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3 Sports Scheduling Tips for 2017
When one season ends, it's already time to start scheduling for next year. This can be a time-consuming and challenging process, and can create some headaches along the way. Here are some ideas for how to schedule your youth sports season efficiently and effectively.
Release the Schedule ASAP
Members of your sports organization like to know everything ahead of time – especially the parents. The best way to accomplish this is to release the game schedule well before the season starts. This requires a couple of things.
First, the planning should be done well in advance. This involves getting everyone together during the off-season, and hammering out the details early on. Doing so allows you to focus on other aspects like registration and practice as the season draws nearer, and it gives you an ample amount of time to make adjustments if scheduling conflicts arise.
Second, the schedule should be distributed to the parents and posted on your sports organization's website right away. Between work, school, and extracurricular activites, families already have a small amount of time together to begin with. Releasing the schedule quickly gives parents the opportunity to adjust their own schedules, and plan accordingly for events like family vacations.
Make Re-Scheduling Less of a Burden
Inclement weather is the natural enemy of schedules. It can cause everything to be thrown into disarray, and it can be a hassle to make changes. A lot of sports organizations post a PDF version of the schedule to their websites, but this can be troublesome when you need to edit game information and rearrange dates. It is also problematic for parents who received a hard copy at the pre-season meeting, and now have to scribble out dates and write-in new ones.
Technology is your biggest ally in the fight against weather and other unforseen events. Using a web application to create your schedule can make the re-scheduling process less painful when the time inevitably comes. It gives you the ability to easily change the date, time, and location of a game, or remove it completely if it will not be made-up. This means you won't have to keep posting a new file to the website each time the schedule is updated, and parents won't have to splash ink all over their paper copies.
Schedule EVERYTHING
The game schedule is one of the most important aspects to figure out before the season starts, but there are a number of other schedules that need to be created for your sports organization to run smoothly.
Staff and volunteers are essential parts of your operation, and they, too, have to schedule their tasks ahead of time. Whether it be for the concession stand, fundraising events, or refereeing, having schedules set in place can ensure that everyone is on the same page for their responsibilities.
Similarly to games, it can be difficult to alter these PDF or paper schedules throughout the season if people are unable to attend. Using something like Google Drive allows you to post the schedules in one location and make any adjustments if needed. You can also set the Google Drive to "private," and invite certain people to view and edit it. This means you can still post a link on your website for easy access, but only the people with permission can get in.
Want to make scheduling easier? Click the "Sign Up Now" button below or send me an email at firstname.lastname@example.com to learn more.


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Gandini Juggling's "4 x 4 Ephemeral Architectures", London, UKAdd to Cart Download
© Licensed to London News Pictures. 13/01/2015. London, England. Dancer Joe Bishop at the centre. Dress rehearsal of Gandini Juggling's new show 4 x 4 Ephemeral Architectures. Four classical dancers, choreographed by former Royal Ballet First Artist Ludovic Ondiviela, join four of Gandini's jugglers. World premiere at Linbury Studio Theatre, Royal Opera House, 13 to 15 January 2015. The show is part of the London International Mime Festival and is followed by a UK tour. Dancers: Kieran Stoneley, Kate Byrne, Erion O'Toole and Joe Bishop, jugglers: Kim Huynh, Sakari Männistö, Owen Reynolds and Kati Ylä-Hokkala. Photo credit: Bettina Strenske/LNP
- Filename
- LNP_GandiniJuggling_BST_017.JPG
- Copyright
- © under licence to London News Pictures. +44 (0)20 8408 0190
- Image Size
- 3000x2000 / 3.6MB
- Contained in galleries

subset: commoncrawl


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"LLAMC is the best place in town for any pet needs. Their DVM's are incredibly experienced, compassionate, and smart. My pup Sadie day boards quite frequently and honestly gets as excited to see the receptionists and techs as she does to see me. They are all so caring it makes parting with her so much easier!"Faith M.
"The Dr.'s and staff are top notch!!!! Our dogs are not only pets, they are also family. Lover's Lane treats them like their own. We cannot thank everyone there for all the excellent service that we have received. The only reason I gave them a 5, is because I could not give them a 6. LOL"Robert H.
"Thank you to Doctor Martin & Doctor Jiranek for being so compassionate and trying to save our dog Truman last week. Sadly he didn't make it but I know they did their best and we really appreciate it. Love this clinic & their whole staff. Highly recommend."Claire Pruitt W.
"LOVE these folks!! And they love my dogs, Goliath and Gunner. :) Free treats all around!!!!"Melanie W.
"It the only place I'll take any of my fur babies. I've been taking my kids there for 20 years."Van M.


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Find Us
Locating our Spa and Shop can be a little tricky as, like many roads in Cambodia, we are on one that hasn't been named yet. Tuk Tuk drivers can strugle to follow Google Maps so that's why we recommend using Grab or PassApp on your smartphone. We will also be adding a list of trusted Tuk Tuk drivers who are familiar with our location here.
Most online maps accurately show Angkor Scrub if you search but please report any problems to us.
GPS Co-ordinates: 13.339679, 103.861087


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Druha Trava, one of the leading bands to emerge from the vibrant Czech Republic bluegrass scene, celebrates its 15th year with the release of its 8th album. Once one gets past the twin incongruities of a standard like "Sitting On Top Of The World" sung with Czech accents and bluegrass versions of "Bad Moon Rising," two Mark Knopfler tunes and a Tom Waits number, there is quite a bit of good music here.
The band is built around the lead vocals of mandolinist Robert Krestan and banjoist Lubos Malina, although Dobroist Lubos Novotni and and guitarist Emil Formįnek are fine pickers as well. Krestan's gravelly voice is not varied in his inflections, but he is an effective singer who is at his best on world-weary tunes. The group's harmonies are solid, although occasionally supplemented by guest vocalists.
The choice of material is a bit scattered, ranging from two obscure Dylan songs ("Ring Them Bells," "Going To Acapulco") to Celtic (Peter Rowan's "Holy Wells of Ireland" joined with Malina's "Ossian") to a pair of excellent instrumentals, "Fall Farm" and "Caurea," written by Malina and Novotni respectively. The latter in particular sounds like a long-lost New Grass Revival track and features hot picking from Novotni, Malina, Formįnek and bassist Petr Sury. Definitely worth checking out.


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Cushion floor tiles are interlocking sections of foam rubber that look a lot like puzzle pieces. They are temporary flooring that can be carried around from room to room and taken outside to be cleaned with the hose. They are a wonderful way to add some temporary cushion to a floor for a number of reasons.
Cushion Floor Tiles
You might have seen multi-colored cushion floor tiles designed for children's playrooms or bedrooms. They make a safer environment in which the children play. Every little fall they take while roughhousing is cushioned and prevents an injury. But there are other styles and uses for these handy tiles.
These interlocking style tiles come in black, too. They can be used in workspaces where the person is on their feet for hours at a time while doing their job. The tiles cushion the feet and that prevents a lot of foot pain, but helps to prevent back pain, as well. Any workspace benefits from these tiles, even the home kitchen!
Once you use this extra cushioning on the floor you'll never want to give it up. They are easy to clean and are the best thing ever for taking the load off of your feet and back. But, this type of cushioning doesn't have to be a temporary thing.
There is flooring that has a good deal of cushioning under it. When you install flooring, you can have a pad underlayment that helps to cushion the floor and muffle sounds. Another choice is cushion vinyl flooring which is one piece of a very cushiony floor that will feel like you're walking on pencil erasers.
When it is time to add some extra cushion in your home, you can try out the cushion floor tiles to see how you like it. When you want to make it permanent, give us a call.
There are a lot of different kinds of hardwood floor stains and each type has a different remedy. But it is not as difficult to repair the wood as you might think. It takes the right tools and some elbow grease and your floor will be beautiful and shiny once again.
Hardwood Floor Stains
When you have hardwood floor stains on your gorgeous flooring you'll be tempted to just put a rug on it and pretend it never happened. There is a better way. You can restore it to like new condition with the right tools.
Water or Liquid Stains
Milk or water stains will leave a white ring on the floor if they aren't wiped up immediately. The white ring can be removed with some patience, steel wool and floor wax. Try #000 steel wool to rub the stain out using a generous portion of floor wax during the rubbing. If that fails, try #00 wool with mineral spirits then let the floor dry. With this 2nd method you will need to stain the area and re-wax.
Black scuff marks from shoes are usually easy to remove with a cleaner made for urethane floors. If they are particularly bad then buy the version of this cleaner that comes with a scrubbing pad.
Mold and Mildew
The bad news is, there is usually no way to rid yourself of mold or mildew in wood floors. Mold has tentacles that grow deep into the wood. Even if you clean it from the surface, it will quickly return. You will need to address the source of the moisture that's feeding the mold and then replace the moldy floor boards.
Flooring Contractor
For all of your flooring needs contact US Hardwood and Carpet. We can recommend a hardwood cleaning service to remove your hardwood floor stains professionally.
April 2019


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Like every year, the holy month of Ramadan, together with its blessings has brought a price cut at government maintained utility stores. To many people's disbelief, Ramadan Relief Package has been finally approved by the Economic Coordination Committee (ECC) of the federal cabinet. The meeting was held on Tuesday and chaired by Finance Minister Ishaq Dar. He revealed that at most 19 commodities with subsidies worth Rs1.6 billion have been agreed as a part of the Ramadan Relief Package.
The commodities claimed to be subsidized include:
Sugar, ghee, rice, squashes, oil, wheat flour, dates, basin, pulses including channa, masoor, mash and moong washed, white grams, milk, tea and all spices.
The public would be able to access these commodities at Utility Stores Corporation (USC) outlets throughout Pakistan at the proposed subsidized rates.
Also Read: Transporters' strike may spoil mango exports this season
Other subsidiaries to be offered
To add to the public's sigh of relief, USC has also claimed to cut their prices on around 2,400 items other than those relieved by the government. Almost double the discount i.e. from 5% to 10% has been promised by the utility outlets as they have decided to reduce their own profit margins and some discounted assistance through their vendors and suppliers.
In addition, ECC has also increased urea exports to 0.6 million metric ton. It is augmented from the current export quantity of 0.3 million metric ton. The export of urea has also received an extension in the deadline i.e. from April 28th to 31st of October.
The decisions of ECC were an upshot to the recommendations suggested by the Fertilizer Review Committee after they were put forward by the Ministry of Commerce.
Read More: The online store that lets you sell anything from anywhere in the world
The ECC learnt that Kharif 2017 is expected to bring about ample production of urea of domestic consumption that would allow for increased exports of the resource.
ECC also approved the restoration of import duty and sales tax on cotton imports. The step has been taken on the Finance Division's proposal which would come to action by July, 2017. This decision is sought to impart some confidence to the domestic cotton growers to get them ready for the upcoming sowing season.


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My name is Sarah, I am a 32 year old Australian living life in the south of France with my husband and two small children. These days I work in the Sports Nutrition industry, where I am the UK Regional Manager for a large company (based in Monaco) that sells health foods and sports supplements. In the past I have worked as a model, an Elementary school teacher and also in the Motor Sport Industry.
I have lived with Primary Lymphedema in my left leg since April 2013; initially seeing signs of the condition when I was three months into my first pregnancy. I noticed some slight swelling in my left groin area and put it down to "normal pregnancy swelling." However within a few weeks my whole leg had blown up and I knew something wasn't right. After a few visits to the ER, blood tests, doctors assessments and ultrasound scans, the doctors were still unsure what was causing my swelling and basically told me it was just a weird thing that would go away after I gave birth. A few months after the birth of my daughter, I still had a very swollen leg and was eventually diagnosed with Primary Lymphedema.
I managed my condition to the best of my ability, doing all the right things (compression, bandaging, MLD), and my leg responded well. I would constantly ask my doctor if there was more I could be doing, or if surgery could help, but I was always told that surgeries did not exist and to just 'live with it.'
It was around August 2015 that my Lymphedema suddenly worsened (during my second pregnancy) and I became really worried. I started to feel very isolated, depressed and like the only person in the world who suffered this condition. I decided to search on Instagram the hashtag #lymphedema to see what would come up. This was the moment things would change for me. I found a community of people, like me, who came from all the different corners of the world, who suffered from LE! They were asking the same questions as me, searching for the same answers, sharing information and supporting each other. Connecting with these people gave me hope, finally! It showed me that Lymphedema doesn't discriminate; it doesn't care who you are or what you do, because it can happen to anyone.
The information, support and guidance I found on Instagram was much more than I could find in my local community and from caregivers. Although it was wonderful, it also deeply worried me and made me question why our health system was lacking so badly in this area? Why didn't my doctor know vital information that seemed so "common knowledge" amongst the people online? Why didn't I have access to the same treatment and care as others did?
My online journey connected me with more and more people living with LE who were struggling, feeling hopeless and unaware of the treatment options available to them. Many people did not understand the basics of compression garments, how to correctly bandage their limbs or that there exists surgeries for Lymphedema that can greatly improves ones condition. It was very upsetting for me to know that people (including myself) were so clueless about how to manage their condition. This motivated me to take action and work towards changing this in the LE community.
These days, I am heavily involved in the Lymphedema movement by acting as a voice to change the way we talk about, perceive and treat this condition. I truly believe that we have come to a point in time that things must change. Caring for limbs with Lymphedema should be based on what individuals need, rather than what works best (or easiest) for the health and social care system to provide. Our already stretched health care systems are currently failing to deliver this; and the demands on it are only increasing. We urgently need to keep the conversation moving, to tackle the big issues and find solutions.
I am Chronically Motivated in many ways:
- Lymphedema Surgery Advocate- using my social media platforms (Instagram and blogging) I work to raise awareness of the current surgical options available to treat Lymphedema. I believe we should all be informed about the surgical choices available to us and its up to our caregivers to give us this information as soon as we are diagnosed with Lymphedema. Early intervention is they key to successfully treating Lymphedema, therefore patients need access to this information immediately.
- I use my own experience of surgery to promote awareness and share information on surgeries that a lot of people are unaware of. It's a matter of raising awareness, not just amongst people who live with this condition but also our caregivers and doctors- to make them aware and give them the power to pass this information onto their patients. We also need to de-stigmatize the world of Lymphatic surgery, as tremendous results are being had and not recognized by the rest of the world.
- Advocate for raising awareness of congenital Lymphedema in Children- I find this an extremely interesting area of Lymphedema and am extremely motivated to help improve the lives of children at risk and who lack the financial means, access to basic materials and education to manage their lymphedema.
- Campaigner for change towards funding and health legislation for LE.
- Social media Lymphedema ambassador.
These days, I no longer feel like it's the end of the world to have LE. Understanding my condition and discovering the treatment solutions that are available gave me back a sense of control. I will always have Lymphedema and that is okay, I accept this and am thankful to be given this path to walk in life.
I know the road to recovery is long and requires a lot (and I mean, a lot!!!!) of hard work and patience, but that is fine by me! I'm enjoying the journey and learning so much more I could ever have thought I would from this experience. I have met amazing people who have changed MY life and I know that by sharing my experiences I have changed other people's lives. For this I am thankful.
Top 5 "cant live without" Lymphedema materials:
1) Toe caps- these help a lot to keep my toes from turning into little sausages.
2) MalleoTrain ankle support- seriously amazing for stopping the liquid build up around my ankle.
3) Jobst Relax night garment- very comfortable alternative to bandaging at night and easy to travel with.
4) Maxi dresses in the summer time
5) Knee high boots (with zips on the sides!) and tights in the winter time.
My best Lymphedema tip:
Recently I discovered the activity of Aqua Biking- it's an exercise bike in the water. It's a great alternative to swimming and combines the benefits of water sport and cycling to help with lymphatic flow.
Where to find Sarah:
Instagram: @lymphosaurus_rex
Blog: www.lymphosaurusrex.wordpress.com


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Daniel William Kriss
1928 - 2019
Funeral Service
Obituary of Daniel William Kriss
Daniel William Kriss age 91, died peacefully on Friday, March 22, 2019 at the Arbor Terrace assisted living facility in Manassas, Virginia. He was a retired captain on the Fairfax County Police Department and a longtime resident of Herndon.
Dan was born February 9, 1928 in Passaic, New Jersey. He began his life of service to others very early by joining the U.S Navy at age 14 in December, 1942. He had lost some older friends who were serving in World War II, and their loss coupled with his patriotism motivated him to devise a clever plan to join the Navy. Dan served aboard the USS Chauncey (DD-667) from May 1943 to January 1946 and experienced significant combat action in the South Pacific, including the Marshall and Gilbert Islands, the Philippines, and near the home island of Japan. He was 17 years, 11 months old when he was discharged, but then had to register for the draft when he turned 18. Dan was one of the youngest Navy combat veterans of World War II and was a member of a distinctive organization, the Veterans of Underage Military Service. Dan's service is documented in volume one of the book, America's Youngest Warriors.
After the war, Dan married his childhood sweetheart Julia Sudol (1927 -1997) and attended school on the GI bill obtaining a college degree. Together they had three children, twins, Daniel John Kriss, 1951 to 1967 and Carolyn Ann Kriss (Burns) 1951 to 2011, and Raymond K. Kriss of Centreville, Va.
At age 21, Dan became a police officer with the Washington D.C. Metropolitan Police Department. He served 61/2 years. In October 1956, Dan joined the Fairfax County Police Department. He rose rapidly through the ranks attaining the rank of Captain in 1970.
Dan was very involved with police training and in 1965 he was instrumental in establishing the Northern Virginia Police Academy, the first regional police training academy in Virginia. That organization exists today as the Northern Virginia Criminal Justice Academy. Dan's education and professionalism distinguished him among his peers and resulted in numerous special assignments throughout his career. He was assigned as the Director of the Virginia Central Police Academy in Richmond for one year and performed instructional duties for the International Association of Chiefs of Police. He is credited with authoring the first set of General Orders documenting process and procedure for the FCPD.
In 1968, Dan was assigned by then Chief William Durrer to assist the Prince William Board of County Supervisors in conducting a study to determine if the County should transition law enforcement from a Sheriff's office to a police department. Then Lieutenant Kriss' report cited, "The project staff concludes that the only sound course of action for Prince William County to follow, is to begin a carefully planned procedural program aimed at establishing an organized county police department." On July 1, 1970 the Prince William County Police Department began operations.
In 1972 Captain Dan Kriss became the commander of the McLean District Station and two years later he became the commander of the Chantilly District Station. While at Chantilly, he was additionally assigned to plan a new station in the rapidly growing Reston area of Fairfax County. The Reston District Station opened in 1976, with Dan serving as the Commander until his retirement in June, 1979.
In retirement, Dan became the Chief of Police at both Dulles International and National Airports, what today is known as the Washington Metropolitan Airports Authority Police (WMAA). He served in that capacity until he accepted a position managing security for the Mellon family in Upperville, Virginia, from which he retired a final time.
Dan remained involved with military alumni associations, including the USS Kidd DESRON 48 Association, the "Tin Can Sailors" National Association of Destroyer Veterans. He served on the board of directors of the Fairfax County Retired Police Association.
Dan leaves behind a long legacy of community service, gaining the respect of all that knew him. In addition to his public service, he was a dedicated family man and a caring friend to many. Survivors include his son Raymond K. Kriss and his wife Meg of Centreville and son-in-law Gerald P Burns (Norma Jean) of Jeffersonton. Grandchildren: Wendi M. Banther (Eric) of Warrenton, Kathleen M. Burns of Manassas, Daniel P. Burns (Jenn) of Fairfax and Alexandra E. Kriss of Reston, and one great grandchild, Haley Verrilli-Banther.
Tuesday, April 2, 2019, 2-4 pm and 6-8pm
Adams Green Funeral Home
721 Eldon St, Herndon, VA 20170
Wednesday, April 3, 2019, 11am
Adams Green Funeral Home
721 Eldon St, Herndon, VA 20170
Immediately Following the Service
Chestnut Grove Cemetery
831Dranesville Rd, Herndon, VA
Reception Immediately following the Internment
Donations may be made to:
St Jude Children's Research Hospital


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AFCO ELIMINATOR Twin Tube Adjustable Shocks are built specifically for drag racing. Our onboard drag car data acquisition systems helps us know what your car is doing every 0.001 of a second. We've engineered the ELIMINATOR to control it at every point. This gives you, the racer, unsurpassed traction.
AFCO DOUBLE Adjustable Shocks are the ultimate tool in the chassis tuner's arsenal. The DOUBLE adjustments of both compression and rebound damping are completely independent. Changing the setting of one has no affect on the setting of the other. By allowing full control over both
compression and rebound forces, this shock gives the tuner the ability to fine tune every aspect of chassis movement to provide the ultimate in acceleration control and overall driveability.
AFCO Twin Tube - ELIMINATOR Shocks
- Widest range of adjustment on the market.
- Make a click and feel the change.
- 100% dyno tested.
- Best for cars up to 1,000+ HP Applications
- Please call us if custom colors are desired
Price is per shock.


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Basic fibroblast growth factor (FGF-2) is involved in the development and maintenance of the nervous system. Exogenous administration of FGF-2 increased dopaminergic (DA) graft survival in different animal models of Parkinson's disease. To study the physiological function of the endogenous FGF-2 system, we analyzed the nigrostriatal system of mice lacking FGF-2, mice overexpressing FGF-2, and FGF-receptor-3 (FGFR3)-deficient mice both after development and after 6-hydroxydopamine lesion. FGFR3-deficient mice (+/−) displayed a reduced number of DA neurons compared with the respective wild type. Whereas absence of FGF-2 led to significantly increased numbers of DA neurons, enhanced amount of the growth factor in mice overexpressing FGF-2 resulted in less tyrosine hydroxylase expression and a reduced DA cell density. The volumes of the substantia nigra were enlarged in both FGF-2−/− and in FGF-2 transgenic mice, suggesting an important role of FGF-2 for the establishment of the proper number of DA neurons and a normal sized substantia nigra during development. In a second set of experiments, the putative relevance of endogenous FGF-2 after neurotoxin application was investigated regarding the number of rescued DA neurons after partial 6-OHDA lesion. Interestingly, the results after lesion were directly opposed to the results after development: significantly less DA neurons survived in FGF-2−/− mice compared with wild-type mice. Together, the results indicate that FGFR3 is crucially involved in regulating the number of DA neurons. The lack of FGF-2 seems to be (over)compensated during development, but, after lesion, compensation mechanisms fail. The transgenic mice showed that endogenous FGF-2 protects DA neurons from 6-OHDA neurotoxicity.
- basic fibroblast growth factor
- midbrain
- mutant mice
- Parkinson's disease
- stereology
- substantia nigra
- terminal 6-OHDA lesion
Parkinson's disease (PD) is the most common neurodegenerative disease affecting the nigrostriatal system. The disorder is characterized by the progressive degeneration of dopaminergic (DA) neurons in the pars compacta of the substantia nigra (SNpc), resulting in severe motor dysfunctions. The etiology of PD is associated with genetic defects (e.g., α-synuclein, parkin) in only ∼10% of the cases (Feany and Bender, 2000; Shimura et al., 2000; Giasson and Lee, 2001), whereas in the majority of patients its pathogenesis is unknown (Goldman and Tanner, 1998; Lotharius and Brundin, 2002). All clinical approaches for the therapy of PD, including drug treatment, deep brain stimulation, and striatal cell transplantation, are symptomatic and do not prevent the progression of the disease. To develop new causal therapeutic strategies and/or to prevent the progression of the disease, one has to identify the molecular mechanisms that regulate the generation, survival, and differentiation of nigral DA neurons, both during development and after lesion.
Basic fibroblast growth factor (FGF-2) is involved in the development, maintenance, and survival of the nervous system and, as one of the most potent survival factors, exerts neurotrophic activity on DA neurons in vitro and in vivo. In vitro, exogenous administration of FGF-2 or cocultivation with FGF-2-producing Schwann cells stimulated survival and neurite formation of DA neuronal cultures and protected them from 6-hydroxydopamine (6-OHDA)-induced neurotoxicity (Ferrari et al., 1989; Knusel et al., 1990; Engele and Bohn, 1991; Beck et al., 1993; Mayer et al., 1993a; Bouvier and Mytilineou, 1995; Grothe et al., 2000). With regard to the in vivo situation, FGF-2 has been efficiently used in different treatment protocols in mouse and rat PD models. Intrastriatal infusions of FGF-2 induced recovery of striatal DA fibers and DA content in the mouse 1-methyl-1,2,3,6-tetrahydopyridine (MPTP)-model and increased the protection of the neurotoxicity-induced lesioned nigrostriatal DA system (Date et al., 1993). In addition, survival of grafted DA neurons was also enhanced after repeated intracerebral infusions or pretreatment of transplanted cells with FGF-2 (Mayer et al., 1993b). Furthermore, cografting of FGF-2-producing fibroblasts (Takayama et al., 1995) or Schwann cells (Timmer et al., 2004) with DA neurons mediated growth-promoting effects on the transplants and improved the behavioral deficits of parkinsonian rats.
Interestingly, in the SNpc of control brains, ∼82% of the melanin containing nigral cells are FGF-2 positive, whereas in the SNpc of patients suffering from PD only, ∼13% of the remaining DA neurons are positive for FGF-2, suggesting a profound depletion of FGF-2 in the remaining DA neurons, and this depletion may be related to the disease process (Tooyama et al., 1993, 1994).
The FGF-2 signal is mediated via four high-affinity tyrosine kinase receptors (R) that are crucial for development and regeneration of the CNS and PNS. In the dopaminergic nigrostriatal system, FGF-2 and three of its respective receptors (FGFR1–FGFR3) are endogenously expressed (Claus et al., 2004; Timmer et al., 2004).
The physiological roles of FGF-2 and its receptors can be investigated because mutant mice (1) lacking FGF-2 (Dono et al., 1998; Zhou et al., 1998), (2) overexpressing FGF-2 (Coffin et al., 1995), and (3) with a reduced level of FGFR3 (Colvin et al., 1996) have been generated.
Using these mice, it has been shown previously that different parts of the CNS and PNS are affected. FGF-2 null mutants are viable and the phenotype looked quite normal (Dono et al., 1998; Zhou et al., 1998), but more detailed evaluation revealed that FGF-2−/− displayed cerebral neocortex defects at birth (Dono et al., 1998).
In addition, we have shown that targeted disruption of the FGF-2 gene affects the response to peripheral nerve injury, whereas the PNS development showed no defects (Jungnickel et al., 2004b). In mice overexpressing FGF-2, we found faster nerve regeneration after sciatic nerve injury (Jungnickel et al., 2006) and a regulation of injury-related effects in the peripheral nervous system via FGFR3 signaling (Jungnickel et al., 2004a). Moreover, FGF-2 and FGFR3 play an important role in the regulation of neuronal death after peripheral nerve injury (Jungnickel et al., 2005; for review, see Grothe et al., 2006).
In the present study, we quantified the nigrostriatal system of the above mentioned mice strains after both development and lesion to analyze the physiological role of FGF-2 in the CNS. Our results show an increase of the DA neuron number in FGF-2−/− mice after development but an increased cell death after lesion, suggesting different functions or, more probably, failing compensation mechanisms within the adult nigrostriatal system. Moreover, mice overexpressing FGF-2 protected DA neurons from 6-OHDA toxicity. Reduced DA neuron numbers in mice deleted for FGFR3 (genotype +/−) during development indicate a major role of released FGF-2 compared with intracellular one. These alterations remain during adulthood.
Materials and Methods
Mutant mice
All experimental protocols were done in accordance with the German law for the protection of animals with a permit by the local authority (Bezirksregierung Hannover; guidelines of the Tierschutzgesetz i.d.F.v. December 25, 1998). Three mutant mice of the FGF-2 system were used for the present study, including (1) receptor-deficient mice (FGFR3+/−), (2) knock-out (ko) mice (FGF-2−/−), and (3) transgene mice (overexpressing FGF-2; TgFGF-2+/−) (Table 1):
FGFR3-deficient mice.
FGFR3 heterozygous mice (Colvin et al., 1996) (background in this study, C57BL/6) were obtained from heterozygous crosses of breeders received from Dr. A. Vortkamp (Max-Planck-Institute of Molecular Genetics, Berlin, Germany). The original colony created in the laboratory of Dr. D. Ornitz (Washington University, St. Louis, MO) was founded in a mixed C57BL/6 × 129 background and maintained via successive C57BL/6 backcrosses. In the present study, FGFR3 heterozygotes were used, because the viability of FGFR3-deleted homozygous animals is low. The heterozygous mice exhibit nearly the same characteristic phenotype as FGFR3 null mice, including kyphotic and scoliotic deformations of the vertebral column at different extent (Colvin et al., 1996; Oh et al., 2003; Jungnickel et al., 2004a). FGFR3 is expressed before embryonic day 16.5 (E16.5). At this stage, the first phenotypical alterations were found in FGFR3+/− mice (Colvin et al., 1996). Genotypes were determined by PCR of the tail DNA.
FGF-2-deleted mice.
FGF-2 ko and wild-type (wt) mice (Zhou et al., 1998) were obtained from The Jackson Laboratory (Bar Harbor, ME). Wild-type (FGF-2+/+; 129P2/OlaHsd:Black Swiss) and homozygous knock-out (FGF-2−/−) mice were studied, and genotypes were determined by PCR of the tail DNA. The lack of the protein was proven by Western blot (Jungnickel et al., 2004b). FGF-2+/+ littermates served as the control group.
Mice overexpressing FGF-2.
Heterozygous TgFGF-2 mice (Coffin et al., 1995) were received from Dr. S. Wolfram (Max-Planck-Institute, Bad Nauheim, Germany) and maintained on a FVB/N background. Wild-type and transgenic littermates were studied, and genotypes were determined by PCR of the tail DNA (Jungnickel et al., 2006). Overexpression of the different FGF-2 isoforms was verified in brain and lung tissue from transgenic mice by Western blot (data not shown).
Experimental design.
For the first part of the experiments (nonlesioned), 9- to 12- week-old male wild-type (FGFR3 wt, n = 5; FGF-2 ko wt, n = 6; TgFGF-2 wt, n = 6) and male mutant mice (FGFR3, n = 6; FGF-2 ko, n = 6; TgFGF-2, n = 6) were studied. Male mice older than 3 months were used for the lesion studies, because the cranial bone must be thick enough for putting the mice into the stereotactical frame (n > 8 per group). Female wild-type and mutant mice were taken for Western blot analysis (n = 3). The genetically altered animals represent wild-type littermates (animals born together from the same female). Wild-type and knock-out animals were obtained by crossbreeding with heterozygous genetically altered animals.
Tyrosine hydroxylase immunohistochemistry
The mice were deeply anesthetized with ketamine and Rompun and perfused transcardially with 20 ml of 0.9% saline, followed by 40 ml of 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4. The brains were postfixed overnight and dehydrated in 20% sucrose. Serial coronal sections were cut on a freezing microtome at 30 μm thickness. To quantify the substantia nigra morphologically, every third section was processed for tyrosine hydroxylase (TH) immunohistochemistry (mouse anti-rat TH monoclonal antibody, 1:2500; catalog #T1299; Sigma, St. Louis, MO) using the avidin–biotin complex kit and 3′,3-diaminobenzidine for visualization, as described previously (Nikkhah et al., 1994). Before putting the first antibody, the sections were preincubated using null serum.
Morphological evaluation of the substantia nigra
All morphological procedures were performed using an Olympus Optical (Tokyo, Japan) microscope (BX 60). We quantified the substantia nigra with regard to the number of DA neurons, the volumes, and the cell density (Fig. 1).
Neuron counts.
Stereological methods were used to evaluate the number of neurons in the SNpc. Total numbers of TH-positive (TH+)cells were estimated with the optical disector method (Gundersen, 1977, 1986; Gundersen and Jensen, 1987; Gundersen et al., 1988; West and Gundersen, 1990; West et al., 1991) according to Oorschot (1996). Sections used for counting covered the entire substantia nigra. The borders of the SNpc at all levels in the rostrocaudal axis were defined at 4× magnification. The medial border was defined by a vertical line passing through the medial tip of the cerebral peduncle and by the medial terminal nucleus of the accessory nucleus of the optic tract, when present in sections, thereby excluding the TH-positive cells in the ventral tegmental area (VTA). Ventral border followed the dorsal border of the cerebral peduncle, thereby including the TH-positive cells in pars reticularis (SNpr), and the area extended laterally to include the pars lateralis in addition to pars compacta (Kirik et al., 2001). One-third of the sections through the substantia nigra stained with TH immunohistochemistry was evaluated under 40× magnification (objective: numerical aperture, 0.85). The measurements in the z-axis were done using the electric microcator (ND 281; Heidenhain, Traunreut, Germany) of the Olympus Optical CAST-Grid system. The counting frame was placed randomly on the first counting area and systematically moved through all fields. Only the profiles that came into focus within the counting volume were counted. The estimate of the total number of neurons was calculated according to the optical disector formula (for more details, see Oorschot, 1996). The coefficient of error was calculated according to Gundersen and Jensen (1987), and values <0.10 were accepted. TH-positive cells were counted only when the nucleus could be identified.
The x-step length of 320 μm and a y-step length of 210 μm between the samples were chosen, and a rectangular counting frame of 26.3% (exact size of the counting frame, 0.01728 mm2) of the total visual field was used. All neurons that were found inside of the counting frame or touching the inclusion lines (upper and right sides) were counted, and neurons touching the exclusion lines (bottom and left sides) or located outside the counting frame were excluded. All of the results (number of counted neurons, the number of samples, and the area of the counting frame) were collected in a table for additional analysis. The formula for the calculation of the total number of neurons in a selected region was based on stereological methods. The formula used was V(ref) × NV = N (unbiased estimate of the total number of neurons) (for details, see Oorschot, 1996). Our mathematical approach was N = I × II × III × IV, where I is total number of counted neurons in the selected area. Thus, I = NV. Hence, II × III × IV = Vref: I, the total number of counted neurons in the selected area was multiplied with II. II, The total number of histological series in which the nucleus was divided was multiplied with III. III, Optical disector is the result from the division between the real physical thickness of the evaluated slice, which is given by the electronic microcator from the actual evaluated slice thickness (real physical slice thickness − excluded bottom and top areas) (the guard zones were 5 μm from the bottom and 3 μm from the top) was multiplied with IV. IV, The area evaluated was estimated as the product of the x-step length and y-step length divided by the area of the counting frame. This resulted in the total estimated DA neuron number within this basal ganglia.
Volume measurement.
The volume of the SNpc was measured by outlining the areas of the SNpc on each section (magnification 10×). The SN was outlined on the computer screen using the software AnalySIS Pro (version 3.00; Soft Imaging System, Münster, Germany). Afterward, the total volume was calculated by multiplying the area of each section with three (because we only used every third section) and multiplying the result with 18 μm (thickness of the slice after shrinkage; before, 30 μm). Both the results and figures show the volume after shrinkage, and, therefore, to get the physiological volumes, one has to multiply all shown values with 1.7. In addition to the volume measurement, the area of both SNpc and SNpr were measured using the same system but only at three different coronal levels with regard to the bregma. The latter approach was performed to compare both parts of the SN.
Cell density.
The cell density was calculated by dividing the counted cell number by the measured volume.
TH Western blot
Western blotting (n = 3) was done after homogenization of the tissue (one hemisphere per probe, excluding the rostral part of the brain and the cerebellum) and separation of it using an 8% SDS-PAGE. The same amount of protein was loaded on each gel. Western blot analysis was performed as described previously (Meisinger et al., 1996). Detection of TH was done using a monoclonal anti-TH antibody (1:25,000; Sigma) and the enhanced chemiluminescence system from Amersham Biosciences (Braunschweig, Germany). Coomassie gels and β-III-tubulin lanes (on the same blot as TH; blot was cut before primary antibody) were used as a loading control (each lane, 100 μg of total protein). Blot bands were photographed and quantified using Scion (Frederick, MD) Image by density measurements.
Terminal 6-OHDA lesion
For the second part of the experiment, FGF-2 ko and TgFGF-2 mice received terminal unilateral 6-OHDA lesions to quantify the number of DA neurons, which survive this kind of intrastriatal lesion comparing mutant and wild-type mice. All surgical operations were performed under intraperitoneal anesthesia by using 10% ketamine/2% Rompun (0.1 ml/10 g). The animals received two stereotaxic injections of 6-OHDA at a concentration of 3 μg/μl (in 0.2 mg/ml l-ascorbate saline) using a 10 μl Hamilton syringe plus glass capillary into the right striatum (for coordinates, see Table 2) (Lundblad et al., 2004). The injection rate was 0.5 μl/min, and the cannula was left in place for an additional 3 min before slowly retracting it. The amount and concentration of the toxin was evaluated before the main experiment to fulfill the following two criteria: (1) reduce the number of DA neurons in wt mice to ∼50%; (2) get the lowest possible mortality rate at the same time. The mice were killed 2.5 months after lesion.
Data are expressed as means ± SEM. Statistical analysis was performed using StatView 5.0 for Windows (SAS, Cary, NC). Comparisons between wild-type and mutant animals were performed with an unpaired Student's t test after testing normality. A p value of <0.05 was defined as significant.
DA neuron numbers differ between mouse strains
We used three different mouse mutants to analyze the influence of FGF-2 on the nigrostriatal system during development: (1) FGF-2 knock-out mice (FGF-2−/−); (2) FGF-2 overexpressing mice (TgFGF-2), and (3) mice with a reduced level of FGFR3. Because the different mouse mutants of the FGF-2 system displayed different genetic backgrounds, the morphometric parameters of the SNpc were compared between the different strains (Table 1).
The number of DA neurons was quantified in ∼2.5-month-old adult mice. Comparing the wild types of these three strains, the results displayed different DA cell numbers depending on the mouse strain analyzed (Table 3). FVB/N mice (TgFGF-2 wt) have 8032.25 ± 414.49 nigral DA neurons, whereas 129P2 mice (FGF-2 ko wt) only have 6057.2 ± 283.50 DA neurons. This difference is significant (p < 0.001). C57BL/6 mice (FGFR3 wt) showed the highest number of nigral neurons with 8350.5 ± 448.86 neurons per SNpc, which was significantly higher than FGF-2 ko wt (p < 0.0005). TgFGF-2 wt mice and FGFR3 wt ones do not differ and show similar DA cell numbers (difference of 3.9%; p = 0.6; F = 5.684).
The volumes of the SNpc were nearly equal between the different strains (p > 0.05). The exact values are depicted in Table 3. These data suggest that the volume of the SNpc is subject to stronger adjustments than the neuron number. Consequently, the cell density also shows significant differences between the three strains. The lowest density was found in FGF-2 ko wt mice. TgFGF-2 wt mice DA neuron density was 1.06-fold greater (p > 0.05, not significant), and the density in FGFR3 wt mice was 1.28-fold greater than in FGF-2 ko wt mice (p < 0.0001; F = 13.756). The DA densitometric difference between FGFR3 wt and TgFGF-2 wt was significant (p = 0.0005) (absolute values, see below).
Number of DA neurons reduced in FGFR3-deficient mice
Recent studies showed an important functional relevance of FGFR3 in the PNS. After peripheral nerve injury, FGFR3 was upregulated in contrast to the other FGF-2 receptors (Ji et al., 1995; Grothe and Nikkhah, 2001). Furthermore, this receptor is crucially involved in the regulation of survival of sensory neurons after lesion but not during peripheral nerve development (Jungnickel et al., 2004a, 2005). Regarding the CNS, it has been shown that the high-affinity tyrosine kinase receptor FGFR3 is expressed in the substantia nigra (Claus et al., 2004). In contrast to the PNS, FGFR3 expression displayed no alterations after 6-OHDA medial forebrain bundle lesion (Claus et al., 2004).
Therefore, we wanted to analyze the role of FGFR3 concerning the development of the nigrostriatal system. The SNpc was evaluated in terms of (1) DA neuron number, (2) SNpc volume, and (3) the density of DA neurons within the SNpc. Interestingly, FGFR3+/− mice (n = 6) displayed a significant lower number of DA neurons compared with their wild-type littermates [+/−, 6692.94 ± 307.3 (n = 5) vs wt, 8350.50 ± 448.9 (n = 6); p < 0.01; F = 8.541] (Fig. 2A,C,E). This decrease in DA neuron number after development in mice with a reduced level of FGFR3 suggests a physiological function for this receptor during development. The volume of the SNpc was not altered in FGFR3 mutant mice: the nigral volume of FGFR3+/− mice was 0.12 ± 0.005 mm3, and the volume of FGFR3wt was 0.12 ± 0.007 mm3 (p > 0.05; not significant; F = 0.001) (Fig. 2A,C,F). Consequently, the neuronal cell density was significantly decreased in FGFR3+/− (56,568.17 ± 2,764.9 DA neurons/mm3) compared with the corresponding wild type (FGFR3wt, 65,451.33 ± 2078.2 DA neurons/mm3; p < 0.05; F = 6.842) (Fig. 2B,D,G).
FGF-2 level is reciprocal to number of DA neurons
FGF-2 is a potent survival factor and is, for example, used as mitogen for the expansion of DA progenitor cells in vitro (Studer et al., 1998; Timmer et al., 2006) and promotes the survival of grafted embryonic DA neurons in vivo (Mayer et al., 1993b; Timmer et al. 2004). However, only little is known about the physiological role of FGF-2 on DA neurons and the nigrostriatal system. To study the influence of FGF-2 on the development of the nigrostriatal system, we quantified the number of DA neurons. Mice lacking FGF-2 (FGF-2 ko; n = 6) resulted in 7170.60 ± 228.9 DA neurons, whereas wild-type mice (FGF-2 ko wt; n = 6) only resulted in 6057.20 ± 283.5 (Fig. 3A,C,E). This result suggests that mice without the important mitogen and survival factor FGF-2 show a significant increase in the number of nigral neurons during development, which remains during adulthood (p < 0.01; F = 9.336). Moreover, mice overexpressing FGF-2 displayed a reduction of dopaminergic neurons after development [TgFGF-2 (n = 6), 6902.23 ± 414.2 DA neurons compared with TgFGF-2 wt (n = 6), 8032.25 ± 414.5] (Fig. 4A,C,E). However, the latter difference was not significant (p = 0.06; F = 3.719). Summarizing these results, it appears that an inverse correlation exists between the number of DA neurons and the level of FGF-2 during development.
Volume of SNpc enlarged in both FGF-2 ko and Tg mice
The volume of the SNpc was relatively constant between the different mouse strains (see above). Therefore, the question whether endogenous FGF-2 can change the nigral volume is of importance. The volume of the SNpc in wild-type FGF-2-deleted mice was 0.120 ± 0.009 mm3 (Fig. 3C,F). The volume in the corresponding mutant strain (FGF-2 ko) was significantly enlarged (0.143 ± 0.006 mm3; p < 0.05; F = 4.628) (Fig. 3A,F). These data suggest that FGF-2 is crucially involved in the development of a proper sized SN. Moreover, the nigral volume was also significantly bigger in mice overexpressing FGF-2. The volume in TgFGF-2 mice was 0.177 ± 0.007 (wt, 0.134 ± 0.005; p < 0.001; F = 24.438) (Fig. 4A,C,F). An alteration of the endogenous FGF-2 concentration seems to result in an enlargement of the SNpc regardless of whether more or less FGF-2 is present.
TH-positive cell densities in the SNpc in FGF-2 mouse mutants
The DA neuron density was unchanged in FGF-2 knock-out mice. The cell density within the SN in FGF-2 ko mice was 50,733.97 ± 2038.9 DA neurons/mm3 and 51,088.51 ± 1987.5 cells/mm3 in the corresponding wild type (p = 0.9; F = 0.016) (Fig. 3B,D,G). In contrast, transgenic mice displayed a highly significant difference (p < 0.0001; F = 23.407). TgFGF-2 wt mice showed a DA neuron density of 54,286.74 ± 1743.6 (Fig. 4D,G). TgFGF-2 have a reduced DA cell density of 39,536.72 ± 2500.9 (Fig. 4B,G). This difference in TH-positive cell density further supports the hypothesis that FGF-2 is important for the development of the substantia nigra.
TH expression in FGF-2 mutant mice
The expression of the DA rate-limiting enzyme TH was evaluated using Western blot analysis. The densities of the bands were measured (n = 3 animals per group) compared with the respective wild-type animals (set as 100%). The same amount of protein was loaded on each lane. This was determined before and confirmed by a Coomassie gel. FGF-2−/− mice displayed increased TH expression levels (112 ± 8.8%) compared with wild-type mice (100 ± 5.9%; p > 0.05) (Fig. 3H). Two examples of TH Western blot of transgenic mice are depicted in Figure 4H (of n = 3). Transgenic mice show decreased TH expression levels of 73 ± 10.9% compared with wt animals (100 ± 8.6%). This difference was significant (p < 0.05) (Fig. 4I). These data confirm the results of DA neuron counting and also suggest an inverse relationship between the TH and the FGF-2 levels in adult mice.
Substantia nigra: compacta and reticularis
In addition to the measurement of the volume of the SNpc of the whole midbrain, we measured the area of the SNpc and of the SNpr at three different coronal levels of the midbrain in FGF-2 ko mice (Fig. 5A). The question was whether the increased nigral size was attributable to a change in the rostral, medial, or occipital part of the SN and whether the pars reticularis was also affected. Compared with wt mice (wt was set as 100%), FGF-2 ko mice displayed a 16% increase of the SNpc area at −2.9 mm, a 26.9% increase at −3.3 mm, and a 23.7% increase at −3.8 mm from the bregma. All three levels showed a significant area increase of the SNpc (p < 0.05), indicating that the SNpc is enlarged throughout the midbrain (Fig. 5B). In contrast, the pars reticularis was not affected. FGF-2 ko mice displayed the following area values of the SNpr: rostral, 108.3 ± 9%; medial, 104.2 ± 11%; and occipital, 110.9 ± 9% (p > 0.05).
Less surviving DA neurons in FGF-2 ko mice after lesion
To understand the physiological role of FGF-2 after CNS lesion could have impact on the development of new therapeutic strategies. Here, we addressed the question of whether endogenous FGF-2 could protect DA neurons from 6-OHDA neurotoxicity. For this purpose, the number of DA nigral neurons was quantified after striatal 6-OHDA lesion.
It has been shown recently that FGF-2 immunoreactivity increases in reactive astrocytes after a striatal 6-OHDA-induced partial lesion of the nigrostriatal system (Chadi and Gomide, 2004). In addition, FGF-2-producing fibroblasts protect the nigrostriatal DA system from 6-OHDA-lesioned rats (Shults et al., 2000). In the present study, the lesion parameters (6-OHDA concentration used and coordinates) were chosen to destroy ∼50% of DA neurons in wild-type mice. FGF-2 ko wt mice result in 2749.6 ± 151.1 surviving DA neurons, whereas in FGF-2−/−, only 2144.3 ± 137.0 survived the terminal lesion (Fig. 6A–C). This significant difference (p < 0.05) suggests that other trophic factors are not able to compensate for the missing endogenous FGF-2 after lesion. The difference becomes even more significant when the lesioned hemisphere is compared with the nonlesioned side, because unlesioned wild-type mice display significantly less DA neurons than FGF-deleted mice (see above): 45.3% of the DA neurons survived in FGF-2 ko wild-type mice, whereas only 29.9% survived in FGF-2 ko mice lacking the protein (Fig. 6C).
DA neurons rescued in TgFGF-2 mice after 6-OHDA lesion
Mice overexpressing FGF-2 were able to protect DA neurons from 6-OHDA neurotoxicity, because 3657.1 ± 128.4 DA neurons survived the 6-OHDA lesion in wild-type mice, whereas in TgFGF-2 mice, 4023.9 ± 166.7 DA neurons survived the lesion (Fig. 7A–C). This significant difference (p < 0.05) also becomes more significant when the lesioned hemisphere is compared with the nonlesioned side, because unlesioned wild-type mice displayed more DA neurons than transgenic mice (see above). A total of 45.5% of the DA neurons survived in TgFGF-2 wt mice, whereas 58.3% survived in TgFGF-2 overexpressing the protein (Fig. 7C). These results suggest that endogenous FGF-2 can protect DA neurons from lesion-induced cell death.
TH expression levels after striatal 6-OHDA lesion
The TH Western blots confirmed the quantification of DA neurons after 6-OHDA lesion. A profound reduction of TH was found after terminal lesion (Figs. 6D, 7D). In addition, TH expression after 6-OHDA lesion was lowered in FGF-2 ko animals compared with wt mice (Fig. 6D). Furthermore, FGF-2 transgenic mice showed higher TH expression after lesion than the corresponding wt mice (Fig. 7D).
To study the relevance of the FGF-2 system in SNpc development, we used mice overexpressing FGF-2, without FGF-2 (ko) and lacking FGFR3, one of the high-affinity receptors of FGF-2. The present study revealed crucial functions of the endogenous FGF-2 system in the developing SNpc and after lesion, including four main findings: (1) an increase in DA neuron number in mice lacking FGF-2 and, moreover, a TH decrease in mice overexpressing the factor; (2) a vice versa situation under lesioning conditions in which more DA neurons were rescued in TgFGF-2 mice and less in FGF-2 ko ones; (3) an enlargement of the volume of the SNpc when the FGF-2 level was altered regardless whether this change was an increase or a decrease; and (4) a significantly lower number of DA neurons in FGFR3+/− deficient mice, which suggests an involvement of FGFR3 signaling during development (Table 4).
Striatal 6-OHDA lesion provides a useful tool
Inducing PD in animal models can be achieved by different methods such as 6-OHDA, rotenone, paraquat, and maneb (Betarbet et al., 2002; Soderstrom et al., 2006). The most common one in mice is MPTP treatment. Most studies analyzing mutant mice regarding the nigrostriatal system use this approach (Sriram et al., 2002; Zechel et al., 2006). This technique is easily applicable but has several disadvantages, including a recovery of motor impairments after some days and enhanced de novo DA neurogenesis after MPTP lesion (Shan et al., 2006). Furthermore, mice are less sensitive to MPTP treatment compared with primates, and different mouse strains are differently sensitive to MPTP toxicity, with the highest sensitivity in C57BL/6. The degree of the damage is also age related (Gerlach et al., 1991; Schober, 2004). To overcome these limitations, we used the 6-OHDA lesion, which has to be performed stereotactically. Normally, this method is only used in rats, because they are insensitive against systemic MPTP applications attributable to a high endothelial monoamine oxidase-B activity. The common 6-OHDA medial forebrain bundle lesion completely destroys all DA projections (Ungerstedt and Arbuthnott, 1970). More recently, terminal lesions of the rat nigrostriatal pathway induced by intrastriatally injected 6-OHDA have been developed (Sauer and Oertel, 1994; Lee et al., 1996; Kirik et al., 1998; Rodter et al., 2000). This animal model of PD has the advantages that (1) one can directly influence the extent of the lesion by altering the dose given, and (2) this kind of lesion mimics early stages of the disease and is therefore useful for the development of neuroprotective therapies.
FGF-2 as a key factor: influence of the FGF-2 level on DA neurons
FGF-2 and its high-affinity tyrosine kinase receptors (FGFR1–FGFR3) are expressed in the developing and adult mesencephalon and especially in the SN (Bean et al., 1991; Belluardo et al., 1997; Bansal et al., 2003). FGF-2 displays different isoforms with different subcellular distributions and functions (Stachowiak et al., 1994; Timmer et al., 2004). In the mutant mice analyzed (transgenic and knock-out), all three FGF-2 isoforms (18, 21, and 23 kDa) are deleted or overexpressed, respectively.
FGF-2 mainly acts as a survival factor and mitogen in many tissues, including the PNS and CNS (Xu et al., 2005). On the one hand, it is upregulated in several carcinogenic cells (Ueba et al., 1994), and, on the other hand, it is used to stimulate the survival of grafted (stem) cells in regenerative medicine. In PD, it was successfully applied to enhance the survival and differentiation of transplanted embryonic DA cells (Takayama et al., 1995; Timmer et al., 2004), and it was taken as mitogen to expand neural progenitor cells before differentiation (Studer et al., 1998; Timmer et al., 2006). Moreover, FGF-2 promotes neurogenesis and neuroprotection (Jin et al., 2005). On the basis of these facts, an increase in DA neuron number in mice overexpressing FGF-2 and a decrease in FGF-2-deleted mice could be expected. Interestingly, the opposite was found in this study. Significantly more DA neurons were found in FGF-2 ko mice. A possible mechanism explaining this finding could be that the lack of FGF-2 is (over) compensated during development by other trophic factors, which could be upregulated. Such compensatory regulations are reported from many knock-out mice displaying only some deficits and nearly normal phenotypes.
The compensation of FGF-2 in FGF-2−/− mice could theoretically occur through (1) other members of the FGF family or (2) other trophic factors [for example, brain-derived neurotrophic factor (BDNF)]. The latter possibility is unlikely, because no compensation takes place when the FGF receptor is deleted, suggesting that other FGFs are responsible for the compensation (see below). To date, the FGF proteins are encoded by 22 distinct genes known as Fgf1–Fgf18 and Fgf20–Fgf23 in the mouse species (Dono, 2003). Human and chick Fgf19 is the ortholog of mouse Fgf15 (Wright et al., 2004). To evaluate a putative candidate for the compensation of FGF-2, first all FGF members have to be excluded that are not expressed in the SN. Most FGFs are expressed in the brain, whereas FGF-21 is preferentially expressed in the liver (Nishimura et al., 2000) and FGF-22 in the inner root sheath of the hair follicle (Nakatake et al., 2001). Other FGF family members are only expressed in distinct parts of the brain, such as FGF-23, which is expressed in the ventrolateral thalamic nucleus (Yamashita et al., 2000). Many studies showed that FGF-4 is not expressed in the brain, but a recent study revealed localization of mouse Fgf4 mRNA in the hippocampus, subventricular zone, and the rostral migratory stream in which new neurons are generated and migrate (Kosaka et al., 2006). The Fgf8/Fgf17/Fgf18 subgroup of Fgfs mainly have midbrain and cerebellum regulatory properties in very early stages of development (Reifers et al., 2000; Liu et al., 2003), but there is some evidence that they are also expressed during later stages in the SN. In addition, FGF-1, FGF-9, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, and FGF-20 are expressed in the SN and could be responsible for the compensation when FGF-2 is deleted (Todo et al., 1998; Ohmachi et al., 2000; Dono, 2003). FGF-3, FGF-5, FGF-6, FGF-7, FGF-10, and FGF-16 could theoretically also compensate FGF-2, but, so far, no evidence has been published that these FGF members are expressed in the SN.
Concerning the volume of the SN, it seems that overcompensation of the deleted FGF-2 by other FGFs leads to a similar effect as overexpressed FGF-2 in transgenic FGF-2 mice. That both FGF-2 and other FGFs (in FGF-2 knock-out mice) play a crucial role on the size (in terms of an enlargement) of the SN is very likely.
In mice overexpressing FGF-2, we found a nonsignificant decreased DA cell number and, in addition, a significantly lower TH expression level, suggesting that too much FGF-2 may be harmful for DA neurons during development. A recent finding supports this hypothesis because it was shown in vitro that increased concentration of exogenous FGF-2 leads to reduced outgrowth of human embryonic stem cell (hESC) colonies with time in culture (Dvorak et al., 2005). In addition, we have shown recently that FGF-2 can exhibit toxic effects on transfected DA progenitor cells overexpressing FGF-2 (Cesnulevicius et al., 2006). Moreover, continuous exposure of glial cell line-derived neurotrophic factor (GDNF) to mature DA transplants impairs the ability of the graft to improve spontaneous motor behavior in parkinsonian rats (Winkler et al., 2006).
The SNpc and its trophic factors
Several growth factors promote the survival of mesencephalic dopaminergic neurons such as GDNF, a member of the transforming growth factors β superfamily, neurotrophins including brain-derived neurotrophic factor (BDNF), and low molecular compounds mimicking growth factor activities (Krieglstein, 2004). Currently, the available data support GDNF to be a highly promising candidate for the treatment of PD in terms of exogenous application [e.g., viral (Kirik et al., 2004)]. However, GDNF-null mutants lack a nigral DA phenotype (Granholm et al., 1997). In another experimental approach by transplanting fetal neural tissues from GDNF−/− into wild-type mice, it was demonstrated that the continued postnatal development of these grafted DA neurons was severely disturbed in terms of reduced DA neuron numbers and fiber outgrowth (Granholm et al., 2000).
Wnt-BDNFko mice revealed a persistent 23% reduction of TH+ cells at postnatal day 21 (P21) compared with controls. The reduced number of TH-expressing neurons was present at birth and continued through P120 (Baquet et al., 2005). Heterozygous mice (BDNF+/−) show no reduction in TH+ cell numbers, and BDNF−/− mice are postnatal lethal.
It was reported recently that FGF-2 deficiency alters neither the DA cell density nor the vulnerability of DA neurons toward MPTP intoxication, which was evaluated on the basis of TH+ cell density (Zechel et al., 2006). In accordance with our results, they found no difference between unlesioned FGF-2 null mutants and wild-type mice. As we could show in this study, the unaltered cell density in FGF−/− is attributable to an increased overall DA neuron number and, simultaneously, an enlarged volume. After lesion, Zechel et al. (2006) found no difference of the TH+ cell density, whereas we found a significant lower number of TH+ cells afterward. This apparent discrepancy could be attributable to (1) different lesion models (Zechel et al., 2006, MPTP; this study, unilateral, terminal 6-OHDA) and (2) different kinds of analysis (Zechel et al., 2006, TH+ cell density; present study, stereologically evaluated absolute TH+ cell number). Our results suggest that endogenous FGF-2 is a physiological key player within the nigrostriatal system important for the development and protection/repair during adulthood. This finding is supported by previous studies in which the most striking difference in patients suffering from PD was found in FGF-2 expression: a profound depletion of FGF-2 [from 82% (control brains) down to 12.7% (PD brains)] in the remaining DA neurons was found (Tooyama et al., 1994), whereas BDNF was only reduced from 65 to 43% (Parain et al., 1999). In situ hybridization failed to show any detectable levels of GDNF in PD or in control cases, suggesting that GDNF is not a major physiological factor during adulthood (Hunot et al., 1996). These postmortem studies are in good correlation with the present null mutant studies. FGF-2 seems to be one of the first growth factors that affects the nigrostriatal system during development, with an effect throughout adulthood.
It is not very likely that a single neurotrophic factor supports survival of a specific cell population. Neuron losses in BDNF/GDNF double mutants are not additive compared with the losses in single null mutants, indicating that in vivo survival in many cells, including DA neurons, is supported by more than one factor (Erickson et al., 2001). However, additional studies are needed to understand the interplay between the different growth factors and to exactly evaluate which factor is able to compensate or support each other. Double-knock-out mice could help to address this question.
FGFR3 involved in postnatal DA neuron survival
The present study shows a significant decrease of DA midbrain neurons in unlesioned FGFR3 knock-out mice (+/−), suggesting an involvement of FGFR3 during DA neuron development. This finding could mean that FGFR3 is involved in DA neuron development. Expression analyses of FGFR3 during embryonic development revealed that FGFR3 displayed a diffuse expression throughout the brain at E14 (Claus and Grothe, 2001). Postpartum, there is a wave of FGFR3 mRNA expression in the normal postnatal brain that spreads from the hindbrain to the forebrain. At P1, FGFR3 was expressed diffusely and localized in cells (Peters et al., 1993). At P2, FGFR3 showed little expression in the midbrain. By P4, the FGFR3 mRNA expression increased in the midbrain (Oh et al., 2003), where it remained during adulthood (Belluardo et al., 1997; Oh et al., 2003; Claus et al., 2004; Timmer et al., 2004). This FGFR3 expression pattern suggests that FGFR3 does not play an important role during embryonic midbrain development but could be an important player after birth. A similar result was shown recently in which the postnatal expression pattern of FGFR3 parallels the appearance of differentiated oligodendrocytes (OLs), and the absence of FGFR3 expression in FGFR3 null mutants resulted in reduced numbers of differentiated OLs (Oh et al., 2003). Interestingly, significant neurophysiological and neuropharmacological changes, including DA neuron cell death, take place within the SN in the first postnatal month (Tepper et al., 1994). Tepper et al. could show that the number of TH+ neurons decreased significantly in the SN from P1 to P14, with the most striking effect between P1 and P7. This time frame correlates with the increase of FGFR3 expression in the SN (see above), indicating that FGFR3 could have the highest impact on the development of DA neurons during the first week after birth as survival-promoting factor. Nevertheless, the alterations must also have an effect during embryonic stages because the effect is too big and can therefore not only be explained by postnatal changes. The ∼20% reduced DA neuron number in FGFR3+/− knock-out mice demonstrates that FGFR3 can, in contrast to FGF-2 (see above), not be compensated (e.g., by FGFR1 or FGFR2).
One major question is whether this dramatic effect is mediated via FGF-2 or via another ligand. FGF-1–FGF-3, FGF-5–FGF-18, and FGF-20 are possible candidates because of their expression pattern (see above; this list includes all possible candidates). The intracellular FGF-11 to FGF-14 do not bind to FGFRs and can therefore be excluded (Dono, 2003). FGF-19, the ortholog of FGF-15, displays unique specificity for FGFR4 (Xie et al., 1999). FGF-6 is only expressed at very low levels in the brain, making this candidate very unlikely (Ozawa et al., 1996). The FGF ligands bind to the different FGF receptors with different affinities. FGF-3, FGF-7, FGF-10, and FGF-18 do not bind to FGFR3 or only with a low affinity (Ford-Perriss et al., 2001). However, a more recent publication showed that FGF-18 can bind to FGFR3 (Zhang et al., 2006). One of the most promising protein is FGF-20, because it is preferentially expressed in the substantia nigra pars compacta (Ohmachi et al., 2000), and it promotes the differentiation of Nurr1-overexpressing neural stem cells into TH+ neurons (Grothe et al., 2004). Up to now, it was thought that FGF-20 preferentially acts via FGFR1c (Ohmachi et al., 2003), but a new binding study revealed that the binding affinity between FGF-20 and FGFR3 is ∼90% (Zhang et al., 2006), making it a "high potential" candidate for compensating FGF-2 in FGF-2 ko mice. Thus, FGF-1, FGF-2, FGF-5, FGF-8, FGF-9, FGF-16, FGF-17, FGF-18, and FGF-20 could act via FGFR3 in the SN during postnatal development. However, to date, no published data showed FGF-5 and FGF-16 in the SN, making both candidates unlikely. FGF1 would be a good candidate for compensating FGF2, but it has been shown previously that FGF1 does not compensate FGF2 during development because FGF1–FGF2 double-knock-out mice were viable and fertile and did not display any gross phenotypic defects other than seen in FGF2 knock-out mice (Miller et al., 2000). Taking all of these results from literature and this study together suggest that the FGF-8/FGF-17/FGF-18 and the FGF-9/FGF-20 subfamilies might be the candidates that (over)compensate FGF-2 in FGF-2 ko mice (see above).
FGFR3 is a transmembrane tyrosine kinase receptor mediating the extracellular FGF signal. The lacking receptor prevents secreted FGFs (2, 8, 17, 18, 9, 20) from entering the cytoplasm and acting in an autocrine/paracrine manner. Thus, deleted FGFR3 knocks down most FGFs at the same time. This would explain why no compensation, like in FGF-2 ko mice, is possible and at the same time confirms the hypothesis that, in FGF-2 ko mice, other FGFs come into play. The results also suggest that the other two FGF receptors [FGFR1 and FGFR2; FGFR4 is not present in the nigrostriatal system (Timmer et al., 2004)] cannot fully compensate the lack of FGFR3 or may fulfill different functions (Corso et al., 2005; Klejbor et al., 2006). This idea is supported by an oligodendrocyte study showing that distinct FGF/FGFR signaling pairs initiate diverse cellular responses in cell lineage (Fortin et al., 2005).
FGFR3c is preferentially found in glia but also in DA neurons (Yazaki et al., 1994), suggesting an indirect effect. We have shown previously that FGFR3 was not reduced after 6-OHDA lesion (Claus et al., 2004), suggesting that the majority of the FGFR3 population might be located on cells other than DA neurons. This finding further supports the hypothesis of an indirect (i.e., glia-mediated) effect. Moreover, released FGF (e.g., FGF-2) must be crucially involved in DA neuron development and survival, because intracellular FGF-2 is not affected in FGFR3 knock-out mice but the extracellular cannot act as ligand without its receptor. This consideration is supported by a recent study in which FGFRs were inhibited. The resultant inhibition of FGFR suppresses activation of downstream protein kinases and causes rapid cell differentiation, suggesting an involvement of autocrine FGF signals in the maintenance of proliferating hESCs in the undifferentiated state (Dvorak et al., 2005).
The finding of the present study, that released FGF is important, is supported by a recent publication showing that autocrine FGF-2 signaling is critical for self-renewal and differentiation of mesenchymal stem cells (Zaragosi et al., 2006). Moreover, extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway is involved in FGF-2-mediated self-renewal but not in differentiation. Another study showed that rotenone-induced apoptosis in DA cells was attenuated by pretreatment with FGF-2 via activation of ERK1/2 and phosphatidylinositol-3 kinase (PI3-kinase) pathways (Hsuan et al., 2006). Ectopic activation of ERK1/2 or PI3-kinase protected against rotenone, whereas inhibition of either pathway attenuated FGF-2 protection. These data indicate that second-messenger systems play a crucial role in this scenario. FGFR3 is expressed as two main splice isoforms (IIIb or IIIc). We have shown previously that FGFR3 mediates neuronal death in the PNS (Jungnickel et al., 2004a), whereas this study suggests that FGFR3 supports survival and inhibits naturally occurring apoptosis during development. How signaling through FGFR3 can lead to apoptosis, differentiation, or cell proliferation is not fully understood. It seems that STAT1 (signal transducer and activator of transcription 1) phosphorylation is involved in inhibition of proliferation, whereas activation of the ERK pathway inhibits differentiation (L'Hote and Knowles, 2005). Some of the cell specificity may arise via modulation of signaling by crosstalk with other signaling pathways. Together, FGFR3 seems to be essential for (at least) a ∼20% subpopulation of DA neurons during development.
Different mechanisms during development and after lesion?
Our data after terminal 6-OHDA lesion clearly indicate a physiological neuroprotective function of FGF-2 because significantly more DA neurons survived after lesion in mice overexpressing FGF-2 compared with wild-type mice. This result together with the finding that released FGF-2 seems to be important in this context (see above) confirms the physiological trophic effects of FGF-2 on DA neurons and justifies the exogenous application of FGF-2 to (1) prevent the degeneration of the remaining DA neurons in PD patients or (2) enhance DA graft survival and benefit. Moreover, these findings encourage the development of new FGF-2-mediated neuroprotective therapies for neurodegenerative disorders. The observed effects, at least in FGF-2 ko mice, seem to be specific to DA neurons and not systemic/general because other studies showed different results for other brain regions/neuronal populations [e.g., impaired cerebral cortex development (Dono et al., 1998)].
After lesion, significant more DA neurons died in FGF-2 ko mice than in wild-type mice. This further underlies the survival-promoting and neuroprotective effect of FGF-2. In contrast to the situation during development, the compensation mechanisms (e.g., upregulation of other members of the FGF family) seem to fail or at least are not as sufficient as in fetal/neonatal animals. These results verify that plasticity and repair mechanisms are much more powerful in developing systems. Furthermore, this is a fundamental finding for neuroprotective and restorative therapies because it shows restrictions in terms of limited endogenous repair functions and could help to overcome these limitations.
The present findings not only depict the function and relevance of FGF-2 but also support the hypothesis that most biological systems are redundant, and even one of the most important survival factors seems to be (over)compensated. However, this compensation is by far not perfect and fails when lesioning adult mice.
Thus, FGFR3 seems to be crucial during (postnatal) development of DA neurons, and FGF-2 seems to fulfill an unique physiological function within the nigrostriatal system after lesion. It has to be further analyzed whether FGF-2 is supportive or essential. This gain in knowledge could help for a better understanding of how FGF-2 regulates DA survival and differentiation and how compensation mechanisms come into play or fail. In addition to development, the neuronal changes observed in adult mice could also reflect an altered cell homeostasis.
Moreover, these findings are also relevant to exogenous FGF-2 applications [e.g., transplantation experiments (Cesnulevicius et al., 2006)] because detailed information about the growth factors used is necessary for a proper application. Together, these data could help to develop new therapeutic approaches for PD, including protective ones. Moreover, these results exemplify how narrow the range between toxic and beneficial effects of e.g., growth factors is.
We are very grateful to Dr. J. D. Coffin (University of Montana, Missoula, MN) and Dr. S. Wolfram (Max-Planck-Institute, Bad Nauheim, Germany) for providing the TgFGF-2 breeders. We acknowledge the excellent technical assistance of G. Braun, H. Brinkmann, N. Heidrich, K. Kuhlemann, and J. Wittek. We thank Dr. K. Haastert for critical reading and comments on this manuscript.
- Correspondence should be addressed to Prof. Dr. Claudia Grothe, Hannover Medical School, Department of Neuroanatomy, Center of Anatomy, OE 4140, Carl-Neuberg-Strasse 1, 30623 Hannover, Germany. grothe.claudia{at}mh-hannover.de


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Ramirez out of lineup, day after Uggla squabble
MIAMI -- Hanley Ramirez was sitting at his locker in the Florida Marlins clubhouse, earphones on, stoic and staring straight ahead at the movie "Scarface" playing on his laptop.
If anyone was questioning the NL batting leader's effort and desire to win, he couldn't hear it.
Ramirez was out of the lineup with a tight left hamstring for the second straight game against Atlanta on Thursday, a day after teammate Dan Uggla openly argued with the All-Star shortstop over a perceived lack of commitment.
Ramirez could miss a few more games, but the public bashing has at least stopped for now.
"Ever since we got everything in the open yesterday, we're fine," said Uggla, sitting at his locker about 10 feet away from Ramirez, in the same spot the verbal spat occurred almost exactly 24 hours earlier.
That was a far different scene than the one that unfolded Wednesday.
Ramirez had said he "got some people upset" for leaving Tuesday night's game with the injury. Overhearing the comments, Uggla called out Ramirez by saying he was one of them.
The two exchanged words, and Uggla went on to question how hurt Ramirez really was.
He also said Ramirez wasn't caring because he's already secured a $70 million, six-year contract -- by far the biggest contract on baseball's thriftiest team.
The squabble prompted manager Fredi Gonzalez to close the clubhouse for a closed-door meeting. Ramirez turned down interview request Thursday, but Gonzalez said he met with his double-play tandem separately and worked out any differences.
Gonzalez also said Ramirez could miss a few more games.
"(Ramirez) feels a little better. Hopefully, we can get him on the field and he can take some (practice) balls," Gonzalez said.
The Marlins were 4½ behind Colorado for the NL wild card before the game. But even in the midst of a playoff race, teammates said the feud won't have a lasting impact.
Center fielder Cody Ross, whose locker is between the pair, compared the incident to a fight with his wife that was better to get out in the open.
"We're constantly screaming at each other," Ross joked. "It's good. We get on each other, and I think it builds camaraderie. We all love each other and support each other. Hopefully, maybe it lights a little fire under us and we can get on a roll."
Wes Helms, who hit the game-ending home run in Florida's 8-7 win Wednesday night that provided some temporary relief, also downplayed any tension.
"We're a big family. It's just like us and kids. We have to punish our kids, but they love us. It's one of those things about going as a team. It's a part of team chemistry," Helms said. "If you love something that much, you're going to fight."
The Marlins got some relief with first baseman Nick Johnson activated from the disabled list. He was in the lineup Thursday night. Right fielder Jeremy Hermida was out with a rib injury and will likely miss a few more games.
Still, it will be difficult to replace Ramirez's bat.
Ramirez has a league-leading .355 batting average with 19 home runs and 85 RBIs. He is perhaps the biggest reason Florida has a winning record with a lineup full of deficiencies and a rotation that has been a disappointment.
Ramirez's struggles of late -- he is in an 0-for-14 slump -- have only prompted speculation about how serious his injury is, which were part of Uggla's comments. But Uggla has since changed his tune, saying Ramirez is "like a brother" and that they have patched things up.
Uggla said he was glad he made the comments but should have kept them between teammates.
"I'm a firm believer that you do things like that," Uggla said, "and get them out in the open."


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The best womenswear street style from Milan Fashion Week — F/W 2019
Exclusive gallery: The best street style from Milan Fashion Week
Words— Marc Richardson
After hitting the shows in London and Florence, the fashion world has turned to Milan where it's always an interesting time. Marc Richardson spent the weekend there and documented what the most stylish women are wearing on the streets. Scroll below for a little dead-of-winter inspiration.
Don't miss Marc's exclusive shots from London and Florence. You can also follow him on Instagram.
Marc Richardson is a writer and photographer based in Montreal. Follow him on Twitter.


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Mid-Engine Corvette Comes Alive for $500,000
A company has decided to develop a mid-engine Corvette. It will produce 100 of them and they will cost $500,000. They are calling the Corvette, "Fast Eddy".
Photo from: http://www.carbuzz.com/resizeimg/imageshandler.ashx?w=640&h=480&url=http://db.carbuzz.com/images2/620000/2000/800/622806.jpg
Key Takeaways:
1. A company has decided to develop a mid-engine Corvette.
2. The car is limited as there will only be 100 of them made.
3. The car will cost $500,000.
Quote: "Aria hopes to build 100 units of the FE, priced around $500,000." (Lynton, 2017)
Link to article: http://www.carbuzz.com/news/2017/3/18/The-Mid-Engined-Corvette-Is-Already-Here-And-It-Looks-Sublime-7738297/


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Banknotes Misproduction security & biometric weakness
In the last months vulnerability lab team reviewed the new 20€ & 50€ banknotes of the european central bank. One of our core team researchers identified that for the security sign of the holograms are different components in usage. The security signs are build by the European Central Bank with several high profile elements in the signs to ensure, that the banknotes has a serious level of protection again fraud or fake money. After processing some time to identify an impact, we were finally able to identify the following security problematic.
Due to the last days "Eric Fanning" secretary of the us army announced, that the new "Hack the Army" Bug Bounty Program will start soon. In 2016 the "Hack the Pentagon" Program was disclosing about 140 vulnerabilities to the infrastructure of the department of defense and pentagon. All the valuable information of the successful program made the decision more easy for representatives to lunch the next new bug bounty program called "Hack the Army".
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Pezzo launches Teriyaki Salmon and brings back Durian King!~Invited Session~
The kind folks at Pezzo sent over a couple of pizzas and we were in for a treat! Unveiling their first pizza creation of 2018!
Teriyaki Salmon is a sweet savoury creation on a handcrafted pink dough topped with tender salmon chunks, cheese, teriyaki sauce and creamy mayonnaise!
On top of the very pretty pink hues, this creation is really tasty. Truth be told, my usual orders at any pizza joint are pepperoni and Hawaiian if curiosity does not get the better of me and I head straight for their specials.
Limited for a short period, this creation is served as a full 14" pan ($29.90) or sold in 3 slices or individually at $5.90 each. New sides this year include tempura chicken fries with nacho cheese that taste uncannily like luncheon meat and crispy garlic bread!
And they also reprised Durian King pizza this year! Made with a charcoal pizza dough and topped with a mix of 100% pure durian flesh of Mao Shan Wang and D24, this is the island's cheapest durian dessert I'd say!
Available in limited quantities, these go at $32.90 for a 14" pan, $16.90 for 3 slices or $5.90 a slice. Grab them whilst they last - durians aren't available the whole year long anyway!
Have you decided which to try? How about both!
Available at all Pezzo outlets.


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Iconic, high-tech men's sneaker that's ultra-breathable and extremely lightweight, boasting a youthful, dynamic design with box outsole.
Nebula™ has taken the traditional breathability concept one step further by adding a special lined interior with cavities to the characteristic large holes on the sole and combining this with the special breathable membrane: Nebula™ breathes in three different directions.
Allow yourself to be carried away by Nebula™'s lightweightness and breathable comfort. Its versatile design and streetwear vibe will take you far and keep you walking confidently.


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It may be a little bit complicated for all those just getting started with multilevel marketing. You will possibly not know very much about multilevel marketing or perhaps the ideal way to ensure your success. This short article will present you with many ways and suggestions to help you get started off. Take note of the recommendations under to have the guidance you need.
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You may travel lots of targeted traffic to your websites for multi-degree advertising and marketing when you use marketing with video. A youtube video is actually a cost effective strategy to create marketing activities which are content-wealthy.
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The 10 Best BBQ Joints in Louisiana! By
Penny Fox
Everyone loves the taste of barbecue, whether you enjoy a thick, bold flavor, mesquite, slightly tangy, or sweet sauce on your favorite beef, pork, chicken, or fish. Louisiana has some of the best in the country, and below are the 10 best barbecue joints in the state where you can satisfy your taste for some finger-licking, mouth-watering deliciousness.
1. The Joint, New Orleans, LA
If the thought of juicy slow-cooked pulled pork and beef brisket, chicken, sausage, and ribs make your mouth water, then you should visit the Joint in New Orleans to get your hunger satisfied. With great sides that compliment these great smoked barbecue choices like mac & cheese, cole slaw, and baked beans, you won't go home hungry from this national award-winning eatery that opened in 2004.
2. Lil' Daddy's Bar-B-Q, Lafayette, LA
Are you looking for hot, fresh, and tasty barbecue? Lil' Daddy's in Lafayette has the answer to your quest. The cooks here know how to cook up great barbecued meats because they know you can't hurry flavor. You can find dry rub ribs, tender brisket, juicy brisket, and melt-in-your-mouth chicken with a special house sauce that will make you lick your fingers and your lips. They'll even cater your next event if you want to share.
3. McClure's Barbecue, New Orleans, LA
You'll find your favorite barbecue sauce and your new favorite barbecue joint at McClure's in New Orleans. Open every day from 11 a.m. until 10 p.m., Texas, St. Louis, and Carolina-style barbecued meats and a variety of traditional sides like baked beans, cole slaw, greens, and macaroni and cheese awaits you and your taste buds will thank you.
4. Pimanyoli's Smokehouse Café, Baton Rouge, LA
You won't find a better barbecue joint in the state than Pimanyoli's Smokehouse Café in Baton Rouge. With a unique sauce that uses 21 key ingredients, this is a very special (and secret) combination that will make you beg for more. When you visit Pimanyoli's, you'll hear the sounds of blues, and favorites from the past, and everything makes the whole dining experience one you won't soon forget. Open every day but Sunday.
5. Grayson's Bar-B-Q, Clarence, LA
If you want authentic Southern barbecue, you have to try Grayson's in Clarence, Louisiana. A social media favorite, when people try this little eatery, they leave talking about it. And it's no wonder because they take their time making their meats flavorful by letting them cook slowly to lock in the flavor. A third-generation family-owned business established in 1959, you won't go wrong with anything on the menu. Open every day except Monday.
6. Eddie's Smokehouse & Seafood, Alexandria, LA
Good barbecue plus good people plus good location equals awesome experience. That's what you'll get at Eddie's in Alexandria, Louisiana. The wood-smoked boldness of this Southern treat is really smokin' and you can get your choice of beef, ribs, port, ham, turkey, chicken or sausage with sides like mac & cheese, potato salad, corn maque choux, okra, onion rings, and more. They can cater your next event and also have a kid-friendly menu.
7. Big Mike's BBQ Smokehouse, Houma, LA
The day starts early at Big Mike's BBQ Smokehouse in Homan, Louisiana. Briskets get started at 4 a.m. every day and are allowed to cook slowly for 16-20 hours to seal in the delicious smoked flavor. Chicken and ribs are started a little later, but have the same great taste. This award-winning family-owned business is a dream come true for owners Mike and Judith, who opened the eatery in true traditional barbecue style. Dine in, take home, or have them cater your next party.
8. Hickory Stick, Shreveport, LA
Simple and delicious food is what you can expect at Hickory Stick in Shreveport, Louisiana. Offering single plates, po'boys, smoked meats, soups, salads, and family packs, there are daily specials like chicken & dumplings and red beans & rice that are hearty and filling.
9. The York Chop, Coushatta, LA
This is truly a little hidden gem of a barbecue place. The York Chop in Coushatta, Louisiana, serves up some mighty fine barbecue. Besides sandwiches, burgers, seafood, and bulk meats, they serve Southern hospitality with a smile in an environment that feels like home. Open daily, you'll love the York Chop.
10. Cou-Yon's Bar-B-Q, Port Allen, LA
What do you get when you combine Texas barbecue and Cajun flavor? You get Cou-yon's Bar-B-Q. Located in Port Allen, Louisiana, Cou-Yon's has the best ribs, seafood around, not to mention the chopped beef, pulled port, sausage, turkey, and fish. Winner of TripAdvisor's Certificate of Excellence, this barbecue joint might just be the best in country! This is simple, serious food where people come together for fun and conversation and leave satisfied. Open every day.


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Mileeven Irish Cream Liqueur Honey 225g
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Mileeven Irish Cream Liqueur Honey 225g
Creamy honey flavoured with Irish Mist - an Irish whiskey liqueur, and Irish whiskey.
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The family company Mileeven had the great idea to combine pure Irish honey with the delicate, smooth and creamy flavours of Irish Mist - the famous Irish whiskey liqueur, and Irish whiskey. The result is superb and the Great Taste Awards agreed as the company received a prize for its creation.
This "Irishness" is a must try!
|Net Weight
|0.225 kg
Mileeven is an Irish family company born in Kilkenny in 1988 from a passion for beekeeping. The company is run by a mother-daughter duo. Pure honey was their first product marketed and is still an important product in the range, but Mileeven has diversified and offers today new quality products: flavored honeys, fruit cakes, jams...
Awards that the company has been regularly receiving attest of their products' quality. By tradition, each family had its own fruit cake recipe that was transmitted from generation to generation. Mileeven, with its generous fruit and butter cakes offers the quality and taste inherited from these traditional recipes. The company's range of jams and marmalades offers fruity flavours that blend perfectly with authentic Irish products.