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Lindsay is an Associate with Harper Grey and is a member of our Critical Injury, Professional Regulation and Health Law Groups.
firstname.lastname@example.com 604.895.2865
AssistantMonica Yu firstname.lastname@example.com
Read about Lindsay Johnston's critical injury expertise here.
Lindsay has appeared before the British Columbia Supreme Court, the Provincial Court of British Columbia and the Health Professions Review Board administrative tribunal.
In her critical injury practice, Lindsay represents clients who have been injured in motor vehicle accidents, such as those who have sustained brain injuries, spinal cord injuries and chronic pain injuries with a psychological element.
Lindsay also assists a wide range of health care professionals with respect to regulatory matters. A large part of her professional regulation practice is devoted to representing clients in billing audits, College files, and dispositions reviewed by the Health Professions Review Board.
Lindsay was born and raised in Powell River B.C. on the Sunshine Coast.
Critical Injury
- Representing clients in personal injury actions stemming from motor vehicle collisions
- Representing clients with critically severe injuries including brain injuries, spinal cord injuries and chronic pain injuries with a psychological element
- Representing clients in wrongful death actions
Professional Regulation
- Representing professionals in regulatory matters including:
- Billing audits before the Medical Services Commission of British Columbia
- Prescribing practices
- Complaints
- Registration and licensing issues
- Substance abuse/diverting
- Privacy/confidentiality matters (including Freedom of Information queries)
- Ethical breaches
- Certification under the Mental Health Act
- Informed Consent
- Sexual misconduct
- Quality of care concerns
- General advice matters
- Mandatory reporting
- Alleged incompetence, and
- A broad range of issues before the Health Professions Review Board
- University of Edinburgh, LL.B. (With Distinction), 2007
- Simon Fraser University, B.A., 2003
Bar Admission
- British Columbia, 2010
- Volunteer Adviser, Life In Law
- Contributing Author, LexisNexis® Harper Grey Administrative Law Netletter™
- Contributing Author, Harper Grey Canadian Administrative Law Blog
- Contributing Author, Harper Grey Administrative Law Observer Newsletter
- Contributing Author, Administrative Justice: A Practitioner's Guide, 2016, Published by LexisNexis® Canada
- Volunteer supervising lawyer for the Law Students' Legal Advice Program Clinic at Britannia Community Centre
- Member, Vancouver Bar Association
- Member, Trial Lawyer Association of BC
- Member, Downtown Vancouver Toastmasters Club
- Member, The Law Society of British Columbia
- Member, Canadian Bar Association, BC Branch Subsections:
- Health Law
- Administrative Law
- Civil Litigation Law
- Workplace Investigations


text: +Randy58 6 Posted April 14, 2017 I have a Garmin Montera GPSr and I use the GDAK app. It's a great app but I am having problems with their offline maps. The maps have no detail and I would like to obtain a fairly detailed map with streets and some topo lines. Any suggestions? Am I using it in correctly and missing a feature that is already built in? Quote Share this post Link to post

subset: commoncrawl


text: 10 things that SERIOUSLY annoy rugby players It doesn't matter at which level you play rugby, these 10 things will always annoy you until the day you finally hang up your boots…and probably after. 1. Keeping a Cool Head Standing there respectfully whilst the referee berates you, even though you know that you are in the right and they are completely wrong. 2. Just This When you dive over to score a try, not realising it's a bog. 3. The Whistle There's nothing more heartbreaking than when you run the length of the pitch to score a glorious try only to have to sprint back to where you started from because you didn't hear the referee's whistle. 4. Empty Bottles Trying to get a drink of water from every bottle during a break in play only to find they're all empty! via GIPHY CONTINUES ON PAGE TWO Pages: 1 2 3 Share


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PocketFullOfApps' Coverage of MacWorld 2013 [Sticky, Updated x2]
If you don't know by now, PocketFullOfApps will be at MacWorld 2013 bringing you coverage and photos of the events, tech talks, and everything else. So with that said, here is the one stop location for all of our updates that we'll be bringing you from the event. Below we have it sectioned off for each day plus a section for any pre-MacWorld updates due to the fact we'll be there a day before it starts.
Are you a fan or reader of PocketFullOfApps? How about a developer or renting out a booth at MacWorld? Awesome! Contact us and we'd love to stop by and say hi! Click on the following link to see updates and photos! Continue reading this post ->


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Including me.
I got a card from Victoria( I call her by her first name) for a free panty…(stop laughing like a school girl!!! We can talk about delicates here! We all wear them:0) These just happen to be super cute! and comfy and um FREE…lol. Which makes it 10x better. Sign up on their mailing list…go…go ahead.
The Ruched Bloomer:0)
Along with that they sent me a $10.00 off any purchase. So after dragging my husband in the most uncomfortable store…for him…lol and making him smell just about EVERY scent there was, I picked out this YUMMY scent to add to my little perfume collection. This is actually a body spray, but you know what I mean.
Fresh Strawberry and Almond Milk….the Beauty Rush Line
I like to smell like food..sweet food, for some reason. I think it all started with Jessica Simpsons line when I was in college:0) Remember that line she came out with and everything smelled like dessert!…OMGosh golly goodness. OBSESSION!
Well I picked up a bottle for you too:0) Because, well, I like you and want you to smell good to. Then you will be irresistible..to all who come in contact with you…lol
So here is the dealeeeO.
1.) Leave me a comment about YOUR favorite scent. So then I can write them down and try them all out…lol
2.) Blog about my giveaway on your BLOG and link back to this post.
3.) Be a NEW follower…its time. Leave me a comment telling me so!
4.)Hit "Like" on our facebook page to the right…and leaving me a me comment saying so!
Giveaways ENDS Saturday a 12:00pm PHX Time:0)
Everyone loves a little secret;0)
Including me.


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The RNA genome of human immunodeficiency virus type 1 (HIV-1) is converted into DNA after infection in order to integrate into the host cell DNA. However, a large number of these reverse-transcribed genomes remain unintegrated in the nucleus of infected cells. Currently, there are no data available about the intranuclear distribution pattern of unintegrated HIV-1 DNA in relation to nuclear structures as observed on the single-cell level. In the present study, we investigated the intranuclear fate of unintegrated viral DNA in cell lines expressing CD4 and coreceptors (HOS-CD4.CCR5 and U373-MAGI-CXCR4CEM) infected with HIV-1 (strain 89.6). We used a novel approach to distinguish in situ unintegrated from integrated viral DNA by performing fluorescent in situ hybridization on cells in which stress-induced chromosome condensation had been induced, a procedure that contracts chromosomes independent of the cell cycle. Cells infected for 15 h accumulated large amounts of HIV-1 DNA which was located between the condensed chromosome strands, allowing the identification of this viral DNA as unintegrated. In contrast, in HeLa/LAV, a cell line carrying integrated HIV-1 genomes, the great majority of viral DNA colocalized with the cellular DNA. We show that unintegrated HIV-1 DNA does not evenly distribute within the host cell nucleus but tends to aggregate into clusters containing many copies of the viral genomes. The formation of these DNA clusters was independent of viral DNA replication and thus appeared to result solely from multiple infections. The DNA aggregates remained in the nuclei of infected cells for at least 25 h after the infection was stopped. The emergence of transcription sites, which most likely denote sites of the integrated provirus, lagged clearly behind the accumulation of viral DNA. These transcription foci could not be linked to unintegrated DNA molecules, suggesting that this DNA type is unable to transcribe, at least at levels comparable to those of integrated DNA. Neither unintegrated HIV-1 DNA nor transcription foci nor integrated DNA was observed to associate with nuclear domain 10 (ND10), a nuclear structure known to represent the site where several DNA viruses replicate and transcribe. Also, HIV-1 does not modify ND10 at early or late times of infection. There was no specific association of HIV-1 transcripts with splicing factor SC35 domains, in contrast to what has been reported for a number of both cellular and viral genes. Surprisingly, unintegrated HIV-1 DNA was found to accumulate within or in close association with SC35 domains, demonstrating a specific distribution of the viral DNA within the host cell nucleus. Taken together, our results demonstrate that unintegrated proviral HIV-1 DNA does not randomly localize within infected cells but preferentially aggregates in the nucleus within SC35 domains.
Upon infection, the RNA genome of human immunodeficiency virus type 1 (HIV-1) is reverse transcribed into a linear, double-stranded DNA molecule which is destined to integrate into the cellular genome to produce new viral RNA. The proteins involved in reverse transcription are assembled in the cytoplasm together with the viral genome into the preintegration complex, which is actively transported into the nucleus through the nuclear pores, a process unique to lentiviruses which allows them to access the host cell DNA independent of the cell cycle (10). Nuclear localization signals within the matrix, Vpr, and integrase proteins have been described (for a review, see references 9 and 25), and recently a central DNA flap of the HIV-1 genome was reported to be essential for nuclear uptake (62). Once in the nucleus, the proviral DNA has to integrate into the host cell genome for viral replication. The degree to which integration is a prerequisite for viral transcription at all has been widely discussed, but it appears that productive infection, at least, requires integrated viral genomes (12, 13, 21, 22, 54, 56, 59).
However, not all of the reverse-transcribed HIV-1 DNA will actually become integrated into the chromosomal DNA. A large proportion of the viral DNA molecules that are synthesized after infection remain unintegrated, a phenomenon that seems to be common to all cytopathic animal retroviruses (for a review, see references 8 and14). Unintegrated HIV-1 DNA has gained clinical importance because its accumulation within infected cells might be associated with cytopathic effects or might be used as a marker for disease progression (28, 46, 47, 49, 50, 61). Whereas the cellular distribution of HIV-1 RNA has been investigated in several studies by in situ hybridization (6, 7, 23, 24, 41, 63), to our knowledge no data are currently available about the intranuclear localization of HIV-1 DNA, integrated as well as unintegrated, in relation to nuclear domains. Two types of domains within the cell nucleus are of particular interest when studying the distribution of viral genomes and transcripts, i.e., nuclear domain 10 (ND10) (also termed the promyelocytic leukemia protein [PML] oncogenic domain or PML body) and SC35 domains (also known as interchromatinic granule clusters).
The ND10 contain several interferon-upregulated proteins such as PML and Sp100 and have gained increasing interest as the nuclear site where DNA viruses start transcription and replication. Although these viruses apparently enter the nucleus randomly, they transcribe and replicate only in immediate association with ND10 (for a review, see reference37). Moreover, several DNA viruses have the ability to modify or disperse ND10 after starting their transcription at this site (30, 31, 38). RNA viruses also appear to interact with ND10. Hepatitis delta virus, a satellite virus of human hepatitis B virus, aggregates its antigenomic RNA preferentially at ND10 and thereby causes a segregation of ND10 proteins (3). Overexpression of PML, an essential constituent of ND10, reduces replication of vesicular stomatitis virus and influenza A virus (15), suggesting that ND10 are involved in an antiviral defense mechanism.
SC35 domains represent the second nuclear component which is often used as a landmark in intranuclear localization studies. These domains are defined by aggregations of splicing factor SC35 (26) but also contain other components of the splicing machinery (for a review, see reference 43). SC35 domains are located in the interchromosomal space; transcripts of many genes, both cellular and viral, show a specific type of association with this domain (20, 31, 40, 55). The relationship between HIV-1 transcripts and SC35 domains has been studied mainly by transfecting cells with plasmids containing the proviral genome. Unspliced HIV-1 RNA and SC35 domains appear to be more or less randomly distributed within the nucleus without being specifically associated with each other (6, 7, 63), although a certain degree of specific association of both components has been described (23). In contrast, spliced HIV-1 RNAs may show a different localization pattern by accumulating within the splicing factor domains as has been shown for the multiply spliced transcripts encoding the tat gene (6).
In this report, we studied the fate of unintegrated HIV-1 DNA within the nuclei of infected cells by in situ hybridization and compared its nuclear distribution with the localization pattern of integrated DNA and viral transcripts as well as the distribution of ND10 and SC35 domains. Our studies were performed at a single-cell level allowing the direct visualization of viral genomes instead of studying whole cell populations. Also, we freshly infected cells with infectious virus particles rather than using transfections, as in earlier studies addressing the intranuclear distribution of HIV-1 RNA. To distinguish in situ unintegrated from integrated HIV-1 DNA, we employed stress-induced chromosome condensation (SICC) (51), which is a combination of heat shock and osmotic stress that leads to the contraction of interphase chromosomes within 15 to 20 min and thus reveals the interchromosomal space which is otherwise not visible during interphase. We show that unintegrated DNA accumulates between the chromosomes, a distribution pattern that is in sharp contrast to the specific chromosomal localization of integrated DNA. We demonstrate that unintegrated DNA aggregates into clusters and accumulates in the nucleus before viral transcripts are detectable. In contrast to several other viruses, neither HIV-1 RNA nor DNA affiliates with ND10. However, unintegrated HIV-1 DNA genomes, but not integrated genomes or transcription sites, specifically associate with SC35 domains. The implications of these localization patterns with regard to the possible cytopathic effect of unintegrated HIV-1 DNA are discussed.
Cell culture and infection.The following cell lines were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Program: HeLa/LAV, a permanently HIV-1-infected HeLa cell line that carries integrated viral genomes (5); HOS-CD4.CCR5, a cell line that expresses CD4 and CCR5 (19); and U373-MAGI-CXCR4CEM, which expresses CD4 and CXCR4 (58). Cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO2. For the maintenance of HOS-CD4.CCR5 and U373-MAGI-CXCR4CEM cells, puromycin (Sigma) was added to the medium at a concentration of 1 μg/ml.
The dualtropic HIV-1 strain 89.6 (16) was used for the infection of HOS-CD4.CCR5 and U373-MAGI-CXCR4CEMcells. Viruses were produced in U937 cells, and the culture supernatants were collected, supplemented with MgCl2 (5.4 mM), and treated with DNase I (300 U/ml; Boehringer) for 30 min at room temperature. The virus stocks had a 50% tissue culture infective dose of 5 × 103/ml as determined by p24 staining of U373-MAGI-CXCR4CEM cells infected for 2 days with a 1:3 serial dilution of virus stock. For infection, HOS-CD4.CCR5 and U373-MAGI-CXCR4CEM cells were grown on glass coverslips at a density of 200,000 cells per well in 24-well plates, and culture medium was replaced for 15 h with HIV-1 inoculum at a multiplicity of infection of 0.005. After infection, cells were washed three times with sterile phosphate-buffered saline (PBS) and either fixed immediately or further cultivated in virus-free medium for various times. In some experiments, the infection was carried out in the presence of aphidicolin at 10 μg/ml (ICN) or zidovudine (AZT)-5′-triphosphate at variable concentrations (obtained through the NIH AIDS Research and Reference Program from R. F. Schinazi).
SICC was induced as previously described (51). In brief, cells grown on coverslips were incubated at 41°C for 15 to 20 min while covered only with a thin film of medium. Cells were then immediately fixed and processed as described below.
Immunofluorescence and microscopy.ND10 were visualized with rabbit sera against Sp100 or PML (34), and SC35 domains were detected using a monoclonal antibody against splicing factor SC35 (Sigma-Aldrich) (26). Monoclonal antibody HIV.OT 34A specific for p24 was obtained from ICN.
Cells were grown on coverslips in 24-well plates and fixed in 1% paraformaldehyde (PFA) for 10 min at room temperature. The fixed cells were permeabilized by incubation in 0.2% Triton X-100 (20 min on ice) and incubated with primary (1 h) and Texas red-labeled secondary (30 min) antibodies (all solutions were in PBS). Finally, cells were stained for DNA with Hoechst 33258 (0.5 μg/ml) and mounted with Fluoromount G (Fisher Scientific).
Confocal images of cells were obtained using a Leica confocal laser scanning microscope. The two channels were recorded simultaneously if no cross talk was detected. In the case of strong fluorescein isothiocyanate (FITC) labeling, sequential images were acquired with more-restrictive filters to prevent possible breakthrough of the FITC signal into the red channel. Both acquisition modes resulted in the same images. The Leica enhancement software was used in balancing the signal strength, and images were scanned eightfold to separate signal from noise. Alternatively, cells were analyzed with a Leitz Fluovert inverted microscope equipped with a digital camera. Images were obtained with software from QED Imaging (Pittsburgh, Pa.).
FISH.Cells were fixed in 4% PFA or, when fluorescent in situ hybridization (FISH) was combined with immunocytochemistry, were first immunostained as described above and then refixed in 4% PFA to cross-link bound antibodies (39). Cells were then permeabilized in 0.2% Triton X-100 and, for detection of DNA, treated with RNase (Boehringer) at a concentration of 100 μg/ml in PBS for 30 min at 37°C. After equilibration in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), cells were dehydrated in an ethanol series (70, 80, and 100% ethanol for 3 min each at −20°C), air dried, and incubated overnight at 37°C with the hybridization mixture. Plasmid p89.6 FS− (kindly provided by R. Collman) (16), which contains the HIV-1 89.6 genome, was labeled with biotin-16-dUTP by nick translation and used as a probe. The DNase concentration was adjusted to yield probe DNA with a fragment length of 200 to 500 bp. Probe DNA was dissolved at a concentration of 10 ng/μl in 50% formamide in 2× SSC containing 10% dextran sulfate, 100 ng of salmon sperm DNA (Gibco BRL)/μl, 1 μg of yeast tRNA (Sigma)/μl, and 0.5 mg of cot1 DNA (Gibco BRL)/μl. For DNA detection, probe and cells were simultaneously heated at 94°C for 4 min to denature DNA. To detect RNA, only the probe DNA was denatured at 90°C for 5 min. After hybridization, specimens were washed at 37°C with 55% formamide in 2× SSC (twice for 15 min each time), 2× SSC (10 min), and 0.25× SSC (twice for 5 min each time). Hybridized probes were labeled with FITC-avidin (1:500 in 4× SSC plus 0.5% bovine serum albumin; Vector Laboratories), and signals were amplified with biotinylated anti-avidin (1:250; Vector Laboratories), followed by another round of FITC-avidin staining. Finally, cells were equilibrated in PBS, stained for DNA with Hoechst 33258 (0.5 μg/ml) or propidium iodide (1 μg/ml), and mounted with Fluoromount G (Fisher Scientific).
To quantify the size of in situ signals, nonconfocal images of cells were obtained with the Leitz Fluovert microscope (100× objective) under identical conditions with the QED imaging program. The number of pixels of each signal was counted at an image resolution of 72 pixels per in. with Adobe Photoshop software.
Unintegrated HIV-1 DNA accumulates within the interchromosomal space.Flat, adherent cell lines are far more amenable to the in situ hybridization procedure than round suspension cells such as lymphocytes. To visualize HIV-1 genomes by FISH, we therefore used cell lines derived from human glioblastoma cells (U373-MAGI-CXCR4CEM) and human osteosarcoma cells (HOS-CD4.CCR5) which express CD4 and the coreceptors CXCR4 and CCR5, respectively. Staining with antibodies against p24 2 to 3 days after a 15-h infection with HIV-1 strain 89.6 demonstrated high infection rates in both cell lines with up to 95% p24-positive cells (data not shown). Generally, U373-MAGI-CXCR4CEM cells yielded slightly higher infection rates than HOS-CD4.CCR5 cells. When hybridized with an HIV-1-specific probe to detect reverse-transcribed DNA, cells incubated for 15 h in HIV-1-containing culture supernatant usually displayed 10 to 50 nuclear signals of variable size (Fig. 1A). As controls, FISH was performed with uninfected cells, or infected cells were hybridized with a probe not specific for HIV (biotinylated bacteriophage λ DNA). In either case, no nuclear or cytoplasmic signals could be detected. DNase treatment prior to hybridization also eliminated any detectable signals, demonstrating that all visible spots actually represented HIV-1 DNA and not viral RNA. We further infected cells in the presence of AZT-5′-triphosphate at concentrations of 0.01, 0.1, and 1 μM to see whether the detected viral DNA molecules were synthesized de novo by reverse transcription. As expected, AZT-treated cells showed a dose-dependent reduction of detectable DNA signals. Most nuclei of cells treated with 1 μM AZT lacked viral DNA. In most of the untreated cells, viral DNA could be found only within the nucleus, whereas cells which usually displayed 3 to 10 cytoplasmic signals were observed only occasionally (data not shown).
To find out where input HIV-1 DNA is located in the nucleus, we performed SICC, which causes a reversible nonmitotic as well as nonapoptotic chromosome condensation within 20 to 30 min (51). SICC allows us to distinguish clearly between chromosomes and the interchromosomal space, an observation which is otherwise not possible during interphase. When we subjected HOS-CD4.CCR5 and U373-MAGI-CXCR4CEM cells to the SICC procedure after infection and performed FISH to visualize HIV-1 DNA, about 90% of all DNA signals were found between or at the outer periphery of the contracted chromatin strands (Fig. 1C), obviously representing DNA which had not been integrated into the cellular genome. Only 10% of the HIV-1-specific signals directly colocalized with the cellular DNA, denoting either integrated proviral genomes or unintegrated DNA trapped between chromatin loops. To show that the presence of HIV-1 DNA between the condensed interphase chromosomes was not an artifact of the SICC procedure, we performed the same experiment with HeLa/LAV cells, a cell line that carries integrated proviral DNA usually visible as one to three signals per nucleus (Fig. 1B). Since reinfection appears to be low in this cell line, most of the detectable HIV-1 DNA should be found in colocalization with chromosomes rather than within the interchromosomal space. As expected, 80% of the HIV-1-specific signals localized on the condensed chromatin, often in the form of close pairs of dots denoting the presence of sister chromatids after S phase (Fig. 1D). Visualization of total HIV-1 RNA by FISH in infected HOS-CD4.CCR5 cells usually revealed several RNA foci which most likely denote the sites of the integrated viral DNA (see Fig. 1G and I). Upon SICC, those RNA foci were found to be located in the interchromosomal space but attached to or overlapping with the cellular chromosomes, obviously showing the release of transcripts from the provirus into the interchromosomal space (Fig. 1E). Thus, SICC demonstrates that unintegrated HIV-1 DNA accumulates in the interchromosomal space during infection and allows a distinction in situ between integrated and unintegrated viral DNA. Since the great majority of all detectable HIV-1 DNA in HOS-CD4.CCR5 and U373-MAGI-CXCR4CEM cells represents unintegrated forms, we can be confident that observations made in the absence of SICC of nuclear viral DNA in these two cell lines apply mostly to unintegrated DNA. In contrast, HIV-1 DNA detected in HeLa/LAV cells can be considered to represent mostly integrated viral genomes and thus serve as a control to compare integrated with unintegrated HIV-1 DNA.
Nuclear unintegrated HIV-1 DNA aggregates into clusters.A striking feature of nuclear HIV-1 DNA in HOS-CD4.CCR5 and U373-MAGI-CXCR4CEM cells was the variable size of signals obtained by FISH, since the size of in situ signals strictly correlates with size and/or number of the target molecules. Unintegrated DNA in both cell lines could be visualized as dots with a wide range of different sizes, often in the form of irregularly shaped aggregates up to several microns in diameter (Fig. 1F). In contrast, integrated viral DNA in HeLa/LAV cells was visible as more or less uniform signals of smaller size (Fig. 1B). Quantification by counting the number of pixels for each signal confirmed that integrated DNA was represented only by small-sized signals (mostly 2 to 4 pixels with an average signal size of 2.7 pixels), whereas unintegrated DNA yielded both small and large dots without any obvious size limit (average signal size, 7.8 pixels; the largest signal observed comprised 155 pixels) (Fig. 2). Since integrated DNA corresponds to single proviral genomes, we assume that the small signals observed in HeLa/LAV cells (two to four pixels) represent the size of one individual HIV-1 DNA molecule. Therefore, about half of the signals from unintegrated viral DNA (53%) appear to represent single HIV-1 genomes, whereas 47% of the DNA dots seem to be composed of multiple, closely associated copies of unintegrated DNA. The great majority of the total amount of unintegrated DNA molecules is therefore aggregated as clusters in the nucleus. The mean size of those DNA clusters correlated to the length of the infection time, with an average of 3.0 pixels per signal after 6 h of infection and 7.8 pixels per signal after 15 h of infection in HOS-CD4.CCR5 cells, indicating that viral DNA entering the nucleus is continuously forming aggregates.
We wondered whether these DNA aggregates were formed by input viral DNA alone upon multiple infection or whether HIV DNA possesses the ability to replicate within the nucleus and thus to form clusters of DNA. Those replication domains consisting of replicating viral DNA and associated proteins are a typical feature of DNA viruses after lytic infection (e.g., see references 1, 30, 35, and 57). When we infected cells for 15 h in the presence of the DNA replication inhibitor aphidicolin (29), no difference in size and number of unintegrated DNA accumulations between aphidicolin-treated and untreated cells could be observed (data not shown). We therefore conclude that HIV-1 input DNA molecules are unable to replicate in the nucleus and thus to form replication domains but that they have the ability to associate with each other and to form clusters within the interchromosomal space.
HIV-1 DNA and transcripts are not specifically associated with ND10.To study the localization of HIV-1 genomes in relation to ND10, we performed immunohistochemistry combined with FISH with infected HOS-CD4.CCR5 and HeLa/LAV cells. HOS-CD4.CCR5 cells possess considerably larger ND10 than U373-MAGI-CXCR4CEMcells and thus allowed an easier identification of the ND10 domain by immunostaining. When we identified ND10 with antibodies against PML or Sp100 and simultaneously stained unintegrated HIV-1 DNA by FISH, cells which contained HIV-1 DNA still possessed intact ND10, whose number and appearance were not different from those in uninfected cells. ND10 remained unaffected both at early times after infection (30 min to 6 h) and after infection periods of 15 to 18 h. Unintegrated DNA was not specifically associated with ND10, and only occasionally were ND10 and viral DNA found in close association, obviously reflecting a random distribution of both components (Fig. 1H). Thus, in contrast to DNA viruses, HIV-1 DNA neither destroys nor associates with ND10. Since most of the unintegrated HIV-1 DNA probably is transcriptionally inactive or supports only low-level transcription, we wondered whether transcribing (i.e., integrated) viral genomes behaved differently and interacted with ND10. Visualization of total HIV-1 RNA by FISH of infected HOS-CD4.CCR5 cells usually revealed one to four RNA foci, most likely denoting sites of integrated HIV-1 genomes, and lower concentrations of transcripts more or less evenly distributed within the nucleus except for the nucleoli which lacked any visible signals. As in the case of HIV-1 DNA, cells positive for HIV-1 transcripts possessed intact ND10, as shown by costaining for PML or Sp100. Also, the transcription foci could not be found to be specifically associated with ND10 (Fig. 1G). We furthermore tested viral DNA in HeLa/LAV cells, i.e., integrated DNA, for its association with ND10. As expected from the absence of transcription sites from ND10 in HOS-CD4.CCR5 cells, integrated proviral DNA also did not show any specific localization pattern in relation to ND10 (data not shown). We conclude that HIV-1, in contrast to many other viruses, does not interact with ND10 irrespective of its status as DNA or RNA genome.
Unintegrated HIV-1 DNA, but not RNA, associates with SC35 domains.The interaction between unspliced HIV-1 transcripts and SC35 domains has been previously investigated, mostly by transfection of cells with plasmids containing the HIV-1 genome. Both colocalization (23) and a random distribution of both components (6, 7, 63) have been described. We reexamined the behavior of HIV-1 RNA in relation to SC35 domains in infected instead of transfected cells using a probe that comprises the whole HIV-1 genome and also tested whether HIV-1 DNA interacted with this domain. Concentrations of viral RNA denoting integrated genomes after infection of HOS-CD4.CCR5 or U373-MAGI-CXCR4CEM cells did not specifically associate with SC35 domains. Although about 50% of the RNA foci were observed in direct contact with the splicing factor domains, this finding obviously reflects a random distribution of both components, given the relatively large nuclear volume occupied by these domains (Fig. 1I). Nucleoli lacked both SC35 domains and detectable HIV-1 transcripts and therefore appeared as empty holes within the nucleus (Fig. 1I). Likewise, hybridization signals in HeLa/LAV cells representing integrated HIV genomes were also associated only in a random fashion with SC35 domains (data not shown).
In contrast, unintegrated viral DNA showed quite a different distribution pattern. Approximately 95% of the DNA aggregations were either associated with or completely enclosed in the SC35 domains (Fig.1J). This affiliation was independent of the size of the DNA signals, i.e., both small signals probably representing single copies of unintegrated DNA and large DNA clusters were found in direct contact with the splicing factor domains. Also, cells infected at lower virus concentrations displayed a lower number of signals without a change in their distribution pattern. Unintegrated HIV-1 DNA therefore appears to be targeted to SC35 domains where it accumulates and forms aggregates.
Accumulation of HIV-1 DNA precedes viral transcription.Although unintegrated HIV-1 DNA is probably not able to support productive viral replication, several studies suggest that it is able to transcribe (for a review, see reference 14). Since we observed high levels of unintegrated DNA in infected cells, we wondered how much the accumulation of unintegrated DNA correlated with the production of HIV-1 RNA. We incubated U373-MAGI-CXCR4CEM cells in HIV-1-containing culture supernatant for 15 h and then further in virus-free medium. Viral DNA could be detected in 15% of the cells 6 h after the infection was started; however, no HIV-1 transcripts were visible at this time point. After a 15-h incubation with HIV-1, virtually all nuclei contained viral DNA, whereas viral RNA was observed in only 13% of the cells (Fig. 1K and L). Forty hours after infection, the number of HIV-1 RNA-positive cells finally reached levels similar to those of cells displaying viral DNA with 95% RNA-positive and 100% DNA-positive cells (Fig. 3). These findings suggest that the production of HIV-1 transcripts is a relatively slow process compared to the accumulation of viral DNA. Even after 40 h, although 100% of the cells contained high numbers of viral DNA for at least 25 h, 5% of the cells still lacked detectable transcripts.
The fact that we found a high number of unintegrated DNA molecules in the nuclei but (in the case of cells that were positive for HIV-1 RNA) a much lower number of RNA foci per nucleus indicates that at least the majority of the unintegrated DNA molecules are unable to transcribe or to produce transcripts at levels comparable to those of integrated DNA. As already mentioned, viral RNA was usually visible both in the form of RNA foci, which most likely denote the site of the integrated provirus, and as numerous small dots distributed throughout the nucleus (see Fig.1G and I). These small signals were only observable in cells which also contained the RNA foci, suggesting that these transcripts originated from the foci. If unintegrated DNA molecules are able to produce detectable amounts of transcripts, many RNA signals should be present in nuclei which lack the larger transcription foci as well.
Although we removed the HIV-1-containing culture supernatant after 15 h and incubated the cells for an additional 25 h in virus-free medium (= 40 h after infection start), HIV-1 DNA was still present in 100% of the cells. The number of RNA-positive cells increased further from 13 to 95%, despite the absence of (at least) high virus concentrations after 15 h. This suggests that integrations occurred earlier or that part of the unintegrated DNA molecules served as a reservoir for later integrations.
Since efforts are currently being made to develop integrase inhibitors for AIDS therapy (18, 48, 52), a model system that allows the visualization of unintegrated HIV-1 DNA at the single-cell level might represent a useful tool to study the dynamics of the integration process. Here, we employed a novel method to distinguish in situ integrated from unintegrated HIV-1 DNA. Cells treated to perform SICC (51) contract their chromatin strands and thus clearly show the two major nuclear compartments, i.e., chromosomes and the interchromosomal space. In contrast to mitotic cells, SICC-treated cells are still in interphase and thus retain their nuclear envelope, allowing a distinction between nucleus and cytoplasm. FISH revealed the great majority of the nuclear proviral genomes either in the interchromosomal space in infected HOS-CD4.CCR5 and U373-MAGI-CXCR4CEM cells or colocalizing with the cellular DNA in HeLa/LAV cells. Therefore, using this approach, we showed that nearly all HIV-1 DNA visible in freshly infected cells represents the unintegrated form. Here, we demonstrated specific localization features of unintegrated HIV-1 DNA, i.e., it aggregated into clusters within the nucleus and localized within or in close association with SC35 domains but showed no affiliation with ND10.
Transcription and replication of many DNA viruses is visible only at ND10. This localization pattern is believed to represent a cellular segregation mechanism to confine or restrict these viral activities within the nucleus. This view is supported by the fact that ND10 contain several interferon-upregulated proteins and that herpesviruses as well as adenovirus type 5 destroy or modify ND10 after having started their replication at these sites. Alternatively, ND10 might represent nuclear sites required by viruses to replicate (for a review, see reference 37). These nuclear domains have therefore gained interest as a possible target to interfere with or manipulate viral replication. However, with HIV-1 we never observed unintegrated or integrated DNA or viral transcription foci affiliated with ND10, except for what appeared to be random associations. This domain, therefore, appears not to be directly involved in the replicative cycle of HIV.
In contrast, unintegrated HIV-1 DNA showed a clear association with splicing factor domains containing the marker protein SC35. Over 90% of the unintegrated DNA molecules were observed to colocalize with SC35 domains, whereas only about 50% of viral transcription foci (or integrated viral DNA) were found to associate with this domain. The association between HIV-1 transcription sites and SC35 domains appears to be the result of a random distribution of both structures, due to the relatively large nuclear volume occupied by the splicing factor domains. In contrast, the almost exclusive localization of unintegrated DNA in or at these domains obviously reflects a nonrandom distribution pattern. A similar behavior has been described for microinjected simian virus 40 DNA, which also accumulated within the SC35 domains (17). However, viral DNA is not generally targeted into SC35 domains, although viral transcripts may show a clear association with this structure (31). The specificity of the colocalization of HIV-1 DNA with SC35 domains becomes even more evident when compared with the nuclear distribution of Epstein-Barr virus episomes. As we have shown previously, the circular, unintegrated genomes of Epstein-Barr virus localize almost exclusively outside of SC35 domains in latently infected cells (4). Moreover, they do not form aggregates but remain as individual molecules in the host cell nucleus, in contrast to unintegrated HIV-1 DNA, which is prone to form clusters containing many copies of the viral genomes.
It is noteworthy in this context that morphological changes in the three-dimensional structure of SC35 domains have been described for HIV-1-infected cells (36). SC35 domains contain high concentrations of splicing factors and small nuclear RNAs (snRNAs), as well as transcription factors, although splicing itself appears not to occur within these domains. SC35 domains are therefore believed to be necessary for the regulation of splicing factor ratios and concentrations, for recycling of splicing components, and for the assembly of the transcription and processing machinery (42-44). The presence of large numbers of unintegrated HIV-1 molecules within this domain could therefore interfere with the regulation of splicing factor concentrations in the nucleus.
The large amount of unintegrated HIV-1 DNA in the nuclei of infected cells is generally believed to be the result of multiple infections (50, 53). When we infected cells in the presence of the DNA replication inhibitor aphidicolin, replication-arrested and control cells displayed equal amounts of unintegrated DNA, also indicating that infection but not viral DNA replication is the only source for HIV-1 DNA. The first appearance of reverse-transcribed DNA has been reported at 4 h (2, 32) or even at 1 to 2 h (27) after infection. Together with our results showing that after 6 h viral DNA is already present in 15% of U373-MAGI-CXCR4CEM cells, these data indicate that the generation of HIV-1 DNA represents a rapid process probably starting immediately after infection. In a lymphocyte cell line, the amount of viral DNA was found to gradually increase under single-cycle growth conditions for HIV-1 until a peak was reached at 8 to 12 h postinfection (32). Interestingly, the amount of HIV-1 DNA decreased after having reached the peak (32), whereas in our system the viral DNA appeared to be more or less stable at least until 40 h after infection. The number of unintegrated DNA molecules in infected lymphocytes has been estimated to be between 20 and more than 400 copies per cell (32, 45, 53), similar to our observations of infected HOS-CD4.CCR5 and U373-MAGI-CXCR4CEM cells. The advantage of in situ hybridization combined with SICC is the direct visualization of unintegrated HIV-1 DNA molecules, although due to the aggregation of the viral DNA, the exact number of molecules contained in clusters can only be roughly estimated.
After a 15-h infection, virtually all U373-MAGI-CXCR4CEM cells contained unintegrated DNA, but only a relatively small fraction of these cells displayed viral transcripts. Our FISH procedure allows the visualization of individual full-length HIV-1 transcripts; however, smaller RNA species may escape detection at the single-molecule level. We therefore conclude that unintegrated DNA molecules are unable to produce full-length transcripts or, alternatively, high concentrations of smaller transcripts which would be equally detectable. Since the first appearance of viral RNA from certain genes (tat,nef) could already be observed by PCR 2 to 3 h after infection of H9 cells (27), the generation of low levels of small RNA species of subgenomic size may have remained undetectable in our study. The number of transcription foci (which most likely denote the site of the integrated provirus) was considerably lower than the number of viral DNA molecules; integration, as a prerequisite for detectable transcription, therefore appeared to be a relatively rare event. Also, the generation of detectable transcripts clearly lagged behind the first appearance of unintegrated DNA, suggesting that integration or at least production of full-length viral RNA represents a relatively slow process. Using a coculture system for infection of MT4 cells, it has been estimated that at least one third of all input reverse-transcribed DNA will become integrated (2), whereas our data suggest a much lower integration rate. We further observed a considerable increase (from 15 to 95%) of cells containing viral transcripts in the absence of HIV-1-containing supernatants after an initial infection, indicating either that previously formed unintegrated DNA molecules (present in 100% of the U373-MAGI-CXCR4CEM cells) may serve as a reservoir for later integration events or that integrated DNA may not immediately start transcription, at least at high levels. Earlier reports support both possibilities. In quiescent T cells, unintegrated DNA was found to retain the ability to integrate later upon T-cell activation in vitro (11). In a lymphocyte cell line with single-cycle growth conditions for HIV-1, the level of unspliced transcripts remained low until after 24 h of infection; whereas subgenomic RNAs, which might be less easier to detect by FISH, accumulated earlier (32). Although there seems to exist a considerable amount of integration-competent DNA, it has also been shown that newly synthesized viral DNA may be highly labile at least before being imported into the nucleus (33, 60). This might account for our observation that HIV-1 DNA was only occasionally detectable within the cytoplasm despite being present in the nucleus in large amounts. It remains to be determined whether future therapeutic interventions which block the HIV-1 integrase will be affected by a stable pool of integration-competent DNA.
This study was supported by NIH grants AI 41136 and GM 57599, NSF grant MCB9728398, the G. Harold and Leila Y. Mathers Charitable Foundation, the W. W. Smith Foundation (G.G.M.), NIH grant AI 47760, and by M. Stengel-Miller and H. Miller, Jr. (L.J.M.). NIH core grant CA-10815 is acknowledged for the support of the microscopy and sequencing facility, and the Philadelphia Foundation funded the BSL-3 laboratory.
We thank R. Collman for providing plasmid p89.6 FS−.
- Received 13 February 2001.
- Accepted 9 May 2001.
- Copyright © 2001 American Society for Microbiology


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4 Week Fast Action Coaching Package
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Jenn spent 23 years teaching communication skills to college students, helping them to transition from student to employee in the healthcare field. She is a writer and the author of "What They See: How to Stand Out and Shine in Your New Job", and speaks to audiences all over North America.
She is a coach, a skilled facilitator, creator/host of a podcast called Careers by Jenn that focuses on career and self-growth topics. Jenn has a BC Provincial Instructor's Diploma, a Master's Degree in Public and Pastoral Leadership, is certified as a conflict coach, and (when not online) is serving a vibrant and growing congregation as an ordained United Church of Canada minister.
On Sundays, she can be found preaching on the shores of Burrard Inlet in beautiful British Columbia, Canada.
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Girls' Generation's Tiffany and Sooyoung Are Cute Juice Buddies
Girls' Generation's Tiffany revealed an adorable selfie video, taken with her teammate Sooyoung.
On July 25, Tiffany posted a short video, with the comment "To Osaka!" In the quartered video, Tiffany and Sooyoung are drinking fruit juice and making cute faces. Tiffany's trademark smile in which her eyes curve into a beautiful crescent shape, and Sooyoung's funny expressions are visible.
Girls' Generation left for Osaka to perform at "SMTOWN Osaka Concert." The group recently released their latest song "Party" and are currently promoting it. They are also filming for their variety show "Channel Girls' Generation."
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Jc Rv Repair Llc is located at the address 175 1/2 Birch St in Junction City, Oregon 97448. They can be contacted via phone at (541) 998-8402 for pricing, hours and directions.
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text: Under the direction of Dr. Andrzej W. Kawecki, SK&S actively participates a wide range of transactions encompassing company admission to stock market trading, on-going services for public companies, the issue of shares and debt instruments on Polish and foreign markets, mergers and takeovers of public companies, services for investment funds, as well as advice on public trade regulations and other capital market regulations. We have extensive experience in asset securitization projects. We have floated several companies on the Warsaw Stock Exchange and advised numerous public companies on issues concerning securities law. Furthermore, for many years, SK&S has provided legal advice on capital markets and securities to several renowned international investment banks.


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Gabriela Jankauskaite
Gabriela Jankauskaite is currently studying media and communications at the University of Greenwich. While still studying at school in Lithuania, Gabriela worked on TV shows, presentations and radio programmes. She is a media influencer and regularly contributes to her Instagram blog which has 64,000 followers https://www.instagram.com/gabr
She is coming from a varied international background, being multilingual (she speaks Russian, English, Polish, Lithuanian) and is a young professional musician. Gabriela finished Karoliniskiu Music School and is a winner of many music competitions and awards, one of them being a prestigious Mikolo Konstantino Ciurlionio contest. She is interested in exploring different cultures, loves fashion, photography and art.


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For me there is no better time to go hiking than when fall hits and the leaves begin to change color. As the seasons change so too do many of the best hiking routes in the US and in truth there are some which only appear once fall begins. Each year my friends and I love nothing more than wrapping up with a warm jacket, picking up the best GPS trackers that we can find and heading out into nature. I wanted to talk through my 5 favorite routes in the hope of inspiring you to get out and breathe in that glorious and fresh fall air. Without further ado then, here are my top 5.
Aspen, Colorado
We often think of Aspen as being a perfect place to go skiing but when the fall comes and the snow dissipates, Aspen presents itself as a fine location to watch the trees change color and sicker this beautiful part of Colorado. The Maroon Bells trail is my preferred favorite but get there early to avoid any crowds.
June Lake California
California may be a sunny all year round but if you are smart you can still experience the very best that fall has to offer here. The Sierra Mountains are beautiful to hike at any time the year but my personal favorite is during fall, when the scenery becomes a beautiful combination of jagged rocks with beautiful colors poking up from in between the crags.
Stowe, Vermont
The Stowe trail in Vermont is very short at just 2,8 miles yet despite its short distance it is a fair old slog. The trail is very steep as it leads up to the summit, which is where all of your hard work will pay off. At the top of the trail you will be presented with stunning fall views across what seems like the whole state, with some of the most fascinating foliage that you are likely to see.
Portland, Oregon
The Wildwood Trail in Portland is a great place to hike throughout the year but it really comes alive in the fall months. The entire trail is actually 30 miles so you may want to take a tent with you if you plan to complete the whole thing. Alternatively you could take the smaller four mile hike, which takes you through the Japanese garden and the sequoia grove, much more manageable and still just as beautiful.
Sandpoint, Idaho
The trail that I would recommend here in Idaho is the Mickinick Trail, which takes you through the Idaho Panhandle National Forest. Beginners may struggle with this one but moderate hikers and above can enjoy this 7 mile trail which climbs a huge 2,000 feet. As you wind up the steep incline you'll see the whole array of fall colors, and as you get to the top you may even get to see a smattering of snow.
Where are your favorite places to hike in the fall? Let us know in the comments below.


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Sheffield Utd have signed a ten-year catering deal with the Compass Group, the Championship club's second deal in a week following on from their agreement with brewer Molson Coors.
It is estimated the The Blades will pay Compass around £30m over the course of the contract to provide services at their Brammall Lane stadium. Compass will operate 16 retail outlets, 31 corporate boxes and three suites.
Sheffield United operations director Simon Argall said: "We were impressed by the sales processes that Compass had to offer us, as we felt that we had exhausted the local and regional markets and wanted to get more national business, which through Lime Venues they seem positioned to get."
Matthew Thompson, who is managing director of sport, leisure and hospitality for Compass Group UK & Ireland, added: "We know that fans and visitors to Bramall Lane are there for the football but food and drink is an increasingly important contributor to the day's experience. It is for this reason that we will be looking to introduce new technologies and innovations, such as fast pour, to enable visitors to be served more quickly and efficiently, and to cut down queuing time."
Compass will also provide sales, marketing, catering and hospitality for non-matchday events held at the stadium and the on-site hotel, while Brammall Lane will be added to the Lime Venue Portfolio's collection of meetings and events venues.


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Resources for Medical Professionals
Anti-depressants, Pregnancy and Lactation
The following article was published in the newsletter Journal Watch in January 2007.
Antidepressants in Pregnancy: The Ongoing Story
The optimal approach for pregnant women with severe or longstanding depression may be to continue antidepressant treatment, but risks to fetuses are emerging.
Although pregnancy was once thought to protect against psychiatric disorders, gravid and nongravid women have similar risks for major depression, at 10% to 15%. Both depression and antidepressant treatment during pregnancy have been associated with risks. This year, several studies have provided new information regarding depression and antidepressant use during pregnancy.
Two studies add to concerns about undertreating depression during pregnancy beyond the risks for preterm birth and intrauterine growth retardation. In one study, researchers naturalistically followed 201 pregnant eurhymic women with histories of major depression; many women had highly recurrent illness. Of those who discontinued antidepressants during pregnancy, 68% relapsed, versus 26% of those remaining on antidepressants; half the relapses occurred during the first trimester. Relapse risk was significantly higher in women whose illness was highly recurrent (>4 episodes) or lengthy (>5 years). In the other study, concerning a smoking cessation program for pregnant smokers, women with dysthymia or minor depression tended to be less likely than nondepressed women to quit smoking. Dysthymic women increased their smoking, a worrisome finding because smoking is associated with preterm birth and Sudden Infant Death Syndrome.
Other studies in 2006 increased the concerns about the effects of antidepressants, especially SSRIs, on the fetus. In one study, amniotic-fluid concentrations of several SRIs, venlafaxine, and their metabolites were highly variable and in some cases vastly elevated above maternal serum levels. In another report, levels of nortiptyline and clomipramine in cord blood at delivery were close to maternal levels.
One year ago, the FDA warned of an increased rate of cardiac defects following in utero exposure to paroxetine and changed paroxetine's fetal risk classification from Class C to Class D. This warning was based on two large, but unpublished and non-peer reviewed, studies showing a 1.5 to 2 times increased risk of cardiac (primarily ventricular and atrial septal) defects in newborns exposed to paroxetine in the first trimeseter. In December, the American College of Obstetrics and Gynecology advised against using paroxetine during pregnancy (Obstetrics & Gynecology 2006 Dec; 108:1601-3).
Other potential risks were also reported in 2006. A registry study of 119,547 live births showed higher rates of respiratory distress and birth weight below the 10th percentile in SSRI-exposed infants than in nonexposed infants. In another case-control study, SSRI exposure after week 20 was significantly associated with persistent pulmonary hypertension of the newborn (PPHN), a severe respiratory illness that ordinarily occurs in 1 to 2 of 100 births. The risk with SSRI exposure was significantly increased (adjusted odds ration, 6.1) although, given the low base rate, the number of SSRI-exposed babies with PPHN would remain very small (about 6-12 per 1000). Finally, in a study of 120 term infants, 30% of the 60 infants with prolonged in utero exposure to SSRIs, versus 0% of nonexposed infants, developed a neonatal abstinence syndrome, including a high-pitched cry, sleep disturbance, tremor, hypertonicity or myoclonus, tachypnea, and gastrointestinal symptoms. Symptoms peaked within 48 hours after birth and generally resolved within 4 days (Arch. Pediatr. Adolesc. Med. 2006 Feb; 160:173-6).
These findings complicate decisions about prescribing antidepressants in pregnancy. Women with longstanding, severe, or recurrent depression risk relapse during pregnancy with antidepressant discontinuation; the optimal approach may be to continue their antidepressants. However, increasing reports of fetal risks argue for caution in prescribing SSRIs during pregnancy, especially for less severely ill women. Until more data are available, it would be prudent to avoid paroxetine during pregnancy, if possible.
Alternative treatments for depression during pregnancy include psychotherapy (e.g., cognitive-behavioral or interpersonal therapy) and ECT. Therapeutic light and repetitive transcranial magnetic stimulation have occasionally been reported effective for treating depression in some pregnant women. Earlier concerns about risks for cardiac defects with bupropion have not been borne out (Pharmacoepidemiol Drug Saf, in press; AM J Obstet Gynecol 2550 Mar;192:932-6). Mirtazapine and venlafaxine have been examined in only one study each (J Clin Psychiartry 2006 Aug; 67:1280-4 and Am J Psychiatry 2001 Oct; 158:1728-30), but to date have not been associated with major malformations. Data are scant regarding duloxetine. All antidepressants have been associated with higher rates of spontaneous abortion. Overall, it remains crucial to individualize treatment and to discuss risks and benefits of antidepressants in pregnancy with depressed women how are, or may become, pregnant.
Deborah Cowley, MD


text: Brand Marketing Chevy Gets Selective With Tagline By Tanya Gazdik|March 1, 1999 Share By Tanya Gazdik|March 1, 1999 Share Bow Tie Icon to Cover in Some Ads; Jingle Retooled for 3 Models DETROIT–General Motors' Chevrolet division has used "Genuine Chevrolet" as the tagline for its cars for almost four years, but the line is notably absent from a new divisional TV spot. The 60-second spot, "American Snapshots," ends with the Chevrolet bow tie emblem and a voiceover: "Wherever America's going, we'll be there." The ad was created by Campbell-Ewald Advertising, Warren, Mich., Chevrolet's national agency. Media is placed by GM Mediaworks, also in Warren. The "Genuine Chevrolet" tag will now only be used "as we see appropriate," said Jim Jandasek, Chevrolet's advertising/promotions director for passenger cars. "It's a sizable graphic element when you put all those letters together. Sometimes it doesn't work as well as just the bow tie." Chevrolet spent $556 million on advertising through the first 11 months of 1998, according to Competitive Media Reporting. Of that, $228 million went to car advertising. The spot, which features a new rendition of the "See the USA in Your Chevrolet" jingle popularized by Dinah Shore in the 1950s, debuted last week during the Grammys. It will air through mid-May on network and cable TV and reappear in December for the millennium, Jandasek said. The commercial features historic American images interspersed with shots of Chevys through the decades. Digital editing made it possible to add Chevrolets to the famous pictures, said Bill Ludwig, Campbell-Ewald's chief creative officer. Folk/pop singer Shawn Colvin recorded the new version of the jingle, but renditions by other singers in different musical styles are planned, Jandasek said. "We can tailor this to everything from a pretty hard punky rock to whatever appropriately matches the positioning," Jandasek said. The jingle will only be used for the Impala, Malibu and Cavalier models, Jandasek said. Monte Carlo advertising breaking in October will not feature the music, he said. The work is part of an effort to put GM's biggest division back on top in sales–a position it hasn't held since the Celebrity was the top-selling vehicle in 1984. Share http://adweek.it/2kPJzsi copy Adweek Adweek


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Department of Biological, Geological, and Environmental Sciences
Academic discipline: BIO/03 Environmental and Applied Botany
Director of Second Cycle Degree of Sciences and Management of Nature
Full Professor at the University of Bologna from 2014. Botanist and ecologist with research activities in vegetation science, community ecology, biogeography, biodiversity assessment and conservation biology. Coordinator of the Plant Diversity, Ecology and Conservation Research Team.
My research mission is to understand the patterns and processes of species coexistence in relation to the spatial and temporal scales and to improve the methods adopted for biodiversity assessment. I collaborate with several European and extra-European Research Groups, within the frame of various theoretical, empirical and applied research projects.
I consider Science as a contribution to a better world, peaceful and healthy for the human beings and the other organisms living on the Earth.
Dipartimento di Scienze Biologiche, Geologiche e Ambientali
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Student reception: Tuesday from 14:00 to 16:00
Since the possible overlap with other working business I recommend booking by telephone or email
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Wednesday, September 25, 2019
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Four Points by Sheraton London
Early Bird Rate: Ends August 31st at 23:59 p.m.
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General Academic Paediatricians, Community Paediatricians, Family Medicine Physicians, Nurses, Nurse Practitioners, and Allied Health Professionals,
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Skiing is one of the most exciting and rewarding sports of all. While some people might see cold weather as a reason to head inside, skiers know better. When the air becomes chilly and the snow starts to fall, skiers have more fun than just about anyone else.
One key to satisfying skiing sometimes goes overlooked, though. Having the right equipment can make every bit as much of a difference as the snow conditions that so many focus on. An improperly fitted pair of skis can drag down the experience just as icy snow or a lack of cover might. Making good use of an online ski size calculator, though, is typically all that it takes to make sure this cannot happen.
Ski size chart styles vary quite a bit, but some basics tend to crop up again and again. For one, skis will generally need to grow longer as the person meant for them becomes taller. The same basic idea holds with regard to weight, where a heavier skier will normally want longer skis than a lighter one.
If this is all there were to the matter, then any old ski size chart would be able to give an answer quickly and without fail. In fact, though, other considerations mean that these static figures can only provide a baseline, with skier preferences and other factors also figuring in to the equation.
For skiers who prefer to make quick, snappy turns all day long, for example, a ski length calculator will typically suggest some shorter skis than might otherwise be recommended. For those who would rather cruise at higher speeds carving longer arcs in the snow, on the other hand, going with a somewhat longer ski will often make more sense.
The reason for this is simple and easy enough to understand. A longer ski will tend to offer more stability, a feature that can make it easier to stay in control at higher speeds. Likewise will increased length also lend a ski more to making turns of similarly expanded radius.
The opposite tends to hold for shorter skis, too. Skis that are short relative to what might normally be recommended for a skier of a particular height and weight will be easier to turn quickly, making them more appropriate for slalom-style carving than for high-speed downhill bombing. While it can take a bit of effort to figure out exactly what length of ski will best suit a given skier, doing so often turns out to be part of the fun.


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Sunday Smiles- Reflections on Alaska
My ActiveRain Friends in Colorado have been posting about the blue skies they have 300 days of the year. Though we have lots of sunshine in Tennessee, I'm pretty sure we can't boast of having that many days of blue skies. There are some hazy and overcast days that set in upon us.
There is one place I visited that had the most gorgeous blue skies even with some fluffy white clouds. The blue skies of Alaska are crisp and reflect off of the lakes, rivers and streams. Even the ice in the glaciers refracts the light in a beautiful way.
What a wonderful place to visit and so is Clarksville TN. If you move here to Clarksville you will need a Real Estate Agent. Call The Real Debbie Reynolds and I will help you find a home. 931-320-6730
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15gr 100% Pure Nembutal (Pentobarbital) Active Ingredient - Dignitas Grade (Lab Tested)
Quick OverviewWe have a large stock of 100% Pure Nembutal (Pentobarbital) Active Ingredient - Dignitas Grade. We do source our Nembutal (Pentobarbital) Active Ingredient directly from Lundbeck A/S and Akorn Pharmaceuticals laboratories meaning the quality is genuine and sealed are untouched, unbroken or altered. We guaranteed all our Nembutal (Pentobarbital) Active Ingredient to be 100% Pharmaceutical Grade (works perfectly well in "Peaceful, Painless and Certain" end of life), and secure more than 550 bottles of Nembutal (Pentobarbital) Active Ingredient every month from producers laboratories for our valued customers. We also do wholesale Nembutal (Pentobarbital) Active Ingredient, contact us for more details.
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30 grams: $ 580 USD
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♥ African wedding guest and evening wear ♥
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1787. One weary man and one weary teenager have been walking north.
1787. One weary man and one weary teenager have been walking north for days towards the headwaters of the Savannah River. With little to eat, they spend a long, wet night with only a small wooden cart to protect them from a torrential early summer rainstorm. On the sixth day of June, father and son arrive at Hopewell, a plantation along the Seneca River in a settlement on the outskirts of present day Clemson. The bedraggled pair are received graciously by General Andrew Pickens himself, and they present a proper letter of introduction from one Colonel Le Roy Hammond. They are, presumably, wined and dined, and rejuvenated by a comfortable night's sleep. Father and son travel on to reach the settlement of Seneca, situated beside the lovely Keowee River, before following the faintest of trails into the vale of Jocassee, a place whose name may be forever linked to that of Andre Michaux. ~K


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Each specific objective has associated a strategy to follow, a relevant step in the formulation of the PMI, since it allows specific institutional improvement.
Strategies associated with 1 objective
- Strengthening of the processes installed for the curricular redesign of Pedagogies
- Redesign of the practice component of teacher training programs
- Integration of disciplinary and pedagogical training in the educational process of Pedagogies
- Development of a quality assurance model for initial teacher training, based on diagnostic, intermediate and final evaluations regulated by a plan designed for this purpose.
Strategies associated with 2 objective
- Design of a collaborative work program with the regional educational system, to incorporate talented students of the school system into pedagogical careers, ensuring their retention.
- Optimization of the existent institutional strategies and devices of the Basic Skills Leveling Program, for the integral diagnosis and leveling of the competences of the future teachers.
- Optimization of the methodological and evaluative procedures, of development and deepening of basic competences for the improvement of the learning and retention levels of the pedagogy students, on the basis of the support of Chilean and foreign institutions that have successful experiences to the respect.
Strategies associated with 3 objective
- Implementation of a focused recruitment plan (4 doctores) according to two modalities: i) incorporation of new capacities, not available in the Institution, ii) improvement of the contractual relationship of part-time academics, pre-existing in the Institution, currently in doctoral programs.
- Creation and maintenance of a permanent teacher training and updating program, in the field of pedagogies, that responds to criteria of integration between didactic and disciplinary aspects, from which criteria and indicators are derived that allow progress in the improvement of the evaluation system of the existing academic performance, considering the requirement of the innovative curricular proposal.
- Design and implementation of institutional strategies for collaboration with higher education institutions, national and international, that have effective programs in teacher training, forming networks to share experiences and research related to continuous improvement, strengthening of the profession and articulation with the labor context.
- Incentive for applied pedagogical research, carried out jointly with actors of the school system and with teachers in training, as a relevant instance of the professional development of the academic body.
Strategies associated with 4 objective
- Review and adaptation of existing regulatory frameworks that affect the achievement of timely titling.
- Management of the progression of the students ensuring the improvement of the competences for the teaching exercise, in coherence with the teaching career policies promoted by the MINEDUC.
Focus and strengthening of the institutional system of follow-up to the graduate, so that it is permanent, periodic, diachronically comparable and efficiently responds to the specificity and requirements of the labor insertion and the professional development of the graduates of the pedagogical careers of the ULS.


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By LARRY VAUGHT
For some Kentucky football players, UK's 31-year losing streak to Florida is not that significant because they did not grow up in this state and have not been part of more than two or three losses — or maybe none.
For in-state players like Drake Jackson, Kash Daniel and David Bouvier, it's a bit different. They all grew up UK fans and have dealt with the losing streak for years.
"A lot of the guys on the team know about it but they don't know like the Kentucky guys do," Bouvier, a Lexington Catholic product, said about the streak. "We have been around.
"I have had season tickets for the games. I have been through heartache and we have been close the last several years. It's really a different feeling for us. I am always thinking about the streak."
One difference for him this year is that he will be on the field as a starting receiver and starting punt returner. He's not played against Florida before.
"It is definitely different than previous years. I don't want to over think it. I just want to get out there and do my job. Just make sure I am giving it all for my teammates so we can win," Bouvier said.
He had a touchdown catch in the Central Michigan win, but says it was far from a perfect performance from him.
"When I saw the film, there were areas I could have done a lot better on," Bouvier said. "I did well on that (touchdown) drive but there were many things I can improve on. Gunnar (Hoak) made me look very good. We have worked — and same with Terry (Wilson) — on those throws a lot and I knew where the ball was going to be. We all trust Terry and Gunnar both. They are both great guys, great friends. I know they'll play great at Florida, too."


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Hey all,
So yesterday I had my pouchoscopy to make sure everything is healed so I can get my second (takedown) surgery done. I'm 8 weeks post op from my first surgery. I did the scope without sedation and it wasn't too bad. I'm a little frustrated though. Doctor said the pouch was a "little more inflamed than she would like to see at this point". I have UC. Had it for 18 years. Pathology says UC when they removed my colon. Doc took some biopsies to check if any of this inflammation could be Crohns or if it's just diversion colitis. Did any of you experience this? I'm not having any problems. I feel good after healing up these past 8 weeks. My pouch is all healed. No leaks and whatnot. Just some inflammation. What do u guys think? I know I need to wait for pathology results but it's frustrating. If it's not Crohns then I get my surgery in 1.5 weeks!!! But if it's Crohns she said we will have to figure out meds and surgery postponed for maybe 3 additional months (Definitely frustrated about that).


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HTRK – Nostalgia LP (Reissue)
*Orders ship September 27th 2019 – White Vinyl
HTRK's seminal debut 'Nostalgia' (2007) gets a new cut to white vinyl with white sleeve and gloss cover.
An abrasive record shrouded in sonorous pulses, deafening feedback and tension-soaked reverb that immerses you deep into their world.
HTRK's lo-fi post-punk, post-industrial noise-electronica echoes Suicide, Throbbing Gristle and Joy Division.
'Nostalgia' took inspiration from the sound and imagery of David Lynch, The Birthday Party and electronic artist Pita. Rowland S Howard's 'Teenage Snuff Film' was also cited as a big influence, HTRK notably caught the post-punk legend's attention with their live gigs which saw him endorse the band and go on to co-produce their next release 'Marry Me Tonight'.
A compelling introductory statement that delves into their guilty nostalgia for the nihilistic energies rampant in the 80s underground.
"An agitated haze of addictive ambivalence instead of the swagger and violence of their influences, the overall feeling is of beautiful disharmony" All Music
"Somewhere between Throbbing Gristle and Sonic Youth. Imagine if the Cocteau Twins moved to the dark side of the force." Norman Records
You may also like…
Vanishing Twin – The Age Of Immunology£12.00 – £20.00 Select options
Infinite Music – A Tribute To La Monte Young£12.00 – £18.00 Select options


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Healthcare Consultants Pharmacy Staffing (HCC) is seeking a well-qualified Florida Pharmacy Technician Biller for the full time opportunity of Pharmacy Technician Biller with an established specialty pharmacy in the West Palm Beach, FL and North Palm Beach, Florida areas. Technicians who currently work in the Miami Florida area are encouraged to apply.
The ideal candidates will have a background in pharmacy billing; prefer two (2) or more years' experience as a Biller. Experience with PioneerRX software is a real plus. The ideal candidate will have great people skills along with accurate attention to detail.
Competitive compensation and the benefits of Paid Time Off accruing after thirty (30) days along with 401k eligibility after one year.
To start your next step in your pursuit of opportunities as a Pharmacy Technician Biller, call Healthcare Consultants Pharmacy Staffing, today at (800) 642-1652, or contact us now online.
Linton G. Creel
Senior Recruiter
Healthcare Consultants
Pharmacy Staffing
E-Mail: email@example.com
Web Site: www.pharmacy-staffing.com
Tel: 800-642-1652 or 321-397-4968


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Check out our weekly guide to TV this week, and make sure you're catching the big premieres, crucial episodes and the stuff you won't admit you watch when no one's looking. — Class is in session on … [Read more...] about What To Watch On TV This Week: 'O.J.,' 'Fashion Police', Nate Berkus on 'Oprah: Where are They Now?'
Chaz Bono
Gay Iconography: What Is It About Cher?
One attribute regularly associated with gay icons is an outsider status. Cher's hit "Half-Breed" tells one such story of a girl shunned by both her white and Native American … [Read more...] about Gay Iconography: What Is It About Cher?
Cher Talks About the 'Amazing' DOMA Ruling: VIDEO
Visit NBCNews.com for breaking news, world news, and news about the economy … [Read more...] about Cher Talks About the 'Amazing' DOMA Ruling: VIDEO
LGBT Celebrities Speak Out for Obama: VIDEO
Cher Makes Surprise Appearance at GLAAD Awards to Honor Chaz: VIDEO
Fox Psychiatrist On Newt Gingrich: Infidelity A Sign Of Awesomeness
South Carolina's big primary is today, and Newt Gingrich is the most talked about man in America. He's been up, he's been down, and then he was up and then down and now up again, and the … [Read more...] about Fox Psychiatrist On Newt Gingrich: Infidelity A Sign Of Awesomeness
Chaz Bono Proposes To Girlfriend
WATCH HERE as Chaz Bono proposes to girlriend Jennifer Elia atop Seattle's Space Needle. (Sorry I couldn't embed the vid. That function's disabled.)Chaz has a new special, Being Chaz, … [Read more...] about Chaz Bono Proposes To Girlfriend
Towleroad Guide to the Tube #1005
KATORI HALL: Is an American for Marriage Equality. CHAZ BONO: Surprises a young transgender fan. SHERRY VINE: We Found Sperm. PROTEIN: What's the grossest thing you've ever found in a salad? … [Read more...] about Towleroad Guide to the Tube #1005
Chaz Bono Exits 'DWTS', Says 'I Wanted to Show America a Different Kind of Man': VIDEO
Cher at 'Dancing with the Stars': VIDEO
Here's the Chaz performance: … [Read more...] about Cher at 'Dancing with the Stars': VIDEO


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.002 Ballet Short Disposable Needles
These Ballet F-shank sterilized disposable stainless steel epilation needles come in a 50-unit blister-pack. It is specifically designed to fit all modern epilators with F-shank probe holders. These needles are ideal for clients who respond normally to electrolysis treatments.
Specifications: sculpted of 1/8 nickel-chrome Swedish stainless steel, which is used in fine surgical instruments, disposable, pre-sterilized
*Individual results may vary. The statements above have not been evaluated by the FDA. Advice on treatments or the proper products for an individual patient should be obtained through consultation with a physician or trained health care practitioner who has examined that patient or is familiar with that patient's medical history.


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Lungu A.C., Constantinescu Iulia, Stoica Cristina
Abstract: Childhood and adolescence are critical times for the development of all organs and systems. Achievement of optimal bone mass in this period is thought to be the best predictor for bone health in the adult life1. In children, nutrition, physical activity, growth, endocrine and metabolic function is mandatory for a normal skeleton development and cardiovascular system. Disordered mineral and bone metabolism zassociated with chronic kidney disease causes important obstacles to final adult height, bone strength and cardiovascular integrity that may contribute to chronic morbidity[2, 3]. As kidney function decreases, a progressive deterioration in mineral homeostasis emerge, with an abnormal serum and tissue concentrations of phosphorus and calcium, and changes in circulating levels of hormones[4]. Therefore, treatment target of MBD includes maintaining optimal serum parameters for calcium, phosphorus and parathyroid hormone according to stage of CKD, in order to improve growth, high-turnover bone disease and prevent cardiovascular calcifications.
Keywords: chronic kidney disease, metabolic bone disorder, children
Keywords: chronic kidney disease, metabolic bone disorder, children


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Kevin McIntyre School Of Motoring provides personalised and specialised driving lessons in and around Runcorn. A lot of learners are nervous and anxious about approaching someone to teach them how to drive. We understand your concerns and address them in the best possible way! Kevin provides friendly instruction and timely advice so you can be driving around on your own in no time!
Rest assured that our intensive theory lessons and specialised driving lessons will prepare you to get your driving license.
• On time pick up and drop off
• Intensive driving lessons
• Experienced driving instructors
• Mock theory and driving tests
• Quality practice vehicle - Ford Fiesta
• Friendly learning environment
• Lessons in Runcorn, Widnes, Frodsham and Warrington
Along with a free first lesson you also get a feel for our method of instruction. This allows us to personalise the training to your specific needs. Kevin can teach you speed awareness, defensive driving, better navigation skills and also theory lessons. You can also utilise block booking to get the most out of a single day of training.
Pass your driving test with ease with Kevin McIntyre School Of Motoring


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Will Exxon clean all these tar sands oil spills like BP "cleaned up" the Gulf? Meanwhile, a solar spill is called a nice day.
June 2013, pipeline ruptured in Alberta: 250,000 gallons spilled into the Red Deer River.
27 March 2013, train derailment in Minnesota: 15,000 gallons spilled.
"Only about 1,000 gallons has been recovered," said Dan Olson, spokesman for the Minnesota Pollution Control Agency. "The remaining oil on the ground has thickened into a heavy tar-like consistency."
30 March 2013, pipleline rupture, Mayflower, Arkansas: "thousands of gallons" spilled.
Kimberly Brasington, an Exxon spokeswoman, confirmed the oil from the ruptured Pegasus pipeline originated in Canada. The oil is "Wabasca Heavy Crude from Western Canada," she said in an e-mail Sunday. Canadian group CrudeMonitor describes Wabasca as a blend of heavy oil production from the Athabasca region.
Aerial footage of the Arkansas crude seeping through woods, waterways, streets, and yards:
Video by Lee Camp, Moment of Clarity
Graphic by Jim Harris:
Paul Koring wrote for The Globe and Mail 31 March 3013, Exxon oil spill in Arkansas seeps into Keystone debate,
Canada's Energy Minister, Joe Oliver, said he didn't think the Arkansas spill would have a big impact on the Keystone debate.
"Americans understand that incidents are not caused by the origin of oil transported through a pipe," he said, adding the Canadian government "strongly supports the Keystone XL pipeline because it would advance our mutual interest in attaining North American energy security and create jobs in both countries."
Well, he's right the origin of the oil isn't the basic problem. The problem is using more fossil fuels when we have right now a much cleaner and more distributed path to energy independence for Canada, the U.S., and the world: wind and solar power. If New Jersey and Toronto can do it (more later), any place in the world can do it. And the U.S. installed 76% more new solar power in 2012 than in 2011, continuing solar deployment like compound interest. Nothing else shows that kind of growth rate: not oil, not gas, not nuclear. Only wind and solar power grow like money in the bank and power on your roof.
Doubling down on disaster is not the answer. Sun, wind, and water are the answer to power the world.
Short Link:


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Meet the League
The Admissions Committee will host a "Meet the League" event at Portico. Potential Provisional members are invited to mix and mingle to learn about JLM membership and the application process. Appetizers will be provided.
For more information, please contact Jessica, prior to November 14, 2018, at (318)267-5521 or email@example.com.


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READ.GOV is website from the Center for the Book at the Library of Congress.
The web resource links to key Library of Congress resources connected with reading. In addition to materials related to books and authors, users will also find many ways to participate with Library of Congress activities such as contests, events, and awards.
Students will particularly enjoy learning about the National Ambassador for Young People's Literature and watching author webcasts.
Librarians will find this website to be a great way to weave literature, author, and reading activities into the classroom.
To learn more, go to http://www.read.gov/.


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Do you daydream and wish you were a pastel Princess or Prince living high above the clouds in your very own dripping candy castle? This is the piece of fancy flair for you!
Pin this fantasy grape castle with pink icing and fun tiny details on your backpack, purse, jacket, hat~ anyplace that needs a little touch of cute flair!
- Original Miss Kika design
- Approx 40mm
- 7 color metal enamel pin in gold
- Smooth crisp finish with sweet candy colors


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Sign Up for promotional emails and never miss a sale! New subscribers get 20% off their next order!
Paws a Walking
Hexagonal Gold
Stock Sample See this product with our default options. This is a great way to touch and feel this product's paper and print quality. One Click and your Stock Sample will be added to your cart.
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"Add to Current Cart" will merge this item into the above user's cart. If this item is for a different user, click "Logout & Start New Cart" below.
Order notes entered here must be reviewed by a customer service specialist and may cause delays in processing your order.
The weather may be cold in many places, but tropical Christmas cards are a great way to convey a message of warm winter wishes from your sunny location. Whether you are spending your Christmas vacation somewhere hot, permanently live beside the ocean, or just want to capture the serene feelings displayed by your favorite beach scene, tropical Christmas cards are an excellent way to do this. Beach-themed Christmas cards will put your recipients in a pleasant mood even as the snow falls.
Is your business located somewhere tropical? Or do you run a seasonal business that deals in summertime products? Tropical-themed Christmas cards are a great way to showcase your unique company profile while remaining top-of-mind with clients. Send beach Christmas cards to your employees and coworkers too!
You can also send coastal-themed Christmas cards to your family and friends from places like California, Florida, Cape Cod, and many others. Coastal Christmas cards are enjoyed by boating enthusiasts, fishers, surfers, beachgoers, scuba divers, and those who simply love to relax by the ocean.
Check out our Miami palm tree photo Christmas card lit up with white lights – personalize the message and create your very own Florida Christmas cards! See Santa driving down the coastline, and design the ideal California Christmas cards. Go for a different vibe and craft Cape Shore Christmas cards. You can customize any of our tropical Christmas cards and beach-themed holiday cards with your name, signature, logo, graphics, photographs, and messaging. We also offer a number of pre-crafted beach Christmas card sayings to include in your cards. You can even choose a blank create-your-own template to upload your own tropical Christmas card design or make entirely new personalized beach themed Christmas cards. Create Santa snowman Christmas cards, southern Christmas cards, and beach-themed photo Christmas cards too! Unlike boxed tropical Christmas cards from a store, our beachy Christmas cards come with endless customization options. Our custom beach Xmas cards will make great branded marketing pieces for your business and are perfect for your personal mailing list as well.
We provide quality made in the USA Christmas cards, but feel free to send these beach cards from anywhere! Whether you're in Mexico, the Caribbean, or somewhere closer to home, our tropical Christmas cards are sure to make waves this season.
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J. G. Ikeorgu awarded PhD. He is a faculty from Department of Agronomy, Institute for Agricultural Research Farm (IAR),Ahmadu Bello University, Zaria, Nigeria. His international experience includes various programs, contributions and participation in different countries for diverse fields of study. He is the Member of the Scientific / Professional Organizations. His research interests reflect in his wide range of publications in various national and international journals
Research Interest
Agriculture, crop plant breeding, horticulture, crop techniques, post harvesting.


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Tips till Avl�gsna Application.DealAgent.AYZC
Mer fel whic Application.DealAgent.AYZC orsakar
0x80244022 WU_E_PT_HTTP_STATUS_SERVICE_UNAVAIL Same as HTTP status 503 – the service is temporarily overloaded., 0x80244033 WU_E_PT_ECP_FAILURE_TO_EXTRACT_DIGEST The file digest could not be extracted from an external cab file., 0x0000003D, 0x000000EA, 0x80240032 WU_E_INVALID_CRITERIA The search criteria string was invalid., 0x80247FFF WU_E_OL_UNEXPECTED Search using the scan package failed. , 0x8024001E WU_E_SERVICE_STOP Operation did not complete because the service or system was being shut down., 0x0000005E, 0x00000045, 0x000000D7, 0x0000007E, 0x8024D001 WU_E_SETUP_INVALID_INFDATA Windows Update Agent could not be updated because an INF file contains invalid information., 0x80240011 WU_E_INVALID_RELATIONSHIP An invalid update relationship was detected.
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