text:
Our friends can be funny. Very funny, if you ask me. I am talking about our uncles by the main mall. Well, to be fair it could be those by Botswana Unified Revenue Service (BURS).
Both have the capabilities to interfere with our communication and are not known to be very enthusiastic about law and ethics. Somehow, one is supposed to live with the reality of voices popping up in their telephone conversations.
The other day they were at it for about five seconds. I laughed it off and they quickly realised their mistake and cut off. Learned Counsel Lecha says that once upon a time, they mistakenly sent him a text message. I swear I am not lying. Just ask him. Imagine an SMS from them. It must have been like a scene from a horror movie, even a Ludlum tale. Except, a local production.
Somehow, people are supposed to be afraid. People are supposed to be afraid of their government. People must be afraid of what their government could do to them. God forbid. I have always said that democracy is not when the people fear their government.
Democracy is when a government fears the people. The worst disfavour any people can do for themselves is to fear their creation. Governments are supposed to keep us secure, not terrified. Governments have no mandate but the happiness of the people.
When an individual infuses fear in you, it is because they have their own fear(s). They instill fear in order that you may personally censor yourself. Fear is a cruel and inexpensive way of governance. A government needs not send the police after you.
No, you police yourself. Fear confers an advantage to your tormentor. You do half their job; all against yourself. A government cannot be with you everywhere.
But you are with yourself everywhere. You must be afraid that your office is bugged; that the car following you belongs to a spy agency; that your home is bugged; that your cellphone is bugged; that they have your bedroom pictures and that they have gadgets all over your car.
You must be afraid that they will have your conversations on tape; that they are monitoring your movements; that they could hurt your family. You must be afraid they could ruin your job or business; that they could block your career paths.
Most importantly, you must be afraid that they could kill you. That's what sick governments want.
My university roommate and I had a collection of Martin Luther King audio tapes and speeches. We studied them whenever we were off the books. "If physical death is the price we must pay", he once said, "to free our children from a permanent psychological death, then nothing shall
Never fear being killed if it is for the course of truth. As in the letter to that soldier in the movie, Letters from Iwo Jima, "do what is right because it is right". Seek no popularity. Seek not the applause of the multitudes or the favours of your government. Seek only the applause of your conscience.
When you lie back on your bed at the end of a long day, you can enjoy the peace that comes with knowing you have done the right thing. I say to you fellow countrymen and women; never fear. Fine, obey the law and respect authority but never fear. Even where your communication is being interfered with never fear. Fear not your government.
Having said that, I am told there is a new gospel in town. Citizens must lick their way to success. Sycophancy has ceased to be a vice but a virtue. In Mahalapye, where I grew up, if anyone called you lelope, they were guaranteed a fistfight. And we didn't know half what the word meant. Now, you must be a member of your local KMA club. Merit is the reason for your poverty.
I can imagine sitting with my little boy, looking down upon the clear canvas that is his formative conscience, attempting to write upon it the lessons for success; "Lick boots son, lick them till kingdom come. Do it better than your peers. And you will be a man my son".
Sorry, I reject that gospel. It is exactly what is wrong with the country today. We have way too much fear and way too many bootlickers.
People can't look at those in authority and have some honest talk. When men with vile motives seek to hush you they say you have no respect. Respect is earned, not commanded. Only human dignity is conferred at birth. Our notion of respect is a perverted one. One of sycophancy and unqualified obedience.
My kids and I have an understanding. So long as they are respectful, they are free to engage me on any subject and they need not fear anger or reprisals.
My arguments with my 13-year-old girl go into the sunset. True, the last word is always mine, as it must be. I just don't want her licking boots. Sycophants applaud their own oppression and celebrate every vice of the one to whom their consciences are mortgaged. A sycophant has no soul his own. Fear not, and lick no boots. Fear no man, but God.


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Something's always wrong at {RHC, rather at Near East transmission facilities too, TRT Emirler, or IRIB's Zahedan / Sirjan sites, for example on Jan 9:
TURKEY 5960.004 kHz TRT German.
Glenn Hauser often reports on a dissolute way of working at the TRT Emirler
technicians, i.e. they don't switch TX off after the English broadcast
regularly 23-24 UT, but cheerfully continues to broadcast the TRT GERMAN
service taken from their web livestream Broadcast.
Today Jan 9th at 00.22-00.24 UT a travel report from their folklore feature
corner, belongs to the remote Perseus in New Jersey's eastern US coast
with S = 9 + 20dB signal level.
MOLDOVA/TURKEY 13730even Voice of Radio Denge Welat Kurdish radio program
via Grigoriopol Maiac Moldova at 09.10 UT on Jan 9,
originate from Brussels Belgium,
hit by co-channel of - possibly -
13730.008 kHz TRT Radio Emirler something px taken from
Turkish security patriotic sce.
S=9+25dB both equal level, noted in Doha Qatar remote SDR unit.
9.6 kHz wideband audio block seen.
[selected SDR options, span 12.5 kHz RBW 15.3 Hertz]
(wb df5sx, wwdxc BC-DX TopNews Jan 9)
IRAN 13820.005 VoIRIB Arabic sce was in the clear at 09.18 UT check,
S=9+15dB strong sidelobe in remote Doha Qatar SDR rx.
13820 0830-1030 Arabic 500 282 Sirjan 293548N0554707E
had weak 3 x strings accompanied on either sideband,
50, 100, 150 Hertz apart distance.
But at 09.23 UT heard also another station testing TEST tones of
8 x 767 Hertz apart distance strings on either sideband,
also on S=9+15dB strength level.
At 09.55 UT of 824 Hertz apart tones heard ... but no IRIB Arabic anymore.
My only guess:
13820 0530-0730 Arabic 500 289 Zahedan 292835N0605150E
the technician on Zahedan site - scheduled on air at 05.30-07.30 UT slot -
tested the IRIB Zahedan tx with different test tone procedure ???
15129.959 much ODD frequency from Sirjan outlet.
S=9+15dB in short distance in Doha Qatar.
0930-1430 UT Arabic 500 kW 216degr azimuth
towards Saudi Arabia, NoEaAF, Sahel, WeAF target.
[selected SDR options, span 12.5 kHz RBW 15.3 Hertz]
(wb df5sx, wwdxc BC-DX TopNews Jan 9)

subset: commoncrawl


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If you're a typical musician, you lack one or both of these critical qualities. Let's see how important they are to your success.
If you're a typical musician, you lack one or both of these critical qualities. Let's see how important they are to your success.
I do as much pre-production as humanly possible at home. When you`re ready to record, and I can`t stress this enough, have everything rehearsed to death and a production plan ready. I did my latest CD at a commercial place, but my dream is to do my next one at home.
The bottom line is: I believe in my music. As an instrumental artist you`re not going to land a huge deal with a major label. If you do get a deal the label is going to take a huge chunk of money, and even if they do sign you, they may not even distribute your CD. Some friends of mine signed with a major label, and the label has not done a thing with them. So they have a great product that they can`t do anything with.
Self-assessement time. Can you go it alone? You might need the synergy of two people working together to kick start your label.
Even though these days it takes a lot of idealism to produce an instrumental record, I strongly encourage everybody who`s playing around with the idea to do so. Just don`t expect to get any money out of it, regardless of the quality of your product. It might happen, but it`s very likely that it won`t. But you will have the best musical business card there is and a unique document of your own creativity.
The one thing I always try to do is be in a creative state of mind when I compose. It seems that the ideas flow better if you are working on it on an ongoing basis. For a lot of my instrumental tunes, I sometimes get a mental picture of something in my head, and then ask myself what music goes with this scene. This is much like scoring for film--except the film is in my head!
Completely subjective, straightforward tips in the areas of promotion, publicity and your career.
13 additional tips you can use today to improve your tracks.
I record at home in my 16 track (analog) studio. It took me lots of time, energy and money but I`m glad that I`ve got one. Nothing can beat a home studio. You can go there anytime you want and not be bothered by anyone other than yourself. You don`t have to book time, and pay by the hour. Unless you`re a lunatic, that is.
You have to have a good promotion budget and don`t skimp corners when it comes to this. You can have the greatest album and if no one knows that you have one out and available, it really is no good. Utilize all that you can with local publications and national ones as well. Always think of the nation as your market and not just your town. You can be the biggest thing in your hometown and unless they know you around, it really is not going to help you.
Are you a half-finisher? Always eighty percent complete? How you can finish what ya started.
Dan McAvinchey helps you to eliminate those pesty sonic gremlins from your home or project studio.
Dan McAvinchey is a guitarist and composer living in Raleigh, NC.
He believes every musician or composer has the power to write, record and release their own music.
His 1997 CD release on Guitar Nine was entitled "Guitar Haus".
Send comments or questions to:


text:
Are you between the ages of 16-24, and looking for a job? NCWorks and Western Piedmont Workforce Development invite you to be a part of the 2nd Annual NextGen Job Fair on May 11 from 10:00 a.m. to 12:00 noon at Alexander Central High School.
This is an excellent opportunity to connect with local companies that are hiring those ages 16-24. Participating employers include: Kincaid Furniture, Royale Comfort Seating, Taylorsville Glass, Cricket Wireless, Wendy's, Village Inn Pizza, Food Lion, North Carolina Department of Public Safety/Alexander Correctional Institution, Turf Pro/Fence Pro, Tyson Foods, US Census Bureau, and Turning Point Services.
For more information, please contact Jessica Miller at (828) 315-1712 or firstname.lastname@example.com.


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- © 2011 American Society of Plant Biologists
Cell-to-cell trafficking of RNA is an emerging biological principle that integrates systemic gene regulation, viral infection, antiviral response, and cell-to-cell communication. A key mechanistic question is how an RNA is specifically selected for trafficking from one type of cell into another type. Here, we report the identification of an RNA motif in Potato spindle tuber viroid (PSTVd) required for trafficking from palisade mesophyll to spongy mesophyll in Nicotiana benthamiana leaves. This motif, called loop 6, has the sequence 5′-CGA-3′...5′-GAC-3′ flanked on both sides by cis Watson-Crick G/C and G/U wobble base pairs. We present a three-dimensional (3D) structural model of loop 6 that specifies all non-Watson-Crick base pair interactions, derived by isostericity-based sequence comparisons with 3D RNA motifs from the RNA x-ray crystal structure database. The model is supported by available chemical modification patterns, natural sequence conservation/variations in PSTVd isolates and related species, and functional characterization of all possible mutants for each of the loop 6 base pairs. Our findings and approaches have broad implications for studying the 3D RNA structural motifs mediating trafficking of diverse RNA species across specific cellular boundaries and for studying the structure-function relationships of RNA motifs in other biological processes.
A central question in current biology concerns how basic processes in individual cells are integrated to support development and function of the whole multicellular organism. Cellular boundaries play a pivotal role in this integration by maintaining a certain level of cellular autonomy while enabling communication between cells to achieve coordinated gene expression and metabolism within an organism. Cell-to-cell trafficking of specific RNA and protein molecules is emerging as a new paradigm of gene regulation at the whole-organism level in plants, in addition to its fundamental role in the systemic spread of viral infection and defense responses (Giakountis and Coupland, 2008; Kehr and Buhtz, 2008; Lucas et al., 2009; Turgeon and Wolf, 2009; Chitwood and Timmermans, 2010; Chuck and O'Connor, 2010; Hannapel, 2010; Lehesranta et al., 2010). Such trafficking requires rethinking of RNAs and proteins as functioning solely within the cells in which they are produced.
Infectious agents, such as viroids and viruses, must move from cell to cell and throughout a plant to establish systemic infections. All viroids and many plant viruses have RNA genomes, so that their systemic infections represent classical examples of cell-to-cell and long-distance RNA trafficking (Flores et al., 2005; Scholthof, 2005; Lucas, 2006; Ding and Itaya, 2007; Ding, 2009). Many cellular RNAs also traffic between cells and between organs. Analyses of phloem sap collected from various plant species revealed many different mRNAs (Sasaki et al., 1998; Ruiz-Medrano et al., 1999; Doering-Saad et al., 2006; Omid et al., 2007; Deeken et al., 2008), small RNAs, including microRNAs (miRNAs) and short interfering RNAs (siRNAs) (Yoo et al., 2004; Buhtz et al., 2008, 2010; Varkonyi-Gasic et al., 2010), and some other types of RNAs (Zhang et al., 2009). Grafting experiments have shown that some mRNA species are transported over long distances in the phloem to regulate developmental processes, such as leaf morphogenesis in tomato (Solanum lycopersicum; Kim et al., 2001; Haywood et al., 2005) and tuber formation in potato (Solanum tuberosum; Banerjee et al., 2006). Trafficking of a miRNA from shoot to root has been implicated in the regulation of a gene involved in phosphate homeostasis in Arabidopsis thaliana (Lin et al., 2008; Pant et al., 2008). Intercellular trafficking of some miRNAs contributes to regulation of root vascular development (Carlsbecker et al., 2010). siRNAs can act as mobile signals that traffic from cell to cell and from organ to organ to mediate gene silencing including RNA-dependent DNA methylation (Chitwood et al., 2009; Schwab et al., 2009; Dunoyer et al., 2010a, 2010b; Molnar et al., 2010). Gene silencing signals containing siRNAs also traffic within a plant to mediate systemic RNA silencing as a means of antiviral defense (Ding and Voinnet, 2007; Kalantidis et al., 2008). RNA trafficking also occurs between some parasitic plants and their hosts, with biological functions yet to be understood (Roney et al., 2007; David-Schwartz et al., 2008).
Cell-to-cell RNA trafficking also occurs in animals. Numerous circulating nucleic acids, including RNAs, have been found in human plasma and serum under healthy and diseased conditions (Fleischhacker and Schmidt, 2007; Vlassov et al., 2007). Gene silencing signals in animals traffic intercellularly as a means of gene regulation and antiviral defense (Ding and Voinnet, 2007; Jose and Hunter, 2007). Exosomes, which are membrane vesicles released into extracellular spaces by many types of mammalian cells, enclose and mobilize mRNAs and miRNAs between different cells (Valadi et al., 2007; Kosaka et al., 2010). Viral miRNAs can also be transfered via exosomes between certain cells of the immune system and function therein (Pegtel et al., 2010). In addition to this secretory pathway, cell contact also enables intercellular transport of small RNAs between mammalian immune cells (Rechavi et al., 2009). These and other examples support the idea that interecellular RNA trafficking plays a role in signaling (Vlassov et al., 2007; Dinger et al., 2008; Simons and Raposo, 2009).
An outstanding question is how different RNAs are recognized for transport between distinct types of cells, which is essential for controlling the specificity of trafficking. We have been using Potato spindle tuber viroid (PSTVd) infection as a model system to test the hypothesis that unique RNA structural motifs mediate trafficking across different cellular boundaries. Viroids are single-stranded, circular, and noncoding RNAs that infect plants. They are the smallest plant pathogens known to date, with sizes ranging from 250 to 400 nucleotides (Flores et al., 2005; Ding and Itaya, 2007; Tsagris et al., 2008; Ding, 2009). Lacking protein-coding capacity and helper viruses, viroid RNAs most likely interact directly with preexisting cellular machineries to move from cell to cell and from organ to organ to establish systemic infections. Therefore, viroids provide simple models to investigate the RNA structural elements critical for cell-to-cell trafficking (Ding and Wang, 2009; Wang and Ding, 2010). The 359-nucleotide genome of PSTVd folds into a rod-like secondary structure that has been supported by biophysical, structural and mutational studies (Flores et al., 2005; Owens, 2007; Ding, 2009). As shown in Figure 1, this secondary structure comprises 27 loops/bulges (collectively refered to as loops hereafter, for simplicity of description), numbered 1 to 27, starting from the left in Figure 1, that are flanked by short double-stranded helices. With the PSTVd model, our previous studies obtained genetic evidence for the role of RNA loops in mediating RNA trafficking between specific cells (Qi et al., 2004; Zhong et al., 2007).
Computational analyses, such as minimum free energy calculations (e.g., mfold) (Zuker, 2003), generally show RNA secondary structures as comprising short double helices punctuated by loops. In the absence of additional information, such loops are usually depicted as single-stranded regions lacking structure. However, many x-ray crystallographic and NMR studies in the last decade have provided evidence that nucleotides within most loops form distinct three-dimensional (3D) motifs via non-Watson-Crick (non-WC) base pairing and base stacking and that such motifs comprise the primary binding sites for RNA–RNA, RNA–protein, and RNA–small ligand interactions (Leontis et al., 2002a, 2002b, 2006; Noller, 2005; Steitz, 2008). RNA nucleobases interact with each other by hydrogen bonding at any one of three edges, the Watson-Crick (WC), Hoogsteen (H), and Sugar (S) edges (see Supplemental Figure 1 online). Non-WC base pairs are categorized into 12 geometric base pairing families according to the interacting edges and relative orientation (cis or trans) of glycosidic bonds of the paired bases (see Supplemental Figure 1 online) (Leontis and Westhof, 2001).
Many RNA 3D motifs recur in nonhomologous RNA molecules or in distinct sites of the same RNA molecule (Simons and Grunberg-Manago, 1998; Leontis et al., 2002a). Recurrent 3D motifs comprise sets of nucleotides with similar spatial arrangements, including the same non-WC base pairs. The 3D structures of recurrent motifs are more conserved than their sequences; in other words, different RNA sequences can fold into the same 3D structure by forming the same geometric non-WC base pairs (Leontis and Westhof, 1998a, 1998b; Leontis et al., 2002b; Schudoma et al., 2010). The set of base substitutions compatible with the 3D structure of a motif is its sequence signature. Thus, certain non-WC base pairs can substitute for each other in a motif without distorting the 3D structure of the motif; such base pairs are isosteric or nearly so (Michel and Westhof, 1990; Leontis and Westhof, 1998b; Leontis et al., 2002b). More specifically, isosteric pairs form hydrogen bonds using the same base edges and have the same glycosidic bond orientation and identical or similar C1'-C1' (i.e., first carbon of ribose) distances. Isostericity matrices summarize the isosteric relationships for each geometric base pairing family and provide the basis for analyzing the sequence signatures of RNA motifs (Leontis et al., 2002b). Furthermore, isostericity matrices allow predictions of base substitutions in an RNA motif that will either disrupt or maintain the isostericity of a base pair and, consequently, the structural integrity of the motif. We used these principles to successfully predict, and then experimentally confirm by mutational and functional analyses, the 3D structures of PSTVd loop E, which is critical for replication (Zhong et al., 2006), and loop 7, which mediates trafficking from bundle sheath to phloem (Zhong et al., 2007).
We used mutational analysis to identify at least 11 loops in PSTVd critical for systemic trafficking in Nicotiana benthamiana plants (Zhong et al., 2007, 2008). This study investigates loop 6, which comprises the six nucleotides G36, A37, C38, C323, G324, and A325 in strain PSTVdInt (accession number NC002030; Figure 1). When we mutated three nucleotides (G36U/A37C/C38G) to form WC base pairs and thereby closed loop 6, as predicted by mfold (Zuker, 2003), systemic trafficking of the mutant PSTVd, but not its replication, was abolished (Zhong et al., 2008). To gain mechanistic insights into the function of loop 6, we investigated its 3D structure (i.e., specific non-WC base pairing) as well as the cellular boundary at which it functions to mediate trafficking. Here, we provide evidence that loop 6 plays a decisive role in PSTVd trafficking from palisade mesophyll to spongy mesophyll cells of N. benthamiana leaves. We also present a 3D structural model of loop 6 that specifies all non-WC base pair interactions. Our findings and approaches have broad implications for studying the 3D RNA structural motifs mediating trafficking of diverse RNA species across cellular boundaries and for studying the structure-function relationships of RNA motifs in other biological processes.
A 3D Structural Model for PSTVd Loop 6
To infer the 3D structure of PSTVd loop 6, we used the Find RNA 3D (FR3D) program suite (Sarver et al., 2008) to search a nonredundant list of atomic-resolution Protein Database (PDB) files, updated on May 5, 2010. The goal was to find internal loop sequence matches of the PSTVd loop 6 sequence 5′-CGA-3′...5′-GAC-3′ flanked on both sides by WC (including near-isosteric G/U wobble) base pairs as templates for modeling the structure of loop 6. Although we did not find an exact sequence match in the 3D database, we did find two internal loops that each differ from PSTVd loop 6 at single base positions. One of these loops occurs in helix 89 (H89) of bacterial and archaeal 23S rRNAs. The sequence and 3D structure of this motif appear to be broadly conserved in bacterial and archaeal 23S rRNAs. The sequence of this internal loop is 5′-CAA-3′..5′-GAC-3′ in the 23S rRNAs of both the archaeon Haloarcula marismortui and the distantly related bacterium Escherichia coli, having the same crystal structure (Table 1). The boldface A in this bacterial and archaeal loop sequence differs from G at the equivalent position of PSTVd loop 6. The corresponding H89 motif in the 23S rRNAs of bacterium Deinococcus radiodurans has the sequence 5′-CGA-3′...5′-GGC-3′, with G being different from the A at position 37 of the PSTVd loop 6 (Table 1). The structure of this H89 motif is nearly identical to the E. coli and H. marismortui versions. In the following paragraphs, a boldface base in a loop sequence indicates that it is different from the base at the equivalent position in PSTVd loop 6. The other internal loop we found in the database has the sequence 5′-CAA-3′...5′-GAC-3′ in helix 41 (h41) of E. coli 16S rRNA (Table 1). It has the same 3D structure as the H89 internal loop but is not conserved in other 16S rRNAs.
To further explore the sequence variability that can produce the same geometry, we used the H89 internal loop structure to carry out geometric searches using FR3D to find geometrically similar 3D motifs. In addition to recovering the original motifs, we identified an internal loop in the 3D structure of the Azoarcus group I intron that adopts a 3D structure similar to that of the 23S H89 motif, even though its sequence, 5′-CAA-3′...5′-AAA-3′, is considerably different (three base changes) (Table 1). The results of this search, including 3D structure superpositions and detailed structure annotations, can be accessed at the interactive website http://rna.bgsu.edu/WebFR3D/Results/pstvd. A view of the web link is shown in Supplemental Figure 2 online.
All of these motifs comprise a conserved set of three stacked non-WC base pairs flanked by WC base pairs, as shown in Figure 2. According to the Leontis-Westhof nomenclature of base pair classification (Leontis and Westhof, 2001), the first non-WC base pair is a cis Watson-Crick bifurcated pair, the second is a trans Sugar-Hoogsteen (tSH) pair, and the third is a trans Hoogsteen-Sugar (tHS) pair. Note that tSH GA and tHS AG indicate the same base pair geometry, with hydrogen bonding between the Hoogsteen edge of A and the Sugar edge of G.
The sequence variability observed in the instances of this recurrent 3D motif recovered from the structure database is consistent with the base pair isostericity matrices defined for base pair families (Leontis et al., 2002b). GA, AA, and GG are mutually isosteric or near-isosteric tSH base pairs, and CC and AC are near-isosteric cWC bifurcated pairs. Most importantly, these same interactions can also be formed by the PSTVd sequence 5′-CGA-3′...5′-GAC-3′. Thus, we hypothesize that PSTVd loop 6 follows the same base pair pattern as seen in this 3D motif. Specifically, we predict that a GA tSH base pair forms between nucleotides 36 and 325, a tHS base pair between A37 and G324, and a bifurcated cWC pair between C38 and C323 (Figure 2). As can be seen in Figure 2, the only difference between the PSTVd sequence 5′-CGA-3′..5′-GAC-3′ and the 23S rRNA sequence 5′-CAA-3′..5′-GAC-3′ is the substitution of G for A in the second base pair (tSH). This implies the presence of a GA tSH pair in the viroid where an AA tSH pair is found in the H89 23S motif. As tSH AA and GA base pairs are isosteric (Stombaugh et al., 2009), which means that they occupy the same 3D space, this change does not significantly perturb the 3D structure of the motif. In fact, AA substitutions for GA tSH base pairs are very common in homologous RNA molecules (Stombaugh et al., 2009). Furthermore, the rRNA alignments show that GA is found at this position in some 23S rRNAs, as will be discussed below. Finally, we will show that a PSTVd loop 6 mutant with G-to-A substitution at this position, a sequence identical to the 23S rRNA motif, is indeed capable of systemic infection (see below). Figure 2C shows the structural similarity of the AA and GA tSH base pairs.
As mentioned above, the internal loop that we propose as a model for PSTVd loop 6 is highly conserved in H89 of archaeal and bacterial 23S rRNAs. The crystal structure shows that this motif provides a specific protein binding site for the conserved ribosomal protein L16 (called L10e in archaeal and eukaryal ribosomes). More specifically, Lys-123 from L16 protein in E. coli and the equivalent Lys-156 from L10e protein in H. marismortui interact with the conserved CC bifurcated pair, which provides an ideal docking site for the positively charged Lys amino group. Figure 3 shows the electrostatic complementarity between the terminal -NH3(+) group of Lys and the minor groove of the bifurcated CC pair. Examination of the L16/L10e alignment in the Pfam database (Finn et al., 2010) (Pfam id PF00252) showed this Lys to be almost universally conserved. Conservation of this docking interaction in bacterial and archaeal rRNA 3D structures as well as in sequence alignments indicates the biological significance of this docking interaction, which could also be relevant to other RNAs, including PSTVd.
The Proposed PSTVd Loop 6 Structural Model Is Compatible with Chemical Probing Data
Our structural model for PSTVd loop 6 is also consistent with existing chemical probing data, which show that A37, A325, and C38 are highly modified by dimethylsulfide (DMS) (Gast et al., 1996). Figure 4A shows the proposed base-pairing model for loop 6 with the bases accessible to DMS marked by red arrows. DMS attacks the imino nitrogens of adenine (atom A-N1) and cytosine (atom C-N3) when the WC edges of these bases are exposed. If the WC edges were involved in base pairing, these imino nitrogens would not be reactive. In the proposed PSTVd loop 6 structural model, the As that form tHS base pairs using their Hoogsteen edges have exposed WC edges, as shown in Figure 4C, which are predicted to react with DMS, consistent with the chemical probing data. In fact, tHS pairs are among the most common non-WC base pairs, and As forming these base pairs are generally highly reactive to DMS probing unless such As use their WC edges to form another interaction, such as a base triple. Furthermore, the fact that C38 is DMS-reactive, whereas C323 is not, is also consistent with the proposed loop 6 model. As shown in Figure 4D, the bifurcated base pair between C38 and C323 is not symmetrical, as the WC edge of C323 forms bifurcated hydrogen bonds with the amino group of C38. This interaction protects the WC edge of C323 from DMS modification, while leaving the WC edge of C38 exposed and accessible to the probe.
Loop 6 is also susceptible to RNase T1 cleavage between G36 and A37 (Gast et al., 1996). RNase T1 cleaves on the 3′-side of guanines that are not engaged in WC base pairing. This is also consistent with the proposed base-pairing model of PSTVd loop 6, as G36 forms a non-WC tHS base pair with A325.
We also compared the chemical modification pattern of the PSTVd loop 6 with that observed in the h41 motif of 16S E. coli rRNA (Moazed et al., 1986) in the region corresponding to the proposed structural motif (nucleotides 1273:1275 and 1260:1262; Table 1). The pattern of reactivity in the h41 motif of 16S rRNA is essentially identical to that of PSTVd loop 6 (Figure 4B). All bases, except C1273, are accessible to chemical probes targeting atoms on the WC edges of the nucleotides. C1273 is the only base making an interaction that protects its N3 atom in this structure. These data support our PSTVd loop 6 model, especially for the bifurcated CC base pair. Unlike the h41 motif, PSTVd was not probed with kethoxal. Our model predicts that both G36 and G324 of PSTVd loop 6 should be accessible to kethoxal.
Pospiviroid and 23S rRNA Sequence Alignment Supports the PSTVd Loop 6 Structural Model
To further test the proposed PSTVd loop 6 structural model, we analyzed the sequence variability of this loop in all natural PSTVd variants as well as in different pospiviroid species. The question was whether the proposed model could accommodate the observed sequence variations. First, in the 138 unique sequences of PSTVd natural variants (among 156 isolates) registered in the Subviral RNA Database (Rocheleau and Pelchat, 2006) (http://subviral.med.uottawa.ca), PSTVd loop 6 sequence 5′-GAC-3′…5′-CGA-3′ is conserved in the equivalent genomic positions of all the variants. The alignment of PSTVd sequences was conducted by ClustalW program available at the Subviral RNA Database website. The result of this search is provided in Supplemental Figure 3 online and also can be accessed at the following website: http://subviral.med.uottawa.ca/cgi-bin/clustalw-results.cgi?jobID=clustalw-1283877994. Second, we downloaded sequences of eight species of the pospiviroid group from the NCBI Refseq collection (Pruitt et al., 2007) and aligned them using MAFFT (Katoh and Toh, 2008). Sequence variants identified in the pospiviroid alignment are summarized in Table 2. The details of sequence alignments are shown in Supplemental Figure 4 online. We excluded Pepper chat fruit viroid (Verhoeven et al., 2009) from the analysis because its sequence was poorly aligned with other pospiviroid sequences in the region of loop 6 due to low nucleotide sequence identity.
We analyzed the alignment using the Jalview program (Waterhouse et al., 2009). By looking at the columns corresponding to the location of PSTVd loop 6, we observed that the only difference in the pospiviroid sequences of loop 6 was a change from G to C in Chrysanthemum stunt viroid, at a position corresponding to G324 in PSTVd (Table 1). This substitution results in a change of an AG tHS pair to an isosteric AC tHS pair (Stombaugh et al., 2009), suggesting that the 3D structure of loop 6 may be conserved across all or most pospiviroid species. Intriguingly, the sequence of loop 6 equivalent from CSVd is the same as the sequence of an essential part of l-Trp binding motif (Majerfeld et al., 2010), again suggesting the possibility that PSTVd loop 6 and its equivalent in other pospiviroid species serve as protein binding sites.
We also examined rRNA alignments to gain additional insight into sequence variants capable of forming the proposed 3D motif. We used the Ribostral program (Mokdad and Leontis, 2006) to analyze nonredundant alignments of rRNA sequences (Stombaugh et al., 2009). We focused on the H89 motif from 23S rRNA because its structure is conserved in archaea and bacteria and because it binds a conserved protein. The h41 motif from 16S rRNA is not conserved, even within bacteria, and the sequence alignments show high variability, indicating variation in its 3D structure.
Table 3 summarizes the sequence variability observed for the H89 motif. Most species have the same sequence as that observed for E. coli, 5′-CAA-3′…5′-GAC-3′, which differs from PSTVd loop 6 at one position, as discussed above. However, five species have exactly the same sequence as PSTVd loop 6, providing additional support for the loop 6 structural model.
Mutational Analysis Provided Genetic Evidence in Support of the PSTVd Loop 6 Structural Model
To experimentally test the 3D structural model of PSTVd loop 6, we generated all possible mutations for each base pair in the PSTVdInt (wild type) template and then assayed their systemic infection capabilities. The goal was to determine whether the functional consequence of a mutation could be accounted for by the structural model. For the systemic infection assay, in vitro transcripts were prepared for each mutant and used to inoculate the first two true leaves of N. benthamiana seedlings as described in Methods (Figure 5A). The systemic infection ability of each mutant was assayed by RNA gel blot analysis of RNA extracted from upper, noninoculated leaves (10th and 11th true leaves, collectively termed systemic leaves in this study) at 28 d after inoculation (DAI). The presence of (+)-circular PSTVd in the upper leaves, followed by sequencing to verify maintenance of the mutant sequence, demonstrates the ability of a mutant to mount a systemic infection. By contrast, absence of a mutant viroid in systemic leaves indicates a failed systemic infection. The experiments were repeated with at least eight biological replicates (i.e., individually inoculated plants) for each mutant. Inoculation with wild-type PSTVd and water served as positive and negative controls, respectively.
A total of 45 mutants were analyzed. The systemic infection system and representative RNA gel blots are shown in Figure 5A. The results from multiple experiments are summarized in the three 4 × 4 matrices shown in Figure 5B, one for all mutants derived from each proposed base pair of the loop 6 3D model. The sequencing results of mutant progenies extracted from systemic leaves are presented in Supplemental Table 1 online. We categorized a mutant as viable only when the mutation(s) was maintained in systemic leaves. Additional representative RNA gel blots showing the systemic infection capacities of different mutants are presented in Supplemental Figure 5 online.
The first thing to note is that no viable variants (i.e., those capable of systemic infection) were observed for base combinations that cannot form a base pair of the indicated geometric type (no BP in the matrices in Figure 5). The second thing to note is that in each matrix, all viable sequence variants are able to form base pairs belonging to the same isosteric group as those predicted to occur in the wild-type PSTVd loop 6 sequence.
For the G36/A325 tSH base pair, only those mutants in which G36 was changed while retaining A at position 325 were capable of systemic infection. In all of these variants, the bases at positions 36 and 325 can form tSH base pairs, isosteric to the wild-type tSH GA base pair (Stombaugh et al., 2009). While tSH pairs in which C replaces A325 are also isosteric to GA, they are not viable. Evidently, A is required at position 325, perhaps for sequence-specific protein binding in the minor groove. Thus, besides the 3D structure of a motif, its primary nucleotide sequence can also be important for function.
Sequence variation is even more restricted for the A37/G324 tHS base pair, which only tolerates AA and AG. We observed reversion of three mutants (A37G, A37U, and G324U) to the wild type in infected plants in all the progeny clones that were sequenced (see Supplemental Table 1 online). This suggested that these mutants were incapable of systemic infection. In terms of the proposed model, we note that the tHS AG base pair is calculated to be the most energetically favorable pair of this type (Mládek et al., 2009). Interestingly, although the AA combination did not appear in the pospiviroid alignments, it was common among the bacterial 23S rRNA sequence alignments at the corresponding location. Here, it should be noted that G324U was reported as a viable sequence variant, and its reversion to the wild type also was observed in a previous study (Zhong et al., 2008).
The third pair, C38/C323, is required for PSTVd to traffic throughout the plant. All pospiviroid sequences analyzed also have two Cs at these positions, which, taken together with the fact that in the 23S rRNA the H89 motif interacts with a conserved protein, suggests that PSTVd loop 6 serves as a binding site for a protein responsible for trafficking.
All Systemic Infection-Defective Mutants of Loop 6 Fail to Exit the Inoculated Leaves
The experiments described above showed that most mutants of loop 6 were incapable of systemic infection. There could be two possible explanations: either the mutants failed to exit the inoculated leaves or they failed to traffic long distances after they left the inoculated leaves. To investigate this, we conducted RT-PCR (40 cycles) with RNA samples collected from the petioles of inoculated leaves for all mutants. The RNA samples were collected at 8 DAI, the same time point when RNA was collected from inoculated leaves for in planta replication assay as described below. Use of the same time point for these two types of assays ensured consistency of data interpretation. As shown in Figure 6, the progenies of these mutants were not detected in the petioles except for three mutants, G324C, A37G, and A37U. As described above, sequencing of the progenies of these three mutants from systemic leaves revealed reversion back to the wild-type PSTVd sequences. As expected, the four loop 6 mutants that showed systemic infection while maintaining their mutated sequences (blue color in Figures 5 and 6) were detected in the petioles.
The Failure of Some Loop 6 Mutants to Establish Systemic Infection Is Not Due to Deficiency in Replication
Given that the systemic infection-defective loop 6 mutants did not exit the inoculated leaves, the next question was whether these mutants failed to replicate in the inoculated leaves. To address this question, in vitro transcripts of each loop 6 mutant were inoculated onto young N. benthamiana leaves as for the systemic infection assays described above. Wild-type PSTVd and water inoculations served as positive and negative controls, respectively. The inoculated leaves were harvested at 8 DAI and chemically fixed for whole-mount in situ hybridization using digoxigenin-labeled PSTVd-specific riboprobes. Here, whole-mount refers to the fact that pieces of a leaf, rather than thin sections, were used for in situ hybridization. We established that wild-type PSTVd could be detected in the petioles of inoculated leaves at 5 DAI; therefore, 8 DAI was a sufficiently long time interval to analyze the replication patterns. The assay was repeated with seven to eight biological replicates (i.e., individual plants inoculated) for each mutant.
Figure 7 shows typical images of PSTVd localization patterns from such analyses. The dark purple spots represent PSTVd hybridization signals in the nuclei. Because of overlap of cells and compression of the tissues incurred during the fixation/in situ hybridization processes, it was not feasible to identify clearly the types of cells containing the PSTVd signals. This issue will be addressed below by in situ hybridization on thin sections. However, our objective here was to detect signs of viroid replication/accumulation, regardless of cell type. Most mutants showed hybridization signals, suggesting that they were capable of replication. However, there were generally fewer cells exhibiting hybridization signals for the mutants than for the wild-type PSTVd. This could be due to low infection efficiency (i.e., low capacity to initiate replication in all inoculated cells), low levels of replication in some cells, or limited trafficking between cells.
The next question was whether the failed systemic infection of a mutant was due to fewer cells supporting its replication/accumulation. To address this, we determined the percentages of cells showing visible accumulation of all mutants as well as the wild type. We counted the number of PSTVd-accumulating cells per 6.25 mm2 (2.5 × 2.5 mm) area of inoculated leaves. The number of cells accumulating a mutant was calculated as a percentage of that accumulating the wild type, to represent a measure of relative replication efficiency. The data are shown in Figure 8. A key observation is that mutant G36C, which is capable of systemic infection (Figure 5), was detected in <10% of the cells relative to the wild type. We therefore postulated that a 10% infection efficiency was sufficient to enable a mutant to achieve systemic trafficking. Then, for any mutants showing 10% or higher infection efficiency in the inoculated leaves, their absence from systemic leaves could be interpreted as chiefly the consequence of failed cell-to-cell trafficking. Fifteen mutants fit this criterion, including G36C/A325C, A325C, A325G, G36U/A325G, G324C, G324U, A37C/G324A, A37C/G324C, A37C, A37C/G324U, A37G/G324A, A37G, A37U, C323U, and C38G/C323U. For mutants that exhibited <10% of the wild-type infection efficiency, it is unclear whether their failure to develop systemic infection is due to impaired intracellular trafficking such as nuclear import/export, replication, in vivo stability, or cell-to-cell trafficking.
We recognize that using 10% of infection efficiency as a threshold level sufficient for systemic infection is arbitrary. However, it is a useful and practical approach in the context of this investigation. What is important is that a relatively low infection efficiency compared with the wild type, in terms of the percentage of cells infected, could still enable a viroid mutant to achieve successful systemic infection if trafficking is not impaired.
Loop 6 Is Required for Trafficking from Palisade to Spongy Mesophyll
As described above, the whole-mount in situ hybridization images do not allow assessment of infection efficiencies of loop 6 mutants in distinct cell types and therefore do not reveal the cellular boundary at which loop 6 functions to mediate trafficking. However, the data allowed us to choose particular mutants for further in situ hybridization on thin sections of inoculated leaves to identify this cellular boundary. Mutant A325G was first chosen because its 15% of the wild-type infection efficiency represents nearly the median level among all mutants. Furthermore, we did not observe any reversion of this mutant to the wild type in our extensive infection experiments, which ensures consistency of observations in multiple samples.
The A325G-inoculated N. benthamiana leaves were chemically fixed at 8 DAI to obtain paraffin sections for in situ hybridization. Figure 9A illustrates the leaf cell types in a transverse view and shows that viroid inocula were applied to the upper epidermis. Figure 9B shows typical in situ hybridization images of transverse sections of N. benthamiana leaves inoculated with wild-type PSTVd (positive control), mutant A325G, and water (mock negative control). The wild-type PSTVd hybridization signal was detected in the nuclei in upper epidermal, palisade mesophyll, and spongy mesophyll cells. It was also detected in the phloem cells (see Supplemental Figure 6 online). Mutant A325G was localized in the nuclei of upper epidermal and palisade mesophyll cells but very rarely in spongy mesophyll cells. Furthermore, the mutant was not observed in any bundle sheath or phloem cells (see Supplemental Figure 6 online). Hybridization signal was completely absent from any cells in mock-inoculated leaves (Figure 9B). These observations suggest preliminarily that loop 6 plays a critical role in PSTVd trafficking from the palisade mesophyll to spongy mesophyll.
To further test this possibility, we analyzed the number of epidermal, palisade and spongy mesophyll cells exhibiting mutant A325G signal compared with the wild-type control. We analyzed 88 sections from 17 leaves individually inoculated with mutant A325G and showing viroid hybridization signals, and 103 sections from 14 leaves individually inoculated with wild-type PSTVd and showing viroid hybridization signals. As demonstrated in Figure 9C, the numbers of viroid-accumulating epidermal cells per section (~1 × 1 mm for each section) are not significantly different between A325G- and wild type–inoculated leaves (t test, P > 0.05). However, the numbers of A325G-infected cells are significantly lower for palisade (P < 0.05) and spongy mesophyll (P < 0.05) compared with the wild-type infected cells. These quantitative data demonstrate, importantly, that mutant A325G has similar replication/accumulation capacity as the wild-type PSTVd, as shown in epidermal cells. The lower number of palisade mesophyll cells showing A325G accumulation may then be due to impaired trafficking from the epidermis into these cells. It is possible that comprised lateral trafficking between palisade mesophyll cells also contributed to the lower number of infected cells. Most significantly, however, the rare presence of mutant A325G in spongy mesophyll cells is best explained by the requirement of loop 6 for PSTVd trafficking from the palisade to spongy mesophyll.
In addition to A325G, we analyzed A37U, which had a relatively high infection efficiency (50% of the wild-type level) as shown by whole-mount in situ hybridization. In some leaves, A37U was localized only in the upper epidermal and palisade mesophyll cells, similar to mutant A325G (see Supplemental Figure 7 online). In other leaves, it was detected in a few spongy mesophyll as well as upper epidermal and palisade mesophyll cells (see Supplemental Figure 7 online). Because mutant A37U often reverted to the wild type (see Supplemental Table 1 online), we postulate that this instability may well account for the localization of A37U in the spongy mesophyll in some cases, although direct evidence was difficult to obtain from the fixed leaf samples. This reversion may also lead to an arbitrarily high infection efficiency of A37U among all mutants. Overall, exclusive localization of A37U in epidermal and palisade mesophyll cells in many cases is consistent with a decisive role of loop 6 in trafficking from palisade to spongy mesophyll as revealed by the A325G mutant.
Although our current data support a critical role of loop 6 in PSTVd trafficking from palisade to spongy mesophyll cells in young N. benthamiana leaves, we do not know whether this motif is also required for trafficking in the reverse direction (i.e., from spongy to palisade) or whether leaf development impacts loop 6 function at a specific cellular boundary. This is an important question to be addressed in future studies, given our previous finding that a bipartitie PSTVd motif, whose 3D structure remains to be determined, is required for unidirectional trafficking from mesophyll to bundle sheath in a leaf development-dependent manner in tobacco (Nicotiana tabacum) (Qi et al., 2004).
These thin-section in situ hybridization data may provide an explanation for the low infection efficiencies for many mutants as shown from whole-mount in situ hybridization. Specifically, some mutants, like A325G, may have similar infection efficiency as the wild type in the epidermis. Their impaired trafficking into mesophyll cells, especially the spongy mesophyll, would then lead to the overall lower infection efficiency when infected cells are counted from whole-mount in situ hybridization samples. In support of this interpretation, our counting of epidermal cells infected by A37U, on in situ–hybridized thin sections of leaf samples in which this mutant was absent from the spongy mesophyll, led to a number not significantly different from those for A325G- and wild type–inoculated leaves (see Supplemental Figure 8 online). For other mutants, other possibilities may also be considered. First, their low infection efficiency may suggest an additional role of loop 6 in replication. However, it is possible that any mutations in a loop, while disrupting the major biological function of the loop, leads to disturbance to some degree the overall viroid structure so as to indirectly impact other biological functions of the RNA as a whole. This would not be surprising if we consider any one motif to function as an integral part of the whole RNA, rather than an isolated entity. Second, for those mutations that caused reduction in infection efficiency to barely detectable levels (e.g., A37G/G324C, A37G/G324U, A37U/G324C, A37U/G324U, C323A/C38A, C323A/C38G, C323G/C38G, and C323U/C38U), we cannot exclude the possibility that some of these mutations produce a dominant-negative effect by interacting with some cellular factors to interfere with intracellular trafficking or replication. Finally, whether any mutations cause or enhance RNA instability in vivo is an open question. These issues may provide opportunities for new learning about RNA structures in relation to function and for developing more comprehensive research tools to advance studies on RNA structure-function relationships.
In summary, our work provides evidence that the PSTVd loop 6 plays a decisive role in mediating RNA trafficking from palisade mesophyll to spongy mesophyll in N. benthamiana leaves. Our structural and bioinformatic analyses, together with the experimental data including structure probing data of PSTVd and the functional data on the loop 6 mutants, support the proposed 3D structural model of PSTVd loop 6. While it is formally possible that other 3D models could also be consistent with these data, at present we are not aware of any such models. Whether the primary sequence and proposed 3D structure of this loop also function similarly in other host plant species remains to be investigated. Together with previous identification of PSTVd U43/C318 (loop 7) 3D motif mediating trafficking from the bundle-sheath to phloem in N. benthamiana (Zhong et al., 2007), our findings provide compelling evidence to support the hypothesis that distinct 3D RNA motifs mediate trafficking across specific cellular boundaries. Further studies will determine whether one or more motifs are involved in trafficking across a particular boundary. This hypothesis and our experimental approaches may be useful for identifying the 3D motifs in other RNAs that mediate trafficking between specific cells. A cis-element in the 5′-untranslated region (UTR) of potexviral RNA, which plays a role in replication (Miller et al., 1998), was found to mediate cell-to-cell transport of a fused green fluorescent protein reporter RNA (Lough et al., 2006). A green fluorescent protein reporter system also was used to show the existence of RNA trafficking signals in nucleotides 1 to 102 of the Arabidopsis FLOWERING LOCUS T mRNA (Li et al., 2009) and in the 3′-UTR of GIBBERELLIC ACID-INSENSITIVE mRNA (Huang and Yu, 2009). The UTRs of potato BEL5 mRNA are important for long-distance movement (Banerjee et al., 2006, 2009). Systemic spread of Brome mosaic virus RNAs in the absence of replication suggests that they have sequence or structural elements recognized by cellular factors (Gopinath and Kao, 2007). The identification of 3D structural motifs in these RNAs mediating trafficking between specific cells should significantly advance our mechanistic knowledge of RNA trafficking.
How might PSTVd loop 6 or other motifs mediate trafficking between specific cells? A simple working model predicts that these motifs are recognized by distinct cellular factors that are components of the cell-specific RNA trafficking machinery. There is evidence for cellular proteins involved in trafficking cellular RNAs. Xoconostle-Cázares et al. (1999) isolated CmPP16-1 from pumpkin (Cucurbita maxima) phloem exudates that binds and traffics endogenous RNAs. Yoo et al. (2004) identified pumpkin Phloem SMALL RNA BINDING PROTEIN1, which binds synthetic single-stranded siRNAs and potentiates their trafficking in N. benthamiana mesophyll as shown by microinjection. Recent studies identified a mobile 50-kD RNA binding protein (RBP50) from the pumpkin phloem exudates that binds polypyrimidine tract in mRNAs, providing a platform for additional protein interactions to form a ribonucleoprotein complex in the phloem translocation stream (Ham et al., 2009). While the putative cellular factors recognizing any particular PSTVd motifs are yet to be identified, the observations that rRNA 3D motifs similar or identical to PSTVd loop 6 and loop 7 represent protein binding sites support the proposition that these motifs are recognized by palisade- and bundle sheath-specific factors, respectively, to initiate trafficking.
cDNA Construction of PSTVd Loop 6 Mutants
Plasmid pRZ6-2 containing the cDNA of PSTVdInt (pRZ6-2-Int) was constructed by Hu et al. (1997) and was a gift from Robert Owens. All PSTVd mutants described in this article were generated by site-directed mutagenesis based on this template, as previously described (Zhong et al., 2008). All introduced mutations were verified by DNA sequencing with a 3730 DNA Analyzer (Applied Biosystems) at the Plant-Microbe Genomics Facility of Ohio State University. Plasmid pInter (−) was constructed as previously described (Qi and Ding, 2002).
Plant Growth and Infection
Nicotiana benthamiana plants were grown in a growth chamber maintained at 16-h-light/8-h-dark (28°C) cycles. The in vitro transcripts of PSTVd mutants were mechanically inoculated onto the first two true leaves of 2-week-old N. benthamiana plants (200 ng/leaf) dusted with carborundum powder. Diethylpyrocarbonate-treated water was used for mock inoculation. All inocula were applied exclusively to the upper surfaces of leaves.
In Vitro Transcription
To prepare in vitro transcripts of wild-type and mutant PSTVd for systemic infection and replication experiments, HindIII-linearized pRZ6-2-Int and all mutant derivatives were transcribed using the T7 Megascript kit according to the instructions provided by the manufacturer (Ambion). Antisense PSTVd riboprobes for in situ hybridization and RNA gel blots were prepared by in vitro transcription of SpeI-linearized pInter (−) plasmid using T7 Maxiscript kit (Ambion). [α-32P]UTP-labeled riboprobes were used for RNA gel blots, and digoxigenin (DIG)-UTP labeled riboprobes were used for in situ hybridization. All RNA transcripts were purified by the MEGAClear kit (Ambion) after DNase I digestion.
RNA Extraction and RNA Gel Blotting
Total RNA from leaves of PSTVd- or mock-inoculated N. benthamiana plants was extracted by Ribozol reagent (Amresco) by following the manufacturer's instructions. RNA gel blotting was performed essentially as described in Zhong et al. (2006).
Total RNA from petioles of PSTVd- or mock-inoculated N. benthamiana leaves was extracted using Ribozol reagent. The extracted RNA was purified by RNeasy mini (Qiagen) followed by RNA cleanup using the manufacturer's protocol. A total of 500 ng of pooled RNA was reverse transcribed using Superscript III (Invitrogen). The synthesized cDNAs were amplified with Taq polymerase using PSTVd-specific forward primer (5′-CGGAACTAAACTCGTGGTTCCT-3′) and reverse primer (5′-AGGAACCAACTGCGGTTCCA-3). As an internal control, actin mRNA was RT-PCR amplified using actin-specific forward primer (5′-CACCATGGCAGATGGAGAGGATATTCAGCC-3′) and reverse primer (5′-AGGAACCAACTGCGGTTCCA-3′). The PCR products were run on 1.2% agarose gel and visualized with ethidium bromide staining.
Sequencing of PSTVd Progenies
The PSTVd progenies were sequenced in a manner similar to that described by Qi and Ding (2002). Briefly, after the PSTVd signal detection either by RNA gel blotting or RT-PCR, the PSTVd RNA was RT-PCR amplified as described above except using Pfu Turbo DNA polymerase (Agilent Technologies) instead of Taq polymerase. Next, the Pfu-amplified product was incubated with Taq for 20 min at 72°C and ligated into pGEM-T vector (Promega). The inserted DNA was sequenced as described above.
Tissue Processing and in Situ Hybridization
The tissue fixation and processing followed the protocols previously described (Zhu et al., 2001; Qi et al., 2004) with slight modifications. Briefly, 8 DAI leaf samples were fixed in a solution of 50% ethanol/5% formaldehyde/5% acetic acid for 30 min and then dehydrated by a step-wise gradient of ethanol solutions. The samples for whole-mount in situ hybridization were washed by PBS and treated with 10 μg/mL of proteinase K for 20 min at 37°C. Then, the samples were hybridized with DIG-labeled PSTVd antisense riboprobes at 55°C overnight. The samples were washed and incubated with alkaline phosphatase–conjugated DIG antibodies (Roche) followed by visualization with NBT/BCIP (Roche) color reaction.
To obtain thin sections, fixed and dehydrated samples were infiltrated with paraffin and embedded in paraffin blocks. Sections of 14 μm in thickness were obtained with a HM 340 E rotary microtome (Microm International). PSTVd accumulation was detected by in situ hybridization followed by the color reaction as describe above.
In situ hybridized samples were examined and photographed under a Nikon Eclipse E600 light microscope. Images were captured and processed with a SPOT RT KE Slider camera and the associated software (SPOT Advanced; Diagnostic Instruments). The size bar in each picture was stamped on each image after magnification calibration using SPOT Advanced and cut and pasted to the intended location of the same picture, if needed, by Adobe Photoshop.
Quantitative Analysis of PSTVd-Accumulating Cells
PSTVd-accumulating cells were counted by visual inspection under the light microscope. For whole-mount in situ hybridization samples, the counting was conducted in a 2.5 × 2.5-mm area per leaf sample. For thin-section in situ hybridization samples, the number of PSTVd-accumulating cells was determined separately for different cell types: epidermis, palisade mesophyll, and spongy mesophyll. The number was counted per section, which was ~1 × 1 mm. For the wild type, 103 sections from 14 leaves (each from an individual plant) were used. For mutant A325G, 88 sections from 17 leaves were used. For each leaf, at least four different sections were analyzed, and the average of numbers from these sections was used to represent the number from each leaf for statistical analysis, so that comparisons were made among biological replicates, rather than among individual sections. The data were analyzed by t test using Microsoft Excel (two-tailed distribution, two-sample unequal variance). P value < 0.05 is treated as a significantly different value and is indicated in the text.
Accession Number
The PSTVdInt sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under accession number NC002030.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. RNA Nucleobase Interactions.
Supplemental Figure 2. RNA 3D Motifs Used for Modeling of PSTVd Loop 6.
Supplemental Figure 3. The Alignment of PSTVd Natural Variants Showing Conservation of Loop 6 Sequence.
Supplemental Figure 4. Pospiviroid Sequence Alignments Provide Support to the PSTVd Loop 6 Structural Model.
Supplemental Figure 5. Representative RNA Gel Blots Showing Systemic Infection of PSTVd Loop 6 Mutants.
Supplemental Figure 6. In Situ Hybridization on 14-μm Paraffin Sections of N. benthamiana Leaves Inoculated with PSTVd or Water (Mock Control).
Supplemental Figure 7. In Situ Hybridization 14-μm Paraffin Sections of N. benthamiana Leaves Inoculated with A37U Mutant.
Supplemental Figure 8. Comparison of Infection Efficiencies of the Wild Type, A325G, and A37U in Leaf Epidermis of Inoculated N. benthamiana.
Supplemental Table 1. Sequencing of PSTVd Loop 6 Mutant Progenies in Systemically Infected Plants.
We thank Jesse Stombaugh and Xuehua Zhong for their initial insights into the analysis of loop 6 structure and function, Mark Forster for helpful discussions, and Woong June Park, Ying Wang, Xiaorui Yang, Yuan Tian, Carter Mason, and Farah Khan for critical reading of this manuscript. This work was supported by grants from the U.S. National Science Foundation (IOB-0840906 to B.D. and MCB-0443508 to N.B.L.) and from the National Institutes of Health (2 R15 GM055898-05 to N.B.L.).
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Biao Ding (ding.35{at}osu.edu).
↵1 These authors contributed equally to this work.
↵[W] Online version contains Web-only data.
- Received November 15, 2010.
- Revised December 13, 2010.
- Accepted December 25, 2010.
- Published January 21, 2011.


text:
Message from Wraff in Vibrant Diversity #general
Hey, the guy in Jazz and Jesse right now has a parody name of one if my childhood games
trying to figure out if this uber-SJW on my feed is what he seems, or a parody trolling the shit out of libs
I don't know who is SJWing who any more
looks like a troll acc
Poe's Law
the summary smells of *fucking white male*
also retweets of bantz
Hard to tell the parody from the reality any more
is his daughter a lib shit or something
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thanks houston
You know what's funny?
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Did Laura Loomer ever get a new tire?
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Im worried about her car failing inspection.
I had to check if that tweet was real
It is
Fucking street shitter
@Wraff "so much for the pro family conservatives"
Someone with a big account tweet that.
He failed as a father if she thinks Mussolini was a bad guy
Might as well disown and save face
so what would be a good book to send cantwell
He'll write his own Im sure.
I hope
So is daca not ending now? I was so excited
Yeah whats the deal with daca


text:
All events for West Coast Swing Dance Classes (Adult Class)
January 2019
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What is MomsTRI? MomsTRI is a sprint triathlon designed for all women, with a focus on encouraging first-time triathletes, beginners and "non-athletes". MomsTRI is less of a race and more of an experience, an accomplishment, a chance to do something hard, instead of wishing you could do it!
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What is body marking? We'll be stamping your bib number on each of your arms and your age on your calf. This helps us identify you during the race since we can't always see your bib. Your age on your calf makes it easy to tell who is in your age group. Race results will be posted in age brackets as well as in total order of finishing. Wear your age proudly, this is a day to be proud to be a woman of any age, in any stage of life!
What should I wear? Whatever you feel comfortable doing all three events in! We recommend trying out what you're going to wear sometime before the event so you're most comfortable during the event. Triathlon shorts are recommended. Some women wear tri suits but those can be expensive.
What is a transition? A transition is the time between each event used to get ready for the next event. In the first transition, you'll be switching from swimming to cycling. This can include toweling off quickly if you want and putting on your cycling shoes (or running shoes if you don't have specific cycling shoes), and your helmet, then grab your bike. You'll walk your bike through the transition area to the bike mount area and begin your cycling event. After completing the bike course, you'll start the second transition. Dismount your bike and walk it back through the transition area to your rack. Switch shoes if necessary and take your helmet off. Then you're ready to start the run! Please remember you can only enter the transition area through the south opening and exit through the north opening. The timing mats are placed at each opening and will activate the next timed section of your event. If you need to use the porta potties between events, they are located at the north end of the transition area. Be sure to take everything with you for your next event before you exit to use the porta potties so you don't need to return to the transition area.
What is a transition area? The transition area is where you will set up your bike, towel, shoes and other equipment needed to transition from one event to the next. The transition area will be open at 5:30am on race day to begin setting up and it will close 15 minutes before the event begins at 7:00. Nobody other than participants is allowed in the transition area at any time. This is strictly enforced for the safety of our participants as well as your fans! There is fencing along the perimeter where your proud spectators can cheer you on during transitions and you can show them, "you've got this"! When you have finished your event and you are ready to take your gear home, we ask that you still do not bring non-participants into the transition area. It's a big job for us and our volunteers to keep you and your gear safe. Thanks for being understanding if our volunteers need to give you a polite reminder!
How do I set up my bike & gear in the transition area? There are lots of great articles on the internet about transition set up and even a great video on our Facebook page from our Trek Transition 101 class. Be sure to keep your gear to approximately 18 inches wide so there is room for everyone!
What are the rankings for the swim? When you check in for the race at packet pickup or in the morning, we'll ask you to rank yourself for the swim. The rankings are as follows:
Expert -under 7 minutes
Advanced 7:00-9:00 minutes
Intermediate 9:00-11:00 minutes
Beginner 11:00-17:00 minutes
Novice 17:00 +minutes
We'll give you a wristband corresponding to your ranking, making it easy to find your group when you line up at the pool.
What should I bring, what equipment do I need? When preparing for MomsTRI, think about walking through the stages of each event and what kinds of things you will need. Starting with the swim, you'll need goggles and most people like a swim cap. You'll run from the pool to the transition area to get ready for the bike ride. Some people bring a bucket or an extra towel to sit on while putting shoes on. You'll need your helmet and your bike and whatever shoes you ride in. Any bike will do, bring your beach cruiser, mountain bike, or grandpa's tricycle! After the ride, you'll transition into running shoes and you're set to go! This is a most basic list to get through a triathlon. Consider bringing other items that might make you more comfortable like sunglasses, water bottles for your bike, or anything else you think will help you. Please remember, headphones are only allowed during the run.


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Immaculate modern inner city home located mere steps to the river pathway system & minutes to DT. This home offers a functional bright space w/quality finishings such as 9ft ceilings, solid core doors, & custom built-ins. Open concept main is warm & inviting w/maple stained hardwood t/o. Front den greets you upon entry & flows to a dream kitchen boasting maple cabinets, granite counters, ss appliances, & impressive island. Adjacent to the gourmet kitchen is the dining area w/a custom built-in wine cabinet, & an elegant living space w/gas FP, storage cabinets, & access to the back East facing back deck. A 4ft wide staircase leads you up to a surprising flex room that overlooks the front foyer, along w/4pc bath, laundry, & 2 bedrooms, one of which showcases a private balcony. The master oasis will amaze w/large picturesque window, his/her walk-in closets, & ensuite feat a glass shower & soaker tub. Fully devel'd lower welcomes your family w/an expansive rec room, 4pc bath, & 3rd bedroom. See add'l notes... East facing backyard is low maintenance & great for entertaining. Poured aggregate walkway in both front & back, plus in ground irrigation system. This spectacular infill is at an exceptional price & in close proximity to shopping, the new University District, & the Foothills/Children's Hospital.
Bookcases, High Ceilings, Vaulted Ceiling(s)
Back Lane, Near Shopping Center, Landscaped, Level, Near Public Transit, Rectangular Lot
Stucco, Wood Frame, Wood Siding
Disclaimer: Information herein deemed reliable but not guaranteed by CREB®.
Listing information last updated on August 12th, 2020 at 5:00am CDT.


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It is estimated that 5% of cancer is genetic and 95% is a result of lifestyle and environmental factors. Over 6 million (and growing) dogs die from cancer each year! More than 1 in 2 dogs and 1 in 3 cats are diagnosed with cancer.
A new turmeric study reveals that one of the world's most extensively researched herbs has the ability to selectively target and destroy cancer stem cells,


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NEVADA CITY (CBS13) — Sound is being used to put out fires.
It's from a simple device created by college students for a class project. Now it's getting the attention of the Nevada City mayor, who wants to use her entire town as a test location.
Co-founders Seth Robertson and Viet Tran developed the technology as students at George Mason University.
"We did like a legal fire in a lab, we were so surprised that we miniaturized it and just put it out," Tran said.
They stumbled across the idea after reading the theory to put out fires, even wildfires, with sound.
"We're really here help people, and really here to prevent the catastrophes that come from fires," Robertson said.
The set up is easy. It's basically a speaker with a funnel for the sound to come out. So the secret ingredient to put out the fire is all about that bass. For it to work, the bass must be between 30 to 60 hertz.
Their goal is to place these speakers in wooded areas or connect them to drones.
Nevada City Mayor Reinette Senum invited the creators to town to test the device.
"This town is a town of activists and we really galvanized after Paradise," Senum said.
The mayor wants to use her entire town as a test location as the device needs more research.
"Having possibly land available for doing prescription burns, so they can actually do some trials on the devices," Senum said.
CBS13 spoke with local fire department who said the devices require more research.
When I heard of the research by the two George Mason students, I thought it should be studied as a way of putting out forest fires before they are out of control. Thanks to the Nevada City mayor (and of course to the students) it may be developed. Wishing them the best of luck.


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Ariel Herrmann
Aperçu des activitésLatest activity by Ariel Herrmann
Ariel Herrmann a ajouté un commentaire,
Similar error here -- attempting to upload an activity after it has been deleted online gives the "activity already exists" error. The deleted activity does not show up in my training log or calen...
Ariel Herrmann a ajouté un commentaire,
In addition to ensuring basic search functionality works properly... Filtering / search should be possible by any header or summary field. (Equipment, device, activity distance or time, ...) Lacki...


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Tony Orvis - Tony Orvis
My current work is based based on an amalgam of previous themes with aspects of the sea, coast, weathered rocks, distressed wood and metal being used as starting points for my pots.
Simple forms with a muted, limited palette and random surface textures are at the heart of my work. Although each of the pots are unique they share a number of common or similar elements that hopefully binds them together as a body of work.


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Biggest winners and losers from college football week 2
The 2017 college football season is off and running and has already provided many memorable moments. Let's take a look at my biggest winners and losers from week 2:
- Oklahoma. The Sooners went into Columbus and boat-raced the defending Big 10 champs a year after the Buckeyes did the same to OU in Norman. Quarterback Baker Mayfield inserted himself into early Heisman conversations after throwing for 386 yards and 3 touchdowns. Mayfield made a lasting impact on Ohio State fans as he spiked the OU flag on the 50-yard line following the impressive victory.
- Anyone that saw this play. When it was all said and done…Louisiana Tech would be faced with a 3rd down and…93.
3rd & 93 upcoming…#HailState pic.twitter.com/ysNOd1QpmM
— MSU Football ? (@HailStateFB) September 10, 2017
- Georgia kicker Rodrigo Blankenship. He kicked the go-ahead field goal that allowed Georgia to knock off Notre Dame in South Bend, then announced to the team afterwards that he was given a scholarship. Seriously, college football always bringing it with all the feels!
How do you make a great night even better? Here's how.
Congratulations, Rod.#GoDawgs pic.twitter.com/SnUgkngNJj
— Georgia Football (@FootballUGA) September 10, 2017
- Clemson. The defending champions but on a defensive clinic in a massive home victory over Auburn. The Tigers may not be as explosive without DeSean Watson at the helm but the ACC had better be on notice that no one is unseating them just yet. Speaking of the ACC, Lamar Jackson is still the best player in America.
- Sexual assault university. Sometimes things just have a way of taking care of themselves. The Baylor Bears are currently 0-2 following losses to start the season against Liberty University and UTSA. It's good to know that sometimes, just sometimes, the world finds a way to right itself.
- Arkansas. Bret Bielema's Razorbacks got thumped at home 28-7 by TCU; a team they defeated in overtime in Fort Worth last September. That wouldn't be so bad really, but factoring in the teams' disastrous finish to the 2016 season and things could be getting uncomfortable really quick down in Fayetteville.
- Missouri. The stage was set. "SEC Nation" in town, solid home crowd and a night game. The Tigers even opened with a 10-0 lead before things completely fell apart, leading to a 31-13 South Carolina win. If you are a Missouri fan right now you are wondering how things got so bad so quickly and it's obvious coach Barry Odom has a massive hill to climb from here.
- Kansas. The Jayhawks went from 0 wins in 2015 to 2 in 2016 which led to the school excitedly giving coach David Beaty an extension. With a bit of recruiting momentum occurring this past summer, the Jayhawks are likely in the process of seeing those players run for the hills following a 45-27 thumping at home to Central Michigan. The good news is Kanas will get to play both Baylor and Texas this year again.
- People that fell asleep before Boise State/Washington State was over. These two teams provided viewers with a 3 overtime thriller that saw Wazzu come roaring back from a 21-point deficit in the fourth quarter to force the overtime. Good win for Mike Leach who continues to have success in Pullman.


text: August 15, 2019 - 10:29 BST Sharnaz Shahid In honour of Princess Anne's 69th birthday, HELLO! has decided to take an in-depth look into her close father-and-daughter bond with Prince Philip... Princess Anne is the only daughter of the Queen and Prince Philip, so it comes as no surprise that the royal has a special place in her parents' hearts. In honour of her 69th birthday on Thursday, HELLO! has decided to take a closer look at her bond with her father. The Princess Royal was born in 1950, a year after the Queen and the Duke of Edinburgh welcomed Prince Charles into the world. The royal couple went on to have their third and fourth children - Princes Andrew and Edward - almost a decade later. Princess Anne is believed to be Prince Philip's favourite child Close friends and family have previously been open about Philip's relationship with his children - with many claiming that Anne is his favourite child. The Princess has inherited many qualities from her father, showing her love for sports and dedicating her life as a working royal. Eileen Parker, wife of Philip's close friend Mike Parker, previously revealed via Daily Mail: "[Philip] always had more fun with Anne. Charles is more like the Queen, while Anne is very like Prince Philip." WATCH: Princess Anne's sweetest moments with her grandchildren On the day Princess Anne was born - 15 August 1950 - Philip was promoted to the rank of lieutenant-commander in the Royal Navy. After the birth, he allegedly told everyone: "It's the sweetest girl." When she was growing up in the royal household, Anne confessed that she didn't mind that her parents weren't around much. "They were supportive and never really quibbled about what you wanted to do," she said, according to royal biographer Ingrid Seward. "There were occasional comments like - 'Was that really a good idea?' I don't think that any of us for a second thought that she [the Queen] didn't care for us in the same way as any other mother did." Prince Philip and his daughter share a love of sports Having entered the public life at age 18, Anne has dedicated the majority of her time to serving her country. She is often hailed as one of the most hardest-working royals and also topped the list last year carrying out an astonishing 540 official duties both at home and overseas. In 2017, Prince Philip announced his retirement at the age of 96 after 65 years of service. When Anne was asked about whether she would ever slow down on her commitments, she told the BBC in 2010: "Look around at the members of my family who are considerably older than me and tell me whether they have set an example which suggests I might - unlikely." Out of all the Queen's children, Anne has been the most successful in leading an independent identity, both as a professional horsewoman and in her charity work. And in order to lead a relatively normal life, Anne turned down royal titles for her own children, Peter Phillips and Zara Tindall, knowing that her position in the line of succession would be moved down once her brothers had children. Make sure you never miss a ROYAL story! Sign up to our newsletter to get all of our celebrity, royal and lifestyle news delivered directly to your inbox.


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Today, M. Swann, Gilberte's father, has suddenly left the queue of the second water intake and has gone "for a walk", which has caused a commotion among the disciplined clients of the spa, who have interpreted this act of free will as a bad omen: a signal that an insurrection is looming or, God forbid, a war. After the second climb of the day, I went to see Gilberte who was queuing in front of the Sprudel geyser and she has pointed out to me that seven or eight individuals follow me everywhere. I wanted to play a joke on her and replied that she was being followed by eighty-six individuals, referring to those who followed her in the queue. But she didn't find it all that funny and she has sent me packing. Willing to check if Gilberte's statement was true, I turned suddenly. Indeed, a lot of people queued behind me. I don't understand what their purpose may be. I am not a source of healing like hot mineral waters, as far as I know. Maybe I have become, without intending to, a style guide in terms of men's fashion. (The truth is that the Tyrolean hat included in the climbing equipment, looks real nice on me.) While I was going to climb the rock for the third time today, I heard a thud. In particular, the typical noise that a grand piano makes when falling from a height of three floors on someone with a mustache. And when I returned to the hotel in the evening, I learned with dismay that my hearing impression was correct: My clandestine roommate, General Von Krieghammer, had suffered an accident. When he was leaving the Grandhotel after steaming in a vapor room, a grand piano has fallen from the third floor leaving him flat like a mustachioed sheet of paper.
This is a non-profit blog whose purpose is to raise funds for children in need. So if you want to make a donation in exchange for this story, click on this link to UNICEF. I really appreciate it!


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As of March 20, 2020 and by Decree 297/20, the entire country entered a period of social,
preventive and obligatory isolation.
What does this mean?
That you cannot leave your home except to provide yourself with essential items, such as food,
cleaning supplies and medicines.
If you do not comply with these rules, you are breaking the law.
If you have traveled to a country where the virus is spreading or have been in contact with
someone who tested positive, you must stay in isolation for 14 days.
If you have any of the symptoms, call free of charge at 107.
For more information, send an email to firstname.lastname@example.com
Enjoy Buenos Aires' green spaces
Whether it's to walk, relax, do sport or meet people and drink "mate", enjoy the city in the open air.


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PETROPAVLOVSK-KAMCHATSKY - Only a UK-Russian joint probe can reveal the truth about the Salisbury poisoning incident, however London continues to reject Moscow's offers to cooperate and keeps destroying evidence, Russian Foreign Intelligence Service Director Sergey Naryshkin said on Sunday.
"If a joint professional investigation is launched, only it can probably show what really happened," Naryshkin told the Rossiya 1 broadcaster, when asked to suggest when the world would know the truth about the Skripal case.
He stressed that the Russian authorities saw the UK probe into the case as "a farce, provocation."
"The Russian side, if you remember, immediately offered its assistance and expressed readiness to participate in the investigation of this incident, this provocation. Unfortunately, the UK side is not meeting us halfway. Moreover, the UK side, in our view, keeps destroying evidence, such as [the Skripals'] pets and other items. Nobody has seen the Skripals [since then]. The Porton Down lab is located nearby," he added.


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Added by 5 of our members
White Thorn(2005)
A novel by Bryce Courtenay
From Bryce Courtenay comes a new novel about Africa. The time is 1939. White South Africa is a deeply divided nation with many of the Afrikaner people frantically opposed to the English. The world is also on the brink of war and South Africa elects to fight for the Allied cause against Germany. Six year-old, Tom Fitzsaxby finds himself in The Boys Farm, an orphanage in a remote town in the high mountains, where the Afrikaners side fiercely with Hitler's Germany. Tom's English name proves sufficient for him to be ostracised, marking him as an outsider. And so begin some of life's tougher lessons for the small lonely boy. Like the whitethorn, one of Africa's most enduring plants, Tom learns how to survive in the harsh climate of racial hatred. Then a terrible event sends him on a journey to ensure that justice is done. On the way, his most unexpected discovery is love.
Genre: Historical
Genre: Historical
Similar books by other authors
Used availability for Bryce Courtenay's White Thorn
Hardback Editions
November 2006 : UK Hardback
January 2006 : Canada Hardback
January 2005 : USA Hardback
Paperback Editions
January 2007 : Canada Mass Market Paperback
January 2007 : UK Paperback
May 2006 : USA Paperback
Audio Editions
December 2015 : Australia MP3 CD
May 2012 : Australia Audio CD
May 2012 : Australia Audio CD
May 2012 : Australia MP3 CD
May 2012 : Australia MP3 CD
October 2008 : Australia Audio CD
December 2006 : Australia Audio Cassette
December 2006 : Australia Audio CD
December 2005 : USA Audio edition
Title: Whitethorn
Author(s): Bryce Courtenay
Publisher: Bolinda Publishing Pty Ltd
Availability: Amazon
January 2005 : Australia Audio CD
Title: Whitethorn (MP3)
Author(s): Bryce Courtenay
ISBN: 1-74094-971-4 / 978-1-74094-971-2 (Australia edition)
Publisher: Bolinda Publishing Pty Ltd
Availability: Amazon
Kindle Editions
March 2011 : Canada Kindle edition
Title: Whitethorn
Author(s): Bryce Courtenay
Availability: Amazon CA
August 2007 : UK Kindle edition
Title: Whitethorn
Author(s): Bryce Courtenay
Publisher: Penguin
Availability: Amazon UK


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Sentencing for nail gun accused postponed
The wait for a final sentence will continue for Shawn Wade Hynes after a continued sentencing hearing this morning. Judge Del Atwood withheld his decision in anticipation of the publishing of the R v. Basso case in Halifax. After hearing sentence recommendations from the Crown and defence as well as community impact statements from African […]Continue Reading


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Public notice
'Public notice' comments on the recently introduced playground by-law brought in by Cork and Kerry County Councils which states that only family members may use image recording devices. In controlling who has the right to use image recording devices in playgrounds this new bye-law sets a precedent in relation to the right to privacy in a public place. The artist encourages the viewer to question this right to privacy and the authorization afforded to some camera operators (i.e family members) and not others. In 'Public Notice' Aileen Drohan uses YouTube clips filmed in irish playgrounds and uploaded by the general public.
'Public Notice', 2012, screen projection still
'Public Notice', 2012, sign


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more correct
Synonyms for more correct
- appropriate
- equitable
- factual
- legitimate
- perfect
- precise
- proper
- strict
- true
- okay
- right
- stone
- according to Hoyle
- actual
- amen
- cooking with gas
- dead on
- faithful
- faultless
- flawless
- for sure
- free of error
- impeccable
- just
- nice
- on target
- on the ball
- on the beam
- on the button
- on the money
- on the nose
- on track
- regular
- right as rain
- right on
- right stuff
- righteous
- rigorous
- undistorted
- unmistaken
- veracious
- veridical
SWAP more correct IN A SENTENCE
Join our early testers! See how your sentence looks with different synonyms.
Antonyms for more correct
- biased
- false
- imprecise
- improper
- inaccurate
- incorrect
- invalid
- unsuitable
- wrong
- flawed
- inappropriate
- inexact
- unfitting
Perhaps I was wrong in saying you saw me; it would be more correct to say that I saw you.
It would be more correct to say "Look for the sexual motive!"
The conception of Plato, in the days before logic, seems to be more correct than this.
It would be more correct to say that Gustavus deserted England.
Perhaps it would be more correct to say "opened the door and was down."
And it is their judgments which seem to be the more correct.
"Just reverse the proposition, and it would be more correct," said Marian.
So it is more correct to call the jaguar the American leopard.
Again, 'awfully' is quite wrong, 'very' would be more correct, dear.
The imagination is more correct and powerful than the pen in such cases.


text: search Search keywords City, state, zip code Find a job Please select an auto suggested location. Sort By Relevance Date within 25 miles Category Industrial & Manufacturing Industrial & Manufacturing 8 Assembly / Production 8 Warehouse 2 State New Hampshire Connecticut 1 Delaware 5 New Hampshire 7 Wisconsin 4 City Milford Bow 1 Brookfield 1 Cromwell 7 Danbury 2 Exeter 17 Guilford 8 Hinsdale 2 Hudson 7 Keene 6 Londonderry 4 Manchester 9 Meriden 3 Middleton 1 Milford 8 Newington 17 Newport 2 North Haven 1 Norwich 2 Portsmouth 2 Rochester 11 Seabrook 1 Stamford 1 Stratford 1 Stratham 7 Trumbull 5 Wallingford 1 Contract Type Contract/Temporary Contract/Temporary 8 Category: industrial-manufacturing State: connecticut City: milford City: Milford Contract Type: Contract/Temporary State: New Hampshire Reset Searching for Milford New Hampshire Industrial & Manufacturing jobs? We currently have 8 jobs available. Create a job alert Related Searches For - Tap here to refine jobs Show more Show 10 20 30 Results Per page Assembly Manufacturing New Milford, New Hampshire Contract/Temporary $ 13 - $ 13.5 / Hour Adecco is seeking Assembly Manufacturing for temp to hire workers at Alene Candles in Milford, NH. These are entry level opportunities. Training will be provided, and no previous experience is necessary. Apply now for instant consideration! ... See job description Manufacturing Assembly Milford, Connecticut Contract/Temporary $ 12.5 - $ 13 / Hour Adecco is searching for reliable candidates to work as Cable Reel Assemblers for our client located in Milford, CT. These positions are on 1st shift 7:00a-3:30p with daily and weekend OT available. Pay starts at $12.50/hr. with an increase to $13... See job description Operations Assembly Worker Milford, New Hampshire Contract/Temporary $ 13 - $ 13.5 / Hour Partnerships between Adecco and Alene Candles always give great benefits! While working as an Operations Assembly Worker for Adecco with Alene Candles, you will earn $13.00-$13.50/hr. and receive a weekly pay check. This Operations Assembly... See job description AD Emsanblador Milford, New Hampshire Contract/Temporary $ 13 - $ 14 / Hour Adecco actualmente está asistiendo a un cliente de manufactura local y está reclutando para trabajos de ensamblacion en Milford, NH. ¡Estos Trabajos de ensamblador pagan de $13 a $14/hora, más horas extras según sea necesario! Actualmente tenemos... See job description Entry Level Assembler Milford, New Hampshire Contract/Temporary $ 13 - $ 14 / Hour Adecco is assisting a local client recruiting for Assemblers jobs in Milford, NH hese assembler jobs are long term temporary opportunities. Pay Rate - $13.00 - $14.00 per hour Assembler jobs require candidates to trim candle wicks... See job description Manufacturing Production Milford, New Hampshire Contract/Temporary $ 13 - $ 13.5 / Hour You landing a new Manufacturing Production job in Milford, NH would be pretty good, BUT you landing a new Manufacturing Production job with US would be simply AWESOME! It can happen, because we're looking for long-term Manufacturing Production to... See job description Entry Level Manufacturing Assembly Operations Milford, New Hampshire Contract/Temporary $ 13 - $ 13.5 / Hour How would you like to earn $13-$13.50/hr. for Entry Level Manufacturing Assembly Operations work!? If so, you're in luck because Adecco is currently hiring for Entry Level Manufacturing Assembly Operations Jobs with Alene Candles located in... See job description Manufacturing Assembly Milford, New Hampshire Contract/Temporary $ 13 - $ 13.5 / Hour Adecco is assisting Alene Candles, recruiting for Manufacturing Assembly jobs in Milford, NH. These Manufacturing Assembly jobs are temp to hire opportunities! Responsibilities for Manufacturing Assembly jobs include but are not limited to: ... See job description Clear your search criteria to see more results or browse the job categories we service. Send Us Your Resume and we will closely review your information. If we find an opportunity that matches your skills and career goals, we'll reach out to you to get to know you even better. Want new jobs? We'll email you. Name your job alert First name Last name Email How often would you like to see new jobs? Job Alert Duration Get the career advice you need when you need it. Sign up for our Career Advisor newsletter. Send me job alerts! By signing up. you are agreeing to the Terms of Use. Apply.JobAlertsTermsLabel Apply.TermsAndConditionsCheckBoxError Exit without saving × Awesome! Your job alert is saved. You'll see new jobs in your inbox soon. Good luck with your job search!


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Like a Ripple From a Pebble.
Like a ripple from a pebble, Thrown far into a lake,
The ever growing ripple, Increases in it's wake,
And the circles keep on growing, Until they lap the shore,
The influence from the pebble Keeps growing more and more,
Liken it to Jesus, who, Loved deeply for mankind,
He suffered for each living soul, no greater love you'll find.
Let the love to you and me, Indwell our hearts until,
It overflows a rippling tide, Of love, to others fill.
An ever growing ripple, That turns into a flood,
From one who loved so deeply, And offered up his blood,
Lord, our loving Saviour, Fill our lives with love that grows,
And let it be, your love within, To others, from us flows.


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Durban steel plant closed after fatal explosion [So. Africa]
Author: Edwin Tshivhidzo, Bua News [So. Africa], Published on: 25 February 2008
The Department of Labour has closed the Assmang plant in Cato Ridge, Durban following the death of four workers in a massive explosion on Sunday...This weekend's accident took place at a time when the plant was under inquiry for exposing workers to poisonous fumes in 2007. [Department spokesperson Zolisa ] Sigabi said the inquiry...seeks to establish [whether there had been any] negligence or flouting of occupational health and safety measures...The Assmang plant is jointly owned by Assore and African Rainbow Minerals.
Related companies: African Rainbow Minerals Assmang (joint venture between Assore and African Rainbow Minerals) Assore


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Item type:Portable Charger
Type: charger, USB connection, fast charging, smart charging
Screen: no screen
Capacity: 1200 mAh
Output: 4.7 4.2 V DC
Input: 5 V DC
Battery type: lithium ion polymer battery
Material: ABS
Package Include:
1 x product, 1 x Manual, 1 x box packaging
The colors deviation might differ due to different monitor settings.
12-Month Warranty
This product hasn't received any reviews yet. Be the first to review this product!
All prices are in AUD.