and also foreign companies.
Gatis is an active member of the Professional Association for European Restructuring and Insolvency Practitioners (INSOL Europe) and the honorary advisor to the British Ambassador in Latvia on legal issues.
Clients praise Gatis Flinters for his responsiveness and clear advice, describing him as "very solution-oriented and skilful at finding solutions".


Abstract: In this study, 50 female and 50 male 5-month-old captive-bred chukar partridges (Alectoris chukar) were released in 2012 into nature and monitored in the Çamkuyular Cedar Research Forest, Elmalı, Antalya. All individuals were tagged with colored leg tags and 15 also had radio transmitters attached. Over 4 months, the chukars were hunted by their natural predators, with the last dead bird found in January 2013. Of the 33 dead birds, 60.6% (n: 20) had been killed by large mammals and 39.4% (n: 13) by predatory birds. This study indicates that chukars produced in captivity are unable to successfully adapt to nature following release, primarily because they are unable to protect themselves against their natural predators. This suggests that a strategy favoring protection rather than bird release should be implemented in areas with declining partridge stocks.


Hello and thank you for visiting my website! For your convenience I have provided links to information I find valuable as it pertains to physical education and/or physical fitness. All of these may be found to the left under, "Additional Web Resources."
Physical Education will have a place on our school's report card. As part of Senate Bill 210, all public schools in Ohio are required to conduct the, "Physical Education Evaluation." I have created an interactive page which highlights all major details and provides more in-depth information for you to consider. Please click on Mr. Ranger below to find out more!!
The mission of the Lakewood City Schools Physical Education Department is to educate minds, develop healthy bodies, and promote positive attitudes towards lifetime physical activity, fitness, and sports skills. With a quality physical education program in place, each student will be empowered with the knowledge and skills necessary to make responsible lifestyle choices that directly impact his/her health and well-being.


The Commission on Diversity and Inclusion will issue a final set of first year recommendations, together with a first year report, early in 2018. To inform its deliberations toward that final product, the President's Commission seeks the input of the campus community regarding its Recommendations. It invites you to attend one or more of the two face-to-face listening sessions, dates and times TBD.


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07 06 , 2021
Marina Talieva
5 Chocolate Desserts Around The World That You Need To Try
7th of July is celebrated as the World Chocolate Day! The legends say that the date marks the introduction of chocolate to Europe in 1550.Up to that time, chocolate was only known to the natives of Mexico and parts of central and South America.
Today, chocolate is one of the most popular food types and flavors in the world, and many foodstuffs involving chocolate exist, like everyone's favourite chocolate brownies and chocolate chip cookies. To celebrate our love of chocolate, we have decided to find which chocolate desserts around the world are considered to be the prominent culinary staples in the different countries cuisines!
Brazil - Brigadeiro
The brigadeiro is a traditional Brazilian dessert, created by a confectioner from Rio de Janeiro, Heloisa Nabuco de Oliveira. It is made of condensed milk, cocoa powder, butter, and chocolate sprinkles covering the outside layer.
It is a popular confection throughout the country, especially for festive events. Brigadeiros are commonly made at home, and also found in bakeries and snack shops. A brigadeiro is generally shaped into small balls covered in chocolate sprinkles and placed in a small cupcake liner. The mixture may also be poured into a small container, and eaten with a spoon, and this is known as a brigadeiro de colher (literally, "spoon brigadeiro"). Brigadeiro can be found now in different countries as a result of Brazilian immigration.
France - Religieuse
The French word "religieuse" translates to "nun" in English and the classic version of this dessert supposedly has this name because the finished pastry resembles a nun in her habit.
Religieuse chocolat is a pastry comprised of chocolate-flavored choux buns (a light pastry) that anchor creme patissiere (pastry cream), and chocolate ganache.
Austria - Sachertorte
According to the official website of the Hotel Sacher, 16-year-old Franz Sacher was an apprentice chef in 1832 when none other than Prince von Metternich, an Austrian diplomat, asked him to make a special cake for his party — and thus, Sachertorte was born.
Chef Sacher's son went on to found the Hotel Sacher, which still proudly serves the chocolate cake that's filled with apricot jam and plated with a side whipped cream. Fortunately, if you can't make it to Hotel Sacher, this cake is still baked, and served throughout the country.
Spain/Portugal - Churros con Chocolate
You might be familiar with the famous fried, cinnamon-sugar-covered pastries, but you may not have tried them with one of their most popular pairings — chocolate dipping sauce.
While in Portugal or Spain, order these fried sticks with some decadent chocolate dipping sauces. Enjoy this dish warm as a dessert or, popularly, for breakfast.
Japan - Nama Chocolate
Nama chocolate is a type of ganache, which is made from a mix of melted cacao and fresh cream. As the mixture solidifies, the block of chocolate is cut into pieces and sprinkled with cocoa powder on top.
The name comes from the freshness of the cream involved — and any chocolate you don't eat right away should be refrigerated because of its dairy content. Japanese confectioner ROYCE' is said to have created the original Nama chocolate recipe in 1995. They sell a variety of flavors of this signature chocolate and many of them are infused with special liqueurs.


Book the Dubai Self-drive boat Tour to embark on a 90-minute journey off Dubai's coastline and stand the chance to feast your eyes on the incredible attractions that line its banks while filling up on a bit of cultural knowledge. The first activity of your tour is a stroll along the fishing harbour. Observe age-old fishing fishing techniques, a collection of unique fishing boats and dhows (called lanshs), fisherfolk mending fishing nets and the bustling fish market. You will then receive a briefing on safety techniques before letting you out on a test spin. Once you are confident with the controls, head out behind your instructor on to the blue waters of the Dubai coastline. Maneuver your Hero boat parallel to the lengthy stretch of the famous Kite Beach and observe the stunning setting as you make your way to the Burj Al Arab. After a brief photo stop at the iconic landmark cruise off to Logo Island which is home to Dubai Ruler's massive private yacht. Catch a glimpse of Palm Jumeirah and learn more about its secrets and other facts from your guide. Your last attraction will be the Atlantis Palm Hotel where you will stop for a selfie or two before starting the return to Dubai harbour. Some of the last sights you will see on the Dubai Self-drive boat Tour are views of the Dubai skyline with the world famous Burj Khalifa standing tall like a crown.
Enjoy a stroll around the fishing harbour and learn more about Dubai's seafaring history.
After arriving at the meeting point, you will meet our guides and be escorted to the departure point. Roam the harbour to know more about UAE's fishing history. You will then receive a safety briefing and the chance to take the Hero boat on a test spin. Once you are thorough with the skills, follow your guide into the water. You will ride along the entire length of Dubai's kite beach. Continue towards the Burj Al Arab then head towards Logo Island. Cruise past Palm Jumeirah and view the Atlantis hotel as well Return back to the harbour at Dubai and view the skyline on the way.


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We assessed serologic responses to an dental, killed whole-cell enterotoxigenic plus
We assessed serologic responses to an dental, killed whole-cell enterotoxigenic plus cholera toxin B-subunit (ETEC-rCTB) vaccine in 73 Egyptian adults, 105 schoolchildren, and 93 preschool kids. aswell as IgG seroconversion in kids (44 to 75%) and adults (25 to 81%), was greater than the matching percentage in placebo recipients considerably, aside from IgA replies to CS2 in adults. IgA anti-CF titers peaked after one dosage in children, whereas in every age ranges IgG antibodies rose after every dosage incrementally. Independently, both preimmunization IgA titer and age PSC-833 were linked to the magnitude of IgA responses inversely. To conclude, serologic replies towards the ETEC-rCTB vaccine may serve as useful immune outcome procedures in potential pediatric studies in areas where ETEC is certainly endemic. PSC-833 Enterotoxigenic (ETEC) strains will be the leading reason behind years as a child diarrhea in developing countries and traveler's diarrhea (4). Advancement of the wiped out, whole-cell ETEC stress plus recombinant cholera toxin B-subunit (ETEC-rCTB) vaccine into extended clinical studies provides invigorated vaccine advancement initiatives. This vaccine was created to induce immunity to the most typical ETEC colonization elements (CFs), i.e., CFA/I, CFA/II, and CFA/IV, and cross-immunity to heat-labile (LT) enterotoxin (3, 5, 17, 21). In early-phase studies, intestinal lavage antibodies or circulating antibody-secreting cells (ASCs) offered as primary immune system endpoints (1, 2, 10, 14). Extrapolating from research of individuals with ETEC diarrhea (8, 9, 16, 19, 20), we considered whether serology might provide a more applicable way of measuring vaccine immunogenicity broadly. In randomized, managed studies in Egyptian adults, schoolchildren, and preschool kids, we discovered that Rabbit Polyclonal to ARSA. the ETEC-rCTB vaccine was well tolerated and effectively activated immunoglobulin A (IgA)-ASC responses to CTB and CF antigens (14, 15). In the present study, we assessed the frequency and magnitude of systemic IgA and IgG antibody responses to the vaccine in these same cohorts and examined isotype-specific patterns of response. Our aim was to evaluate the usefulness of serologic steps as indicators of vaccine response in a setting where ETEC is usually endemic. MATERIALS AND METHODS Subjects and vaccination. The Egyptian Ministry of Health and institutional review boards of the U.S. Army and National Institute of Child Health and Human Development approved each protocol. Informed consent was obtained from each subject or a parent before screening, and the human use guidelines of the U.S. Department of Defense were followed in the conduct of these trials. Details of trial design, subjects, and study brokers PSC-833 can be found elsewhere (14, 15). In brief, 76 adults (21 to 45 years of age), 107 PSC-833 schoolchildren (6 to 12 years old), and 106 preschool children (2 to 5 years old) from Benha, Qalyubia Governorate, Egypt, were enrolled into three serial trials. Each 4-ml vaccine dose (lot E003) contained 1 mg of rCTB plus a mixture of 2 1010 bacteria each of five strains individually expressing CFA/I, CS1, CS2 plus CS3, CS4, and CS5 (14). Each 4-ml placebo dose contained 1011 heat-killed K-12 cells. Each dose was added to 150 ml of water made up of 4 g of sodium bicarbonate plus 1.45 g of citric acid (Recip AB, Stockholm, Sweden) for adult administration. Preschoolers and Schoolchildren received the same complete dosage of research agent put into one-half and one-fourth, respectively, from the antacid option. Topics were randomly assigned to get two dosages of placebo or vaccine 14 days apart. Sample processing and collection. Topics gave venous bloodstream examples preimmunization and seven days after each dosage. Plasma was produced as previously referred to (14, 15) and kept at ?70C until tested. All assays had been completed in a blinded style at Naval Medical Analysis Device PSC-833 No. Three. Option of purified antigen arrangements dictated the decision of vaccine elements contained in serologic assessments. Based on the protocols, plasma IgA titers against CTB, CFA/I, CS1, CS2, and CS4 had been measured in matched samples from as much as.
Blogging PSC-833, Rabbit Polyclonal to ARSA.
← Rheumatoid arthritis (RA) is a highly heterogeneous disease with respect to
Intro. through the bites of vectors, such mosquitoes or ticks [1]. →


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Miloud(ميلود in Arabic) is a masculine Given name which refers to the birth of the Prophet of Islam Muhamed El Mawlid Ennabawi  (and deriving from the latter). In the Middle-East, the name is Miled, with the same root.

This name is  mainly used in Maghreb (the North African subregion), and in Europe by families from North Africa living in Europe, notably in France.

It is a custom to give a son the name  Miloud or Miled if he happens to be born on the day of the Prophet's Birth.

Variants

A female variant of this name, Milouda, has appeared, mostly in Maghreb and Europe:  it is merely the feminine derivation of Miloud and has the same meaning.

References

Masculine given names
African masculine given names

redpajama_set_name: RedPajamaWikipedia


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Experimental Pharmacology and Drug Discovery
Wolff M. Kirsch
Loma Linda University Medical Center (LLUMC), United States
Salvador Soriano
Loma Linda University, United States
David E. Stec
University of Mississippi Medical Center, United States
Front. Pharmacol., 01 May 2018 | https://doi.org/10.3389/fphar.2018.00436
Mequindox-Induced Kidney Toxicity Is Associated With Oxidative Stress and Apoptosis in the Mouse
Qianying Liu1, Zhixin Lei1, Jingchao Guo2, Aimei Liu2, Qirong Lu2, Zainab Fatima2, Haseeb Khaliq2, Muhammad A. B. Shabbir2, Muhammad Kashif Maan2, Qinghua Wu3,4, Menghong Dai2, Xu Wang2*, Yuanhu Pan1 and Zonghui Yuan1,2,5*
1National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, China
2MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, China
3College of Life Science, Yangtze University, Jingzhou, China
4Department of Chemistry, Faculty of Science, University of Hradec Kralove, Hradec Kralove, Czechia
5Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan, China
Mequindox (MEQ), belonging to quinoxaline-di-N-oxides (QdNOs), is a synthetic antimicrobial agent widely used in China. Previous studies found that the kidney was one of the main toxic target organs of the QdNOs. However, the mechanisms underlying the kidney toxicity caused by QdNOs in vivo still remains unclear. The present study aimed to explore the molecular mechanism of kidney toxicity in mice after chronic exposure to MEQ. MEQ led to the oxidative stress, apoptosis, and mitochondrial damage in the kidney of mice. Meanwhile, MEQ upregulated Bax/Bcl-2 ratio, disrupted mitochondrial permeability transition pores, caused cytochrome c release, and a cascade activation of caspase, eventually induced apoptosis. The oxidative stress mediated by MEQ might led to mitochondria damage and apoptosis in a mitochondrial-dependent apoptotic pathway. Furthermore, upregulation of the Nrf2-Keap1 signaling pathway was also observed. Our findings revealed that the oxidative stress, mitochondrial dysfunction, and the Nrf2-Keap1 signaling pathway were associated with the kidney apoptosis induced by MEQ in vivo.
Quinoxaline-di-N-oxides (QdNOs), consisting of one or two acyclic chain moiety combined with quinoxaline ring, are best known as potential antibacterial agents (Wu et al., 2007; Vicente et al., 2009; Wang et al., 2011a, 2015a, 2016c; Cheng et al., 2015; Liu et al., 2016, 2017). Carbadox (CBX), olaquindox (OLA), quinocetone (QCT), and cyadox (CYA) were the classical members of QdNOs (Wang et al., 2015a,b; Zhang et al., 2015). Due to the potential genotoxic and carcinogenic effect, CBX and OLA had been banned in food-producing animals by European Commission since 1998 (Wang et al., 2016c). Mequindox (MEQ; 3-methyl-2-acetyl-N-1,4-dioxyquinoxaline,C11H10N2O3) (Figure 1), a relatively new QdNOs, can effectively improve growth and feed efficiency in animals for its better than other antimicrobial agents in treatment of swine dysentery (Treponema hyodysenteriae) (Liu et al., 2010; Ihsan et al., 2011, 2013a; Ding et al., 2012). As a synthetic antimicrobial agent, MEQ had significant antibacterial activities against both Gram-positive and Gram-negative species (Huang et al., 2015).
FIGURE 1. Chemical structure of mequindox (MEQ).
Previous studies demonstrated that some of the QdNOs such as OLA and CBX exhibited toxicity in the liver, kidney, and adrenal glands (Huang et al., 2010a,b; Ihsan et al., 2010, 2013a,b; Wang et al., 2016b; Liu et al., 2017). Early findings identified the adrenal glands as one of the main targets of QdNOs in vitro and in vivo (Huang et al., 2009, 2010a,b; Ihsan et al., 2010). MEQ was reported to reduce the output of adrenal aldosterone in pig adrenocortical cells (Huang et al., 2010b), and decreased serum aldosterone in rats (Huang et al., 2010a). The kidney has an important function in some unique physiology and metabolic pathways like the uptake, metabolism, and elimination of drugs (Lohr et al., 1998). Besides adrenal gland, kidney also plays an essential role in fluid-ion balance. However, the previous organ toxicity studies of MEQ only focus on the adrenal gland, while the kidney toxicity is commonly ignored. To date, only one study demonstrated the kidney damage on the male Wistar rats after exposure to MEQ (110 and 275 mg/kg) for up to 180 days, indicating that the kidney was another important target of MEQ (Huang et al., 2010a). However, the mechanism underlying kidney toxicity mediated by MEQ in vivo, especially regarding some linked signaling pathways was still not adequately understood.
A lot of evidence suggested that the oxidative stress was closely related to the damages caused by QdNOs, including apoptosis, DNA, and lipid damage (Chowdhury et al., 2004; Azqueta et al., 2007; Liu et al., 2012, 2017; Yang et al., 2013; Zhang et al., 2014; Wang et al., 2015a). The oxidative stress was also considered as the main factor in adrenal gland (Huang et al., 2009), liver (Wang et al., 2011b; Liu et al., 2017), spleen (Wang et al., 2011b), and testis toxicity (Ihsan et al., 2011) that caused by MEQ in vivo. The previous study concluded that MEQ-induced oxidative damage with the changes of reduced glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) in kidney (Huang et al., 2010a). Moreover, the oxidative stress was regarded as a molecular mechanism for MEQ to induce adrenal toxicity in H295R cells that originated from a human adrenocortical carcinoma (Wang et al., 2016b). Therefore, it was suspected that the oxidative stress may be involved in the toxicity in mice after chronic exposure to MEQ.
The QdNO-mediated oxidative stress is associated with a large number of biological responses and related cell signaling pathways. It was reported that the apoptosis invoked by OLA was always accompanied by the generation of ROS in renal tubular epithelial cells (HK-2 cells) (Li et al., 2015) and HepG2 cells that are from the liver (Zhang et al., 2011; Zhao et al., 2013, 2015). The apoptosis was observed in HepG2 cells after incubation with QCT (Zhang et al., 2013, 2015). However, whether the oxidative stress that triggered by MEQ-induced apoptosis in the kidney in vivo is unknown. Due to the wide use of MEQ in food animal production, it is of great significance to investigate the kidney toxicity mediated by MEQ in vivo.
In the present study, we investigated comprehensively the following parameters: the effects of MEQ exposure on body and kidney weight, morphological and ultrastructural changes in kidney; activity of the uric acid (UA), urea, creatinine (crea), blood urea nitrogen (BUN), GSH, total superoxide dismutase (T-SOD), MDA, 8-OHdG, and lactate dehydrogenase (LDH) in the serum; the mRNA expression of some genes in the kidney linked with apoptosis (e.g., caspase 3, caspase 8, caspase 9, and cychrome c), and correlated with oxidative stress (e.g., Nrf-2, GCLC, NQO1, HO-1, GSH-Px, and GST); the protein expression of cleaved caspase 3 and Nrf-2 in the kidney by immunohistochemistry, Western blot, and immunofluorescence assays. The study aimed at providing insight into molecular mechanism of MEQ-induced kidney toxicity in vivo, which will help to evaluate MEQ in its clinical use and improve the prudent use of QdNOs for public health.
Mequindox (MEQ; C11H10N2O3, CAS No: 60875-16-3, purity 98%) was obtained from Beijing Zhongnongfa Pharmaceutical Co., Ltd. (Huanggang, China). SOD, GSH, 8-OHdG, MDA, and LDH kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Nrf-2 (D1Z9C) XP Rabbit mAb (12721T) and cleaved caspase 3 (Asp175) antibody (9661T) were purchased from Cell Signaling Technology (United States), and a reagent Western Blot Stripping Buffer (T7135A) was purchased from Takara (Japan). All other chemicals and reagents were of analytical grade and obtained from Sigma-Aldrich (St. Louis, MO, United States).
Animals and Treatment
Recent studies revealed that the toxicity to liver (Liu et al., 2017) and testis (Liu et al., 2017a,b) were caused by MEQ in Kunming mice, while the kidney toxicity of MEQ in Kunming mice remains poor understood. Thus, the Kunming mice were chosen in this study. A total of 40 specific pathogen-free (SPF) Kunming mice (6–7 weeks old, weighing 30 ± 5g) were obtained from Center of Laboratory Animals of Hubei Province (Wuhan, China). The individual body weights of the mice were within ± 20% of the average. The mice were maintained in a room conditioned at 22 ± 3°C, a relative humidity of 50 ± 20%, and a 12-h light/dark cycle. The current study was approved by the Ethical Committee of the Faculty of Veterinary Medicine (Huazhong Agricultural University). Before treatment, the mice received basic feed and a standard diet from Nuvital Nutrients (Colombo/PR, Brazil), allowed to access to distilled water ad libitum for 7 days to evaluate for any signs of disease and weight gain.
According to the Organization for Economic Cooperation and Development (OECD) Guideline 453 and Procedures for toxicological assessment of food in China, the high-dose level should cause some toxic effect performance or damage, and the low-dose group may not show any toxic effects, but should be 1–3 times greater than the clinical dose (GB15193.17, 2003; OECD, 2009). In a previous sub-chronic and chronic toxicity study, MEQ in 110 mg/kg diet made an increase in plasma potassium (K+) level without growth inhibition, and this dose was determined to be no-observed-adverse-effect level (Ihsan, 2011). Therefore, the 110 mg/kg diet was selected as the high dose, and 55 mg/kg for the middle and 25 mg/kg for the low dose, respectively.
For the experiments, the mice were randomly divided into four groups (n = 10), including a control group treated with the basic diet without feed additives and three experimental groups treated with the same diet supplemented with 25, 55, and 110 mg/kg MEQ, respectively. The mice were housed five per group per sex in shoebox cages with hardwood shavings as bedding. Food and water were supplied ad libitum and the treatment period lasted for 11 months. During the experimental period, symptoms and mortality were observed and carefully recorded each day. In this study, the use of animals was in compliance with NIH Publication "The Development of Science Based Guidelines for Laboratory Animal Care" (NRC, 2004).
Coefficients and Preparation of Kidney
Following an overnight fast, the mice were weighted and sacrificed. After weighting the body and kidneys, the coefficient of kidney to body weight was calculated as the ratio of kidney (wet weight, mg) to body weight (BW) (g). The kidneys were excised, rinsed in phosphate-buffered saline (PBS), and the left kidney from all mice (n = 10) was quickly frozen at -70°C.
All histopathological tests were performed using standard laboratory procedures. The right kidney from the mice (n = 5) was preserved in 10% neutral-buffered formalin. After fixation, the kidneys were embedded in paraffin blocks, then sliced into 5 μm in thickness and placed onto glass slides with hematoxylin–eosin (HE) staining. Slides were observed and the photos were taken using an optical microscope (Olympus BX 41, Japan) for morphological alterations.
Observation of Nephrocyte Ultrastructure by Transmission Electron Microscope
The right kidney from other mice (n = 5) was fixed by 2.5% glutaraldehyde in 0.1 mol/dm3 cacodylate buffer for 4 h, then followed by wash three times with 0.1 mol dm cacodylate buffer (pH 7.2–7.4) and put in 1% osmium tetraoxide for 1 h. A graded series of ethanol (75, 85, 95, and 100%) was used to dehydrate the specimens and embedded in Epon 812. Ultra-thin sections (70 nm) were contrasted with lead citrate for 10 min and uranyl acetate for 30 min, and then observed with an H-7650 TEM (Hitachi, Japan). The nephrocyte apoptosis was judged according to the nuclear morphology changes (e.g., chromatin fragmentation and condensation).
TUNEL Assay
The right kidney from the mice (n = 5) was performed on 4-μm paraffin sections using antigen retrieval for 10 min of boiling in 10 mM citrate buffer (pH 6.0). They were fixed in 4% paraformaldehyde (pH 7.4) at -20°C for 3 min. After washing with PBS, the sections were permeabilized with 0.1% Triton X-100. Then, the samples were washed in PBS and incubated with a terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) reagent containing terminal deoxynucleotidyl transferase and fluorescent isothiocyanate dUTP. After incubation, they were stained with 1 μg/ml DAPI for 30 min to investigate the cell nucleus by UV light microscopic observations (blue). The kidney samples were analyzed in a drop of PBS under a fluorescence and UV light microscope. All morphometric measurements were observed by at least three independent individuals in a blinded manner.
Biochemical Analysis
For biochemical analysis, the serum aliquots were obtained by placed the blood samples in serum tubes at temperature of 24°C for approximately 30 min. After clotting, the blood tubes were centrifuged at 3000 rpm for 10 min using a Himac CR 21 G centrifuge (Hitachi, Tokyo, Japan). Supernatants were separated out and then stored at -20°C for further analysis. Kidney functions were determined by serum activities of UA, urea, crea, and BUN. All biochemical assays were performed using a Synchron CX4 Clinical System (Beckman Coulter, Brea, CA, United States) according to the manufacturer's protocol (Beijing Leadman Biochemistry Technology Co., Ltd., Beijing, China).
Oxidative Stress Assay
The effects of MEQ on the activity of 8-OHdG, MDA, GSH, T-SOD, and LDH in the serum were examined. Assays of 8-OHdG, MDA, T-SOD, and GSH levels were performed using commercial kits. The release of LDH was assessed using Synchron Clinical System CX4 (Beckman Coulter, Brea, CA, United States) according to the manufacturer's directions (Beijing Leadman Biochemistry Technology Co., Ltd., Beijing, China). The data were analyzed according to the manufacturer's instructions.
RNA Extraction and qPCR
Total RNA from the left kidney from all mice (n = 10) was extracted using the Trizol Reagent according to the manufacturer's instructions. RNA (1 μg) was reverse transcribed to cDNA with the use of ReverTra AceTM First Strand cDNA Synthesis Kit (Promega, United States). Synthesized cDNA was used for quantitative real-time polymerase chain reaction (Bio-Rad, United States) by SYBR® Premix Ex TaqTM RT-PCR kit (Takara, CodeDRR041 A, Japan). The mRNA expression of the apoptotic cytokines (e.g., caspase 3, caspase 8, caspase 9, cytochrome c, Bcl-2, and Bax), and oxidative stress-related genes (e.g., Nrf-2, HO-1, GCLC, NQO-1, GST-Px, and GST) were determined by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Mice specific primers were designed using Primer Express Software according to the software guidelines (Table 1). The primers were manufactured by Nanjing Genescript Co., Ltd. (Nanjing, China). For the 25 μL PCR reaction, 12.5 μL SYBR® Premix Ex TaqTM, 1.0 μL of each primer (10 μm), 2.0 μL of cDNA, and 8.5 μL Rnase Freed H2O were mixed together.
TABLE 1. PCR primers used in the gene expression analysis.
For GSH-Px, HO-1, GCLC, and Caspase 8, the cycling conditions were as follows: step 1, 30 s at 95°C; step 2, 45 cycles at 95°C for 5 s, 55°C for 30 s; step 3, dissociation stage. For Nrf-2, NQO1, GST, caspase 3, Bcl-2, Bax, and cytochrome c, the cycling conditions were as follows: step 1, 30 s at 95°C; step 2, 45 cycles at 95°C for 5 s, 60°C for 30 s; step 3, dissociation stage. For caspase 9, the cycling conditions were as follows: step 1, 30 s at 95°C; step 2, 45 cycles at 95°C for 5 s, 62°C for 30 s; step 3, dissociation stage. In this study, the housekeeping gene β-actin was used as internal calibrator reference gene for expression profiling of apoptotic and oxidative stress-related genes.
Following amplification, the authenticity of the amplified product by its specific melting temperature (Tm) was verified by a melting curve analysis with the complementary computer software. The threshold cycle of gene of interest and housekeeping gene (HK), and the difference between their Ct values (ΔCt) were counted. Relative quantitative analyses of gene expression were calculated using 2-ΔΔCt data analysis method in accordance with the previous literatures (Huang et al., 2009; Wang et al., 2016b; Liu et al., 2017).
The Nrf-2 (D1Z9C) XP® Rabbit mAb (12721T) and cleaved caspase 3 (Asp175) antibody (9661T) were purchased from Cell Signaling Technology. Total protein of the tissues was extracted according to the manufacturer's recommended protocol (Vazyme, Nanjing, China). Phenylmethylsulfonyl fluoride (PMSF) was added into radio immunoprecipitation assay (RIPA) lysis buffer, and the final concentration of PMSF was 1 mM. Total protein from the kidney was isolated in a preparation of RIPA lysis buffer, and then a sonic oscillator was used to break the cells or organelles. Next, it was centrifuged at 12000 g for 30 min at 4°C. The protein concentrations were determined using the BCA Protein Assay Kit (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China). Samples with equal amounts of protein (50 μg) were loaded for 1-dimensional SDS–PAGE using 12% separating gel and 5% stacking gel. The separated proteins were electrophoretically transferred to a polyvinylidene fluoride (PVDF) (Minipore) membranes in Trans-Blot Cells (Liuyi, Beijing, China). The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 1 h and were then immunoblotted with the primary antibody (anti-cleaved caspase 3 antibody at a dilution of 1:1000; anti-Nrf-2 antibody at a dilution of 1:1000) overnight at 4°C. The membranes were washed for 10 min 3 times with TBS-T and then were incubated with horseradish peroxidase-conjugated secondary anti-IgG antibody (diluted 1:5000) (Beyotime Inst. Biotech, Peking, China) at room temperature for 1 h. The membranes were washed for 10 min three times with TBS-T. Immunoreactive bands were visualized with a chemiluminescent substrate (ECL-Plus, Minipore) for 2–5 min. Images were captured with a LAS-4000 luminescent image analyzer (Fujifilm, Tokyo, Japan).
Immunohistochemical Assay
The kidney was dissected out and fixed in 10% neutral-buffered paraformaldehyde, and paraffin blocks were embedded on appropriate glass slides for processing. These were placed into the oven at 58°C, for 10 min, followed by deparaffinization in xylol, rehydration in alcohol at decreasing concentrations, and washing in distilled water and PBS (0.1 M sodium phosphate buffer, pH 7.2) for 5 min. The endogenous peroxidase was blocked with a 3% hydrogen peroxide solution for 30 min, and then washed in distilled water and PBS for 15 min (3 times, 5 min each). The slides were placed in citrate buffer and subjected to steam heat recovery, maintained at 95–100°C for 30 min, and they were then naturally cooled. The glass slides were then washed with PBS (3 times, 5 min each) and incubated with 5% bovine serum albumin (BSA) for 1 h, in a moist chamber at 37°C. After that, the BSA was discarded, and the glass slides were incubated with appropriate primary antibodies (Nrf-2, 1:500; cleaved caspase 3, 1:300), diluted according to the manufacturer's instructions (Santa Cruz or Millipore, United States), overnight at 4°C, in a moist chamber. Negative controls were processed without using the primary antibodies. The glass slides were then washed with PBS (3 times, 5 min each) and incubated with the biotinylated secondary antibody for 1 h at 37°C in the moist chamber. After another wash in PBS (5 times, 5 min each), they were incubated in 0.1% domain antibody (DAB) solution (in 3% hydrogen peroxide). The glass slides were counter stained with hematoxylin for 2 min, and then washed in running water for 10 min. Finally, the glass slides were washed in distilled water, dehydrated in alcohol (at increasing concentrations), diaphanized in xylol, and mounted on Entelan® for optic microscopy examination.
Immunofluorescence Assay
The kidney samples were performed on 4 μm paraffin sections of kidney using antigen retrieval for 10 min of boiling in 10 mM citrate buffer (pH 6.0). They were fixed in 4% paraformaldehyde (pH 7.4) at -20°C for 3 min. After washing 4 times in PBS, the sections were permeabilized with 0.1% Triton X-100, exposed to the blocking solution (PBS/3% BSA) and incubated with the primary antibodies cleaved caspase 3 and Nrf-2 at 4°C overnight. After four washes in PBS, the sections were incubated with secondary fluorescently labeled antibodies Dylight 594 antibodies for 45 min and then were washed 3 times in PBS. Nuclei were stained using DAPI. Fluorescent images were taken using an AX70 widefield microscope (Olympus). All morphometric measurements were observed by at least three independent individuals in a blinded manner.
All results are expressed as means ± SD. Statistical analysis was examined by SPSS 15.0 software. Group difference was assessed by one-way analysis of variance (ANOVA) followed by least significance difference (LSD) test. The p < 0.05 was considered statistically significant.
MEQ-Induced Kidney Injury in Mouse
Body Weight and Kidney Coefficients
During administration, all the mice were growing and the clinical behaviors, such as food and water consumption in the MEQ-treated groups were as normal as in the control group. The coefficient of kidney to body weight was expressed as milligrams (wet weight of kidneys, mg)/(grams body weight, g). The final body weight and kidney coefficient of mice after administration of MEQ for 11 months were shown in Figure 2I. Compared with the controls, significant reductions in body weight were observed in 25, 55, and 110 mg/kg groups (p < 0.05 or p < 0.01), while the significant increase in kidney coefficients were noted in 25, 55, and 110 mg/kg MEQ groups (p < 0.01).
FIGURE 2. (I) Effects of MEQ on body weight (g) and kidney coefficients (mg/g) in different groups. (II) Selected microphotographs of kidney (200×, 400×). (A) Kidney from the control group (400×). (B) Kidney from the 25 mg/kg MEQ group showing swelling and hyperplasia of the renal capsule wall of cell (400×). (C) Kidney from the 55 mg/kg MEQ group exhibited aggregation of the lymphocyte into a group around the central veins (200×). (D) Kidney from the 110 mg/kg MEQ group, renal tubular epithelial cells was marked with degeneration and necrosis (400×). (III) Ultrastructure of kidney in mice after the administration of MEQ for 11 months (Scale bar = 1 μm). (A) The kidney from control group showing normal nucleus and completed mitochondrial membrane; (B) the kidney from 25 mg/kg MEQ group showing vacuolization of organelles and nuclei; (C) the kidney from 55 mg/kg MEQ group showing cell swelling and vacuolization; (D) the kidney from 110 mg/kg MEQ group showing nuclei dissolution and cells loss. ∗p < 0.05, and ∗∗p < 0.01. Values represent means ± SD (n = 10).
Histopathological Evaluation
The frame of reference as to where in the kidney these slides were obtained in the Supplementary Figure S1. As shown in Figure 2II, the significant histopathological changes in kidneys were observed in MEQ-treated groups. The glomerular capillaries exhibited expansion, and the renal capsule wall of cell showed swelling and hyperplasia in 25 mg/kg MEQ group (Figure 2IIB). Lymphocyte aggregated into a group around the central veins in 55 mg/kg MEQ group (Figure 2IIC). In 110 mg/kg MEQ group, renal changes were characterized by degeneration and necrosis of renal tubular epithelial cells with the appearance of protein casts in the tubular lumen (Figure 2IID). This result suggested that the kidney damage was induced by MEQ in vivo.
Ultrastructural Changes and Apoptosis Induced by MEQ
The TEM analysis and TUNEL staining were carried out to investigate the ultrastructural changes and apoptosis of kidney induced by MEQ (Figures 2III, 3). The kidney in the control group presented normal nuclear morphology and completed mitochondrial membrane (Figure 2IIIA). In MEQ-treated groups, cells loss, cell swelling, nuclei dissolution, and mitochondria appeared vacuolization were noted (Figures 2IIIB,C,D). Additionally, the obvious apoptosis was also observed in MEQ-treated groups (Figures 3B,C,D). These findings revealed the obvious apoptosis and mitochondria damage caused by MEQ in the mouse.
FIGURE 3. TUNEL staining of kidney tissue. Cell nuclei (Blue), TUNEL-positive cells (Green) (Scale bar = 50 μm). The green spots represent TUNEL-positive cells and the white arrows indicate the apoptotic region. (A) The kidney from control group showing normal cells; (B–D) the kidney from MEQ-treated groups showing tissue lesion and apoptosis.
The Changed Serum Biochemical Levels and Oxidative Stress Induced by MEQ
The effect of MEQ on the serum biochemical parameters was presented in Figure 4I. In comparison with the control group, the UA and BUN had shown a greatly decreased in all the MEQ-treated groups (p < 0.05 or p < 0.01). The significant increased levels of urea and crea was found in 25 mg/kg MEQ group (p < 0.05), and in 55 and 110 mg/kg MEQ groups (p < 0.05 or p < 0.01), respectively. The changed serum biochemical parameters demonstrated the chronic kidney disease occurred in mice after exposure to MEQ.
FIGURE 4. (I) Effects of MEQ on the activity of (A) UA, (B) Urea, (C) Crea, and (D) BUN in the serum of mice after administration of MEQ for 11 months. (II) Effects of MEQ on the levels of (A) T-SOD, (B) 8-OHdG, (C) GSH, (D) MDA, and (E) LDH in serum of mice after administration of MEQ for 11 months. ∗p < 0.05, and ∗∗p < 0.01. Values represent means ± SD (n = 10).
As shown in Figure 4II, the levels of 8-OHdG and MDA were markedly increased in the MEQ-treated groups (p < 0.05 or p < 0.01). The levels of T-SOD were significantly increased on females after exposure to MEQ at 25, 55, and 110 mg/kg groups (p < 0.05), and on males at 110 mg/kg (p < 0.05), respectively. For the effects of MEQ on the activity of GSH, the significant increased levels were observed in the female at 110 mg/kg group (p < 0.01), and significant decreased in the male at 25 mg/kg group (p < 0.01). These results show that the oxidative stress was triggered in mice after chronic administration of MEQ.
MEQ-Induced Apoptosis and Activation Nrf2-Keap1 Signaling Pathway
Apoptosis plays an important role in both the physiological process of kidney growth and in different human renal diseases. To determine the role of apoptosis signal pathway in the mouse kidney after administration of MEQ for 11 months, real-time quantitative RT-PCR was used to demonstrate the changes of the genes including caspase 3, caspase 8, caspase 9, Bcl-2, Bax, and cytochrome c (Figure 5). With increased MEQ doses, there was a significant increase in caspase 3 and caspase 9 (p < 0.01). Exposure to MEQ significantly induced the expression of cytochrome c, Bcl-2, and Bax in 55 and 110 mg/kg groups (p < 0.05 or p < 0.01). A significant increased expression of caspase 8 was observed in 55 and 110 mg/kg MEQ groups (p < 0.01), and a marked reduction expression of caspase 8 was observed in 25 mg/kg MEQ group (p < 0.01).
FIGURE 5. Alterations of Bax, Bcl-2, Caspase 3, Caspase 8, Caspase 9, Cychrome c, Nrf-2, NQO1, HO-1, GCLC, GSH-Px, and GST mRNA expression by RT-PCR in mouse kidney after administration of MEQ for 11 months. ∗p < 0.05, and ∗∗p < 0.01. Values represent means ± SD (n = 10).
The frame of reference as to where in the kidney these slides were obtained in the Supplementary Figures S2–S7. Cleaved caspase 3 is a well-characterized cell apoptotic marker, the protein level of cleaved caspase 3 was detected by Western blot (Figure 6I), immunohistochemical analyses (Figure 6II), and immunofluorescence assay (Figure 7), indicating that compared to the control group, treating with MEQ caused increased cleaved caspase 3 expression. Taken together, these results suggested that the apoptosis induced by MEQ in kidney, and the mitochondrial dysfunction and caspase activation was involved in apoptosis mediated by MEQ.
FIGURE 6. (I) The protein levels of phosphorylated Nrf-2 and cleaved caspases 3 were determined by Western blotting. β-actin was used as an internal control. (II) The protein expression of cleaved caspase 3 and Nrf-2 were detected by immunohistochemical assays (the positive reaction of anti-cleaved caspase 3 and anti-Nrf-2 antibody were brown); (a) the kidney from the control group, (b–d) the kidney from M25, M55, and M110 groups, respectively, showed a positive reaction in the glomerulus and medulla kidney.
FIGURE 7. Translocation of the Nrf-2 from the cytoplasm into the nucleus and the expression of cleaved caspase 3 were evaluated by immunofluorescence (Scale bar = 50 μm). Blue spots represent cell nuclei, green spots represent Nrf-2 staining, and red spots represent cleaved caspase 3 staining. The integrated option density (IOD) of DAPI was used as an internal control. (A) Indicates the kidney from the control group, and (B–D) indicate the kidney from M25, M55, and M110 groups, respectively.
Some oxidative stress-related genes (e.g., Nrf-2, HO-1, NQO1, GCLC, GST-Px, and GST) were determined using real-time quantitative RT-PCR (Figure 5). Exposure to MEQ significantly induced the expression of Nrf-2 and GSH-Px in the MEQ-treated groups (p < 0.05 or p < 0.01). An increased expression mRNA level of NQO1 and GST were observed on the doses of 55 and 110 mg/kg MEQ group (p < 0.01). There was a significant increased expression of HO-1 and GCLC in 110 mg/kg MEQ group (p < 0.01), and an obvious reduction expression of HO-1 and GCLC in 25 and 55 mg/kg MEQ groups (p < 0.01).
Accordingly, Western blot (Figure 6I), immunohistochemical analyses (Figure 6II), and immunofluorescence assay (Figure 7) also showed that MEQ-treated groups significantly increased the expression of Nrf-2 compared with those in the control group. These results showed that the activation of Nrf2/Keap1 signaling pathways mediated by MEQ might be a cellular protection response to oxidative stress in kidney.
The role of oxidative stress associated with organ toxicity mediated by QdNOs was extensively studied in rats (Huang et al., 2009, 2010a; Ihsan et al., 2010, 2011; Wang et al., 2011b; Yu et al., 2013, 2014) and mice (Wang D. et al., 2011; Zhao et al., 2011; Liu et al., 2017). The kidney has been identified as the main toxic organ target of QdNOs including MEQ. However, unlike other toxicities caused by QdNOs, their effect on kidney is not well studied. The results of present study demonstrated that the oxidative damage was triggered by MEQ, which might be responsible for apoptosis and mitochondrial dysfunction in kidney of mice after the administration of MEQ for 11 months. Additionally, MEQ-induced apoptosis via upregulated Bax/Bcl-2 ratio, caused cytochrome c release and a cascade activation of caspase. Furthermore, the Nrf2-Keap1 signaling pathway might be a protective response to the apoptosis in mice kidney caused by MEQ. This study is of great significance to evaluate MEQ in its clinical use and improve the prudent use of QdNOs for public health.
The kidney was reported to be an important target organ of MEQ, and the main characters of the kidney damage included the cell loss, cell swelling, nuclei shrinkage, hemorrhages, cell atrophy, and vacuolation in the Wistar rats after chronic exposure to MEQ (Huang et al., 2010a). In the present study, a significant reduction in body weight and a significant increase in the kidney coefficients were observed in mice after treatment of MEQ at doses of 25, 55, and 110 mg/kg for 11 months. In serum biochemical analysis, MEQ increased the levels of urea and crea, and reduced the concentration of BUN and UA. The altered levels of these parameters indicated chronic kidney disease in mice. Histopathological evaluation showed obvious kidney damage in MEQ-treated groups, including expansion of glomerular capillaries, swelling, degeneration, and necrosis of renal tubular epithelial cells. In the TUNEL staining and TEM analysis, the obvious apoptosis and ultrastructural changes including cells loss, cell swelling, nuclei dissolution, and vacuolization of mitochondria were caused by MEQ. The histopathological and TEM observations revealed that MEQ caused damage to cortex and medulla of the kidney. The effect of MEQ was primary on renal tubule and collecting duct. These results demonstrated that apoptosis in kidney occurred after chronic oral administration of MEQ, which confirmed the earlier findings that the kidney was an important toxicity organ target for MEQ in vivo.
Previous studies documented that the oxidative stress was closely related to the in vitro toxicity caused by QdNOs, such as cytotoxicity, adrenal toxicity, genotoxicity, and apoptosis (Liu et al., 2016, 2017). Long-term MEQ treatment–induced endocrine and reproductive toxicity via oxidative stress with the significant changed levels of SOD, GSH, MDA, and 8-OHdG in male Wistar rats (Ihsan et al., 2011; Liu et al., 2016). A recent study revealed that the oxidative stress played a critical role in the liver toxicity after chronic exposure to MEQ for 11 months (Liu et al., 2017). Herein, our results showed that the levels of MDA, 8-OHdG, T-SOD, GSH, and LDH were significantly increased in the MEQ-treated groups (Figure 5), suggesting an imbalance of redox in mice. This finding revealed that chronic exposure to MEQ invoked oxidative stress in mice, and the oxidative stress was closely related to the organ toxicity mediated by MEQ in vivo.
Cleaved caspase 3 is a well-characterized cell apoptotic marker and caspase 9 is a biomarker of mitochondrial apoptosis pathway. The multimeric complex formation of cytochrome c, caspase 8, and caspase 9 activates downstream caspases, leading to apoptosis cell death (Alabsi et al., 2016). Simultaneously, the Bcl-2 family proteins function as central regulators of apoptosis in mammals (Alabsi et al., 2016). In this study, MEQ significantly increased the protein expression of cleaved caspase 3 and mRNA expression of caspase 3, suggesting that the apoptosis was induced by chronic exposure to MEQ in the kidney of mice. Additionally, the mRNA expression of caspase 8 and caspase 9 were significantly increased, indicating a mitochondrial apoptosis pathway invoked by chronic exposure to MEQ. Furthermore, the expression of cytochrome c was increased in MEQ-treated groups, demonstrating that cytochrome c was possibly released into the cytosol, and subsequently, induced the changed of mitochondrial permeability transition pores. In response to a variety of apoptosis stimuli, over-expression of Bcl-2 or Bax blocks cytochrome c release (Li and Wang, 2006). In our study, Bcl-2 and Bax gene expression were overwhelmingly increased and the ratio of Bax/Bcl-2 markedly elevated by MEQ. These results were consistent with the ultrastructural changes of kidney under TEM observation. Taking together, these findings indicated that the mitochondrial-dependent apoptotic pathway was triggered by MEQ, and MEQ upregulated Bax/Bcl-2 ratio, caused cytochrome c release and a cascade activation of caspase, eventually induced apoptosis.
Apoptosis can be triggered by oxidative stress and the damage to DNA (Skipper et al., 2016). MEQ was reported to induce DNA damage in testis of rats in vivo, and increased the level of 8-OHdG in rats (Ihsan et al., 2011) and mice (Liu et al., 2017). In this study, the apoptosis in kidney might be resulted from the oxidative stress and mitochondrial dysfunction mediated by MEQ. Currently, there are two well-characterized caspase-activating cascades: one is initiated by the cell surface death receptor and the other is triggered by changes in mitochondrial integrity (Alabsi et al., 2016). Mitochondria were reported to be a target organelle of QdNOs and extra production of ROS might be result in mitochondrial damage, following with mitochondrial apoptotic pathway (Dai et al., 2015). Herein, the organelle included mitochondrial showed vacuolization in the MEQ-treated group, demonstrating the destroyed integrity of mitochondria caused by MEQ. Our data revealed that the mitochondrial damage was induced by MEQ, and the oxidative stress and mitochondrial dysfunction might lead to apoptosis in the kidney of mice after administration of MEQ for a long period (Figure 8). Further study should be conducted to investigate the role of mitochondria in the oxidative stress and apoptosis after chronic exposure to MEQ.
FIGURE 8. The proposed mechanisms of oxidative stress in mouse after chronic exposure to MEQ. The oxidative stress-imbalance occurs, accompanying the increase of 8-OHdG, MDA, T-SOD, and GSH, which lead to mitochondrial damage and apoptosis in a mitochondrial-dependent apoptotic pathway. The oxidative stress activates Nrf2/Keap1. MEQ upregulated Bax/Bcl-2 ratio, disrupted mitochondrial permeability transition pores, and subsequently, caused cytochrome c release and a cascade activation of caspase, eventually induced apoptosis.
The Nrf2-Keap1 pathway was a major cellular defense mechanism against oxidative stress via encoding antioxidant enzymes and phase II detoxifying enzymes (Kobayashi and Yamamoto, 2005; Kimura et al., 2007; Li and Kong, 2009; Pedruzzi et al., 2012). Previous studies suggested that the Nrf-2 was involved in the oxidative damage caused by QCT in rat's liver (Yang et al., 2013; Yu et al., 2013) and NCI-H295R cells (Wang et al., 2016a). Herein, our results demonstrated that the mRNA expression of Nrf-2, GST-Px, NQO1, GST, HO-1, and GCLC were significantly upregulated in the M110 group. Accordingly, the marked increased protein expression of Nrf-2 was noted in MEQ-treated groups. Therefore, the Nrf2-Keap1 signaling pathway was activated by MEQ in mice. Subsequently, the phase II detoxifying enzymes (HO-1, GCLC, and NQO1) and antioxidative enzymes (GST-Px, GST) were generated and these enzymes eventually protected cells from oxidative stress. Interestingly, the mRNA levels of HO-1 and GCLC were significant decreased accompanying the increased of Nrf-2 at the M25 and M55 groups, indicating that the activation of HO-1 and GCLC by Nrf-2 was inhibited, and the further investigation into this phenomenon was needed. Taken together, current study findings revealed that the Nrf2-Keap1 signaling pathway was activated by MEQ, which may be responsible for the protective effect against oxidative damage, apoptosis, and the necrosis in mouse kidney induced by MEQ (Figure 8). Therefore, it was suspected that the Nrf2-Keap1 family was involved in the kidney toxicity caused by MEQ in vivo.
Scientists in 2013 reported that the exposure of rats to QCT for 4 weeks with high dose of QCT (2400 mg/kg b.w.) results in significant increased expression of Nrf2 protein in kidney along with the oxidative stress (Yu et al., 2013). However, after rats were treated with QCT for 13 weeks with high dose of QCT (2400 mg/kg b.w.) results in significantly reduced expression of protein Nrf2 and HO-1 along with excessive ROS generation and irreversible oxidative DNA damage, inflammation, and apoptosis in liver, indicated that persistent QCT exposure will inhibit the expression of Nrf2 and HO-1, and aggravate hepatocyte damage (Yu et al., 2014). These findings also suggested a hormesis in the chronic liver disease. Hormesis is a dose-response phenomenon characterized by a low-dose stimulation and a high-dose inhibition (Calabrese et al., 2010). In the present study, a few toxicity-linked parameters, such as serum urea, caspase-8, and GSH, were changed by MEQ not in a dose-dependent manner. It was well documented that the hormetic dose responses were mediated for endogenous cellular defense pathways, including sirtuin and Nrf2 pathways (Calabrese et al., 2010). Therefore, hormesis was suspected to account for these results, and the further study needed to clarify this hypothesis.
As shown in Figure 8, the current study demonstrated that the kidney was an important toxicity organ target of MEQ in vivo, and the oxidative stress and mitochondrial damage were induced in mice after administration of MEQ for 11 months. Additionally, MEQ upregulated Bax/Bcl-2 ratio, caused cytochrome c release, and a cascade activation of caspase, eventually induced apoptosis. Our findings revealed that the oxidative stress might be responsible for mitochondrial damage and apoptosis in a mitochondrial-dependent apoptotic pathway. Furthermore, the present study illustrated that the Nrf2-Keap1 family played a protective role in MEQ-induced redox imbalance damage in the kidney of mice. These findings may contribute to a better understanding of the molecular organ toxicity of MEQ and other QdNOs in vivo.
ZY conceived the idea; XW analyzed and discussed the data; QLi analyzed and discussed the data and wrote the paper; ZL performed and revised the experiments; AL performed the experiments; QW and MD revised the paper. All the authors discussed the results and contributed to the final manuscript.
This work was supported by the National Key Research and Development Program of China (2017YFD0501405), Grants from 2015 National Risk Assessment of Quality and Safety of Livestock and Poultry Products (GJFP2015008), and Research on the detection standard of veterinary drug residue (2662015PY021), as well as the long-term development plan UHK.
The reviewer SS and handling Editor declared their shared affiliation.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2018.00436/full#supplementary-material
FIGURE S1 | Selected microphotographs of kidney in M110 group.
FIGURE S2 | Protein expression of cleaved-caspase 3 detected by immunohistochemical assays in M25 group.
FIGURE S4 | Protein expression of cleaved-caspase 3 detected by immunohistochemical assays in M110 group.
FIGURE S5 | Protein expression of Nrf-2 detected by immunohistochemical assays in M25 group.
FIGURE S7 | Protein expression of Nrf-2 detected by immunohistochemical assays in M110 group.
8-OHdG, 8-hydroxy deoxyguanosine; BUN, blood urea nitrogen; CBX, carbadox; crea, creatinine; CYA, cyadox; GCLC, glutamate-cysteine ligase catalytic subunit; GSH, glutathione; GSH-Px, glutathione peroxidase; GST, glutathione S-transferase; HO-1, hydroxyl radicals; IκB, nuclear factor κB protein; LDH, lactate dehydrogenase; M4, 3-methyl-2-(1-hydroxyethyl) quinoxaline-N4-monoxide; M8, 3-methyl-2-(1-hydroxyethyl) quinoxaline-N1-monoxide; MDA, malondialdehyde; MEQ, mequindox; N1-MEQ, N1-desoxymequindox; NQO1, NAD(P)H:quinoneoxidoreductase; OLA, olaquindox; QCT, quinocetone; QdNOs, quinoxaline-di-N-oxides; ROS, reactive oxygen species; SOD, superoxide dismutase; TEM, transmission electron microscope; T-SOD, total superoxide dismutase; UA, uric acid.
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Keywords: mequindox, oxidative stress, Nrf2-Keap1, apoptosis, quinoxaline-di-N-oxides
Citation: Liu Q, Lei Z, Guo J, Liu A, Lu Q, Fatima Z, Khaliq H, Shabbir MAB, Maan MK, Wu Q, Dai M, Wang X, Pan Y and Yuan Z (2018) Mequindox-Induced Kidney Toxicity Is Associated With Oxidative Stress and Apoptosis in the Mouse. Front. Pharmacol. 9:436. doi: 10.3389/fphar.2018.00436
Received: 20 December 2017; Accepted: 12 April 2018;
Wolff Mayer Kirsch, Loma Linda University Medical Center, United States
David Stec, University of Mississippi Medical Center, United States
Salvador Soriano, Loma Linda University, United States
Copyright © 2018 Liu, Lei, Guo, Liu, Lu, Fatima, Khaliq, Shabbir, Maan, Wu, Dai, Wang, Pan and Yuan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Xu Wang, wangxu@mail.hzau.edu.cn Zonghui Yuan, yuan5802@mail.hzau.edu.cn


The early 90s seem positively ripe with games that are more character creation tool than game, and Nephilim is no exception. Join the ranks of weird energy spirits that have a collection of past lives, possession capability and spells, and honestly, really seem like the bad guys.
Ah, Nephilim. I remember getting this when it came out, reading it, and realizing it did not have enough material about the world, or how stories in the world would go, to make it playable. Needed more fluff! And then came the looooong wait until any other books came out. That essentially killed it. I was interested in it, got the main book, gave up waiting, and moved on. A lot of people did.
Everyone plays as one kind of thing. There's five types of guys. Some curse thingy that prevents you from being part of society. No real obvious goal or thing you're supposed to be doing.
If this isn't White Wolf, then it's clear that they were drawing from the same playbook. Maybe it was just a '90s thing.