text:
Johnny Paycheck (Donald Eugene Lytle)
Johnny PayCheck, the hard-living Nashville guitar slinger who urged the working class to "Take This Job and Shove It," has died.
Although he had been sober for years, decades of boozing and drugging had taken its toll on the honky-tonker. He had been in declining health for the past several years, spending recent months bedridden in a Nashville nursing home battling emphysema and asthma.
He died February 18 at Vanderbilt University Medical Center at the age of 64.
His old partying buddy George Jones remembered PayCheck as "a great country singer." Jones, who gave PayCheck an early break by hiring him as a bass player and later recorded the album Double Trouble with PayCheck, donated a grave site for the singer, who died broke. The site is adjacent to Jones' own.
"The world will miss a great country singer," said Jones, "and I will miss my friend."
Born Donald Eugene Lytle on May 31, 1938, in Greenfield, Ohio, he picked up the guitar at the age of 6 and began singing professionally by 15. After joining Jones' band, he had stints backing Faron Young, Ray Price and Poter Wagoner and, under the name "Donny Young," wrote hit tunes for Tammy Wynette ("Apartment #9) and Price ("Touch My Heart"). He adopted the moniker Johnny Paycheck in the 1960s (he began capitalizing the "c" in the 1990s) to help boost his profile in Nashville when he decided to go from sideman to solo artist.
The diminutive singer (he topped the tape at just 5-foot-5) made up for his short stature with plenty of attitude, both in his songs and his off-stage carousing.
He joined the Navy as a teenager in the 1950s. He married his first wife, Dinorah, in 1956 and they had a daughter, Jacqueline. But he went AWOL and then wound up court-martialed and sentenced to two years in the brig for whacking an officer in the head, fraturing his skull. His growing alcoholism and constant touring took its toll and the couple split in 1965. His career almost ended in 1989, when he began a seven-to-nine-year sentence for shooting a man in the head in an Ohio bar in 1985 (the victim survived). The Ohio governor, however, commuted the sentence to two years and PayCheck returned to recording.
Greenfield, Ohio
February, 19, 2003
emphysema & asthma


I slept for what felt like a hundred hours at the weekend, I've got a million USBs on my desk, and I'm starting to remember what lunch at actual lunch time looks like again. Yes, it must be the week after Formnext and now that the team is done pounding the floors of Messe Frankfurt and presenting yet another first-class TCT conference @ Formnext, I've actually had a chance to gather my thoughts from four days of additive manufacturing (AM) launches and conversations.
With around 35 press events divvied up between the TCT editorial team, I prepared myself for the onslaught of superlatives and "revolution"s which have become commonplace at such events where a 3D printed metal lattice just doesn't have the same pizazz as years gone by. But as I trekked across both halls and back and forth between meeting rooms, I was met with a very different sense of excitement as conversations were more about "hey, look at how this SME is using our machines" compared to "my build plate is bigger than yours".
Of course, this shift didn't mean big hardware launches were completely done away with. GE Additive was just one company with hardware news as it announced the availability of its Concept Laser M Line Factory systems which debuted at the very same event three years ago. The machine was one of the first modular style systems introduced, running autonomously with robots transporting between process stations, a concept which has now become synonymous with the "factory of the future" vision, and now having undergone rigorous testing, is ready to be shipped to initial customers in Q2 next year. Seeing this concept finally coming to fruition is a sign of the industry's ongoing progression and GE also announced software and materials updates, cementing the idea that AM is not simply about the machine.
This was also clear on the TCT Introducing @ Formnext stage where I was able to moderate a number of the materials and software sessions. These talks, served in snappy 15 minute segments are a great way of learning what's new across each sector of the industry whether it's new hardware developments or post processing techniques. To see a talk about software for post processing (often referred to as the industry's dirty little secret) draw in such a crowd was extremely encouraging and speaks volumes about the way people are now thinking about additive. Sure, it's great that you've got the design and printing down, but what happens after? And if additive is truly a digital technology, surely that digitisation should extend to any post processing steps too? Link3D's new Post Processing Management platform addresses those concerns.
Digitisation was a big theme throughout. Machine tool and laser manufacturer, Trumpf, said digitisation is a key term for manufacturing. It's addressing that with its own TruConnect Industry 4.0 concept and also in its Smart Factory in Chicago where people, machines, automation and software all interact together. Though Trumpf has typically used the Frankfurt event to launch AM hardware, there were no major launches here but it did announce updates to its TruPrint 5000 system, which can now be preheated to 500 degrees Celsius, and a new green laser concept, opening AM up to even more materials and applications.
There were less concepts and "coming soon"s and more shipping dates (Desktop Metal, for example, is getting ready to ship an updated version of its Production System which offers incredible detail and productivity) and customer stories which is a welcome change when so very often we come across machines that aren't even close to being on the market or in some cases, we see once and never hear of again. ExOne, 3D Systems, Arburg, Rize and BigRep also exhibited new machines. The exception here was Stratasys which opted to share more details of its gradually teased metal 3D printing technology with some sample parts but no hardware on display. It now has a name, Layered Powder Metallurgy, and is based on a three-step process combining traditional powder metallurgy with Stratasys' PolyJet ink-jet technology.
Though some visitors may have been expecting more from the metals side, Stratasys had some tricks up its sleeve on the materials front with new capabilities and colours for its polymer processes. In addition, we saw new medical grade materials from Solvay, incredibly soft SLS materials from Sinterit, new ceramic material and soluble support from Xjet, and material profiles from Ultimaker with DSM, BASF, DuPont and others.
On software, Materialise and Simufact both showcased updated simulation tools to assist print right first time, saving development time, materials, costs, which are helping to push production, and MachineWorks demoed the latest version of its Polygonica software which is being leveraged by key industry players.
This year felt different, we started seeing evidence of this at TCT Show in September where incremental developments, which may have once gone unnoticed alongside shiny new hardware, are now becoming all the more crucial. We've got the tools now and companies are putting considerable stock into developing the various parts of the value chain to ensure they can be successfully integrated into industry.
I think it's a good thing we're not as focused on AM being a "revolution". In fact, I can't tell you the number of conversations I've had over the years with industry folk who would love nothing more than to do away with the word along with other infamous tropes like "disruptive" and "innovation ecosystem" (sorry, what now?). It might not be a so-called revolution but what we are able to do with this technology now, will be a revelation to those who may have previously thought it didn't apply to them or was inaccessible, too complicated, not the right materials. Even I, who sometimes suffers from the same tedium our Head of Content, Daniel O'Connor described in his recent IMTS review, felt newly invigorated by some of the speeds, materials (lots of ceramics, silicones and new ways of printing precious metals) and application examples I saw.
There was a lot to see and learn year and we will be continuing to bring you news and interviews from the show floor over the next few weeks and in our upcoming Formnext Review. Subscribe here to receive your free print copy.
TCT Deputy Group Editor, Laura Griffiths gives her own unique insight into the additive manufacturing and 3D printing industry.


"Ghost House: A Haunting" delves into the depths of a haunt that is far more than residual. Even the living are left questioning if they are alive or dead, after they buy the ticket to take this fantastic thrill ride. On the last tour of Halloween night, a tour group of unsuspecting visitors quickly realize their haunted house experience is more than jump scares and cheap tricks. Cold cases are drug into the spotlight to unearth dark secrets as history repeats itself and the dead refuse to rest in peace.


text:
Apple Card Family Promo: Add a co-owner and earn $100 in Daily Cash
Tapbots releases new "Ivory" app for Mastodon
iPhone 5S receives security update
Opinion: How Apple Could Bring Apple Pay to The Apple TV
It's no secret that you can buy things on the Apple TV via stores like iTunes or apps like GrubHub. However, unlike the iPhone, iPad, and Mac, you cannot use Apple Pay to purchase these items. Instead, you have to either have to have the payment go through your iTunes account or type in your credit/debit card information when making a purchase.
In all fairness to Apple and other companies, they do at least make you sign in to your account before you can buy something and try to implement Amazon's "one-click and buy" method.
via @JonyIveParody on Twitter
The way how Apple could implement Apple Pay into the Apple TV is by adding a little adapter to the Siri Remote. This would be similar to the Lightning to 30-pin adapter for iPhone, iPad, and iPod Touch. Anytime you want to make a purchase via Apple Pay on your Apple TV, plug in this adapter to the Siri Remote and you could do just that.
This does make it sound like Apple is making customers buy adapters and dongles for their products, which are not only expensive but also unnecessary.
Of course, Apple could always make a new Siri Remote, sell it as a standalone accessory and also have it in "newer" Apple TV's (perhaps release it with a new 4K Apple TV?). It's just another way to bring Apple Pay to another device.
Siri Remote
Today is the Day; April 18th, 2017
Sling TV's Cloud DVR Services Now Available for Apple TV
New MacBook Pro and Mac mini now available in stores and online today
Deal: Get up to 6 months of Apple Music with an NFL+ subscription
My Gift Recommendations for Father's Day 2017
Father's Day is coming up on June 18th, so you now have less than 10 days to get…
Apple confirms a serious problem with the iPhone X – and an unlikely solution
Face ID, Apple's sophisticated 3D-sensing face recognition system, is one of the signature features of the iPhone X.…
Kelvin Tan
UPDATE: Apple Bug Reporter is down for scheduled maintenance.
Apple's 'Bug Reporter' is currently down right now for maintenance. I believe that Apple is updating the Bug…
Holden Satterwhite

redpajama_set_name: RedPajamaCommonCrawl


text:
Worms Revolution – strategiczna gra turowa należąca do cyklu gier komputerowych Worms, wyprodukowana i wydana przez Team17 10 października 2012 na platformach PlayStation 3, Xbox 360 oraz Microsoft Windows. Gra została oparta na trójwymiarowym silniku graficznym z zachowaniem dwuwymiarowej rozgrywki.

Rozgrywka
Gra kontynuuje tradycyjną rozgrywkę z serii, w której drużyny tytułowych robaków na zmianę korzystają z różnych broni i przedmiotów w celu wzajemnej eliminacji. Rozgrywka toczy się na planszy skomponowanej w stylu grafiki 2.5 D. Nowością wprowadzoną do serii jest silniejszy nacisk na dynamiczne obiekty objęte prawami fizyki. W przeciwieństwie do poprzednich odsłon cyklu, w Worms Revolution woda obecna jest nie tylko w dolnej części planszy. Gracze, wykorzystując elementy mapy, mogą zdobyć przewagę nad przeciwnikami. Przebywanie pod wodą powoduje powolne tonięcie i stopniową utratę punktów życia. Do gry wprowadzono również nowe bronie (np. pistolet na wodę), które uwzględniają zachowanie wody zgodne z fizyką gry. W rozgrywce pojawiają się także inne obiekty, które gracze mogą wykorzystywać i używać do zadawania obrażeń. Kolejną nowością w serii jest wprowadzenie czterech różnych klas dla graczy:
 żołnierz – zwykły robak, który jako jedyna z klas nie ma szczególnych zdolności.
 zwiadowca – jest w stanie poruszać się szybciej, skakać wyżej i dalej, ale jego ataki zadają mniej obrażeń.
 naukowiec – może wyprodukować lepszą broń oraz przyznawać innym zawodnikom w drużynie premię w postaci zdrowia.
 strażnik – powolnie się porusza, ale jego ataki wyrządzają więcej obrażeń.

Gra zawiera opcje rozgrywki jednoosobowej oraz rozgrywkę dla wielu graczy (przy jednym komputerze oraz przez sieć). Istnieją również różne opcje dostosowania zasad meczu, wyglądu robaków, liczby dostępnych rodzajów broni oraz obiektów obecnych na mapie.

Rozwój gry
Gra oparta jest na oryginalnych częściach z serii Worms. W dniu 30 marca 2012 roku Team 17 zapowiedział, że pracują oni nad rozgrywką na nowym silniku. 26 maja 2012 roku firma ogłosiła, że Matt Berry i Dean Wilkinson będą odpowiedzialni za jej tworzenie.

Rozszerzenia (DLC)
Do gry zostały wydane cztery rozszerzenia (DLC):
 Worms Revolution: Medieval Tales,
 Worms Revolution: Funfair Pack,
 Worms Revolution: Mars Pack,
 Worms Revolution: Customization Pack.

Przypisy

Gry z serii Worms
Gry na platformę Windows
Strategiczne gry turowe
Komputerowe gry zręcznościowe
2012 w grach komputerowych

redpajama_set_name: RedPajamaWikipedia


Available at Hennessy Ford in Atlanta, GA. Call 770-621-0200 for Pricing and Specifications. W/ Reading Service Body.
9ft Knapheide Utility Body. Liftmore L-21 7ft Crane w/ 2000lbs Capacity. 1999 Chevrolet C3500 - Service Utility Truck w/ Crane. KA Commercial Trucks, LLC 71062 US Hwy 12 / PO Box 728 Dassel, MN 55325 We are located in Dassel, MN in Central Minnesota.
This 2005 Ford Super Duty F-550 XL DRW Hi-Ranger is proudly offered by TruckMax Homestead CARFAX BuyBack Guarantee is reassurance that any major issues with this vehicle will show on CARFAX report.
Nice truck; up to date on service.
2001 Ford F350 Tool Truck, Parts Truck - Blown Motor, Odometer Reads 303,093 Miles, Ford V8, 7.3L Diesel, Automatic Transmission, Very Good Rear Tires, Visible Issues are: Bent Rear Step, Some Body Rust, Poor Front Tires.
2008 Ford F550 XL - 20ft Crane Truck. Dual Rear Wheels. KA Commercial Trucks, LLC 71062 US Hwy 12 / PO Box 728 Dassel, MN 55325 We are located in Dassel, MN in Central Minnesota. Gray Vinyl Interior.
10,000 pound wireless IMT crane with screw compressor. Tires and wheels have less than 500 miles on them. Truck is in excellent condition, has rare 14 ft bed with partial bed cover enclosure.


THE narrowboat, Helena Knowsley, that was shown sunk in the Cam at Cambridge, will soon be floating again.
THOUGH the mural at Dallow Lock on the Trent & Mersey Canal is only just two years old it needs £5,400 spending to restore it.
THERE'S a chance for you to own part of the Trent this year, as it goes up for auction.
WITH a great risk in the present freezing weather of death should anyone fall into a river, safety alarms have been fitted on the Cam locks as a trial.
THOUGH as we reported there is a scarcity of narrowboats in the hall of the Tullett Prebon London International Boat Show, there are eight on display outside.
IT SEEMS the Thames at Walton is to get its sixth Walton Bridge after a public enquiry approved its design.
THE big London International Boat Show, claims 70 new product launches, but no narrowboats amongst them.
IT HAS been reported that 20 police officers were drafted in to investigate the death of a man found on the towpath of the canal at Newport, which is now half solved.
THE Wendover/Tring Canal Festival will not be going ahead this year as a suitable site cannot be found, Mike Griffin tells us.
AN UNDERWATER search team is searching both the Rother and the Chesterfield Canal for a man missing since Christmas Eve.


text:
41st World Congress of Vine and Wine
3. Results and discussion
A plant regeneration platform to apply new breeding techniques for improving disease resistance in grapevine rootstocks and cultivars
S. Sabbadini1, L. Capriotti1, C. Limera1, O. Navacchi2, G. Tempesta3 and B. Mezzetti1
1 Department of Agricultural, Food and Environmental Sciences, Università Politecnica delle Marche, Ancona, Italy
2 Vitroplant International, Cesena, Italy
3 Vivaio Enotria, Treviso, Italy
Worldwide grapevine cultivation is based on the use of elite cultivars, in many cases strictly linked to local important wine brands. Most of Vitis viniferacultivars have high susceptibility to fungal and viral diseases therefore, new breeding techniques (e.g. Cisgenesis, RNAi and gene editing) offer the possibility to introduce new clones of the main cultivars with increased diseases resistance, in order to reduce environmental impact and improve quality in the intensive wine grape industry. This study is finalized to develop efficient in vitro regeneration and transformation protocols to extend the application of these technologies in wine grape cultivars and rootstocks. With this aim, in vitro regeneration protocols based on the production of meristematic bulks (Mezzetti et al., 2002) were optimized for different grapevine cultivars (Glera, Vermentino, Sangiovese, Thompson Seedless) and rootstocks (1103 Paulsen, and 110 Richter). The meristematic bulks were then used as explants for Agrobacteriummediated genetic transformation protocols, by comparing the use of NPTII and e-GFP as marker genes. Results confirmed the efficiency of meristematic bulks as the regenerating tissue to produce new modified plants in almost all the above genotypes. The highest regeneration efficiency in some genotypes allowed the selection of stable modified lines/calli with only the use of e-GFP marker gene. This protocol can be applied in the use of MYB marker gene for the production of cisgenic lines. Genotypes having the highest regeneration and transformation efficiency were also used for transformation experiments using a hairpin gene construct designed to silence the RNA-dependent RNA polymerase (RpRd) of the GFLV and GLRaV3, which would induce multiple virus resistances, and the Dicer-like protein 1 (Bc-DCL1) and Bc-DCL2 to control B. cinerea infection.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Grapes (Vitis spp.) are among the world's most important fruit crops for their diverse uses. Grapes are consumed fresh commonly referred to as table grapes, dried (sultanas, raisins) or as a high-value drink (juice, wine). However, grapevine cultivation is penalized by pathological problems with significant impact on production, quality and related costs. Traditional breeding techniques are limited in that there is reduced genetic resources and an increase in variability that is unacceptable for the preservation of traditional grapevine clones. Therefore, there is need to improve the quality and resistance to diseases through genetic improvements.
The application of New Breeding Techniques (NBTs), such as cisgenesis, RNAi and gene editing, can be a useful alternative to classical breeding methods to introduce or modify important agronomical traits in grapevine clones [1, 2]. The sequencing of V.vinifera genome has allowed the identification of gene sequences useful for controlling several important agronomical characteristics in grapevine, which has been mostly validated by Agrobacterium-mediated transformation [3–5]. The main bottleneck in the application of biotechnological tools for grapevine genetic improvement is the development of efficient and reproducible in vitro regeneration and transformation protocols, which often need to be optimized for the individual genotypes being studied [5]. In vitro grapevine regeneration has been achieved principally by somatic embryogenesis using different tissues as starting explants, such as anthers, leaves and ovaries [6–8]. In vitro grapevine shoots regeneration was also obtained via organogenesis, which represents a good alternative regeneration method to somatic embryogenesis, avoiding some technical problems related to embryogenetic cultures induction and maintenance [9–12].
Another important key point that is part of genetic engineering protocols applied to plant species, is the availability of an effective selective regime after infection of explants with Agrobacterium, that should lead to a rapid regeneration, which should only be restricted to the putative transgenic cells. In this sense, the combined use of antibiotic-based selectable marker genes and reporter genes, such as green fluorescent protein(GFP), represents an efficient selective strategy, which aids in avoiding the regeneration of chimeric tissues and/or escapes [13, 14]. In grapevine, it is really important to have efficient regeneration and transformation protocols for the most important cultivars and rootstocks for the application of NBTs aimed at achieving some of the main objectives such as, the induction of biotic resistance.
Grapevine is affected by numerous pathogenic diseases including viral diseases, such as "fanleaf (GFLV)" and "leaf roll (GLRaV)" diseases, which are the most diffused in Europe. The application of rigorous certification criteria is the only strategy available to control the diffusion of viruses. However, this method is not efficient in ensuring total control of these diseases thus; application of NBTs such as RNA interference (RNAi) has emerged as alternative tool to induce resistance to virus in several plant species. RNAi is a complex of cell mechanisms that evolved naturally in eukaryotes and used to degrade specific nucleic acid sequences and/or to inhibit gene expression [2, 15]. It has been mainly associated with a natural defence strategy against viruses and transposable elements in plants [16, 17]. This mechanism relies on the presence of non-coding double-stranded RNAs (dsRNAs) molecules, which are processed by the plant-cell machinery and cause the degradation, in a sequence-specific manner, of all the complementary viral transcripts present in the plant cell [17]. RNAi silencing signal is mobile and can move from cell to cell or for longer distance through the plant vascular system [15, 17]. This is of great interest when applied to rootstocks as a source of RNAi controlling plant virus infection into non-genetically modified scion.
In grapevine few examples exist of virus resistance induction by genetic engineering; indeed, the validation of RNAi-based gene constructs has been mainly carried out on model plants like Nicotiana benthamiana [18–23]. Recently it was discovered that a cross-kingdom RNAi mechanism exists, which is used by some plant and animals pathogens to silence host immunity genes [24]. An example is represented by the plant fungal pathogen B.cinerea, whereby mechanism of infection involves the introduction of small RNAs (sRNAs) able to suppress the plant cell immunity defences [25, 26]. Thanks to this discovery, resistance to B.cinerea was obtained in Arabidopsis and Tomato by expressing sRNAs that target B. cinerea Dicer-like protein 1 (Bc-DCL1) and Bc-DCL2; furthermore, gray mold disease was inhibited also by applying Bc-DCL1- and Bc-DCL2-derived sRNAs or dsRNAs molecules on the surface of fruits (tomato, strawberry and grape), vegetables (lettuce and onion), and flowers (rose) [27].
In the present study the regeneration protocol, via organogenesis, based on the induction of meristematic bulks [12], was optimized for different grapevine cultivars (Glera, Vermentino, Sangiovese, Thompson Seedless) and rootstocks (1103 Paulsen, and 110 Richter). To investigate the transformation efficiency of the different genotypes, the meristematic bulks created for each clone were used at the beginning as explants for Agrobacterium-mediated genetic transformation with a gene construct that expresses the enhanced green fluorescent protein (e-GFP) and neomycin phosphotransferase (nptii) as marker genes. The genotypes having the highest regeneration and transformation efficiency were used also to compare two selection strategies for the identification of putatively transformed tissues/shoots: i) the combined use of both nptii and eGFPmarker genes, ii) the visual screening based on the eGFP gene expression alone. The same genotypes were also exploited for transformation trials using two different hairpin gene constructs, one designed to silence the RNA-dependent RNA polymerase (RpRd) of the GFLV and GLRaV3, and another one that silence the Dicer-like protein 1 (Bc-DCL1) and Bc-DCL2 to control B. cinerea infection.
2.1. Plant material
In vitro proliferated shoots of V.vinifera cultivars Thompson seedless, Vermentino, Glera, Sangiovese and the rootstocks 110 Richter (V.berlandieri x V.rupestris) and 1103 Paulsen (V.berlandieri x V.rupestris) were provided by Vitroplant Italia, and used as starting explants to induce meristematic bulks (MBs). The protocol described by Mezzetti et al., 2002 [12] was optimized for the single genotypes; it induces the production of MBs through the addition of increasing concentration of 6-benzylaminopurine (BAP) in the culture medium. The MBs obtained were cut into slices and exploited as vegetative material for the transformation trials (Fig. 1).
Organogenesis-based regeneration method applied to grapevine: a) Grapevine Meristematic bulk; b) Slices obtained from meristematic bulk prepared for Agrobacterium-mediated transformation.
2.2. Plasmid vectors and gene constructs for the transformation trials
Three different plasmids and gene constructs were used to achieve the transformation trials: i) the pK7WG2 binary vector expressing both eGFP and nptii genes under the control of the 35S Cauliflower mosaic virus (CaMV) promoter; ii) a derivative of pBin19 binary vector expressing the hairpin gene construct hp-GFLV-GLRaV, as described by Girolomini et al., 2012 [28], and iii) the phellsgate 8.0 containing the hairpin cassette designed to silence the Bc-DCL1 and Bc-DCL2 sequences [27]. All the gene constructs were inserted in A.tumefaciens strain EHA105.
2.3. Grapevine genetic transformation trials
All grapevine genotypes mentioned above were initially transformed with the gene construct expressing both eGFP and nptii marker genes to identify the most efficient clones in terms of transformation efficiency. The transformation protocol used by Mezzetti et al., 2002 [12] was applied to all the grapevine genotypes in this study. After A.tumefaciens infection all the explants were placed on regeneration medium (MS salt and vitamins [29], 30 g L−1 sucrose, 7 g L−1 plant agar, 13.2 μM BAP, 0.01 μM 1-Naphthaleneacetic acid (NAA)) supplemented with 70 mg L−1 kanamycin, and sub-cultured on fresh media every three weeks. A second transformation trial was carried out using the same gene construct (i.e 35S-eGFP-NPTII) to transform MBs of the genotypes that had shown the best results in term of transformation efficiency from the first transformation. Finally the putatively transformed lines were selected by comparing the use of nptii and e-GFP as marker genes, placing half of the infected explants on media containing kanamycin and half without it. For all transformation trials, data on the number of transformation events ((number of calli expressing e-GFP/total explants) × 100) were acquired, by observing the infected explant under the stereomicroscope Leica MZ10F, to detect the e-GFP fluorescing cells. Genotypes having the highest regeneration and transformation efficiency were also used as starting explants for transformation trials using the hairpin gene constructs hp-GFLV-GLRaV and hp-BcDCL1/DCL2. Identification of the putative transgenic lines was obtained by placing the infected explants on regeneration medium supplemented with 70 mg L−1 kanamycin, and sub-cultured on fresh media every three weeks. The selected explants were isolated and moved to rooting medium (MS salt and vitamins [29], 30 g L−1 sucrose, 7 g L−1 plant agar, 5 μM Indole-3-butyric acid (IBA), kanamycin 70 mg L−1).
3.1. Induction of MBs from different grapevine cultivars and rootstocks
The in vitro regeneration protocol based on the production of meristematic bulks [12] was efficiently optimized for different grapevine cultivars (Glera, Sangiovese, Thompson Seedless, Vermentino) and rootstocks (1103 Paulsen, 110 Richter) (Fig. 2). This in vitro regeneration method, via organogenesis, has been confirmed, also by other studies, to be really effective for the regeneration and genetic transformation, not only of grapevine, but also of other plant species, as Prunus spp. and blueberry [30–33].
Meristematic bulks of grapevine cultivars and rootstocks: a) Thomspon seedless; b) Sangiovese; c) Glera; d) Vermentino; e) 110 Richter; f) 1103 Paulsen.
3.2. Genetic transformation and selection of putative transformed lines using 35S-eGFP-NPTII gene construct
The transformation trials were performed on a total of 100 explants per genotype. Although, all grapevine cultivars and rootstock showed high in vitro regeneration response, transformation competence presented differences between genotypes (Table 1). Thompson seedless and 110 Richter showed the highest transformation competence (among the cultivar and rootstocks under study), calculated as percentage of calli expressing e-GFP on total explants. Whereas, Glera showed the lowest transformation efficiency, by applying the protocol developed by us.
To compare the possibility of selecting transformed lines using a reporter gene (eGFP marker gene) alone, another transformation trial was performed only on MBs from Thompson seedless and 110 Richter. Half of the plant material was selected by both eGFP visual screening and kanamycin selection; the rest of plant explants were placed on the same medium but without the of addition kanamycin, and putative transgenic lines were identified through the detection of eGFP expression only. Results obtained showed that both methods of selection led to the obtainment of transgenic calli and/or shoots. In particular, transgenic shoots of Thompson seedless were isolated by both selection regimes (Fig. 3a–d), while only fluorescent putative transgenic proliferating calli were isolated from MBs of 110 Richter (Fig. 3e–h).
The combined use of e-GFP and nptii marker genes has shown some advantages for instance, a more rapid identification of putative transgenic lines, the reduction of escapes or chimeras regeneration (Fig. 3c, d, g, h). However, the introduction of antibiotic resistance into the plant genome presents some limits from a biosafety point of view and for the public acceptance [34]. The obtainment of transgenic calli/shoots selected only by the use of a reporter gene, demonstrates that this protocol can be applied in the use of grapevine-derived reporter gene system alone, for the production of cisgenic lines [35]. Nevertheless, the use of non-destructive reporter genes, like eGFP, has some main drawbacks, as they don't give a selective advantage to the transformed cells over the non-transformed ones hence, extending the time necessary for the obtainment of transgenic calli and/or shoots.
Transformation efficiency of different grapevine cultivars and rootstocks after 9 weeks of selection on kanamycin 70 mg L−1.
eGFP-fluorescing calli and/or shoots of Thompson seedless and 110 Richter after 9 weeks of culture on media without (a, b, e, f) or with (c, d, g, h) the addition of kanamycin in the medium. (bar = 2 mm).
3.3. Introduction of hairpin gene constructs to induce Post-Transcriptional Gene Silencing (PTGS) in grapevine
A total of 200 MB slices of Thompson seedless and 110 Richter were used to perform transformation trials using a hairpin gene construct designed to silence both GFLV and GLRaV viruses [28]. Another set of transformation trials were carried out on a total of 200 MB slices of Thompson seedless to introduce the hairpin gene construct against the Bc-DCL1 and Bc-DCL2. The infected lines are currently under selection by kanamycin 70 mg L−1 and some putative transgenic lines have been isolated and transferred on rooting medium (Fig. 4) to be submitted to PCR and Southern blot analyses.
Regeneration of putative transgenic lines of Thompson seedless infected with A.tumefaciens harbouring the hairpin construct Bc-DCL1/DCL2, after three months on selective culture medium a) and a putative transgenic line on elongation and rooting medium b).
This study has confirmed the efficiency and adaptability of the in vitro regeneration protocol, developed by Mezzetti et al., 2002, for the obtainment of MBs of different cultivar and rootstock of Vitis, with a high shoot regeneration capability. The results obtained from the transformation trials, using the gene construct 35S-eGFP-NPTII, were useful as they helped to identify, among the genotype tested, Thompson seedless and 110 Richter as the best grapevine cultivar and rootstock, respectively, in terms of Agrobacterium-mediated transformation competence. These preliminary results show the possibility of using Thompson seedless as model genotype for future functional genomic studies, and for the validation of gene constructs, like RNAi-based ones. While, further studies need to be performed on the rootstock to improve its regeneration efficiency from putatively transformed cells; indeed only proliferating eGFP-fluorescent calli of 110 Richter have been obtained from these transformation trials. The use of eGFP gene as selectable marker alone led to the obtainment of transgenic events. The same method can be used in future for the selection of genetically modified plants, using grapevine-derived reporter genes, such as those belonging to the MYB gene family, so as to avoid the introduction of antibiotic-based selectable marker genes.
This work was supported by Vitroplant Italia, Enotria S.S. and Ampelos. We thank Prof. Tiziana Pandolfini and Dr. Barbara Molesini for providing the gene construct 35S-eGFP-NPTII and hairpin gene constructs against GFLV-GLRaV viruses. We thank Prof. Hailing Jin for providing the hairpin gene construct against Bc-DCL1/DCL2 sequences.
D.J. Gray, Z.T. Li, S.A. Dhekney, Plant Sci. 228, 3 (2014) [CrossRef] [PubMed] [Google Scholar]
C. Limera, S. Sabbadini, J.B. Sweet, B. Mezzetti, Frontiers Plant Sci. 8, 1418 (2017) [CrossRef] [Google Scholar]
O. Jaillon, J.M. Aury, B. Noel, A. Policriti, C. Clepet, A. Casagrande, A. Vezzi, Nature 449, 463 (2007) [CrossRef] [PubMed] [Google Scholar]
R. Velasco, A. Zharkikh, M. Troggio, D.A. Cartwright, A. Cestaro, D. Pruss, G. Malacarne, PloS one 2, e1326 (2007) [CrossRef] [PubMed] [Google Scholar]
R. Saporta, S. Pedro-Galan, G. Domenech, M. Carmen, Vitis 55, 173 (2016) [Google Scholar]
C.L. Rosenfield, S. Samuelian, J.R. Vidal, B.I. Reisch, Am. J. Enol. Vitic. 61, 348 (2010) [Google Scholar]
Z.T Li, D.L. Hopkins, D.J. Gray, Transgenic Res. 24, 821 (2015) [CrossRef] [PubMed] [Google Scholar]
L. Dai, Q. Zhou, R. Li, Y. Du, J. He, D. Wang, S. Cheng, J. Zhang, Y. Wang, Plant Cell Tiss. Org. Cult. 121, 397 (2015) [CrossRef] [Google Scholar]
P. Iocco, T. Franks, M.R. Thomas, Transgenic Res. 10, 105 (2001) [CrossRef] [PubMed] [Google Scholar]
K.L. Nicholson, N. Tarlyn, T. Armour, M.E. Swanson, A. Dhingra, PCTOC 111, 123 (2012) [CrossRef] [Google Scholar]
M. Dutt, Z.T. Li, S.A. Dhekney, D.J. Gray, Plant Cell Reports 26, 2101 (2007) [CrossRef] [PubMed] [Google Scholar]
B. Mezzetti, T. Pandolfini, O. Navacchi, L. Landi, BMC Biotechnol. 2, 18 (2002) [CrossRef] [PubMed] [Google Scholar]
S.N. Maximova, A.M Dandekar, M.J. Guiltinan, Plant Mol. Biol. 37, 549 (1998) [CrossRef] [PubMed] [Google Scholar]
A. Ballester, M. Cervera, L. Pena, Plant Cell Rep. 27, 1005 (2008) [Google Scholar]
A.E.M. De Alba, E. Elvira-Matelot, H. Vaucheret, Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms 1829, 1300 (2013) [CrossRef] [Google Scholar]
S.W. Ding, O. Voinnet, Cell 130, 413 (2007) [CrossRef] [PubMed] [Google Scholar]
G.N.Y. Lemgo, S. Sabbadini, T. Pandolfini, B. Mezzetti, Transgenic Res. 22, 1073 (2013) [CrossRef] [PubMed] [Google Scholar]
F. Maghuly, S. Leopold, A. da Câmara Machado, E.B. Fernandez, M.A. Khan, G. Gambino, M. Laimer, Plant Cell Rep. 25, 546 (2006) [Google Scholar]
K.S. Ling, H.Y. Zhu, D. Gonsalves, Transgenic Res. 17, 733 (2008) [CrossRef] [PubMed] [Google Scholar]
C. Monier, P. Barbier, B. Walter, In VII International Symposium on Grapevine Genetics and Breeding 528, 379 (1998) [Google Scholar]
G. Gambino, I. Perrone, A. Carra, W. Chitarra, P. Boccacci, D. Torello Marinoni, M. Barberis, F. Maghuly, M. Laimer, I. Gribaudo, Transgenic Res. 19, 17 (2010) [CrossRef] [PubMed] [Google Scholar]
F. Maghuly, S. Leopold, A.D.C. Machado, E.B. Fernandez, M.A. Khan, G. Gambino, I. Gribaudo, A. Schartl, M. Laimer, Plant Cell Reports 25, 546 (2006) [CrossRef] [PubMed] [Google Scholar]
R. Jardak-Jamoussi, P. Winterhagen, B. Bouamama, C. Dubois, A. Mliki, T. Wetzel, G.M. Reustle, PCTOC 97, 187 (2009) [CrossRef] [Google Scholar]
A. Weiberg, M. Wang, M. Bellinger, H. Jin, Annu. Rev. Phytopathol. 52, 495 (2014) [CrossRef] [PubMed] [Google Scholar]
A. Weiberg, M. Wang, F.M. Lin, H. Zhao, Z. Zhang, I. Kaloshian, H.D. Huang, H. Jin, Science 342, 118 (2013) [Google Scholar]
M. Wang, A. Weiberg, E. Jr. Dellota, D. Yamane, H. Jin, RNA Biol. 1, 8 (2017) [Google Scholar]
M. Wang, A. Weiberg, F.M. Lin, B.P. Thomma, H. Huang, H. Jin, Nature Plants 2, 16151 (2016) [CrossRef] [PubMed] [Google Scholar]
L. Girolomini, S. Sabbadini, B. Mezzetti, D. Palma, T. Pandolfini, A. Polverari, Acta Horticulturae 929, 393 (2012) [Google Scholar]
T. Murashige, F. Skoog 15, 473 (1962) [Google Scholar]
M. Pérez-Jiménez, A. Carrillo-Navarro, J. Cos-Terrer, Plant Cell Tissue Organ Cult. 108, 55 (2012) [Google Scholar]
S. Sabbadini, T. Pandolfini, L. Girolomini, B. Molesini, O. Navacchi, Agrobacterium Protocols edited by K. Wang (Springer, New York, 2015), p. 205 [CrossRef] [Google Scholar]
R. Cappelletti, S. Sabbadini, B. Mezzetti, Scientia Horticulturae 207, 117 (2016) [Google Scholar]
X. Xie, C.B. Aguero, Y. Wang, M.A. Walker, Plant Cell, Tissue Organ Cult. 126, 541 (2016) [CrossRef] [Google Scholar]
D. Breyer, L. Kopertekh, D. Reheul, Critical Rev. Plant Sci. 33, 286 (2014) [CrossRef] [Google Scholar]
R. Kandel, D.R. Bergey, M. Dutt, V. Sitther, Z.T. Li, D.J. Gray, S.A. Dhekney, PCTOC 124, 599 (2016) [CrossRef] [Google Scholar]
Optimization of core phases of biolistic transformation of potato
Transformation of Poaceae and gene expression in transgenic plants
Agronomie 15, 309-319 (1995)
Transgenic resistance of Bulgarian potato cultivars to the Colorado potato beetle based on Bt technology
Agron. Sustain. Dev. 28, 481-488 (2008)
Genetic transformation: a short review of methods and their applications, results and perspectives for forest trees
In vitro organogenesis from adult tissue of 'Bahia' sweet orange (Citrus sinensis L. Osbeck)
Fruits, 2006, vol. 61, p.367-371


text:
https://www.centralofsuccess.com/category/9-11-quotes/ 30% Weekly 2018-10-22 20:53
https://www.centralofsuccess.com/category/a-p-j-abdul-kalam/ 30% Weekly 2018-10-22 21:14
https://www.centralofsuccess.com/category/abraham-lincoln-birthday/ 30% Weekly 2018-10-22 21:49
https://www.centralofsuccess.com/category/abuse-quotes/ 30% Weekly 2018-10-24 06:26
https://www.centralofsuccess.com/category/administrative-professionals-day/ 30% Weekly 2018-10-24 06:44
https://www.centralofsuccess.com/category/famous-author-personalities/adolf-hitler/ 30% Weekly 2018-10-25 13:03
https://www.centralofsuccess.com/category/adoption-slogans/ 30% Weekly 2018-10-25 06:32
https://www.centralofsuccess.com/category/age-quotes/ 30% Weekly 2018-10-26 07:01
https://www.centralofsuccess.com/category/aids-awareness-month/ 30% Weekly 2018-10-27 07:06
https://www.centralofsuccess.com/category/air-crash-quotes/ 30% Weekly 2018-10-27 07:17
https://www.centralofsuccess.com/category/al-pacino/ 30% Weekly 2018-10-30 06:39
https://www.centralofsuccess.com/category/albert-camus/ 30% Weekly 2018-10-30 07:14
https://www.centralofsuccess.com/category/albert-einstein/ 30% Weekly 2018-10-30 21:10
https://www.centralofsuccess.com/category/albino-quotes/ 30% Weekly 2018-10-31 19:11
https://www.centralofsuccess.com/category/alcohol-awareness-month-week/ 30% Weekly 2018-11-02 06:55
https://www.centralofsuccess.com/category/alzheimer-quotes/ 30% Weekly 2018-11-03 06:41
https://www.centralofsuccess.com/category/ambition-quotes/ 30% Weekly 2018-11-03 07:17
https://www.centralofsuccess.com/category/america-quotes/ 30% Weekly 2018-11-03 19:21
https://www.centralofsuccess.com/category/america-recycles-day/ 30% Weekly 2018-11-04 20:32
https://www.centralofsuccess.com/category/american-patriotic-quotes/ 30% Weekly 2018-11-04 19:52
https://www.centralofsuccess.com/category/andy-borowitz/ 30% Weekly 2018-11-05 07:42
https://www.centralofsuccess.com/category/animal-abuse-quotes/ 30% Weekly 2018-11-05 08:13
https://www.centralofsuccess.com/category/animal-quotes/ 30% Weekly 2018-11-05 08:30
https://www.centralofsuccess.com/category/anne-frank/ 30% Weekly 2018-11-06 07:57
https://www.centralofsuccess.com/category/anniversary-of-the-army-chaplain-corps/ 30% Weekly 2018-11-06 08:09
https://www.centralofsuccess.com/category/anti-alcohol-slogans/ 30% Weekly 2018-11-07 18:08
https://www.centralofsuccess.com/category/anti-bullying-slogans/ 30% Weekly 2018-11-07 18:42
https://www.centralofsuccess.com/category/anti-drugs-slogans-weed-cocaine-dope-marijuana-meth-etc/ 30% Weekly 2018-11-07 19:48
https://www.centralofsuccess.com/category/slogans/anti-corruption-slogans/ 30% Weekly 2018-11-08 07:38
https://www.centralofsuccess.com/category/special-days-and-holidays/anti-racism-day/ 30% Weekly 2018-11-08 07:58
https://www.centralofsuccess.com/category/slogans/anti-tobacco-slogans/ 30% Weekly 2018-11-08 08:30
https://www.centralofsuccess.com/category/quotes/apology-quotes-im-sorry/ 30% Weekly 2018-11-09 08:33
https://www.centralofsuccess.com/category/special-days-and-holidays/appreciation-day/ 30% Weekly 2018-11-09 15:41
https://www.centralofsuccess.com/category/special-days-and-holidays/april-fools-day/ 30% Weekly 2018-11-09 15:54
https://www.centralofsuccess.com/category/quotes-calendar/april-holidays-quotes/ 30% Weekly 2018-11-10 07:31
https://www.centralofsuccess.com/category/special-days-and-holidays/arbor-day/ 30% Weekly 2018-11-10 07:38
https://www.centralofsuccess.com/category/author/aristotle/ 30% Weekly 2018-11-10 08:03
https://www.centralofsuccess.com/category/special-days-and-holidays/armed-force-day/ 30% Weekly 2018-11-11 18:39
https://www.centralofsuccess.com/category/special-days-and-holidays/assumption-of-the-virgin-mary/ 30% Weekly 2018-11-11 18:53
https://www.centralofsuccess.com/category/attitude-quotes/ 30% Weekly 2018-10-04 06:29
https://www.centralofsuccess.com/category/quotes/attitude-quotes-quotes/ 30% Weekly 2018-11-12 19:11
https://www.centralofsuccess.com/category/quotes-calendar/august-holidays-quotes/ 30% Weekly 2018-11-12 19:46
https://www.centralofsuccess.com/category/quotes/autism-quotes/ 30% Weekly 2018-11-13 19:53
https://www.centralofsuccess.com/category/quotes/autumn-quotes/ 30% Weekly 2018-11-13 20:00
https://www.centralofsuccess.com/category/greetings-2/baisakhi-wishes/ 30% Weekly 2018-11-14 08:14
https://www.centralofsuccess.com/category/author/barack-obama/ 30% Weekly 2018-11-14 08:28
https://www.centralofsuccess.com/category/quotes/beach-and-oceans-quotes/ 30% Weekly 2018-11-15 08:44
https://www.centralofsuccess.com/category/quotes/beauty-and-the-beast-quotes/ 30% Weekly 2018-11-15 09:04
https://www.centralofsuccess.com/category/quotes/beauty-quotes/ 30% Weekly 2018-11-16 09:14
https://www.centralofsuccess.com/category/quotes/bees-quotes/ 30% Weekly 2018-11-16 09:33
https://www.centralofsuccess.com/category/quotes/being-single-quotes/ 30% Weekly 2018-11-17 10:00
https://www.centralofsuccess.com/category/quotes/believe-in-yourself/ 30% Weekly 2018-11-17 10:15
https://www.centralofsuccess.com/category/author/benjamin-franklin/ 30% Weekly 2018-11-18 10:34
https://www.centralofsuccess.com/category/author/bertrand-russell/ 30% Weekly 2018-11-18 07:48
https://www.centralofsuccess.com/category/best-friend-quotes/ 30% Weekly 2019-07-07 09:04
https://www.centralofsuccess.com/category/quotes/betrayal-quotes/ 30% Weekly 2018-11-19 07:56
https://www.centralofsuccess.com/category/special-days-and-holidays/bhai-duj/ 30% Weekly 2018-11-19 08:39
https://www.centralofsuccess.com/category/quotes/bible-quotes/ 30% Weekly 2018-11-20 09:27
https://www.centralofsuccess.com/category/author/bill-gates/ 30% Weekly 2018-11-20 09:35
https://www.centralofsuccess.com/category/quotes/bio-diversity-quotes/ 30% Weekly 2018-11-21 09:49
https://www.centralofsuccess.com/category/special-days-and-holidays/bird-day-world-migratory-bird-day/ 30% Weekly 2018-11-21 10:11
https://www.centralofsuccess.com/category/greetings-2/birthday-wishes/ 30% Weekly 2018-11-22 07:17
https://www.centralofsuccess.com/category/greetings-2/birthday-wishes-for-father/ 30% Weekly 2018-11-22 07:54
https://www.centralofsuccess.com/category/quotes/blindness-quotes/ 30% Weekly 2018-11-23 06:00
https://www.centralofsuccess.com/category/slogans/blood/ 30% Weekly 2018-11-23 06:44
https://www.centralofsuccess.com/category/author/bob-marley/ 30% Weekly 2018-11-24 06:22
https://www.centralofsuccess.com/category/books-quotes/ 30% Weekly 2018-11-24 09:37
https://www.centralofsuccess.com/category/quotes/boss-quotes/ 30% Weekly 2018-11-25 07:26
https://www.centralofsuccess.com/category/author/brainyquote/ 30% Weekly 2018-11-25 08:08
https://www.centralofsuccess.com/category/quotes/breast-cancer-quotes/ 30% Weekly 2018-11-26 08:33
https://www.centralofsuccess.com/category/quotes/breastfeeding-quotes/ 30% Weekly 2018-11-26 08:36
https://www.centralofsuccess.com/category/author/bruce-lee/ 30% Weekly 2018-11-27 09:14
https://www.centralofsuccess.com/category/special-days-and-holidays/buddha-day/ 30% Weekly 2018-11-27 09:44
https://www.centralofsuccess.com/category/author/c-s-lewis/ 30% Weekly 2018-11-28 09:58
https://www.centralofsuccess.com/category/quotes/camera-quotes/ 30% Weekly 2018-11-28 10:13
https://www.centralofsuccess.com/category/quotes/canada-and-canadians-quotes/ 30% Weekly 2018-11-29 07:38
https://www.centralofsuccess.com/category/quotes/cancer-quotes/ 30% Weekly 2018-11-29 16:42
https://www.centralofsuccess.com/category/slogans/cancer-slogans/ 30% Weekly 2018-11-30 05:35
https://www.centralofsuccess.com/category/author/cardi-b/ 30% Weekly 2018-11-30 05:58
https://www.centralofsuccess.com/category/author/carl-sagan/ 30% Weekly 2018-12-01 07:47
https://www.centralofsuccess.com/category/author/carol-burnett/ 30% Weekly 2018-12-01 08:02
https://www.centralofsuccess.com/category/quotes/change-quotes/ 30% Weekly 2018-12-02 08:14
https://www.centralofsuccess.com/category/quotes/chaplain-corps-quotes/ 30% Weekly 2018-12-02 07:11
https://www.centralofsuccess.com/category/special-days-and-holidays/charity-day/ 30% Weekly 2018-12-03 07:20
https://www.centralofsuccess.com/category/author/charles-dickens/ 30% Weekly 2018-12-03 07:30
https://www.centralofsuccess.com/category/author/charlotte-bronte/ 30% Weekly 2018-12-04 07:50
https://www.centralofsuccess.com/category/quotes/cheating-quotes/ 30% Weekly 2018-12-04 08:29
https://www.centralofsuccess.com/category/author/chetan-bhagat/ 30% Weekly 2018-12-05 08:46
https://www.centralofsuccess.com/category/quotes/chicken-quotes/ 30% Weekly 2018-12-05 09:03
https://www.centralofsuccess.com/category/author/chief-keef/ 30% Weekly 2018-12-06 09:15
https://www.centralofsuccess.com/category/quotes/child-labour-quotes/ 30% Weekly 2018-12-06 09:28
https://www.centralofsuccess.com/category/quotes/childhood-quotes/ 30% Weekly 2018-12-07 09:57
https://www.centralofsuccess.com/category/special-days-and-holidays/childrens-day/ 30% Weekly 2018-12-07 10:18
https://www.centralofsuccess.com/category/quotes/chocolate-quotes/ 30% Weekly 2018-12-09 08:06
https://www.centralofsuccess.com/category/quotes/christmas-quotes/ 30% Weekly 2018-12-09 08:44
https://www.centralofsuccess.com/category/poem/christmas-songs-and-poem/ 30% Weekly 2018-12-10 08:48
https://www.centralofsuccess.com/category/author/christopher-columbus/ 30% Weekly 2018-12-10 09:10
https://www.centralofsuccess.com/category/special-days-and-holidays/citizenship-day/ 30% Weekly 2018-12-11 09:31
https://www.centralofsuccess.com/category/quotes/climbing-mountain-quotes/ 30% Weekly 2018-12-11 10:01
https://www.centralofsuccess.com/category/special-days-and-holidays/coast-guard-day/ 30% Weekly 2018-12-12 19:40
https://www.centralofsuccess.com/category/author/coco-chanel/ 30% Weekly 2018-12-12 19:56
https://www.centralofsuccess.com/category/quotes/coffee-quotes/ 30% Weekly 2018-12-12 20:09
https://www.centralofsuccess.com/category/quotes/company-you-keep-quotes/ 30% Weekly 2018-12-14 19:35
https://www.centralofsuccess.com/category/quotes/compassion-quotes/ 30% Weekly 2018-12-14 19:55
https://www.centralofsuccess.com/category/quotes/confession-quotes/ 30% Weekly 2018-12-15 19:57
https://www.centralofsuccess.com/category/quotes/consistency-quotes/ 30% Weekly 2018-12-15 20:02
https://www.centralofsuccess.com/category/quotes/cooperative-quotes/ 30% Weekly 2018-12-16 20:14
https://www.centralofsuccess.com/category/quotes/couple-quotes/ 30% Weekly 2018-12-16 20:23
https://www.centralofsuccess.com/category/quotes/cousins-quotes/ 30% Weekly 2018-12-17 20:48
https://www.centralofsuccess.com/category/quotes/cricket-quotes/ 30% Weekly 2018-12-17 21:25
https://www.centralofsuccess.com/category/quotes/crime-quotes/ 30% Weekly 2018-12-18 21:51
https://www.centralofsuccess.com/category/quotes/crying-quotes/ 30% Weekly 2018-12-18 07:54
https://www.centralofsuccess.com/category/quotes/cute-quotes/ 30% Weekly 2018-12-20 14:50
https://www.centralofsuccess.com/category/daughter/ 30% Weekly 2017-11-23 17:44
https://www.centralofsuccess.com/category/quotes/dawn-quotes/ 30% Weekly 2018-12-20 15:07
https://www.centralofsuccess.com/category/quotes/deafness-quotes/ 30% Weekly 2018-12-21 15:08
https://www.centralofsuccess.com/category/quotes/death-quotes/ 30% Weekly 2018-12-22 15:34
https://www.centralofsuccess.com/category/quotes/democracy-quotes/ 30% Weekly 2019-01-01 08:12
https://www.centralofsuccess.com/category/quotes/dentist-quotes/ 30% Weekly 2019-01-02 08:47
https://www.centralofsuccess.com/category/quotes/depressing-quotes/ 30% Weekly 2019-01-03 08:10
https://www.centralofsuccess.com/category/quotes/depression-quotes/ 30% Weekly 2019-01-04 19:44
https://www.centralofsuccess.com/category/quotes/desertification-and-drought-quotes/ 30% Weekly 2019-01-05 06:00
https://www.centralofsuccess.com/category/quotes/diabetes-quotes/ 30% Weekly 2019-01-06 06:00
https://www.centralofsuccess.com/category/quotes/dieting-quotes/ 30% Weekly 2019-01-07 06:11
https://www.centralofsuccess.com/category/quotes/disability-quotes/ 30% Weekly 2019-01-09 18:00
https://www.centralofsuccess.com/category/quotes/disappointment-quotes/ 30% Weekly 2019-01-09 07:11
https://www.centralofsuccess.com/category/quotes/disaster-quotes/ 30% Weekly 2019-01-10 06:29
https://www.centralofsuccess.com/category/quotes/dogs-and-puppies-quotes/ 30% Weekly 2019-01-11 06:16
https://www.centralofsuccess.com/category/quotes/earth-quotes/ 30% Weekly 2019-01-12 08:22
https://www.centralofsuccess.com/category/quotes/easter-quotes/ 30% Weekly 2019-01-13 08:14
https://www.centralofsuccess.com/category/author/edgar-allan-poe/ 30% Weekly 2019-01-14 08:20
https://www.centralofsuccess.com/category/special-days-and-holidays/education-quotes/ 30% Weekly 2019-01-15 08:18
https://www.centralofsuccess.com/category/quotes/elder-abuse-quotes/ 30% Weekly 2019-01-18 10:05
https://www.centralofsuccess.com/category/special-days-and-holidays/election-day/ 30% Weekly 2019-01-19 08:48
https://www.centralofsuccess.com/category/special-days-and-holidays/emancipation-day/ 30% Weekly 2019-01-20 16:23
https://www.centralofsuccess.com/category/author/emma-watson/ 30% Weekly 2019-01-21 09:18
https://www.centralofsuccess.com/category/family/ 30% Weekly 2017-12-06 21:07
https://www.centralofsuccess.com/category/farewell-quotes/ 30% Weekly 2018-10-02 06:43
https://www.centralofsuccess.com/category/father/ 30% Weekly 2017-11-26 19:29
https://www.centralofsuccess.com/category/forgiveness-quotes/ 30% Weekly 2019-03-08 06:57
https://www.centralofsuccess.com/category/funny-memes/ 30% Weekly 2019-09-01 16:24
https://www.centralofsuccess.com/category/funny-quotes/ 30% Weekly 2019-11-12 08:07
https://www.centralofsuccess.com/category/good-afternoon-quotes/ 30% Weekly 2019-02-24 07:55
https://www.centralofsuccess.com/category/good-morning-quotes/ 30% Weekly 2019-11-12 08:58
https://www.centralofsuccess.com/category/good-night-quotes/ 30% Weekly 2019-11-10 08:38
https://www.centralofsuccess.com/category/happiness/ 30% Weekly 2019-02-15 15:34
https://www.centralofsuccess.com/category/happy-birthday/ 30% Weekly 2019-11-26 10:29
https://www.centralofsuccess.com/category/happy-birthday-love/ 30% Weekly 2019-11-26 10:29
https://www.centralofsuccess.com/category/health-quotes/ 30% Weekly 2020-01-21 10:27
https://www.centralofsuccess.com/category/hurt-quotes/ 30% Weekly 2018-09-28 06:59
https://www.centralofsuccess.com/category/inspiration/ 30% Weekly 2019-04-01 20:54
https://www.centralofsuccess.com/category/inspirational-quotes/ 30% Weekly 2019-11-08 08:08
https://www.centralofsuccess.com/category/islamic-quotes/ 30% Weekly 2019-03-23 21:02
https://www.centralofsuccess.com/category/life-quotes/ 30% Weekly 2020-01-21 15:23
https://www.centralofsuccess.com/category/love-quotes/ 30% Weekly 2019-11-07 20:01
https://www.centralofsuccess.com/category/mother/ 30% Weekly 2017-12-06 21:07
https://www.centralofsuccess.com/category/motivational-quotes/ 30% Weekly 2019-10-31 07:36
https://www.centralofsuccess.com/category/new-year/ 30% Weekly 2019-08-21 07:10
https://www.centralofsuccess.com/category/quotes/ 30% Weekly 2019-02-15 07:59
https://www.centralofsuccess.com/category/relationship/ 30% Weekly 2019-11-09 08:32
https://www.centralofsuccess.com/category/slogans/ 30% Weekly 2018-11-08 08:30
https://www.centralofsuccess.com/category/smile-quotes/ 30% Weekly 2018-10-05 06:58
https://www.centralofsuccess.com/category/sorry-quotes/ 30% Weekly 2018-10-06 06:57
https://www.centralofsuccess.com/category/success/ 30% Weekly 2019-02-13 20:08
https://www.centralofsuccess.com/category/tattoos/ 30% Weekly 2019-02-24 07:33
https://www.centralofsuccess.com/category/travel-quotes/ 30% Weekly 2019-06-20 07:32
https://www.centralofsuccess.com/category/uncategorized/ 30% Weekly 2019-04-27 20:28
https://www.centralofsuccess.com/category/wallpapers/ 30% Weekly 2019-02-26 22:05


After we got to Arequipa, we settled into our hostel. Here we have two night-time photos, highly desaturated because of the low light (I suspect they're badly underexposed, but I like the effect). I particularly like the one of Mita–you can tell it's a long exposure, because you can see the bench superimposed on her–it means that she held still for a while with the shutter open, and then stood up and walked out of the frame before I closed the shutter.
Then, our tour began: in the pre-dawn hours, we drove out of the city, into the increasingly remote hinterlands.
Some would call these poorly exposed, some would call them "dreamlike," but I just call them reflective of my memory–it was dark, and I was sleepy, and my memory is about as fuzzy as these photos.
Our first stop, however, was not so fuzzy: Petroglyphs and a killer view of an lush green valley. Those photos could use a bit more editing though, so I'll just leave black and whites of the valley, and Mita looking out with our wonderful guide.


text:
Eleutherios Venizelos – politico e primo ministro greco
 Evangelos Venizelos – politico greco
 Sofoklīs Venizelos – politico greco figlio di Eleutherios Venizelos


Strawberry represents the most important berry in the Mediterranean diet. It is the most consumed berry as both fresh and processed products, the most studied berry from the agronomic, genomic and nutritional point of view, and it is also a remarkable source of micronutrients and phytochemicals with a relevant biologically activity on human health. Although individual bioactive compounds present in strawberries have been studied for their biological activities, the mechanisms by which they are metabolized and exert their beneficial effects on human health are still unknown. For this reason, the first objective of this research is the development of an enzymatic in vitro system which simulates the gastrointestinal conditions that occur in vivo during the digestive process. Briefly, the method consists on an initial pepsin-HCl digestion for simulating gastric conditions (pH~1.7) followed by an intestinal digestion with pancreatin and bile salts through a dialysis membrane. The dialysates were concentrated under vacuum, resuspended in H2O2/glacial acetic acid and purified with a C-18 SepPaks Vac 6 cc cartridge. The strawberry cultivar chosen for the study is 'Romina', one of the most promising cultivars developed in the context of the European Euberry project. The total antioxidant capacity of both the fresh fruit and the digested fraction was tested through the DPPH, FRAP and TEAC assays. The results obtained have shown a significant variation in the antioxidant capacity of 'omina', before and after the enzymatic digestion process. Significant decreases were found in all the performed assay, highlighting how the antioxidant capacity present in fresh fruit is drastically reduced following digestion. Further studies are required to elucidate the mechanisms of transport and metabolism of these compounds, with the aim of providing predictive results on their bioavailability and thus their effect on human health.


text:
Home/romania/#Teleormanleaks. A real suitcase with thousands of documents inside the Tel Drum, found by local residents
#Teleormanleaks. A real suitcase with thousands of documents inside the Tel Drum, found by local residents
romania November 4, 2018 romania
In the rural area of Tieorman, a local man even owned a suitcase containing essential information within Tel Drum, writes the RISE Project. The DNA prosecutors did not have access to this suitcase when they physically searched and filled the company's home office last year.
Source: RISE project
The locals have contacted the county RISE project source.
According to this source, the suitcase is as follows:
Black hardcover that saves all 63.39 GB Tel Drum keys, including thousands of bills, contracts, balances, currency, proxies, phantom corporation activities, account statements, etc., but thousands of photo / video images of corporate events, group characters and their standard of living, documents, suicides, screen captures with conversations.
– The Samsung P1000 Galaxy Tab tablet in 2010, the CEO of Tel Drum, was Petre Pitiş. It includes over 1000 photos of exotic excursions with escorts, company events and family moments, as well as the 980 phone number collected in recent years: "Băsescu", "Hrebe", "Dăncilă", "Bănicioiu", "Pieleanu" or "LIVIU".
– Memory card with work pieces from the Tel Drum head.
– Over a thousand newspapers, such as copies of personal Dragnea family documents, or detailed plans for mammoth real estate in Alexandria
Read it:
The accusation of the former ANAF leader: The Dragnea family has gone through tens of millions of dollars
Disclosure: Dragnea House companies have a debt of several million euros to the state
Romania, at the peak of EU counterfeiting. For example, the TelDrum folder
Tel Drum has to pay more than 70 million lei of insolvency debts to at least 120 lenders
Transcripts. The meeting where he voted for CJ Teleorman was not civilian in the Tel Drum file
Your cookie settings do not allow content to be displayed at this stage. You can update your coookie module settings directly from your browser or from here – you will need to accept social media cookies
Romania was stolen. | Who are the people behind Tel Drum and Liviu Dragnea
Liviu Dragnea's charges in the Tel Drum file
In November 2017, Liviu Dragnea was charged with the Tel Drum file for setting up an organized criminal group, improper use or false presentation of documents, or false, inaccurate or incomplete statements and misuse of services for self-help or for another.
According to prosecutors, Liviu Dragnea has initiated an organized criminal group chairman of the Teleorman County Council, which currently operates and has been an official and a member of public institutions and businessmen.
The main objective of the group launched in 2001 was to fraudulently obtain large sums of state-funded contracts for misuse of powers, fraudulent European funds, tax evasion, money laundering and the use of information not intended to be advertised or allow unauthorized persons access to this information, say DNS prosecutors.
According to prosecutors, Liviu Dragnea has initiated an organized criminal group chairman of the Teleorman County Council, which currently operates and has been an official and a member of public institutions and businessmen. The group, initiated in 2001, began to fraudulently obtain large sums of state-funded contracts through abuse of power, fraudulent European funds, tax evasion, money laundering and non-public information or allowing unauthorized persons to access this information , DNS prosecutors say.
The insurer seizes the assets of Liviu Dragnea
The damage in this case is 30 million lei for misuse of the service and 20 million for the fraud of the European funds.
DNA also announced that PSD leader Liviu Dragnea had seized all the assets and seizures, up to 127.5 million lei in the Tel Drum file.
Thus, in the case of the head of the PSD, DNS procurators were ordered to seize and indemnify the insurer for all movable and immovable property, shares and shares in the legal personality of private law, to 127,547,366.34 lei. sums on bank accounts and sums paid by recipients to third parties, including the rent amount.
Liviu Dragnea challenged this measure late last year, but his request was rejected by the court.
Attorney: An institution is not a civil party
Vasii Mária, the lawyer of the leader of the PSD, argued that no institution was a civilian in this document, so no one wanted to claim compensation, the prosecutor would not have the right to take precautionary measures.
"I argued for the introduction of precautionary measures because the prosecutor issued the violation of the essential forms required by law, for example, the attorney-in-law justified the assurance provision in order to dispose of the necessary means to cover the indemnity. There is no civil party so no one claims damages "The prosecutor has no time to automatically declare the lack of a civil party," said Maria Vasii.
In fact, he said that the attack was "after the ear" because the tools of the leader of the PSD were not evaluated.
On January 17, 2018, DNA announced that it had ordered any precautionary measures to be taken against all Tel Drum mobile and real estate assets and that the funds available or to be transferred by third parties should be attached to the account of any third party, up to the amount of 32,118,589.51 lei (which is the sum of unjustified benefits amount of money and the amount of money saved to the state). On the same day, DNA announced the extension of the criminal investigation into society, which was witnessed by the formation of an organized criminal group, the continuation of tax evasion and the complicity of misuse of services.
file liviu dragnea
tel drum
rising project
Exclusive video "Leave the strangers. They're going to bankrupt us." Gabi Balint, after the Gueye scandal
Oppo A93 5G has been launched and can be ordered in advance
To date, more than 30,000 suckling whales have been vaccinated against seasonal flu
Discussions about Szekler Land are back. Hunor: Autonomy is not an exit
Kim Kardashian and Kanye West are separated after almost 7 years of marriage. What wealth they must share
Chinese electric car manufacturer Nio is …


text:
Lesbian Dating in Iowa City
September 19, 2019| | 0 Comments
Iowa City Personals
Meet Iowa Muslims
Two Iowa Cities Made the List of the 'Best Cities for Singles'
Singles from Iowa City
Free Lesbian Dating in Iowa City
Sex in Iowa City: Tinder Part 1-Messages
Iowa City Update: Downtown Block Party
The U. Census Bureau estimated the city's population at 75, in , making it the state's fifth-largest city. Iowa City was the second capital of the Iowa Territory and the first capital city of the State of Iowa. In , Forbes magazine named Iowa City the second-best small metropolitan area for doing business in the United States. This act began:. Commissioners Chauncey Swan and John Ronalds met on May 1 in the small settlement of Napoleon, south of present-day Iowa City, to select a site for the new capital city. The following day the commissioners selected a site on bluffs above the Iowa River north of Napoleon, placed a stake in the center of the proposed site and began planning the new capital city.
We promise to keep your information safe and will never post or share anything on your Facebook page. View Singles Now. Caitrin Standard Member. Seeks something interesting with laughter. Insatiably curious, passionate, intrigued.
I say that as a person that dislikes Iowa City, but I have a great. weather, but I've heard from singles there that the dating scene isn't as good.
Report a Problem. The Department of Education Office on Civil Rights recognizes dating violence, domestic violence, sexual assault, and stalking as forms of sex discrimination. The reauthorization of the VAWA amendments to the Clery Act require institutions to have disciplinary measures in place for dating violence, domestic violence, sexual assault, and stalking.
The team reviewed dating and domestic violence training literature, state and federal dating and domestic violence statutes, and policy language from other universities and private sector institutions. The team chose to ensure dating and domestic violence was prohibited behavior within existing policies rather than recommend a new policy. Shared Governance leadership advised and was consulted on the project.
Given the mandatory nature of the required changes and the determination that the changes did not alter the intent of the policy in significant ways, it was determined that votes of the full governance bodies would not be needed. All policies under review include or reference the draft language below, which is in compliance with the federal legislation. The University of Iowa Search. Office of the Sexual Misconduct Response Coordinator. What happens when a student receives notice that they are under investigation?
The Goosetown Neighborhood is in the north of Iowa City. Goosetown is about a mile from downtown Iowa City, making it a very popular choice with buyers Relocating to Iowa City for the University of Iowa. Forward thinking and tech-savvy, my mission is always to find the best ways to ensure clients enjoy a smooth and stress-free buying or selling experience.
I enjoyed the photos-I grew up in Iowa City and had relatives that were Czech. I have fun tracking down photos on Google-many of which still stand.
Meet & chat with the right Single Women in Iowa City, Iowa, United States on Meetville today! Thousands of local women make online dating near you.
Some forums can only be seen by registered members. UIHC is one of the finest hospitals in the country. It is absolutely spectacular. When it comes to meeting a wife. No, I would not recommend it. Are there eligible and attractive single women?
Ads in may. See your perfect match. Crime news, but we can't take such risk of our boot camp will spend more! First reported the possibility of des moines. Updated aug 25, that there. The risk without jeopardizing all international women on craigslist personals — help you can meet the sale.
We have 6 total listings to choose from in the Iowa City area!!! Fairchild St. – Iowa City – 1 Bedroom 1 Bedroom. Available Date: 9/2/ Rental Price.
Log in Help Post an ad free. Women Seeking Men. Nearby Locations. Cedar Rapids. Quad Cities. Des Moines. It's easy and free! Personals Categories. Here for a few nights Looking for some company I have a hotel room near downtown Madison until Saturday. Looking for a SWM I'm looking for a single white gentleman between the ages of in Central Illinois to get to know. I am 35 year old white lady who lives in
So you met someone on an Iowa dating site. What to do? Ask them out of course! Something more unique, adventurous or romantic is on the cards for the second date. Dating in Iowa can be plenty of fun and we have some Iowa date ideas for you. Many great date ideas will actually be featured in your local magazines as well – festivals, cultural events, shows, concerts and so on happen throughout the year.
Presenter: Jen The third class in the series will help you figure out how to identify potential impostors and stay safe in your interactions online. Once you've sifted.
Police arrested a year-old Iowa City man after a woman he met online told officers he forced her to have sex with him. He's now charged with third degree sexual abuse. Court records show the woman says she and Zachary Berry met through the online dating app "Tinder. But she says he tried to get her to perform sex acts shortly after they met and she declined.
Then when they arrived at the restaurant he parked in a nearby lot and started to bite her and choke her. She says he then forced her to have sex with him. Officers write in the criminal complaint that Barry told them he choked her, but that it was "gentle choking. I9 Investigations. Successful Student.
Get Started 1. Face to face dates. Real dates.
Iowa City Lesbians at Join for FREE and meet hundreds of Lesbian singles in Iowa City and surrounding areas.
Or sign up with email. Already a member? Log in. Start a new group. Sign up. Find a group in Iowa City Imagine what you could do with the right people by your side. Check out groups in the Iowa City area and give one a try. Join Meetup. Let's Meetup! Iowa City, IA. Groups Calendar.
Ourtime is commited to introducing singles over 50 looking to meet someone new. We make it simple and safe for mature singles to find, message, meet and date online. Meet people casually and share your interests at one of the many events we hold across the UK. Cultural outings, nature, musical nights, cooking classes, trips and much more: meet new singles while you have fun.
So, what was the best city for singles in Iowa? Ames ranks as the best place for singles in Iowa if you care about things singles care about like night life.
Either way, we've got you covered here. At least, we'll point you in the right direction – which is why you came here in the first place, right? According to the Pew Research Center , the number of Americans who have remained single not married is growing. That means, people are dating a lot longer than they used to. There are many reasons for that, including financial and cultural. And, we suspect smartphones and social media has a lot to do with it, too.
After all, we're all a lot more distracted these days. If you live in Iowa , there's a pretty good chance there are a lot of singles in your area. The Hawkeye State ranks in the top 10 when it comes to number of people over the age of 18 who aren't married, or in a serious relationship. That's great news for people looking for their match.
So, what was the best city for singles in Iowa?
Databases in Ryan Library: E-F
State-Specific Coronavirus Employer Q&A
Online dating probably will get a boost from pandemic, UAH sociologist says
Dating events in Tacoma, WA
State Child Labor Laws Applicable to Agricultural Employment
PortuguêsالعربيةNorskČeštinaItaliano中文(简体)EspañolFrançaisLëtzebuergeschMagyarPolskiTürkçeNederlandsEnglish日本語Eesti keelDanskSuomiΕλληνικάSvenskaDeutsch


text:
Q: "For each group of results fullfilling condition X, select only result with smallest column value A" I have a table with data and dates like so:
DECLARE @p TABLE (P_key int, P_Data char(1), P_ValidUntil datetime)

INSERT @p VALUES (20, 'T', '2003-02-28')
INSERT @p VALUES (21, 'U', '2005-05-31')
INSERT @p VALUES (30, 'V', '2006-09-30')
INSERT @p VALUES (30, 'W', '2008-04-30')
INSERT @p VALUES (31, 'X', '2007-06-30')
INSERT @p VALUES (32, 'Y', '2005-01-31')
INSERT @p VALUES (32, 'Z', '2007-06-30')
INSERT @p VALUES (33, 'A', '2005-06-30')

Given: qKey (only multiples of 10), qDate
Find: All entries that match qKey in the first digit and have a date larger then qDate. Return only one result per P_key (the one with the next higher P_ValidUntil to qDate).
The current solution is:
DECLARE @qKey AS int;
DECLARE @qDate AS datetime;

SET @qKey=30;
SET @qDate='2006-01-01';

SELECT * FROM @p WHERE
@qKey = (P_Key/10)*10
AND @qDate <= P_ValidUntil

which returns:
30    V    2006-09-30 00:00:00.000
30    W    2008-04-30 00:00:00.000
31    X    2007-06-30 00:00:00.000
32    Z    2007-06-30 00:00:00.000

This is basically correct, except that there are two entries for 30 (both are bigger than @qDate). I only want the smallest date of these multiple hits in the result:
30    V    2006-09-30 00:00:00.000
31    X    2007-06-30 00:00:00.000
32    Z    2007-06-30 00:00:00.000

Thanks in advance!

A: A first fix would be:
SELECT p2.*
from @p p2
JOIN (
    SELECT min(p_validUntil) Min_P_ValidUntil, P_Key
    FROM @p p
    WHERE @qKey = (P_Key/10)*10
    AND @qDate <= P_ValidUntil
    group BY P_Key
) p3 ON p3.Min_P_ValidUntil = p2.P_ValidUntil AND p3.P_Key = p2.P_Key

thanks.

A: Use a Window function
SELECT
    P_Key, P_Data, P_ValidUntil
FROM
    (
    SELECT
        *,
        ROW_NUMBER() OVER (PARTITION BY P_Key ORDER BY P_ValidUntil) AS rn
    FROM
        @p
    WHERE
       P_Key / @qKey = 1 AND @qDate <= P_ValidUntil
    ) p2
WHERE
   rn = 1

FYI: both techniques used in the 2 answers are quite common because it's a common problem. Examples here in this DBA-SE question: How to get the MAX row

redpajama_set_name: RedPajamaStackExchange


text:
WINM Forums :: The Films of Keanu Reeves :: Speed
LucaM 2011-03-16 04:35
Reviews: 13 ... I just realized we don't have a 'Speed' thread.
that error needs to be corrected.
hence this post.
and here, have a link :
http://blogs.amctv.com/movie-blog/2011/03/ten-reasons-we-cant-get-enough-speed.php
lindawn 2011-03-16 05:36
Reviews: 0 Ahhh! The movie that made me fall in love. Can't believe it was 17 years ago. I can probably recite all the dialogue, I've watched it so many times:)
nafsunaek 2011-03-16 08:49
Reviews: 0 Good thinking Luca!
@lindawn
Even after 17 years, it's still a good action movie to watch. And who didn't fall in love with Jack Traven? ;)
Reviews: 13 http://brightwalldarkroom.tumblr.com/post/14120235238/keanu-reeves-week-speed-1994
Was it a hit because the characters were reduced to quick quips? Because "Die Hard on a bus" was an easy sell for audiences? Or was it a hit because of Keanu? He's compelling here in a way that today's movie-star-wannabes — the Chrises and Ryans and Justins — can't touch, a pretty face paired with blunt machismo and given free reign to scream profanities when the situation calls for it. Perhaps it's simply a matter of how much he cares about stopping this bus, saving these people, stopping this psycho that makes him so watchable. Because when he cares, so do we.
ahem ;)
ARYA 2011-12-13 10:45
Reviews: 11 yes it is true. He is so watchable.
BlueGem 2011-12-13 11:24
Because when he cares, so do we.
inkhuldra 2011-12-13 15:33
Reviews: 0 Aw gadz, I just love these two articles. Ani, they're article database worthy, aren't they?
Reviews: 0 Browsing around Reddit I came across this post in the thread about Gentleman Keanu on the subway. It's Speed related, so I thought I'd post it here:
reynard_the_fox:
My mother was actually the set decorator on Speed. Funny story - since they were filming part of it on an emergency landing area for planes, they had to prove they could pack up everything within 5 minutes.
One day, a random security guy made them actually do it. They broke every lightbulb they had throwing everything into trucks - literally throwing - and hightailing it off the set.
http://www.reddit.com/r/videos/comments/n62lf/keanu_reeves_the_dude_has_class/c36n3es
I just had to add this too, from the same sub-thread:
thelogikalone:
In 6th grade I got a gf because I was one of the few kids to own a copy of Speed (ON VHS) & this girl was in loooooooooooooureve with Keanu. I happened to meet him a bit later when he was filming Chain Reaction in Chicago. Super super super nice guy.
Reviews: 11 HD trailer
http://youtu.be/CtoZXZyG44g
Trinity 2012-06-27 03:53
Reviews: 0 It was quite a long time ago I saw Speed and I liked it. A few weeks ago bought it on DVD and it's still great. Keanu is really convincing as a cop...and it is amazing that he did approximately 90% of his own stunts.
Another funny fact is that the director (Jan de Bont) "insisted that Keanu Reeves get a sensible haircut as would befit a hard-working cop. 20th Century Fox were horrified when they saw the buzzcut that he adopted for the film, threatening to postpone the film to allow his hair to grow back."
(Source: http://www.imdb.com/title/tt0111257/trivia)
My favourite interview from Speed times is this one :-)
(note that his shirt is the same as in the film):
http://www.youtube.com/watch?v=aZZX5JE4PFY
Reviews: 0 And yes, I need HELP :-) Hope that somebody is around here.
I need help to understand that part, when Jack is saved from under the bus (where he unsuccessfully tried to disarm the bomb). After that there is a dialogue like this:
Ortiz: You're not too bright, man, but ya got some big round hairy cajones.
Jack: That's very gross, Ortiz.
Ortiz: Huh, can't even pay him a compliment.
O.k., I get what cajones means :-), but I don't get, why Ortiz says something like that.
Can somebody please explain?...was there something R rated that I haven't seen? ;) Or is it a phrase or something?
Reviews: 13 It is a phrase (or something ;)) Ortiz was indeed paying the guy a compliment, but Jack's sense of humor didn't keep up with the bus ;)
http://en.wikipedia.org/wiki/Cojones
http://www.urbandictionary.com/define.php?term=cojones
Reviews: 0 Thanks, LucaM!
Meanwhile it came to my mind that there is a similar phrase in Hungarian, too :-) You say that somebody is "a guy with testicles" if he is tough / spirited. So it's really positive, I think.
Reviews: 13 you're welcome :)
Reviews: 0 I think the English equivalent is 'have the guts'
Reviews: 11 BTW
http://youtu.be/oZcb0A6kWc0
am I the last person to hear that Joss Whedon did uncredited writing for Speed?
Reviews: 11 oh man, I just got done looking at this and already they took it down for copyright violation. Sorry.
Reviews: 11 Here is the bit at IMDB too. About Joss Whedon I mean.
He also worked as a 'script doctor' on various features, notably Speed (1994).
Softie 2012-06-30 13:29
Reviews: 0 If I remember right, Joss Whedon was giving a talk or something at a university several years ago, and somehow Keanu came up. He gave him some high praise. And not the usual backhanded compliments, I remember it was really thoughtful and insightful. Not the usual stuff you hear about Keanu. I only wish I could recall exactly what he said :(
Reviews: 0 Hi Softie, maybe something like these ones?...
Joss began by sharing his thoughts about Keanu Reeves as an actor; sounds like an odd way to start but it was quite lovely. He explained how Keanu has mastered his own career by embodying a rare and authentic style all his own typified by a sense of compassion for humanity and a subtle detachment. He shared a story about when he was "fixing" the script for Speed and working with Keanu on the character, how Keanu had spent some time with detectives and law enforcement to get a sense of the character and he said he was struck by how polite they were. How they all used "sir" and "Ma'am." Joss was impressed by how Keanu objected to certain lines and actions in the script because they seemed outside of what the character would truly do. He explained how Keanu's choices of roles has been indicative of his style and how he has kept his private life private. He described Keanu as having an international appeal both by his "pretty" and by his multi-ethnic aesthetic. Ultimately, Keanu is the perfect Neo.
The Keanu panel was very interesting. I think we all were surprised by how Joss took a topic - which in some ways we were all expecting to be a joke - and created some interesting insights - both about Keanu as an actor and as an example being the type of person who can have a positive influence on the Hollywood machine. By looking at how Keanu embodies a sense of "otherness" (not stereotypically male, having a feminine aspect to his presence, having different intonation, speech patterns, and physicality from what you would normally expect from a Hollywood Action Star), you start to see that it allows people to identify and connect with him as an "everyman" – which is why he is so successful in his performance as Neo in the Matrix. Joss also commended Keanu for his sense of integrity - as a movie star not running after fame, but willing to choose projects that he wants to do, that he believes in. The Hollywood culture is built by those who work in the industry as part of it. The Importance of Being Keanu is that if more people in the industry were thoughtful about their work as an artist, their integrity as a person, their substance as storytellers – rather than chasing fame or that next mega-blockbuster movie – the culture of the Hollywood industry could change for the better. (At least that was my take from it)
WHEDONesque - A Conversation with Joss Whedon '87: Things and Stuff
(Comments from students who were there)
http://whedonesque.com/comments/12997