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Lenexa, KS (PressExposure) August 12, 2015 -- MediaZone Pro®, Keywest Technology's PC-based digital signage system, gets a high-definition input option that captures live video content from multiple sources. The input option processes both digital and analog video formats, enabling users to capture and insert live video feeds into a digital signage playlist from HDMI, DVI, VGA and YUV sources.
Keywest Tech engineered the integration of the AVerMedia® full HD 1080P capture card into the MediaZone Pro platform for seamless use and no-hassle operation. According to the company, the option gives MediaZone Pro users the ability to stream live video directly into the digital signage playlist with absolutely stunning quality. The video input option also supports live stream switching between analog and digital inputs.
Koytt Nichols, Director of R&D for Keywest Technology, commented that this option gives digital signage viewers what they want. He said, "Adding live media to a digital signage zone is one of the best ways to keep content fresh and interesting for viewers. This tactic [a.k.a. sticky content] gives viewers a reason to come back for more."
"The card can capture the video easily and requires minimal configuration. We are satisfied with the hardware combination and look forward to years of use," said Lance Ha, Shell IT A/V Coordinator. Mr. Ha's department manages a multi-channel IPTV distribution system that is used to provide staff local and world news channels combined with other pertinent employee information. "We use the input cards to capture cable television channels from our satellite tuners via the component to serial adapters, and we wrap the channels with zones of relevant information," Ha added.
The MediaZone Pro video input option was released in the 3rd quarter of 2015, and it's available from authorized AV system and architectural integrators throughout North America. Interested parties and resellers are encouraged to visit the MediaZone Pro website for more information: http://www.mediazonepro.com/


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Charter Captain Tiki Bob
Captain "Tiki Bob" Robinson of the Cape Hatteras charter fishing boat "Fin Seeker", which runs offshore and inshore charters out of Teach's Lair in Cape Hatteras, VA.
The Fin Seeker calls Cape Hatteras home for most of the year except for a couple months in the fall for NC peak fishing months, when they move to Hatteras Inlet to run ocean deep drop fishing charters off of the North Carolina coast.
When it comes to choosing an experienced fishing Captain you certainly want to choose one who is professional and knowledgeable about the area fishing grounds. Not only is he friendly but he displays an abundance of patience, a must have quality for a professional fishing Captain.
Captain Tiki Bob has been fishing the waters of Cape Hatteras since he was a kid. He used to fish the Cape Hatteras waters at 13 years old in a small 16ft skiff with a 5 hp Sears and Roebuck motor. Not a luxury cruiser but for a kid that's all Captain Bob needed to get 'hooked'.
Nearly 30 years later Bob is a United States Coast Guard licensed and insured Virginia charter boat Captain. He is also a North Carolina charter Captain fishing out of Hatteras Inlet certain months of the year.
Captain Bobs Other Notables
He is an IGFA approved certified observer
He is an IGFA approved Captain
He has a Coast Guard 100 ton Masters with 4 endorsements
Captain Bob is a tournament tested sportfishing Captain

subset: commoncrawl


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Blast off with your best buds Tim and Tom as we talk about civilian space travel. That's right, some billionaires are trying to make it possible for schlubs like us to go to space, which seems like a really bad idea. We also talk about public drinking laws in New York City, going to the movies with teenagers and we solve a problem from a guy whose roommate hasn't showered in weeks.
Audible.com is offering a free 30 day trial membership! Go to audiblepodcast.com/everything today to start your free trial today! Again, show your support for The Complete Guide To Everything and get a free 30 day trial at audiblepodcast.com/everything
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Oopiri 5th Day Collection 5 Days Total Earning-Business-Thozha :- Oopiri is a one of a top Superhit Telugu movie of this year. After a long time we watched a very demanded actor of South Film Industry in this movie. This is a romantic comedy fun movie. The story of the film is really amazing. All the fans of Telugu movies are loving it and appreciate for all the characters and film story. Oopiri has completed its 5 days Now. Oopiri 5 Days Total Collection is also very impressive.
Oopiri Response on Box Office
Nagarjuna is playing lead role with Karthi and Female Actress Tamannaah. If we talk about the starting of the movie in Domestic + Oversear Market then we can say it is outstanding.
Opening day of the movie was very good in worldwide Market. In Andhra Pradesh and Telangana movie is running on top these days. Nararjuna is getting huge likes on Twitter for this film. Twitter status of Thozha is also very good.
Oopiri 5th Day Collection 5 Days Total Earning-Business-Thozha-BOC
The budget of the movie is $650 Million. This is a big budget movie of Telugu Film Industry. Oopiri also beat a very popular superhit another south movies like Soggade Chinni Nayana and Express Raja.
Oopiri Telugu Movie 2016 Plot
Vikram Aditya (Nagarjuna Akkineni) is an extremely rich person quadriplegic down the maritime of a mishap. He maintains a major business and is helped by capable administrators and a secretary Keerthi (Tammanah), yet he is searching for a decent overseer who can give get zest his dismal, desolate, exhausting life in spite of having everything. Seenu (Karthi) discharges from prison on parole, his mom (Jayasudha) abandons him as she feels he is an awful impact for different children in her white collar class family.
In the mean time, Vikram interviews many men, every one more exhausting and self important than the past competitor, lastly, he chooses Seenu who needs a vocation that will demonstrat to him as a do-gooder. Seenu is awed by the palatial house and Keerti. Seenu's imprudent state of mind and exuberance wins Vikram's heart no doubt and changes his life totally. Be that as it may, life can get entirely intense as it incites issues into Seenu's life through his own issues. Rest of the story is with reference to how both these characters battle their issues and remain for each other in life.
Tamil Thozha Review Response
Tamil Version of this film "Thozha" also positive review by Critics on Box Office. They are very appreciate for the performance of Karthi and Nagarjuna in this Film. Tamil Version also got good response from audience review.
Oopiri Thursday Preview also good response in USA. Oopiri Thursday Preview Business is Thu previews $ 76,635. This is good starting for the picture.
Oopiri First Day Box Office Collection is Fri $ 210,281. This is superb! As we mention here opening day collection of the film. This is very good amount earned in USA.
Oopiri Second Day Box Office Collection is also Amazing. On the Second Day Oopiri Collection increased than First Day in USA and Domestic Market. But this time we are providing here USA Collection of Oopiri. Oopiri 2nd Day Box Office Collection in USA Sat $ 361,247. This is really appreciable amount for the film.
Oopiri 3rd Day is also Wonderful. On 3rd Day Sunday Oopiri Collection very good amount for the movie. Oopiri Third Day Box Office Collection is Sun $ 256,203. This is again increased amount than last days.
If we talk about Oopiri 4th Day Collection. Oopiri Monday Response is very low than past Days in USA. Oopiri 4th Day Box Office Collection in USA is Mon $ 58,306. This is also good amount but less than other days.
Now Oopiri 4 Days Total Collection is Total: $ 965,085 [₹ 6.42 cr]. This amount is very appreciable collected in USA.
Check>>>Oopiri 4 Days Collection Total Earning 4th Day 3rd Business-Thozha
Oopiri 5th Day Box Office Collection
Oopiri 5th Day is also very well for the film in USA. On the 5th Day Oopiri Telugu movie got good response in Overseas Market. With Oopiri Fifth Day Box Office Collection the Total Amount of 5 Day Can touch Rs 6.90 Crore. Oopiri 5 Days Collection in USA is Excellent. Watch and enjoy this film in your near theatres. To get all updates stay tuned with us.


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Miguel Gasca is the newest member of the Rainbow Municipal Water District (RMWD) board.
A 4-0 board vote Feb. 28 selected Gasca to fill the Division 3 seat vacated after Tory Walker moved to Murrieta and resigned from the board. Gasca will fill the remainder of Walker's term, which expires in December 2018.
"I'm glad to have the opportunity to serve the community," said Gasca.
"He'll be a good addition to the board," said Rainbow general manager Tom Kennedy.
Division 3 is the central part of the RMWD service area and includes the Gird Valley and Pala Mesa. Gasca has lived on Wilt Road since 2011 but previously lived in San Bernardino County and served two four-year terms on the Running Springs Water District board.
"He brings experience from serving on a board," Kennedy said. "He brings some understanding of water districts."
Gasca was born in Los Angeles and attended Saint Francis High School in La Canada. In 1974 he received his bachelor's degree in civil engineering from Loyola Marymount University; his field of concentration was water supply and waste treatment. Gasca lived in Running Springs, which is at the junction of State Route 330 and State Route 18, from 1974 to 1987. He was on the board of the Running Springs Water District from 1979 to 1987, including three years as the board president, and he was also a board member and the programs chairman of the San Bernardino County Association of Independent Special Districts.
The Running Springs Water District has not only water and sewer latent powers but also has a regional treatment plant and handles fire, emergency medical, lighting, and parks and recreation services for Running Springs. Gasca worked on master plans for the district's services and also helped secure funding for the replacement of an eight-mile water main.
Gasca received per diem pay for his water district activities, but his full-time job was as an engineer for TRW/Northrup Grumman. He spent two years as the legislative committee chairman of the San Bernardino/Riverside branch of the American Society of Civil Engineers and was also a delegate to the California Legislative Council for Professional Engineering.
Gasca lived in San Bernardino from 1987 to 2011. He retired from TRW/Northrop Grumman in 2009 and founded Apogean Group, LLC, which utilizes senior scientists and engineers to provide consulting services.
The RMWD's committees include the Engineering Committee. Gasca attended a recent Engineering Committee meeting and spoke with Walker, who was in the process of moving out of the district. Gasca was encouraged to seek the board vacancy.
Gasca and Walker still communicate with each other.
"Talking to Tory will enable me to come up to speed fast than I thought I would," Gasca said. "I think he'll be a valuable resource and will continue to help the district in that way."
James Fergus and Christal Punaro also formally applied for the vacant board seat.
"I'm hoping that I'll be able to provide my experience perspective on the issues that we deal with," Gasca said. "I look forward to providing support to all the homeowners and businesses in Division 3. It's my intention to try to get around the division and meet as many people as I can."


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Aggregation of α-synuclein (α-syn), a process that generates oligomeric intermediates, is a common pathological feature of several neurodegenerative disorders. Despite the potential importance of the oligomeric α-syn intermediates in neuron function, their biochemical properties and pathobiological functions in vivo remain vastly unknown. Here we employed two-dimensional analytical separation and an array of biochemical and cell based assays to characterize α-syn oligomers that are present in the nervous system of A53T α-syn transgenic mice. The most prominent species identified were 53 Å detergent soluble oligomers, which preceded neurological symptom onset, and were found at equivalent amounts in regions containing α-syn inclusions as well as histologically unaffected regions. These oligomers were resistant to SDS, heat, and urea, but were sensitive to proteinase-K digestion. Even though the oligomers shared similar basic biochemical properties, those obtained from inclusion bearing regions were prominently reactive to antibodies that recognize oxidized α-syn oligomers, significantly accelerated aggregation of α-syn in vitro, and caused primary cortical neuron degeneration. In contrast, oligomers obtained from non-inclusion bearing regions were not toxic and delayed the in vitro formation of α-syn fibrils. These data indicate that specific conformations of α-syn oligomers are present in distinct brain regions of A53T α-syn transgenic mice. The contribution of these oligomers to the development of neuron dysfunction appears to be independent of their absolute quantities and basic biochemical properties, but is dictated by the composition and conformation of the intermediates as well as unrecognized brain-region specific intrinsic factors.
α-Synuclein (α-syn) is a soluble, acidic protein that typically assumes a random coil structure, however it acquires α-helical conformation upon binding to anionic phospholipids (Davidson et al., 1998; Ulmer et al., 2005). Although the exact function(s) of α-syn remain uncertain, the preferential localization to presynaptic nerve terminals and its interaction with vesicular phospholipids and proteins suggests a regulatory function associated with synaptic activity, dopamine production and metabolism, lipid vesicle trafficking, and chaperone-like activity (Maroteaux et al., 1988; Iwai et al., 1995; Souza et al., 2000b; Cabin et al., 2002; Chandra et al., 2005).
Data from post-mortem evaluations of Parkinson's disease (PD) brains revealed that α-syn aberrantly forms highly organized, linear amyloid fibrils that constitute part of the characteristic inclusions found in neuronal perikarya (Lewy bodies) and dystrophic neurites (Lewy neurites). (Forno, 1996; Goedert, 2001; Norris et al., 2004; Mori et al., 2008). Despite the ubiquitous expression of α-syn throughout the central nervous system, these inclusions are found in certain susceptible neuronal subtypes of specific brain nuclei (Braak et al., 2003). Biochemically, α-syn within inclusions is resistant to extraction with non-ionic detergents. However upon extraction with formic acid or SDS/urea α-syn collapses into monomers and SDS and heat stable oligomers (Baba et al., 1998; Tu et al., 1998; Dickson et al., 1999).
In vitro evidence utilizing purified recombinant α-syn has indicated that the conversion of monomers into amyloid fibrils progresses in a nucleation-dependent manner through an initial polymerization stage involving the formation of oligomeric intermediates (Conway et al., 2000b). The polymerization process is concentration dependent and can be accelerated by the PD-causing mutations A53T, A30P, and E46K (Conway et al., 1998; Giasson et al., 1999; Narhi et al., 1999; Conway et al., 2000b; Greenbaum et al., 2005). Although there is considerable confidence that accumulation and polymerization of α-syn plays an important role in neurodegeneration, the contribution of the different species that emerge through the aggregation process has not been fully delineated. Existing efforts identifying the potential pathogenic formations are based on studies where oligomerization of the protein is forced in vitro (Goldberg and Lansbury Jr, 2000; Volles et al., 2001; Danzer et al., 2007). Characterizing α-syn assemblies that are formed in the brain and in living cells is challenging since unstable conformations may be disrupted during the traditional biochemical extraction processes. Notwithstanding this important concern, there is considerable paucity in the biochemical and biological description of the oligomeric α-syn entities that are formed in mice models and humans, and are stable to standard isolation methodologies with mild non-ionic detergents.
In this study we provide a brain regional-specific biochemical and biological characterization of the relatively stable α-syn oligomeric conformations that are formed in the transgenic mouse line expressing human A53T α-syn driven by the mouse PrP promoter (Giasson et al., 2002). The data indicates that despite similarities in basic biochemical properties, α-syn oligomeric intermediates obtained from different neural regions demonstrated unexpected divergence in promoting α-syn amyloid fibril formation and toxicity.
The mice utilized in this study express human A53T α-syn (line M83) or human WT α-syn (line M20) driven by the murine PrP promoter and have been described previously (Giasson et al., 2002). To generate A53T +/+ and non-transgenic (nTg) control mice used in experiments, A53T +/− females were mated with A53T +/− or +/+ males, since A53T +/+ females were found to produce small litters and exhibit poor motherly behavior. Genotyping was performed by both end-point PCR using GeneAmp PCR system 9700 thermal cycler (PE Applied Biosystems, Sunnyvale, CA) and quantitative PCR using Applied Biosystems 7500 real-time PCR system (PE Applied Biosystems) with the ABI MGB primer-probe set for human SNCA (assay ID Hs00240907_m1). SNCA values were normalized to mouse beta-actin (4352341E). Mice identified as homozygous for the α-syn transgene were verified through backcrosses or validated by quantitative western blot analysis of α-syn protein.
Analysis was performed on 5 month old A53T+/+ males and age-matched nTg male littermates. Motor activity measurements were performed with an Opto-M3 activity meter (Columbus Instruments, Columbus, OH) by quantifying the number of infrared beam-breaks over a 24 hour period using the Multi Device Interface (MDI) software (Version 1.34 Columbus Instruments). Testing was done in the home holding room and home cage under controlled environmental conditions, and food/water was available ad libitum. The animals were acclimated to the apparatus for 2 days, followed by 3 consecutive days of beam-break data collection. The 3-day average was used as n=1 for each mouse, with 5 mice utilized for each group.
Testing was performed on 4–6 month old A53T+/+ males and age-matched nTg male littermates in the home holding room and home cage under controlled environmental conditions. Singly-housed mice were given 100 g of fresh standard animal chow pellets (Laboratory rodent diet, cat#5001; LabDiet Richmond, IN) placed in a suspended wire cage top feeder at the start of the testing period and food consumption was determined by weighing pellets every 24 hr for 5 consecutive days. Mouse body weight was also determined every day during the testing period and used to normalize food intake measurements. The average value of food consumed over the 5 day testing period was used as n=1 for each mouse. The analysis was performed with 6 different mice from two separate litters.
Brain sections from symptomatic A53T+/+ α-syn transgenic mice and age-matched nTg controls were stained with syn505 mouse monoclonal antibody specific for human α-syn (Duda et al., 2002). Perfusions, tissue processing and staining were performed as previously described (Giasson et al., 2002). Adjacent sections were stained with rabbit polyclonal anti-GFAP (ab7260, AbCam, Cambridge MA). Incubation with secondary antibodies conjugated to biotin was followed by addition of avidin-biotin-peroxidase complex (ABC Elite, Vector Laboratories, Burlingame, CA), visualized with 3,3′-diaminobenzidine as the chromogen and counterstained with Hematoxylin (SH30-500D, Fisher Scientific, Hampton, NH).
4–6 month old asymptomatic A53T+/+ α-syn transgenic mice, 11–12 month old symptomatic A53T+/+ α-syn transgenic mice, and 11–12 month old human WT α-syn transgenic mice were utilized for analysis. A53T+/+ symptomatic mice were sacrificed 1–2 days after initial motor symptom onset. A53T+/+ mice were monitored on a daily basis and symptomatic mice were identified by spine stiffness, weight loss, and hind limb paralysis as described previously (Giasson et al., 2002). After removal of the cerebellum (Crb, including the deep cerebellar nuclei; bregma, −5.46 to −7.70), olfactory bulbs (OB; bregma, +4.74 to +3.14), and spinal cord (SC; cervical region), the remainder of the brain was dissected into 1 mm-thick coronal sections using a vibratome (World Precision Instruments, Sarasota, FL) submerged in ice-cold phosphate-buffered saline, to dissect the substantia nigra (SN), hippocampus (Hipp), visual cortex (Ctx) (bregma, −2.44 to −3.88) and the striatum (+0.66 to −1.34). Dissected brain regions were weighed, rapidly frozen on dry ice, and stored at −80 °C until analysis.
Tissues derived from different regions of individual mice were homogenized with 10 volumes of lysis buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 10 % glycerol, 1 % Triton-X 100, 1 mM EGTA and 1.5 mM MgCl2, 1 μM lactacystin, 50 mM NaF, 10 mM NaPyruvate, 2 mM Na3VO4, 1 mM PMSF and protease inhibitor cocktail (P2714, Sigma, St Luis, MO). The tissues were grinded with the aid of mechanical homogenizer and then subjected to four consecutive freeze and thaw cycles of 2 minutes each (−80 °C ethanol bath/37 °C water bath). The Triton-soluble fraction was obtained after ultracentrifugation at 100,000 g for 30 minutes at 4 °C. The resulting pellets were further extracted in 5 volumes of 2 % SDS, 50 mM Tris pH 7.4 and a protease inhibitor cocktail by sonication and then boiling for 20 minutes. Samples were centrifuged for 10 minutes, 16,000 g at 25 °C to obtain the Triton-insoluble (SDS-soluble) fractions. Protein concentration was determined by the microBCA assay. (Pierce, Rockford, IL)
400 μg of Triton-soluble protein from each tissue in a total volume of 250 μl were loaded onto a Superdex 200 HR10/30 column (GE Healthcare Bio-Sciences, Uppsala, Sweden) connected to an Agilent 1100 series HPLC system (Agilent, Palo Alto, CA) for size exclusion chromatographic (SEC) analysis. The mobile phase used to resolve the protein lysates consisted of 25 mM HEPES, 150 mM NaCl, pH 7.25. The fractions were collected and concentrated with 3,000 nominal molecular weight limit (NMWL) Ultracel Microcon filters (Millipore, Bedford, MA). Fractions 2–5 (99-63 Å), 6–9 (63-48 Å), 18–20 (25-20 Å), and 21–24 (20-11 Å) were combined before loaded on the SDS-PAGE gels to enhance the signals. To isolate the 53 Å-size soluble α-syn oligomers the corresponding fractions were separated and concentrated using 3,000 NMWL Ultracel Microcon filters. The gel filtration column was calibrated using a mixture of globular proteins (HMW standards kit, GE Healthcare).
53 Å-size Triton soluble α-syn oligomers isolated by SEC from various neuronal regions were treated with 100μg/ml of proteinase K for 30 and 60 minutes at 37°C and another set of samples was treated with 8 M urea for 30 and 60 minutes at room temperature. The protein concentration for each sample was adjusted to 0.5 mg/ml. The reactions were stopped by heating to 100°C for 10 minutes after adding SDS-sample buffer. The samples were resolved on SDS-polyacrylamide gels.
Human recombinant α-syn protein used for the assay was expressed and purified as described previously (Giasson et al. 1999). In vitro α-syn aggregation was performed as described (Mazzulli et al. 2007). Briefly, 2.5 mg/ml of purified protein was incubated for 4 days in conditions promoting aggregation (shaking at 1000 rpm, 37°C) with 0.1μg/μl of fractionated lysate corresponding to the 53 Å-size Triton-soluble α-syn oligomers from the OB and SC regions. Control experiments were conducted in parallel by using samples immunodepleted from α-syn (immunodepletion described below), the fractions corresponding to the monomer of α-syn, and also using the purified protein with no addition of fractionated lysates. The corresponding fractions from lysates of WT α-syn transgenic mice were also assessed as additional controls. The progress of fibril formation was monitored every 12 hours by addition of 10μM Thioflavin T (Sigma) solution in the reaction and measuring fluorescence emission at 490 nm upon excitation at 450 nm.
For Western blot analysis of samples sequentially extracted, the following primary antibodies were used: monoclonal anti-α-syn LB509 (Baba et al., 1998; Jakes et al., 1999) (1:1000), monoclonal anti-tyrosine hydroxylase (TH) (1:4000; cat# 657010; EMD Biosciences), polyclonal antineuronal specific enolase (NSE) (1:2000, cat# 16625; Polysciences, Inc. Warrington PA) and monoclonal anti-vimentin (1:500; cat# 550513; BD Pharmingen, San Jose, CA). For Western blot analysis of fractions from gel filtration profiles the following antibodies were used: monoclonal anti-α-syn syn211 (Giasson et al., 2000) (1:1000) and polyclonal anti-NSE (1:2000, Polysciences Inc.). Fractionated lysates were also blotted with monoclonal anti-α-syn syn208 and syn303 (1:1000) (Giasson et al., 2000; Duda et al., 2002). Dopaminergic markers DOPA decarboxylase (DDC, EC 22.214.171.124) and dopamine transporter (DAT) were detected with mouse monoclonal antibodies DDC-11-M (1:500; Alpha Diagnostic, San Antonio TX) and MAB369 (1:800; Chemicon, Bedford MA) respectively. Primary antibodies were detected with either goat anti-mouse IgG Alexa Fluor 680 (1:5000; Invitrogen, Eugene, OR) or goat anti-rabbit IgG IRDye 800 (1:5000; Rockland, Gilbertsville, PA) conjugated secondary antibodies and scanned at intensity level 2 with an Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE). The bands from the western blots were quantified by densitometry using Odyssey infrared imaging system software (version 2.1 Odyssey, Li-cor).
All individual α-syn immunoreactive bands from size-exclusion chromatography (SEC)/SDS-PAGE/syn211 western blot analysis were quantified by densitometry using the Odyssey version 2.1 software (see supplemental Fig. 2) (Li-Cor Biosciences). Our previous SEC analysis of WT and A53T α-syn extracted from transfected neuroblastoma cells has shown that in vitro oligomerization does not occur as a result of the extraction and analysis procedure used here (Mazzulli et al, 2006), thus ruling out the possibility that oligomers detected in A53T α-syn mice form in vitro. α-Syn immunoreactive bands that comprised the 53 Å-size α-syn oligomer peaked at a volume corresponding to a 225 kDa globular protein in the gel filtration column, and were found to be made of a 59, 52, and 31 kDa species by SDS-PAGE. A minor oligomer eluted at a volume corresponding to a 36 Å-size particle and was comprised of 64 and 38 kDa bands as determined by SDS-PAGE. The bands eluting at a volume corresponding to a 34 Å-size particle and migrating at 19 kDa in the SDS-PAGE gels were quantified as the α-syn monomer. Each species was quantified and divided by the total α-syn signal (α-syn 53 Å + minor oligomer + α-syn monomer) to determine the percent of each.
Catechols were resolved on a reverse-phase C18 Luna column (150 × 4.6 mm, 5um; Phenomenex, Torrance, CA) connected to an 1100 series Agilent HPLC system (Agilent, Palo Alto, CA) and detected by a Coularray detector (ESA Biosciences, Chemsford, MA) as previously described (Mazzulli et al., 2006).
Cultures were prepared from C57Bl/6 mouse embryos at day 17 (stock# 3704732, Charles River, Kingston, NY). After dissection from embryos in Hepes buffered saline (supplemented with 1 mM sodium pyruvate (Invitrogen), 6 mg/ml D-Glucose (Sigma), 100 units/ml penicillin, 100μg/ml streptomycin), the cortex was mechanically dissociated, trypsinized, and seeded at 50,000 cells per well into 96 well plates (# 35–4640, BD biosciences, Bedford MA) freshly coated with 50μg/ml poly-D-Lysine (Sigma). After seeding, neurons were cultured for 2 hours in serum media (10 % heat inactivated fetal bovine serum, 100 units/ml penicillin, 100μg/ml streptomycin, 2 mM glutamine, in neurobasal media (all from Invitrogen)). After 2 hours, the media was changed to B27 media (neurobasal media containing 1X B-27 supplement, 2 mM glutamine, 100 units/ml penicillin, 100μg/ml streptomycin). At 4 days in vitro (DIV), half of the media was replaced with fresh B27 media.
Triton-soluble lysates from the hippocampus, spinal cord, or olfactory bulbs of symptomatic A53T α-syn were subjected to SEC and fractions corresponding to 53 Å-size α-syn oligomers were utilized for experiments. The fractions were concentrated and protein concentration was determined by the BCA protein assay (Pierce). To immunedeplete α-syn oligomers for control samples, 2μg of syn211 antibody (Sigma) was incubated with 100μg of fractionated lysate in a total volume of 200μl for 6 hours at 4°C. Another set of samples was also incubated with mouse immunoglobulin G (IgG, Santa Cruz Biotechnology, Santa Cruz, CA) in parallel. Protein G-conjugated beads (4 % final, Fast Flow, Sigma) were equilibrated in 25 mM Hepes, 150 mM NaCl pH 7.4 then incubated with the immunocomplexes for 1 hour at 4 °C. α-Syn oligomers were then pulled down by centrifugation at 2000 × g for 2 minutes and the protein concentration of the supernatant was determined. 1μg total protein/well of this supernatant was directly added to primary cultures grown in a 96 well plate at 9 DIV, in a total media volume of 200 μl/well. For samples incubated with IgG, the concentration of α-syn oligomers in the supernatant was estimated by quantitative western blot, using purified recombinant α-syn as a standard, and found to be approximately 10 ng oligomers/μg total protein. In addition to fractions immunodepleted of α-syn, total fractionated hippocampal lysate was utilized as a negative control (1μg lysate/well), since this tissue contains a low level of α-syn oligomers (Fig. 1 and and2).2). Cells were harvested 1 and 3 days after addition of the oligomers or immunodepleted supernatants and analyzed for neurodegeneration.
Primary cortical neurons grown on a 96 well plate were washed briefly in warm PBS, followed by immediate fixation in 4 % formaldehyde for 15 minutes. Cells were incubated with PBS containing 0.3 % TritonX-100 for 20 minutes with gentle shaking and then with Odyssey blocking buffer (Li-cor) for 1 hour. Anti-neurofilament antibody (1:1000, mouse IgG 2H3, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) was then incubated overnight in blocking buffer at 4 °C with gentle shaking, followed by washing in PBS with 0.1 % Tween for 20 minutes. IRdye800-conjugated anti-mouse IgG antibodies (1:1000 dilution, Li-cor) were incubated in blocking buffer for 1 hour. Draq5 (Nuclear stain, 1:10,000 dilution, Li-cor) and Sapphire700 (non-specific cytosolic stain, 1:1000 dilution, Li-cor) were added with the secondary antibody for determination of cell volume and number. Cells were washed four times in PBS 0.1 % Tween and scanned on an odyssey infrared imaging system (Li-cor). Neurofilament intensity was determined using Odyssey software (version 2.1 Li-cor). Toxicity assays were repeated with two separate cell culture preparations with n=6 for each preparation.
One way analysis of variance (ANOVA) with Tukey's post-hoc test was used to assess neurodegeneration in primary cortical cultures as well as SEC analysis of α-syn oligomers, and p< 0.05 was considered significant. For determination of values that varied significantly from the mean, Grubbs test was used to determine Z values. α-Syn oligomer levels that had Z values over 1.15 (n=3), 1.48 (n=4), and 1.71 (n=6) were excluded from the analysis. For direct tissue-to-tissue comparisons as well as asymptomatic/symptomatic comparisons of oligomers from the SC (shown in supplemental Fig. 2), a paired student's t-test was used and p values <0.05 were considered significant. Two way ANOVA with a Bonferoni's post-hoc test was used for the seeding assays shown in Fig. 4, n=4, p values <0.05 were considered significant.
To characterize the regional distribution of aggregated forms of α-syn in A53T α-syn mice, we first sought to biochemically determine the distribution of insoluble α-syn in different neural regions. Tissues from various regions of the nervous system of asymptomatic (4–6 month old) and symptomatic (11–12 month old) mice were sequentially extracted in detergents of increasing strength. Western blot analysis using an antibody specific for human α-syn, LB509, revealed that the spinal cord (SC) and cerebellum (Crb) from symptomatic mice contained more Triton-insoluble α-syn compared to asymptomatic mice (Fig. 1b). Triton-insoluble α-syn from these tissues was present primarily as a 19 kDa monomer, but also contained higher molecular weight bands stable to SDS/heat that likely correspond to dimers, trimers, and multimers. Conversely, in other brain regions such as the substantia nigra (SN), olfactory bulbs (OB), and hippocampus (Hipp), α-syn was mainly extracted in the Triton-soluble fraction with only a modest change in solubility as the mice presented with motor symptoms (Fig. 1a). This is consistent with histological analysis, which demonstrated a paucity of inclusions in these three regions (Giasson et al 2002). Analysis of age-matched transgenic mice expressing human WT α-syn, which do not exhibit α-syn pathology or motor symptoms at any age, revealed undetectable levels of Triton-insoluble α-syn in all brain regions examined (Fig. 1b and supplemental Fig. 1). These biochemical findings were corroborated by histological examination. In accordance with previous reports (Giasson et al 2002) immunohistological analysis revealed abundant α-syn inclusions (both perikaryal and neuritic) in the spinal cord of symptomatic A53T α-syn mice (Fig. 1c, panel H). To assess if indications of neuronal injury are present in areas that contain α-syn inclusions, tissue sections from symptomatic A53T α-syn transgenic mice and nTg mice were histologically examined for gliosis with an anti-GFAP antibody. Robust astrocytic gliosis is observed in the spinal cord of symptomatic A53T α-syn mice (Fig. 1c, panel D), but not in the olfactory bulb (Fig. 1c, panel B) a brain region where inclusions of α-syn are scarcely detected. These data suggest that formation of insoluble α-syn temporally correlates with motor symptom onset and occurs in regions that contain histological α-syn inclusions.
To further investigate α-syn species present in the nervous system of A53T α-syn mice, we initially focused on the SC due to the neurodegenerative changes that occur in this region once the mice develop symptoms (Fig. 1c) (Giasson et al., 2002). Triton-soluble extracts from the SC of symptomatic A53T α-syn transgenic mice were analyzed by size exclusion chromatography (SEC) followed by SDS-PAGE/Western blot analysis. Previous SEC analysis of recombinant purified α-syn had shown that although the monomeric form elutes from a gel filtration column at a volume corresponding to a 34 Å– size particle (or 58 kDa globular protein); in SDS-PAGE it migrates to an apparent molecular weight of nearly 19 kDa (Weinreb et al., 1996). This highly reproducible observation reflects the elongated, unstructured conformation of α-syn in solution. SEC analysis of Triton-soluble SC extracts from A53T α-syn transgenic mice revealed the presence of soluble 53 Å-size oligomers, with apparent molecular weights of 59, 52, and 31 kDa on SDS-PAGE (Fig. 2, and supplemental Fig 2.). These high molecular weight α-syn species were the most abundant oligomeric intermediates detected in the SC of symptomatic mice (Fig. 2). In addition to the 53 Å-size α-syn oligomers, a minor amount of α-syn oligomers eluted at nearly the same volume as the monomeric form, corresponding to a 36 Å-size particle, and migrated at 64 and 38 kDa on SDS-PAGE (Fig. 2, supplemental Fig. 2). Next, SEC profiles of the SC of symptomatic mice were compared to those of asymptomatic mice (supplemental Figs. 2, 3) and revealed a similar SEC profile, indicating that soluble α-syn oligomers accumulate before the formation of insoluble α-syn inclusions and presentation of motor deficits. Additionally, the amounts of oligomers in symptomatic and asymptomatic mice were not significantly different by quantitative densitometry analysis of the SEC/SDS-PAGE blots (Fig. 2). Despite the detection of various oligomeric α-syn species, the SEC analysis did not identify a soluble oligomeric form of α-syn that is specifically correlated with symptom onset (supplemental Fig. 2).
To determine if α-syn oligomers exist in other histologically unaffected regions of symptomatic A53T α-syn transgenic mice, Triton-soluble extracts from the SN, OB, and Hipp were analyzed by SEC. Unexpectedly this analysis revealed a similar elution profile compared to the inclusion bearing regions (SC and Crb), with the 53 Å-size α-syn oligomers as the most prominent species (Fig. 2). Quantification indicated that the relative levels of oligomeric α-syn in the SN, and OB were similar to those in the SC (Fig. 2). Additionally, SEC analysis of hippocampal lysates of A53T α-syn transgenic mice as well as most tissues from control mice expressing WT α-syn showed very low levels of soluble oligomeric α-syn (Fig. 2 and supplemental Fig. 4), indicating that soluble oligomers detected by SEC form as a result of in vivo processes.
A lack of correlation between the relative abundance of soluble oligomers and the formation of insoluble inclusions is unexpected, given the assumption that these oligomeric species are intermediates of the fibril generating process. Therefore, the biochemical nature of the 53 Å-size α-syn Triton-soluble oligomers was further characterized. For this analysis, we focused on the OB and SC because of the clear histological differences between these regions, as well as tissue availability. Following SEC, the fractions corresponding to Stokes radii of 53 Å and higher were separated from the rest of the fractions and concentrated to obtain an enriched solution of oligomeric species. Treatment of 53 Å-size oligomers derived from the SC or OB with urea did not affect the 59, 52, and 31 kDa bands suggesting that these species represent self-associated α-syn molecules that are held together by urea resistant bonds (Fig. 3a). Incubation with proteinase K (PK) was then performed to reveal potential regions of α-syn that might be protected from proteolysis. Oligomers of the SC and OB were equally sensitive to PK treatment, indicating a lack of buried peptide regions protected from digestion (Fig. 3b). Taken together these data indicate that α-syn oligomers in the brain of A53T α-syn transgenic mice are Triton-soluble, SDS and heat stable, resistant to denaturation by urea, and sensitive to PK. Despite the similarity in these biochemical properties, an interesting distinction between oligomers of the OB and SC was evident upon western blot analysis using syn303, a monoclonal antibody generated against oxidized/nitrated recombinant α-syn (Duda et al., 2002). Immunoblotting with syn303 revealed more prominent immunoreactivity with SC oligomers as opposed to the OB in the bands corresponding to dimers and trimers having an apparent molecular weight of 59, 52, and 31 kDa (Fig. 3c). The same samples were analyzed by immunoblot using LB509, a human specific antibody that does not preferentially detect oxidized forms of α-syn, showing roughly equal amounts of oligomers in each region (supplemental Fig. 5). Since syn303 preferentially recognizes oxidized and/or nitrated α-syn in human inclusions (Duda et al., 2002) these data suggest that oxidative modifications to soluble oligomers occur to a greater extent in the SC compared to the OB.
To determine if further differences exist between oligomers of the SC compared to the OB, we compared the ability of these oligomers to influence the kinetics of the in vitro polymerization of α-syn. Based on previous structural analysis of in vitro formed oligomers (Conway et al., 2000a; Apetri et al., 2006) the size of the oligomers detected here (53 Å) is consistent with an intermediate species in the pathway of forming α-syn amyloid fibrils. Therefore "on-pathway" oligomers should act as "seeds" in the in vitro polymerization of α-syn and thus accelerate the initial phase of this process (Jarrett and Lansbury, 1993; Wood et al., 1999). Indeed enriched oligomeric fractions from SC shortened the lag phase and increased the rate of recombinant purified α-syn forming amyloid fibrils as indicated by the changes in Thioflavin T fluorescence (Fig. 4a) when compared to identical SC oligomer-enriched fractions that were immunodepleted for α-syn. In contrast oligomeric α-syn extracted from the OB prolonged the lag phase of purified α-syn amyloid fibril formation (Fig. 4b). These effects on the in vitro kinetics of fibril formation were dependent on α-syn since immunodepletion of the oligomers restored the kinetic profile of the purified α-syn aggregation. Moreover, addition of α-syn monomers extracted from SC or OB of A53T α-syn transgenic mice as well as of SEC fractions from SC or OB of mice expressing WT α-syn corresponding to ≥53 Å did not alter the kinetics of α-syn aggregation (Table 1 and supplemental Fig. 6). Collectively the data indicate that oligomers derived from the OB represent either "off-pathway" oligomers, or contain an oligomer-associated factor that kinetically delays the formation of α-syn fibrils. The absence of the detectable α-syn inclusions in the OB of the A53T α-syn transgenic mice supports these findings.
To determine the toxicity of SC and OB oligomers, SEC fractions containing oligomers were added to primary cortical cultures and neuronal toxicity was assessed at 1 and 3 days after treatment by measuring total cell number using a fluorescent non-specific cell stain in combination with a nuclear stain. Neurodegeneration was also assessed by immunostaining for neurofilament (NF) protein, which is a more sensitive assay that can measure degeneration of neurites prior to occurrence of more severe nuclear toxicity (Zala et al., 2005). While toxicity of α-syn is presumed to occur through intracellular events in PD, recent studies have indicated that α-syn aggregates are taken up by neurons in culture through endocytosis and can be disseminated from cell to cell causing widespread neurotoxicity (Desplats et al., 2009). Furthermore, other studies have shown that in vitro formed α-syn oligomers exhibit toxicity when applied externally to cell cultures, likely through disruptions in lipid bilayers (Kayed et al., 2003; Kayed et al., 2004). Therefore, we considered this experimental paradigm as a reliable proxy for the potential toxicity that may occur in vivo. SEC fractions containing the 53 Å-size oligomers were added to cultures and neurodegeneration was assessed. We found that addition of this SEC fraction containing α-syn oligomers derived from the SC resulted in a significant decline in cell viability as determined by analysis of cell volume and total cell number, whereas the same SEC fraction obtained from the OB had no effect (Fig. 5a). We also observed a decrease in neurofilament immunostain intensity, attributed to neurite degeneration, in cultures treated with SC oligomers but not oligomers obtained from the OB (Fig 5b). The toxicity of SC oligomers was observed 1 day after treatment and persisted until 3 days after addition of the oligomers. This effect is presumed to be due to the presence of oligomeric α-syn since depletion with an anti-α-syn antibody reversed the neurodegeneration observed in the SC treated cultures (Fig. 5a, b). Additionally, the observed toxicity differences between the OB and SC did not result from addition of different quantities of oligomers, since we quantified the amount of α-syn oligomers that were added to the primary cultures by immunoprecipitation and western blot (Fig. 5d). These data suggest that despite similar SEC profiles and biochemical properties, α-syn oligomers display different cellular toxicities. The histological analysis shown in Fig. 1 also supports the neuron toxicity findings, since astrogliosis is more prominent in the SC.
Although previous histological analysis of A53T α-syn transgenic mice at 4–5 months indicated normal cell structure without neurodegenerative changes (Giasson et al., 2002), we sought to determine if the presence of detergent-soluble oligomers detected by SEC/SDS-PAGE is reflected on behavioral changes in the mice. Ambulatory activity measurements revealed that A53T α-syn transgenic mice had similar motor activity compared to nTg mice (Table 2A). Food intake and body weight of A53T α-syn transgenic mice were also assessed, since it has been well established that feeding behavior critically depends on the function of the SNpc→ caudate putamen (CPu) circuit (Zigmond and Stricker, 1972; Szczypka et al., 2001). We found that A53T α-syn transgenic mice had similar body weights and consumed slightly more food compared to nTg age-matched controls, suggesting that the SNpc→CPu circuit is functional (Table 2A). Growth curves, by measuring body weight starting at 2 weeks did not reveal any differences in the development of A53T α-syn transgenic mice for up to 11 months of age (data not shown). After 11 months, A53T α-syn transgenic mice experience motor deficits and a rapid decline in body weight at variable time points. These data show that the presence of detergent-soluble α-syn oligomers in the SN is associated with only mild behavioral changes.
Further biochemical evaluation was also performed to determine the integrity of the dopaminergic system in asymptomatic and symptomatic mice. Enzyme levels related to dopamine synthesis and metabolism were quantified, such as tyrosine hydroxylase (TH), dopamine transporter (DAT), and DOPA decarboxylase (DDC) in symptomatic mice and showed no statistically significant difference compared to nTg mice (supplemental Fig. 7). Additionally, striatal catechol levels were measured in both asymptomatic and symptomatic A53T α-syn transgenic mice, as well as in non-transgenic control mice, by HPLC-ECD. Analysis of 4-month-old mice showed that striatal catechol levels of A53T α-syn transgenic mice were similar to non-transgenic age-matched controls (femtomoles/μg protein, non-transgenic mice: DA=598±82.5, DOPAC=100.4±27.9; A53T α-syn transgenic mice: DA=703±30, DOPAC=89.9±11.5; n=3–4, mean values ± SEM). Analysis of symptomatic A53T α-syn transgenic mice revealed slightly elevated levels of striatal dopamine while the levels of other analytes tested including DOPAC, norepinephrine, and serotonin, did not significantly change (Table 2B). Since the ratio of DOPAC to dopamine reflects the relative rate of DA turnover, these data suggest that the increased concentration of DA may be due to increased synthesis or storage. Catechol levels in the OB of symptomatic A53T α-syn transgenic mice were also similar to age-matched non-transgenic controls (data not shown). Collectively, these data suggest that the presence of Triton-soluble oligomeric α-syn in dopaminergic regions is not associated with alterations in the levels of enzymes involved in dopamine metabolism and coincides with subtle differences in the production of catechols in vivo as the levels of dopamine increase with aging in A53T α-syn transgenic mice.
The documentation that Lewy bodies (LB) and other neuronal inclusions are composed of aggregated α-syn has positioned the process of α-syn aggregation as a critical biochemical event in the pathogenesis of neurodegeneration (Spillantini et al., 1997; Baba et al., 1998; Goedert, 2001). Considerable advances in cell free systems and cell model systems have indicated that the process of aggregation includes a committed step of α-syn dimer formation that is held together by hydrophobic interactions induced by a conformational transition to β-sheet structure (Conway et al., 2000b; Serpell et al., 2000; Uversky et al., 2001). The dimer represents a thermodynamically stable entity allowing the formation of oligomers which will continue to grow in linear β-sheets, forming polarized protofibrils and eventually the typical amyloid fibrils. Alternatively, the transition of oligomers to protofibrils can be kinetically arrested by interactions with small molecules, other proteins or unidentified cellular constituents allowing these oligomers to assume secondary structures such as annular pores or spheres (Conway et al., 2001; Li et al., 2004; Norris et al., 2005).
The emerging appreciation of conformationally distinct oligomers of not only α-syn, but also other proteins that form amyloid structures in human neurodegenerative disorders, has sparked a considerable interest in the pathobiological role of oligomers and mechanism by which they induce neuron dysfunction and neurodegeneration. However, in part due to methodological challenges there is limited insight regarding the biochemical and biological properties of oligomeric species formed in vivo. Therefore the in vivo regional distribution and temporal formation of α-syn oligomeric intermediates was investigated in the previously characterized transgenic mouse model expressing human A53T α-syn (Giasson et al., 2002) that recapitulates the aggregation of α-syn and inclusion formation. Detailed biochemical and biological analysis of the in vivo formed α-syn oligomers revealed unexpected new insights regarding regional differences in conformation and composition that may govern their ability to promote inclusion formation and induce neuron dysfunction.
Using the previously validated two-dimensional analysis, the presence of detergent-soluble oligomeric forms of α-syn was confirmed in brain regions that contain inclusions and unexpectedly in brain regions devoid of α-syn inclusions such as the SN and OB. The presence of oligomeric intermediates in regions containing insoluble α-syn inclusions is expected assuming that these α-syn oligomers represent transient intermediates of the polymerization process. The presence of these oligomers in regions that do not form inclusions, at least up to the life-span of the symptomatic mouse, indicates that the process of aggregation has been initiated in these regions but has not progressed beyond the oligomeric stages. Moreover, the data indicated that oligomers are formed before the formation of inclusions and onset of symptoms (at 4–6 months), and continue to be present at similar levels during aging and symptom onset.
The regional variation is not a result of differences in the levels of human A53T α-syn protein since previous studies utilizing the mouse PrP promoter have shown similar expression levels in various brain regions (Lee et al., 2002), which was confirmed by quantitative western blot analysis for all regions analyzed (Fig. 1 and and2).2). Furthermore, regional variation was not due to generation of oligomers with divergent basic biochemical properties. Detergent-soluble α-syn oligomers isolated from either inclusion bearing or inclusion free regions were composed primarily of 53 Å-size species that are SDS, heat and urea stable, and are sensitive to digestion by proteinase K. However, subtle differences in conformation are evident by the differential reactivity towards oligomer specific antibodies (Fig. 3c). These antibodies were generated against oxidized and tyrosine nitrated α-syn oligomers and were shown to preferentially recognize detergent-soluble α-syn oligomers as well as pathological α-syn inclusions in human brain tissue (Duda et al., 2002). These antibodies also recognize oxidative post-translationally modified α-syn such as dityrosine cross-linked dimers, which has been considered a critical nucleation event for the assembly of α-syn monomers into fibrils (Souza et al., 2000a; Krishnan et al., 2003). It is possible that the oligomers extracted from SC contain a higher proportion of dityrosine cross-linked oligomers that facilitate amyloid fibril formation. This possibility is in part supported by the data documenting that SC-derived oligomers accelerated the aggregation of purified α-syn in a typical fibril formation assay (Fig. 4a and Table 1). In contrast oligomers isolated from the OB prolonged the lag phase of α-syn aggregation in the same assay (Fig. 4b and Table 1). The contrasting biological function of the different oligomeric populations was also evident in primary neuron cultures where oligomers derived from SC but not OB induced cell death and dramatic neurite degeneration as determined by neurofilament stain intensity (Fig. 5).
One candidate pathway that may explain the kinetic deceleration of fiber formation of the OB-extracted oligomers may relate to the previously recognized ability of oxidation products of catechols, such as the ortho-quinones of dopamine and DOPAC, to kinetically arrest the formation of α-syn fibrils (Conway et al., 2001; Li et al., 2005; Norris et al., 2005). This non-covalent interaction has been documented in cell free systems and recently in cellular model systems (Mazzulli et al., 2006; Mazzulli et al., 2007; Mosharov et al., 2009; Outeiro et al., 2009). Interestingly, we found that striatal catechol levels of A53T α-syn transgenic mice increase with age, while they remain stable in non-transgenic mice. This occurred without an apparent increase in the levels of tyrosine hydroxylase, the DA synthesizing rate-limiting enzyme. Tyrosine hydroxylase utilizes tyrosine to catalyze the formation of L-DOPA, a process that is dependent on molecular oxygen, Fe+2, and the cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) (Fitzpatrick, 1989). It has been established that tyrosine hydroxylase activity is intricately regulated through feedback inhibition by catechols, allosteric regulation by various polyanions, and posttranslational modifications such as phosphorylation in the N-terminal region of the protein (Kumer and Vrana, 1996). Therefore, it is possible that the observed increase in striatal DA may result from altered levels of cofactors or posttranslational modifications that increase the enzymatic activity without affecting protein levels. While an increase in striatal DA may provide a biochemical mechanism for preventing the formation of fibrils in the SNpc of A53T α-syn transgenic mice, genetic and pharmacological manipulations of dopamine levels are required to verify the biological importance of a potential α-syn-catechol interaction in vivo. In relation to PD, it is possible that either a decline in steady-state dopamine levels or cleavage of the DA-interacting C-terminus of α-syn contribute to the formation of Lewy bodies in the SNpc, resulting in degeneration of the nigrostriatal pathway.
Whilst the dopamine interaction may be prominent for dopaminergic neurons, other intrinsic factors present in non-dopaminergic neurons must be also considered. Previous studies using cell models have shown that molecular chaperones have the ability to alter the levels of soluble oligomeric species (Tetzlaff et al., 2008). It is possible that some regions of A53T α-syn transgenic mice possess elevated levels molecular chaperones or other intrinsic factors that may engage the soluble oligomers and possibly accelerate clearance or alter their conformation, thus preventing their conversion into insoluble fibrils within the life span of the A53T α-syn transgenic mice. This ability may be sustained with age in some brain regions (such as the Hipp) but decline or become overwhelmed in other susceptible neuronal regions (such as the Crb and SC) which also contain oxidatively modified α-syn species that have the potential to promote the polymerization process resulting in the formation of insoluble inclusions (Souza et al., 2000a).
Overall the data provide a systematic identification and biochemical characterization of in vivo formed oligomeric intermediates of α-syn in the A53T α-syn transgenic mice that exhibit neurodegeneration and α-syn inclusion formation. Although the analysis does not include transient unstable oligomeric conformations that remain unaffected through the extraction process, these more stable and well-represented α-syn assemblies were shown to vary in conformation and possibly composition, based on the region of origin. These differences in addition to other unrecognized regional specific factors may be responsible for the ex vivo and in vivo toxicity of the oligomers. The insights gained by the present analysis highlight the importance of therapeutic interventions with agents that selectively enhance the clearance of α-syn. Those compounds are likely to prevent the formation of both soluble and insoluble toxic α-syn assemblies, providing therapeutic benefit in PD and related disorders.
This work was supported by the National Institutes of Health grants AG09215 (B.I.G), NS053488 (B.I.G.), R01NS051303 (D.K.), USPHS AG13966 (H.I.), ES013508 NIEHS Center of Excellence in Environmental Toxicology (H.I), and award number F32NS066730 to J.R.M. from the National Institute Of Neurological Disorders And Stroke. H.I. is the Gisela and Dennis Alter Research Professor of Pediatrics.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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From all of us at Edelman Sultan Knox Wood / Architects, Happy Holidays and Have a Great New Year!
Last Fall we happily announced the start of construction on the new 60-unit affordable housing building going up in Mt. Eden, the Bronx, under a partnership between Settlement Housing Fund and The Briarwood Organization. 1561 Walton Avenue has progressed steadily ever since and is now more than halfway through plank installation — project team Daughtry, Kerry, and Andrew are very pleased to share a few shots of the progress below.
The building is visually broken up into 4 planes stepping back from the front property line, which will be further articulated with different shades of brick to create a 4-step gradient across the facade.
The project's anticipated completion is Fall 2017.
This Earth Day, we're taking a moment to look back at one of our favorite eco-friendly projects, the Eco Restroom at the Bronx Zoo for the Wildlife Conservation Society. This was a very fun project, and the restrooms far exceeded their initial goal of providing an eco-friendly comfort station to actually become an exhibit and learning opportunity.
The weather is looking great this weekend – it's a great time to go check out the Bronx Zoo, and learn a bit about sustainable water use while you're there.
Photos by David Sundberg for Esto Photography
One of our seasonal traditions here at ESKW/A is the annual holiday gift donation drive. Headed up by illustrious office manager Lauretta daCruz, we collected donations from the office, shopped for gifts, wrapped 'em up, and hand delivered to students at Explore Charter School in Brooklyn. A school social worker and high school placement coordinator identified families who could use some additional cheer this holiday season. The guardians were contacted, and the kids provide a wish list. Each child received special gifts selected just for them off of their list.
We had a lovely haul this year, and we're grateful to everyone who participated. Happy Holidays to all!
Our recently completed 4380 Bronx Boulevard along with current shelter projects at 233 Landing Road, 2570 Fulton, 91 Pitt, 149 W 132, and 8 E 3rd Street, have us thinking about the public perception of shelters and the homeless. Last Wednesday several of us from ESKW/A trouped up to the Bronx Museum to attend and show support for a student presentation on the culmination of a summer spent investigating the ins and outs of the shelter system in NYC. CUP, the Center for Urban Pedagogy, and the College Now program of Hostos Community College brought in teaching artist Patrick Rowe to work with the teens throughout the project and teach them to funnel their newfound knowledge into a visual form (poster shown below).
The students conducted numerous interviews with city council members, the NYC Department of Homeless Services, community board members, and individuals directly associated with local shelters. They also visited a non-profit shelter as well as Picture the Homeless and garnered a better understanding of the system as seen from the inside.
In the resultant presentation, the class was careful to show both sides of the arguments for and against the types and locations of shelters. Many members of the audience were surprised to learn that shelters are not evenly distributed over the boroughs but rather are quite concentrated in the Bronx.
"We like to keep families applying to shelters close to their support systems, their children's school, and to their last known address," said Lisa Black of the NYC Department of Homeless Services. Even with the large number of shelters within the borough, however, they fill up and people can sometimes be relocated across the city. As one student explained, "Let's say you're from here [the Bronx], but let's say all the shelters in the Bronx are full—then they will end up moving to the next closest shelters, which could be in Brooklyn or could be far away, like Staten Island. From that standpoint, you're taking me away from my home, from people I grew up with, where I'm very comfortable."
No discussion of the shelter system can go without touching on the concept of NIMBY, and the class addressed this as well. In his interview with the students, Sam Miller of Picture the Homeless explained, "The Bronx is one of the areas that sends the most families into the shelter system. Many times when communities in the Bronx oppose a shelter, they're opposed to their own neighbors who've been driven out by rising rents."
It was gratifying to hear that on an individual level many of the students felt that their eyes had been opened and their assumptions challenged. As one teen told the audience:
"We were asked the question, 'Who do you think these people are? Where do you think they come from?' and unfortunately I had a very low opinion of them. To be honest, the only homeless people I saw were basically crazy people, drunks, drug addicts, people that didn't really cause me to think about this issue. But as the program went on and as we did our visits, and especially when we went to meet some people from Picture the Homeless, they opened my eyes. Talking to one of the men there taught me through the way that he spoke that not all homeless people are drug addicts, etc. I hope one day to be as smart as him."
Her sentiment was common amongst her classmates, many of whom echoed the assertion that their preconceived notions about the system and especially the shelter inhabitants had been turned around. These students will undoubtedly continue to spread their new knowledge within their neighborhoods, raising awareness and altering the prejudices of those around them. The presentation focused on a serious problem faced by our community, but thanks to the students' enthusiasm the tone of the evening was distinctly hopeful.
Earlier this month we were happy to announce our upcoming partnership with the Bowery Residents' Committee in their revolutionary proposed housing endeavor, Landing Road. Now, as the initial zoning and feasibility studies are complete, it's time for Project Manager Kimberly Murphy and her team to look for the sources of inspiration that will drive the design of this project forward. And inspiration is in no short supply.
Kimberly describes the uplifting event – the BRC fundraising Gala in June – that kicked off the project initially:
"The Bowery Residents' Committee (BRC) held their annual fundraising Gala at the Mondrian Hotel on Columbus Circle. Andrew, Randy, Kimberly, Kerry, and Martin were pleased to support BRC and represent ESKW/A at the event. Emcee Robert Kind (actor from Curb your Enthusiasm and Spin City) shared his dilemma when faced with homeless persons he encounters on the street with his children because he didn't want to just give a hand out. His dilemma was solved when he met BRC because he learned how to give a hand up, not just a hand out.
"A beautifully created short film featuring one of BRC's clients told the story of just one of the many homeless helped by BRC and made the point that all homeless have a story to tell if you're willing to listen. Garland was a special guest at the Gala and was proud to share his journey with all the supporters of the organization.
"Muzzy Rosenblatt, CEO of BRC, thanked all the supporters and described the challenge faced by BRC's formerly homeless clients: affordable housing. Graduates from BRC have done everything they are supposed to do: they are clean, they have jobs (although low-paying), and they are off the streets. However they can't afford to live in this city. Enter BRC's newest project: Project Hope. Located on Landing Road in the Bronx, this new building will provide not only 200 beds and support services to the homeless, but in an innovative model set forth by Mayor Bill De Blasio, the building will also provide affordable housing which will assist in funding the shelter on the ground floor.
"ESKW/A was thrilled to be selected as the architect for the project. Muzzy explained that all but $200,000 has been raised to purchase the site and called on attendees to pony up and donate the balance. And on the spot he was able to raise the remaining funds for Landing Road.
"It was a stellar evening of support for an organization we are proud to be connected to. The view from the 35th floor overlooking Columbus Circle wasn't too shabby either. You know it was an impressive night of fund raising when we recall all of these wonderful details with the distraction of the Lego building block door prizes sitting in front of us. You can't expect Architects to pay much attention to the program when you put Legos in front of them, so well done, BRC!"
The ESKW representatives at the Gala. From the left: Randy Wood, Martin Galindez, Kimberly Murphy, Kerry Zucker, and Andrew Knox


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Blue Bee was established in 2013 by a team of creative professionals, diverse in our skills and abilities but all committed to one main aim - helping our clients achieve their goals.
Based in Salisbury, Wiltshire and Clerkenwell, London, Blue Bee offer a truly diverse range of digital marketing services that are crucial for a business to succeed in the modern world from marketing, design and development.
To make Blue Bee the agency of choice, known as a leader in marketing, driven by great ideas, measured by results.
To grow our positioning in the UK as one of the leading digital marketing agencies.
Encouraged to dream and think big.
Mark is passionate about making Blue Bee one of the foremost digital marketing agencies. With a successful background in marketing combined with creative flair and a great rapport with clients, Mark will advise you on the best ways to promote your business.
When not at Blue Bee HQ, he loves to spend time with his family and watching football.
Christoph oversees our technical department. His attention to detail and vast technical knowledge underpins the quality of our websites and project delivery. Christoph works closely with Dan to ensure client projects are delivered smoothly.
Whilst he loves work, and keeping on top of industry advances, Christoph also enjoys sailing and can be found in and around the Solent at least once a month.
Marketing Manager
Dan has a Marketing degree and is our head of Marketing. He's worked with some of the country's best-known brands and his expertise in developing brand strategy and project delivery has been vital to the smooth running of Blue Bee.
Dan is Chairman of the Federation of Small Businesses in Salisbury , and when not improving the marketing of companies, you can find him at St Marys Stadium supporting the Saints.
It would be quite a list if we wrote down all Vicky's roles. Keeping us organised, on track and paid are her priorities and she loves the variety of her work.
When not in the office, Vicks enjoys cycling, keeping fit and eating. She needs to do the first two to make up for the eating.
Development and Training
Taking projects from design to fruition is what David does best and when that's all done, he helps clients understand their new website and how they can make changes if they want to.
David is sports-mad – like most of us at Blue Bee – and a keen West Ham supporter.
Graphic Design
The newest member of our team, Charlie is already making a big impact on our graphic design department. Her natural artistic talent and great sense of design are helping our clients present their brands in exciting new ways.
Charlie loves staying on top of new industry ideas, and when not doing that, she loves music and open mic nights.
Graphic Design and Development
Our multi-talented designer / developer Liam is quite unique in our industry. Not only is he great at designing beautiful websites, he is also amazing at building them as well.
Liam is a massive comic book fan, and has been to Comic-cons across the world!
Head of Barketing
You'll often find Walt asleep at the office. What he lacks in productivity, he makes up for in cuteness. Walt loves long walks in the park and hates Hoovers.


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Advantages of Using Clothes Lines
Published on July 17th 2012 | Advertising
Published on July 17th 2012 | Advertising
A clothes line or a washing line is a kind of line made of any kind of durable material to dry clothes. The line is stretched between two points and is usually attached to a wall....
Austral Clotheslines – Leading Clothesline Manufacturers in Australia
Published on July 16th 2012 | Shopping
Published on July 16th 2012 | Shopping
Austral Clotheslines are a leading brand of Australian clothesline manufacturers. All the products are 100% indigenously made and there are no imports from other countries like China. The company too ...


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Reliable Binary Options Brokers That Are Fair!
Welcome to our trusted Binary Options brokers page. Having a broker is an essential tool when trading, in fact, its impossible to trade without one! Its crucial that you pick a broker that not only has a good platform, but one that is reliable and fair. Many Binary Options brokers are scams or follow illegitimate or illegal activities. Some of these activities are making it incredibly difficult if not impossible for users to withdraw profits from the Binary Options broker. We don't like this one single bit and we aim to put a stop to it!
We're here to review Binary Options brokers that are not only here to stay, but who are true to their word and pay out to their clients. Below are some of the best Binary Options brokers to date. They all have a proven track record of excellence to traders and many have been around for several years. Please spend time to read the reviews of each broker so that you can be sure you select the best broker for you. When selecting one of the below brokers there are a few things think about. First of all its important to know which assets you would like and match that with which assets are available for trading.
Another vital consideration is the minimum amount required per transition. Depending on the amount of money you are willing to invest, users should always take into consideration good money management. Ideally investors should invest 5-10% per trade, this means that if you are investing $200 you should be looking to invest $10-$20 on each trade. Often people like to be able to trade on the go from their mobile devices and tablets. You might also want take into account if you want your Binary Options broker to have an app, though I would point out that most brokers will allow you to trade through the browser of your mobile device


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Deborah Hanley
Listed by Coldwell Banker Hickok And Boardman
You'll love the extensive updates in this 3 bedroom, 1.5 bath home in sought after Dorset Park. Improvements include a beautiful kitchen with granite counters and some stainless steel appliances, ductless A/C units, most windows replaced a few years ago, including adding 1 new window on each side of the sliding door overlooking the deck and the beautiful pond. You'll also enjoy the updated vanities with Italian White Marble Tops, and the custom built-ins in the family room and some of the closets. The full bath upstairs was tastefully updated as well. You can't beat the convenience of the attached 1-car garage. There is an association pool right down the street as well as a clubhouse. Step inside, you'll love it!
Date Showings Begin:
Delayed Showing: Unknown


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Sometimes I feel like I have fallen so far behind I will never catch up. Then I realize I am the one who sets the standards and only I know whether or not I have fallen behind or not. So, why does this matter?
It matters for a lot of reasons. I feel like I am not being lazy when I get something accomplished. I enjoy walking into a tidy home instead of a messy one. I like to have freshly baked goods for my children instead of one's that will last until next year. A cookie should never be safe to eat a year later!
As my brain scatters about today the word fall has suddenly reminded me of the times I fell on stairs. The darned twisted ankle or being pushed while I was pregnant and when I fainted at the top of the stairs because of a medication and falling backwards down them a lifetime ago.
Then I wonder why on earth I want to jump out of an airplane someday when falling always tends to hurt. I must be crazier than I had thought! Oh, to be free falling through the air being slowed down by the wind as I fall through it at the same time. It sounds magical, scary, exhilarating and even terrifying.
I think a lot of people associate "fall" with failing. The fall of an empire, the rise and fall of a governing force, of rancid people like Hitler, of countries that go from the top to the bottom in a matter of minutes.
Who decides who has fallen though? Is it society or culture or individuals or maybe the news that broadcasts it?
What about the people who have a personal fall that goes unnoticed? Does this mean that the fall never occurred because no one cared? Much like the question "If a tree falls in a forest and no one is around to hear it does it make a sound?"
The best part of a fall is being able to once again rise!


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Share this game with your friends
Did you know?
This game has been added 11.01.2017, has played by 1950 times and has voted by 71 times.
We are sure you have already heard about the BFG games in which you can actually play with giants and that is a very interesting thing to do because, as we well know, we don't have giants in real life and the fact that here you get to know them, to become their friends and of course to play wtogether all kind of funny and silly games, that is a very special thing and that is why we hope you are going to enjoy the BFG games and of course this new one right here which is a really cute one. As you can see, you get to play with your name, that means that in this game you wll find out what is your giant name. All you need to do is type in your name and the game will generate your giant name. Good luck!
Use the MOUSE
You can also read the instructions that Make My Giant Name are given in the game and follow them carefully!
Find the Make My Giant Name game by searching for this items:
the bfg games, games with the bfg, the bfg, the bfg new games, fun games with the bfg, the bfg games for kids, fun games with the bfg, the bfg games for 2017, free games with the bfg, the bfg funny games, the bfg interesting games, cool games with the bfg, the bfg giants games, games with giants, interesting giants games, make my giant name, giant name games, make my giant online, free game, free games, fun game, fun games, funny games, games for kids, interesting games, new games
Here you can say whatever you want if follow a language without swearing and insults. All comments are verified by an administrator to be sure that all our players respect the rules. Have fun!


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It's a known fact that Donald Trump said some unsavory things while filming a segment for Access Hollywood nearly a decade ago, but whether or not he was equally inappropriate while on Celebrity Apprentice is still up for debate.
TV Mark Burnett
It's a known fact that Donald Trump said some unsavory things while filming a segment for Access Hollywood nearly a decade ago, but whether or not he was equally inappropriate while on Celebrity Apprentice is still up for debate.
television jessica rawden 2016-10-11
Donald Trump has been dealing with backlash related to a leaked video of the former reality host making disparaging remarks towards women for a week now. More footage of the same ilk is allegedly out there.
television jessica rawden 2016-10-10
The news is coming just a few weeks after Viacom executive Philippe Dauman made some bold statements, noting that the next year will be "a landmark" for MTV. Go ahead, get a little excited.
television jessica rawden 2016-04-21
If there's a down side to watching all of those cooking shows that air on the various cable channels, it's that all of the delicious food being prepared by chefs -- amateur and professional alike -- are inaccessible to us through our television sets. We can look, but we can't taste.
television Kelly West 2013-10-14
Mark Burnett and Roma Downey are interested in putting together a sequel to the hit mini-series The Bible, and the prolific producers have already found a network for the project. On Monday, NBC announced it had signed on for A.D.: Beyond the Bible, the follow-up project that will follow the years after the death of Jesus Christ.
television jessica rawden 2013-07-01
History Channel's The Bible still has four episodes to air over the next two weeks, so if you haven't caught the TV miniseries, you still have a chance. If you are already a fan of the miniseries, which History is dubbing "the greatest story ever told," you'll be able to purchase the series much sooner than you might guess. The Bible will hit Blu-ray, DVD, and digital HD on April 2.
television jessica rawden 2013-03-21
TBS is putting together two brand new unscipted series with fairly big names involved. The first upcoming program has America's Got Talent's Howie Mandel at the helm and the second has prolific producer Mark Burnett, who has his hands in producing pots as well-known as Survivor and Shark Tank. Both new unscripted shows are expected to premiere sometime in 2013.
television jessica rawden 2013-01-11
Syfy doesn't appear to be shying away from the ghost business anytime soon. The network, which continues to air the popular unscripted series Ghost Hunters, is set to bring another paranormal-themed show to the network, which tells the true stories of potentially haunted schools across the country.
television Kelly West 2011-10-18
Mark Burnett has decided to do his best King James impersonation by bringing his vision of the The Bible to The History Channel. Yes, the man best known for producing such hard-hitting series as Survivor and The Apprentice will bring you the word of GOD... in a 10-part miniseries to air on the History Channel.
television Jesse Carp 2011-05-24
Late night host Carson Daly is also in place as The Voice's host, providing more stability to the offering many suspected would be cast aside as an American Idol also ran
television Mack Rawden 2011-03-02
NBC's The Voice premiers on April 26th at 9 PM EST. Don't expect it to do Idol-like numbers, but it should compete fairly well during its heated Tuesday night timeslot
television Mack Rawden 2011-02-28
Word is officially in. Sarah Palin's getting her own reality show! Discovery Communications signed a deal with the former governor of Alaska for her to star in a series centering on the 49th state.
television Kelly West 2010-03-25
It looks like This is Your Life will be returning to American TV. The series, which has been on the air in various forms in the US and overseas, originated as a radio program back in 1948 when people didn't have TV's and instead, gathered around their giant radios to listen to Little Orphan Annie and other programs.
television Kelly West 2008-10-09
Mark burnett Stories 1 to 13 of 13
Movie Guides


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|Vol 10, No 3: August 2016
|A Combinational Digital Logic Design Tool for Practice and Assessment in Engineering Education
|Abstract PDF
|Rasha Morsi
|Vol 10, No 3: August 2016
|A Descriptive Study of the Critical Thinking Skills of Social Science at Junior High School
|Abstract PDF
|Sri Hapsari
|Vol 7, No 2: May 2013
|A Drama Textbook with Sociodrama Method: Research and Development in Linguistic Education Study Program in Central Java
|Abstract PDF
|Evy Tri Widyahening
|Vol 11, No 3: August 2017
|A Learning Model for Cultivating Self-Awareness on Human Decision-Making in an Emergency Situation
|Abstract PDF
|Chaianun Damrongrat, Mitsuru Ikeda, Alisa Kongthon, Thepchai Supnithi
|Vol 10, No 3: August 2016
|A Model of Women Literacy Preservation through Koran Ibu Program
|Abstract PDF
|Ikka Kartika Abbas Fauzi
|Vol 10, No 3: August 2016
|A Survey of Biology Teachers Use of Activity-Oriented, Laboratory Practical Exercises to Promote Functional Biology Education
|Abstract PDF
|Abigail Mgboyibo Osuafor, Ijeoma A. Amaefuna
|Vol 7, No 3: August 2013
|A Survey of User-Centered System Design for Supporting Online Collaborative Writing
|Abstract PDF
|Nani Sri Handayani
|Vol 11, No 3: August 2017
|Abilities of Reasoning and Mathematics Representation on Guided Inquiry Learning
|Abstract PDF
|Y. Yumiati, Mery Noviyanti
|Vol 11, No 4: November 2017
|Action Reflected and Project Based Combined Methodology for the Appropriate Comprehension of Mechanisms in Industrial Design Education
|H. Güçlü Yavuzcan, Damla Şahin
|Vol 11, No 4: November 2017
|Addressing Reading Instruction to the Issue of Essential Academic Achievements
|Arina Mufrihah, Fitri Aulia
|Vol 6, No 4: November 2012
|Adult Education through Multiple Method For Poor Rural Illiterate Women in Indonesia
|Abstract PDF
|Kamin Sumardi
|Vol 9, No 4: November 2015
|Affirmative Action Application or Black and White in South Africa Higher Education Institutions: Is it the way forward or not?
|Abstract PDF
|Christiena Maria van der Bank, Jafta Mphahlani, Kholeka C Moloi
|Vol 6, No 2: May 2012
|Agricultural Education for Sustainable Rural Development in Developing Countries – Challenges and Policy Options
|Abstract PDF
|Jhansi Seetharam Chittoor, Santosh Kumar Mishra
|Vol 9, No 4: November 2015
|An Epistemological Study on Somantri's Thought about the Social Studies as a Synthetic Discipline
|Abstract PDF
|Mohammad Imam Farisi, Udin S. Winataputra, I.G.A.K. Wardhani
|Vol 7, No 4: November 2013
|An Exploratory Study of the Critical Need for School Health Programs in Lebanon
|Abstract PDF
|Khayrazad kari Jabbour
|Vol 8, No 2: May 2014
|An Intervention Study in Grade 3 Based Upon Reciprocal Teaching
|Abstract PDF
|Monica Reichenberg, Kent Löfgren
|Vol 10, No 3: August 2016
|An Interview with Marcia Tate: Formative Assessment and Brain Based Learning
|Abstract PDF
|Michael Shaughnessy
|Vol 9, No 2: May 2015
|An Investigation on Organizational Charlatan Behaviour and Moral Identity as Predictors of Shame: Importance for Education
|Abstract PDF
|Juneman Abraham, Rahma Putri Noka Berline
|Vol 7, No 1: February 2013
|An Overview of ICT Integration in Nigerian Colleges of Education and the Implications on Social Studies Pre-Service Teacher Training Programme: A Review of the Literature
|Abstract PDF
|Sani Alhaji Garba, Termit Kaur Ranjit Singh, Najeemah Binti Mohammad Yusuf, Azidah Abu Ziden
|Vol 10, No 3: August 2016
|Analysis of Curriculum 2013 Mathematics Textbook for Senior High School X Grade
|Dian Kurniati, Dafik Dafik, Farah Aisadita, Moch. Agus Kurniawan
|Vol 10, No 2: May 2016
|Application of Employability Skills and Contextual Performance Level of Employees in Government Agencies
|Abstract PDF
|Maripaz C. Abas
|Vol 6, No 3: August 2012
|Applying to Study English Course at a College: A Study on a Motivational Aspect
|Abstract PDF
|Thongma Souriyavongsa, Ismail Raob
|Vol 9, No 1: February 2015
|Assessment on Hybrid E-Learning Instrument
|Abstract PDF
|Intan Farahana Kamsin, Rosseni Din
|Vol 8, No 3: August 2014
|Attitudes and Motivation toward Learning the English Language among Students from Islamic Education System Background: Exploring the Views of Teachers
|Abstract PDF
|Ismail Sheikh Ahmad, Hazlina Abdullah, Muhammad Faizal A Ghani
|Vol 11, No 2: May 2017
|Awareness, Knowledge, Proficiency, Training and Expertise of Public Elementary School Teachers in Tacloban City on Education for Sustainable Development
|Abstract PDF
|Generoso Nielo Mazo
|1 - 25 of 246 Items
|1 2 3 4 5 6 7 8 9 10 > >>


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WikiProject Bermuda
From OpenStreetMap Wiki
(Redirected from Bermuda)
| Bermuda, United Kingdom, North Atlantic, North America
|latitude: 32.3, longitude: -64.76
|Browse map of Bermuda 32°18′00.00″ N, 64°45′36.00″ W
|Edit map
| External links:
|Use this template for your city
Bermuda is a territory in United Kingdom, North Atlantic, North America at latitude 32°18′00.00″ North, longitude 64°45′36.00″ West.
|OpenStreetMap images (and underlying map data) are freely available under OpenStreetMap License.
The project of mapping Bermuda can be done successfully with a small number of people.
Update Aug 2010
Major roads & bridges 75% complete; primary airport runways, taxiways and aprons mapped. Most of the fiddly ways still left to do, lots of POIs (docks, hospitals etc) still need tagging. MattD 21/08/2010
Update Oct 2012
- Most of St. George buildings finished.
- Dockyard (most western island) finished.
- Nearly all parks mapped.
- Important golf courses mapped.
- Most addresses, little streets names, and islands names are still missing.
- Now better than Google Maps in most parts !!
hno2 25/09/12
|Town of St. George
|St. George (parish)
Colour and symbol legend
The status of each map region is indicated by a symbol, which describes the type of feature, and a colour, which indicates the completeness of that feature in a map region.
The status is displayed using a template Template:State
- progress - 1/2/3/4/NA
Meaning of symbols
- - Roads for car traffic are present. One way streets and pedestrian streets are present. This means that the map can be used for car navigation - Key: c
- - Street names are labelled. This means that the map can be used to find an address - Key: l
- - All housenumbers are present - Key: h
- - All cycleways, and field and forest roads suitable for bicycles are present - Key: b
- - All footways are present - Key: fo
- - All wheelchair access is present - Key: d
- - All public transports are present (including name of the bus stop/station names) - Key: tr
- - All stop/station/harbour names are present - Key: ha
- - All bus lines are present (including name of the bus stop names) - Key: bu
- - All trolley/guided bus lines are present (including name of the station names) - Key: ty
- - All tram lines are present (including name of the tram station names) - Key: tm
- - All U-Bahn (subway in Germany) lines are present (including name of the station names) - Key: u
- - All S-Bahn (regional commuter train in Germany) lines are present (including name of the station names) - Key: s
- - All fast railway lines transports are present (including name of the train station names) - Key: tm
- - All steam railway lines transports are present (including name of the train station names) - Key: st
- - All ship lines are present (including name of the harbour station names) - Key: sh
- - All public institutions are present (Churches, sports facilities, venues, schools, hospitals, city hall) - Key: p
- - All petrol stations are present - Key: fu
- - All restaurants and hotels are present - Key: r
- - All tourist attractions are present - Key: t
- - All natural resources are mapped (e.g Water, Lakes and Woodland) - Key: n
Meaning of colours
The English translation is Editable here.
|North America – Countries and territories
|Anguilla (UK) · Antigua and Barbuda · Aruba (NL) · The Bahamas · Barbados · Belize · Bermuda (UK) · Bonaire (NL) · The British Virgin Islands (UK) · Canada · The Cayman Islands (UK) · Costa Rica · Cuba · Dominica · Dominican Republic · El Salvador · Greenland (DK) · Grenada · Guadeloupe (FR) · Guatemala · Haiti · Honduras · Jamaica · Martinique (FR) · Mexico · Montserrat (UK) · Navassa Island (US) · Nicaragua · Panama · Puerto Rico (US) · Saba (NL) · Saint Barthelemy (FR) · Saint Kitts and Nevis · Saint Lucia · Saint Martin (FR) · Saint Martin (NL) · Saint-Pierre and Miquelon (FR) · Saint Vincent and the Grenadines · Saint Eustatius (NL) · Trinidad and Tobago · The Turks and Caicos Islands (UK) · United States · The United States Virgin Islands (US)


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Our trainings in the digital workshop
Here we offer you various training units. After completing these modules on the metallurgical process chain, you'll be familiar with the basic tasks, characteristics, and functions of the main plants and machines used in this process:
The learning units (1 day):
- Virtual-reality plant tour
- Introduction
- CSP® plants
- Hot rolling
German Courses (No. 1121):
February 22, 2017(Closing date for reservation: January 25, 2017)
English Courses (No. 1120):
March 29, 2017(Closing date for reservation: March 1, 2017)
SMS group GmbH - Düsseldorf
Traning Fee
€ 900.00
Target group
Anybody who wants to understand the processes of CSP<sup>®</sup> plants and hot rolling.
More Information
You'd like to have more information about this training?Contact


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Welcome to the PDX Trail Community!
We feel lucky to have a wonderful community here in Portland, Oregon and at Wy'east Wolfpack! It's truly the best part of our job to get to work with such awesome, down-to-earth people...
Meet the founders of Wy'east Wolfpack:
Wy'east Wolfpack and the following three local businesses are integral parts of the Portland, Oregon trail community; we have the training & coaching to get prepared, Territory's got the apparel to wear, Trail Butter the all-natural fuel to eat, and Go Beyond the races & events to give us goals to work toward and keep us motivated!
Launched in 2014, Brett Farrell and his Territory Run Co. has made its presence felt in Portland and far beyond, gaining an impressive following of like-minded adventurers who share the companies' philosophy of "wild and free." Many of these individuals, Willie and Yassine included, comprise the "Runners of the Wild" team.
Trail Butter is a product we use and love. Even better, it's a small Portland, Oregon business created by one of our closest friends, Jeff Boggess, and his brother Brad. These guys have created a wonderful community around a delicious product and an impressive group of ambassadors in the form of Team Trail Butter (of which Willie and Yassine are honored to be a part.)
Keep up the good work guys!
Made official in 2013, our friends Todd Janssen, Renee Seker, and Trevor Hostetler have done an amazing job of making Go Beyond Racing the local Portland, Oregon race company. They have been successfully providing the Portland area community and beyond with top-notch events, from the local Portland Trail Series and Trail Factor Half Marathon and 50k in our very own Forest Park to the perennial favorite Mt. Hood 50 miler near Timothy Lake. They've got a number of newer, soon-to-be-classics too, like the Elk-Kings 25k/50k, Mountain Lakes 100, NUT 50k/100k, and Pick Your Poison Relay, to name a few...
We work often with the fine folks at Go Beyond Racing and love hosting aid stations at their events, supporting runners reaching for big goals. See you out there!
We are thankful to be partnered with the official Portland location of Fleet Feet Sports and their team of awesome local folks who are working to make the store a hub for trail runners, especially those heading out to, or returning from, the very nearby Forest Park! Besides the friendly staff and access to a wealth of information, the Fleet Feet PDX store has the biggest map of Forest Park in the entire world! Seriously, it's amazing and has all trails and mileage marked and is hung up on the wall for easy perusal; perfect for route planning, or sitting on their couches afterwards to gaze at what you just ran.
We also team up with Fleet Feet on events and classes such as the popular Blue Diamonds Trail Series and are proud to have their support on our latest community project: Wy'east Trail Sisters.
Other awesome businesses we work with:
Rainshadow Running, helmed by creator/Race Director James Varner and his merry crew of outstanding employees, puts on many quintessential Pacific Northwest races. Known for their down-to-earth atmosphere, awesome vibes, live music, and beer and wood-fired pizza at the finish, you're basically guaranteed to have a good time! From classic Portland area events like Gorge Waterfalls 50k and 100k, Beacon Rock 25k & 50k and their new-ish Oregon Coast 30k and 50k to their popular races in the North Cascades and on Orcas Island, James and gang know how to do it right in some of the most beautiful places in the country.
We host aid stations at a number of Rainshadow events, so get some on your race calendar and we'll see you there!
Extra special thanks to:
Allison Bryan of Open Studio Collective, and Georgia Barnett, for their help with this website! Check out Allison's awesome work, including her beautiful Forest Park Art Print and Western States map.
Our dear friends Lizy Gershenzon and Travis Kochel, founders of the multi-faceted, local Portland business Scribble Tone, for their fine work in designing our new logo and branding and giving us a fresh new face as Wy'east Wolfpack. We are forever grateful for their immense talent and generosity. Check them out!


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Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
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Call us at QuickBooks support phone number (1-800-760-5113) and get an error free and highly function software instantly.


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This has been a hectic few months, but good things are happening and work is moving along. One thing I've been working on a lot is the Upward Anthropology Research Community (also see our Facebook and Twitter page). We have a lot of big plans for the coming months – especially an off-conference workshop during the AAA conference in DC in December. We hope to get a good turnout, and so we'll be doing a lot of publicizing and ramping up for that. As a result, you can expect it to be relatively quiet around here, but if you want to keep track of what I'm up to, you can take a look over at the UARC.
Part of the ramping up of the UARC is another workshop at the American University Public Anthropology Conference. The workshop will be held on October 4 from 2:30-4:30. That's right before the keynote by Carole McGranahan titled "Tibet, Ferguson, Gaza: On Political Crisis and Anthropological Responsibility," so come for the workshop and stay for the keynote – it should be good! I will also be giving a talk earlier in the day on collaborative modeling and environmental management.
Another thing on my agenda is remobilizing the Vulnerability volume that Adam Robbert and I were organizing a while ago. I've been in the process of contacting our previous authors to see if they're still interested, and am also contacting new authors and contributors to see what else we can do with this thing. Hopefully we'll see a book out of it soon! Thanks to everyone who has been tagging along and patiently waiting for me to get back to work. If you want to help or have any ideas for contributions, contact me and we'll work it out.