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dibonacomemorials Group

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Efrem Savin
Efrem Savin

Driven Under ~REPACK~

"This is a complex issue, evidenced in the fact that we're seeing quite a few mixed messages from our outreach," Cole said. "While 40 percent of recreational users said they don't think being under the influence of marijuana affects their ability to drive safely, almost half of all survey participants said driving under the influence of marijuana puts people in danger."

Driven Under

Food and beverage labels occupying a large swath of Gen Z's top 50 most-loved brands reflects another pandemic-driven trend, as young consumers return to packaged foods and snacks they previously shunned for health concerns. Kit Kat landed in the No. 13 spot on Morning Consult's ranking, while Mondelez's Oreo cookies followed at No. 15.

The push and pull of a second love. Both have past pain; more mature. Neither believe the other;love is for idiots. so they never are intimate at the same time. The gun is terminating pregnancy or control because o pregnancy.the world is loveless and we all repeatedly suffer . That my interpretation of driven under

I found an outstanding source, and purchased a breathalyzer. On our next wine tasting trip, I brought it along. After each winery, I had all the people in the van take a test. As the day went on, I would ask them what blood alcohol level they thought they had achieved. Almost everyone underestimated his or her blood alcohol level. After a few wineries, everyone was over the legal limit of .08 yet most felt they were under that number. I found in my brief study most of my friends thought they were under .08 when, in fact, they were at or above a .13 reading.None of these participants have ever been arrested for a DUI and they have often waited after drinking to drive home or even had someone take them home. Their intentions were great yet they were unknowingly getting behind the wheel and if they were pulled over for a bad taillight or other issue they would be at risk for failing a blood alcohol test. The next weekend, my daughter invited my wife to join her and her husband at a wine tasting club event. Each couple brings a bottle of wine wrapped in plain paper. One person pours out the tasting samples so no one knows who brought which bottle. At the end of the evening, after everyone voted for their favorite, the winning bottle earns a prize of a few bottles of wine for the donor of that variety. In addition to my wife, our new associate pastor and his wife, were at the event. I stayed home to write and enjoy a movie or two on TV. I did send along the breathalyzer which was a big hit. At the end of the evening, people would have normally headed home believing they had waited long enough after the wine tasting to be driving safely. Instead, they used the breathalyzer and more than half a dozen people were above a .08 and some of them were over .13! Safety considerations aside, it would be reallyembarrassing to be driving home from an event with friends from church and end up being arrested for a DUI.

&nbsp (a) &nbsp A special registration plate issued under this part: &nbsp &nbsp (1) &nbsp May be used on and for the operation of any vehicle expressly authorized to be driven under that class of registration plate, without registering the specific vehicle with the Administration; and &nbsp &nbsp (2) &nbsp Is interchangeable among these vehicles. &nbsp (b) &nbsp A vehicle may not be driven under a special registration plate issued under this part unless the plate is displayed on the vehicle in the manner required by &#167 13-411 of this title. &nbsp (c) &nbsp A special registration plate issued under this part may not be used on or for the operation of any vehicle other than a vehicle expressly authorized to be driven under that class of registration plate. &nbsp (d) &nbsp If the required license or authorization to do business of a person to whom a special registration plate is issued under this part expires or is canceled, suspended, or revoked, or if the Administration notifies the person that the special registration plates issued to the person have been suspended or revoked under &#167 13-706 of this title: &nbsp &nbsp (1) &nbsp All of the special registration plates issued to that person are invalid; and &nbsp &nbsp (2) &nbsp The person may not drive any vehicle or permit any vehicle to be driven under those plates. This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply. Free Daily Summaries in Your Inbox You're all set! You already receive all suggested Justia Opinion Summary Newsletters. You can explore additional available newsletters here.

The song "Driven Under" by Seether is about someone being driven to desperation by another person, likely a romantic partner. The first verse is about the protagonist questioning whether or not the other person realizes that they're faking their happiness in the relationship, and expresses confusion over the other person's motivations. The chorus shows that the other person has a gun, implying that they are making threats of violence. The bridge talks about the protagonist having to "succumb to the feelings they can never face" and not wanting to go through all this again, likely referring to the emotional pain and turmoil they feel in the relationship. Finally, the outro shows the other person admitting to having a gun and threatening to use it against the protagonist. In the end, the song is a declaration of being driven to the edge by someone they love and a desperate plea to be free from the pain of an unhealthy relationship.

The plasmid constructs containing CaMV::GUS (pMAA-Red) and CaMV::DsRed (pPZP3425) fusions were introduced into A. tumefaciens GV3101 for transformation of Arabidopsis plants by the floral dip method [30]. In case of pMAA-Red, the fluorescent transformed seeds were selected under an inverse microscope (Axiovert 200M; Zeiss, Hallerbergmoos, Germany) equipped with a DsRed fluorescence filter and put on soil to grow the next generation. Homozygous lines were selected based on visual observation as described by Ali et al. [20].

The cysts of the sugar beet cyst nematode Heterodera schachtii were harvested from in vitro stock cultures propagated on mustard (Sinapsis alba cv. Albatros) roots growing on 0.2 concentrated Knop medium supplemented with 2% sucrose [31]. The cysts were soaked in 3 mM ZnCl2 as stimulus for hatching of J2 larvae under sterile conditions. The J2 larvae were then washed three times in sterile water and resuspended in 0.5% (w/v) Gelrite (Duchefa, Haarlem, The Netherlands) before inoculation. Roots of twelve-day-old Arabidopsis seedlings were inoculated under sterile conditions with about 50-60 juveniles per plant.

Histochemical detection of GUS activity was performed by staining [14] using X-gluc (Biomol, Hamburg, Germany) in 0.1 M sodium phosphate buffer pH 7.0 containing 0.1% Triton-X 100, 0.5 mM K3[Fe(CN)6], 0.5 mM K4[Fe(CN)6] and 10 mM Na2EDTA. For GUS staining of syncytia, the infected roots of CaMV::GUS plants were incubated with X-gluc for 6 hours at 37ºC. Staining was performed at 5, 7, 10, and 15 dpi (days post inoculation). Stained syncytia and uninfected roots were counted per line from three promoter::GUS lines and photographed under an inverse microscope (Axiovert 200M; Zeiss, Hallerbergmoos, Germany) having an integrated camera (AxioCam MRc5; Zeiss). The GUS expression in the root cap border cells was also photographed in the same way.

We determined the expression of DsRed and GUS driven by the CaMV promoter at different time points after infection. The lines, CaMV.2x35S + omega::DsRed L1, L2 and L3 contained 80, 101 and 82 syncytia in total, respectively and all of them showed DsRed fluorescence at 5 dpi (Table 1). The percentage decrease of total number of syncytia in fluorescent syncytia at different time points is given in Figure 3A. At 7 dpi, over 90% of the total syncytia were found fluorescent. Similarly, at 10 dpi and 15 dpi on an average from three lines over 80% and 50% of the syncytia showed DsRed fluorescence respectively (Figure 3A). Representative syncytia with DsRed fluorescence were also photographed at different time points (5, 7, 10 and 15 dpi) and are shown in Figure 4A. It is quite obvious from the figure that there was very high fluorescence in the syncytia as compared to the uninfected root segments. However, at 15 dpi, the expression level of DsRed also slightly went down showing decreased activity of the promoter with the passage of time.

For the CaMV.2x35S + omega::GUS construct we used the first 3 transgenic lines harboring the pMAA-Red vector having the highest expression in Ali et al. [20]. The pMAA-Red seedlings were grown on MS medium for 14 days and stained with X-gluc solution (see Materials and Methods) at 37ºC for 6 hours. The seedlings had very high staining, showing that the CaMV.2x35S + omega promoter was highly and constitutively active in the seedlings under the control of this promoter (data not shown). After nematode infection, the transgenic lines CaMV.2x35S + omega::GUS L1, L2 and L3 contained 58, 73 and 95 syncytia in total, respectively and over 80% of them showed GUS staining in syncytia after five days of infection (Table 1 and Figure 3B).

Our results showed that the CaMV.2x35S+omega promoter was active at all the time points in case of DsRed with decreased expression only at 15 dpi. However, in case of GUS, the promoter was active in syncytia at 3, 5 and 7 dpi and was slightly downregulated at 10 dpi and almost inactive at 15 dpi. The molecular weight of GUS gene (UidA) based on the amino acid mentioned by Ali et al. [20] in Genbank is just over 68 KiloDaltons (kDa) which is higher that of DsRed protein is around 25 kDa but this is not the molecular weight difference that the promoter delivered the DsRed (smaller protein) more efficiently into the syncytia as compared to GUS. What could be the reason for the better expression of DsRed in older syncytia as compared to GUS? DsRed is a fluorescent protein which does not need any cofactor or substrate which is needed for GUS staining (i.e. X-gluc) [14]. The substrate thus has to get into the syncytia to be available for the GUS enzyme to interact. Old syncytia have very strong outer cell walls which could make it difficult for the GUS substrate to penetrate into the old syncytia. Similar observations were reported by Goverse et al. [26] who expressed green fluorescent protein (GFP) in transgenic potato (Solanum tuberosum) plants under the control of the CaMV35S promoter. By using confocal laser scanning microscopy (CLSM), they investigated promoter activity in syncytia during potato cyst nematode (Globodera rostochiensis) infection and observed GFP expression up to 13 dpi [26]. 041b061a72


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