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The human neural stem cell line CTX0E03 was developed for the cell based treatment of chronic stroke disability. Derived from fetal cortical brain tissue, CTX0E03 is a clonal cell line that contains a single copy of the c-mycERTAM transgene delivered by retroviral infection. Under the conditional regulation by 4-hydroxytamoxifen (4-OHT), c-mycERTAM enabled large-scale stable banking of the CTX0E03 cells. In this study, we investigated the fate of this transgene following growth arrest (EGF, bFGF and 4-OHT withdrawal) in vitro and following intracerebral implantation into a mid-cerebral artery occluded (MCAo) rat brain. In vitro, 4-weeks after removing growth factors and 4-OHT from the culture medium, c-mycERTAM transgene transcription is reduced by ~75%. Furthermore, immunocytochemistry and western blotting demonstrated a concurrent decrease in the c-MycERTAM protein. To examine the transcription of the transgene in vivo, CTX0E03 cells (450,000) were implanted 4-weeks post MCAo lesion and analysed for human cell survival and c-mycERTAM transcription by qPCR and qRT-PCR, respectively.
The results show that CTX0E03 cells were present in all grafted animal brains ranging from 6.3% to 39.8% of the total cells injected. Prior to implantation, the CTX0E03 cell suspension contained 215.7 (SEM = 13.2) copies of the c-mycERTAM transcript per cell. After implantation the c-mycERTAM transcript copy number per CTX0E03 cell had reduced to 6.9 (SEM = 3.4) at 1-week and 7.7 (SEM = 2.5) at 4-weeks. Bisulfite genomic DNA sequencing of the in vivo samples confirmed c-mycERTAM silencing occurred through methylation of the transgene promoter sequence.
In conclusion the results confirm that CTX0E03 cells downregulated c-mycERTAM transgene expression both in vitro following EGF, bFGF and 4-OHT withdrawal and in vivo following implantation in MCAo rat brain. The silencing of the c-mycERTAM transgene in vivo provides an additional safety feature of CTX0E03 cells for potential clinical application.
Stem cell therapy is a facet of regenerative medicine that aims to ameliorate the damage caused to the brain by the grafting of healthy "reparative" cells. Pioneering studies implanting mouse neural stem cells (NSCs) into the brains of stroke animals have demonstrated significant recovery in motor and cognitive tests [1-5]. These findings provide a rational approach to the development of a cell based therapy for ischemic stroke. A substantial and consistent supply of allogeneic NSCs is required in order to treat a large patient population. Unfortunately, human NSCs are somatic stem cells and susceptible to genetic and phenotypic changes and loss of biological activity following extensive tissue culture expansion [6-8].
Immortalization of NSCs overcomes their restricted expansion potential. The myc proto-oncogene has proven a successful tool for immortalization of NSCs [9-16]. In these research applications, both v- and c-myc promote consistent and enhanced tissue culture expansion of human NSCs while maintaining a stable karyotype and retaining biological activity. The c-mycERTAM technology has proven to be as successful in generating immortalized neural stem cell lines as c-myc alone [17-20]. The c-mycERTAM transgene is better suited as an immortalizing agent for clinical applications because c-Myc protein function is conditional on the presence of the tamoxifen metabolite, 4-hydroxytamoxifen (4-OHT). The translated c-MycERTAM recombinant protein is a conjugation of human c-Myc and modified mouse estradiol receptor (ER) and is present as an inactive monomer in the cytosol of the cell. When added to the tissue culture medium, 4-OHT specifically binds to the ER moiety causing the c-MycERTAM protein to dimerize and subsequently translocate to the cell nucleus where c-Myc is active as a transcription factor [21].
CTX0E03 is a human NSC line that was derived using c-mycERTAM technology and showed recovery in sensorimotor deficits following grafting in MCAo rodent brain [19,22]. One copy of the c-mycERTAM transgene is integrated into the CTX0E03 cell genome (chromosome 13) under the cytomegalovirus (CMV) immediate early promoter [19]. We have shown that the c-mycERTAM transgene is expressed in proliferating CTX0E03 cell cultures in vitro; however, the expression of this transgene following the grafting of CTX0E03 cells in vivo was not characterized. Although neoplasm formation has never been observed with CTX0E03 cells in multiple pre-clinical studies, this information would nonetheless be important in assessing the inherent risk of using a genetically modified cell line for clinical applications.
Analysis of transgene expression following implantation of genetically modified cells is challenging because of the relatively small proportion of implanted cells compared to the host. Using a fluorescent reporter transgene, the CMV promoter has been shown to undergo silencing in grafted hematopoietic cells in vivo [23]. In another example, Lee et al. demonstrated by immunohistochemistry (IHC) that v-myc was silenced following intracerebral implantation of immortalized HBF1.3 cells [24]. Unfortunately, these methodologies were not appropriate for our application. The c-mycERTAM transgene in CTX0E03 cells does not contain a fluorescent reporter that could be monitored. With respect to IHC, we were concerned it would be inconclusive due to the unknown sensitivity and potential cross-reactivity of the antibodies. Furthermore, IHC was ruled out since the analysis would be limited to a finite number of sections and would not encompass all cells present in the graft.
In this study, we developed a novel and sensitive method that is able to analyze absolute gene expression within human genetically modified cells grafted into rodent brain. This method used the human specific Alu genomic sequence to detect and quantify CTX0E03 cells grafted within the rodent brain background [25,26]. Absolute quantification of c-mycERTAM transcription made it possible to calculate the number of c-mycERTAM transcript copies per CTX0E03 cell. Using this methodology, c-mycERTAM transgene silencing was shown following grafting of CTX0E03 cells into MCAo rat brain. Although not intentionally incorporated into the design technology of c-mycERTAM, silencing of the CMV transgene promoter in vivo demonstrates an additional safety feature of this technology and of the CTX0E03 cells.
Alu and c-mycERTAM assay design
The analysis of the c-mycERTAM transcript expression per CTX0E03 cell, both in vivo and in vitro, was dependent upon the ability to sequentially purify genomic DNA (gDNA) and RNA from the same biological sample using the All DNA/RNA Kit (Qiagen). The gDNA preparation yielded one single band by agarose gel electrophoresis with no evidence of degradation or contaminating RNA (Figure 1A). The RNA preparation yielded the expected 28S and 18S bands, again with no degradation or DNA contamination visible (Figure 1B). Standard curves were then generated to determine: 1) the number of CTX0E03 cells present in a gDNA sample by Alu qPCR (Figure 1C); and 2) the number of c-mycERTAM transcripts present in a retro-transcribed RNA reaction sample (Figure 1D). The Alu sequence assay could reproducibly detect human CTX0E03 gDNA with as few as one CTX0E03 cell per reaction; whereas, the c-mycERTAM assay could detect as few as 10 copies per reaction.
Figure 1. Alu sequence and c-mycERTAM assay development and validation. Gels showing the quality and purity of gDNA by agarose gel electrophoresis (A) and RNA by virtual gel produced by Agilent 2100 Bioanalyzer (B, RNA Integrity Number >9.4 as analyzed by Agilent RNA 600 nano kit [32]) isolated from the same sample. Standard curves used to determine: cell number by Alu sequence qPCR (C, Error 0.0300, efficiency 1.993; 3 replicates); absolute c-mycERTAM copy number by c-mycERTAM qRT-PCR (D, Error 0.0837 and efficiency 2.131; 3 replicates). All standard curves were generated from CTX0E03 gDNA diluted in rat gDNA or cDNA. Crossing point refers to the number of PCR cycles required to generate a detectable fluorescent signal generated on a Roche LC480 instrument. Positive control rat brain samples (B1 and B2) were grafted with approximately 300,000 CTX0E03 cells each and harvested immediately (E, F). Data shown is the total number of CTX0E03 cells in each tissue section as determined by Alu, where control is the number of viable cells in the cell suspension prior to injection as determined by counting using a haemocytometer (E); and total c-mycERTAM transcript copy number calculated per CTX0E03 cell detected in brain samples, where control is the number of copies per cell detected in vitro culture (F).
Validation of Alu and c-mycERTAM assays
The Alu sequence and c-mycERTAM transcription assays were developed in the presence of rat brain gDNA or cDNA, as described in the Methods. These experiments involved mixing purified CTX0E03 DNA/cDNA with purified rat brain DNA/cDNA. However, in order to verify that CTX0E03 cells grafted in rat brain could be processed and analysed effectively together a positive control experiment was carried out. This experiment consisted of harvesting and processing the rat brain tissue immediately after a 6 μl injection of CTX0E03 cells was delivered. It was determined by counting using a haemocytometer that the cell suspension prior to delivery contained 46,600 cells/μl – i.e. total of 279,600 cells were present in 6 μl loaded into the syringe. By Alu sequence analysis, the injected rat brain tissue sections contained an average of 241,116 (SEM = 15,487) CTX0E03 cells (Figure 1F). The measured number of CTX0E03 cells is within the range expected. Analysis of the total RNA isolated from these samples showed CTX0E03 cells to express on average 236.9 (SEM = 7.8) c-mycERTAM transcript copies per cell (Figure 1G). Control CTX0E03 cells measured in culture were shown to have a similar value of 242.3 (SEM = 47.8) c-mycERTAM transcript copies per cell.
CTX0E03 cell detection and quantification in vivo
Four weeks after the MCAo, a total of 450,000 CTX0E03 cells were implanted bilaterally into the putamen. The rats were sacrificed 1-week or 4-weeks post-implantation, their brains were removed and sections encompassing the injection tracts were dissected and snap frozen in liquid nitrogen. The Alu and c-mycERTAM assays were then carried out on the gDNA and cDNA isolated from each tissue section, respectively. CTX0E03 cells were detected in all implanted animals at both time points, with a range of 28,151 to 179,184 CTX0E03 cells detected per brain. The total number of CTX0E03 cells detected, per rat brain, is shown at 1-week (Figure 2A) and 4-weeks (Figure 2B) post-implantation. An aliquot of the CTX0E03 cell suspension prepared for implantation was retained and used to determine the pre-implantation c-mycERTAM transcript copy number. The average c-mycERTAM transcription level for the three CTX0E03 cell suspensions prepared in this study was found to be 215.7 (SEM = 13.2) copies per cell (Figure 2C). This is compared to the population of CTX0E03 cells detected in vivo, where the average number of c-mycERTAM transcript copies per CTX0E03 cell was calculated to be 6.9 (SEM = 3.4) at 1-week and 7.7 (SEM = 2.5) at 4-weeks (Figure 2C). The number of CTX0E03 cells, the number c-mycERTAM transcripts calculated per tissue section and the resultant number of c-mycERTAM transcript copies per CTX0E03 cell, are shown in Table 1. c-MycERTAM transcripts were detected within approximately 50% of the tissue sections containing CTX0E03 cells at both time points. It is important to note, in no instance was a c-mycERTAM transcript signal detected where CTX0E03 cells were absent.
Figure 2. Detection of CTX0E03 cells and c-mycERTAM transcription in grafted rat brain. Total cells found at 1-week (A) and 4-weeks (B) post-implantation by Alu qPCR. Animal numbers 103, 304, 401, 402 and 603 were vehicle injected only control brain samples. Absolute quantification of c-mycERTAM transcript level per CTX0E03 cell, in cell suspensions prior to implantation (control) and in vivo (C).
Table 1. c-MycERTAM transcript copy number and number of CTX0E03 cells calculated in the grafted rat brain sections at 1- and 4-weeks post-implantation
Transgene methylation analysis
Direct modification of the DNA by methylation is an epigenetic mechanism to downregulate or silence gene expression. Using bisulfite sequencing, the methylation status of the CMV transgene promoter was investigated in CTX0E03 cells pre- and post-implantation. The CMV promoter of the transgene contains numerous potential methylation sites within a CpG rich island (Figure 3A). Bisulfite sequencing of gDNA clones taken from control non-implanted CTX0E03 cells showed only 2% of these CpG sites were methylated (Figure 3B). However, the same analysis carried out on clones obtained from the CTX0E03 cell implanted MCAo rat brain samples showed that 86.8% (SEM = 7.1) and 75.0% (SEM = 1.4) of the CpG islands were methylated at 1-week and 4-weeks, respectively (Figure 3B). There was no statistical difference between 1- and 4-weeks, p = 0.14, indicating that the methylation had occurred within the first week and was constant to the 4-week time point. Collectively, only four gDNA transgene promoter clones in vivo were found to be devoid of any methylated sites from a total of 99 analysed; in other words, ~96% of the transgene promoter sequences analysed in vivo where found to contain at least one methylated CpG island.
Figure 3. In vivo analysis of CpG methylation within the c-mycERTAM promoter region in CTX0E03 cells. CpG dinucleotide-containing regions (
- underlined
In vitro characterization
Additional experiments were carried out in vitro to determine if a reduction in transgene transcription leads to a concomitant reduction in translated protein. Analyses were carried out on control CTX0E03 cells and those cultured for 1- and 4-weeks in the absence of growth factors and 4-OHT. In this non growth permissive condition, transgene transcription was found to decrease by 59.6% at 1-week and 73.4% at 4-weeks relative to control by qRT-PCR (Figure 4A). Analysis of c-MycERTAM protein was performed by colocalization of c-Myc and ERα antigen staining by immunocytochemistry (ICC; Figure 4B, C, D, E). The c-Myc and ERα antigens were always found to colocalize in the nucleus of a proportion of CTX0E03 cells, indicating that the antibodies were detecting the c-MycERTAM target (Figure 4E). Immunoreactive cells were counted and expressed as a proportion of total cells. These data showed control CTX0E03 cell cultures were approximately 52.0% positive for c-MycERTAM; whereas, following EGF, bFGF and 4-OHT withdrawal the number of CTX0E03 cells positive for c-MycERTAM were 42.5% at 1-week and 12.8% at 4-weeks (Figure 4B). Western blot analysis using the c-Myc and ERα antibodies demonstrated that total c-MycERTAM protein levels followed more closely the progressive drop observed by gene expression and were reduced by 53.8% at 1-week and 73.0% at 4-weeks following EGF, bFGF, and 4-OHT withdrawal (Figure 4F, G).
Figure 4. In vitro characterisation of c-mycERTAM transcript and protein expression. CTX0E03 cells were cultured in growth medium (control) and in non-growth promoting medium (in the absence of growth factors and 4-OHT) for 1- and 4-weeks. Evaluation of c-mycERTAM gene transcript and protein levels were performed by qRT-PCR (A), ICC (B to E) and western blot (F). CTX0E03 cell ICC images shown in panels C to E are representative images of the control c-Myc (green), ERα (red) and overlay (Merge). Scale bars represent 50 μm. The western blots in panel F were quantified using densitometry normalised by α-tubulin (G).
The c-mycERTAM immortalization technology has been successfully utilized to derive a clonal human neural stem cell line from fetal cortical brain. This cell line, referred to as CTX0E03, possess biological attributes which are desirable for clinical applications for stroke therapy. Namely, this cell line can be continuously expanded in vitro, and following implantation in MCAo rodent brain, results in sensorimotor recovery in the absence of cell treatment-related histopathology [19,22]. The in vitro effects of c-mycERTAM in the CTX0E03 cell line have been well characterized. Here we investigated the expression of this transgene following implantation of CTX0E03 cells into MCAo rat brain.
In this paper we report a novel method to examine absolute gene expression within human cells implanted into rodent brain. In this example, we analyzed the number of c-mycERTAM transcript copies per CTX0E03 cell both in vitro and in vivo. The calculated c-mycERTAM copies per CTX0E03 cell prepared directly from fresh culture (242.3; SEM = 47.8), cell suspension prior to implantation (215.7; SEM = 13.2) or immediately following implantation (236.9; SEM = 7.8) had comparable values. This validated that the assays employed were CTX0E03 cell specific and the rodent brain background did not cause any interference. Using these established assays, c-mycERTAM transgene transcription was analyzed in the surviving CTX0E03 cell population at 1- and 4-weeks post-implantation in MCAo rodent brain. At both time points there was a substantial reduction in the calculated number of c-mycERTAM transcript copies per CTX0E03 cell detected.
This molecular biological approach provided many advantages over immunohistochemistry and in situ hybridization techniques. The results obtained by RT-PCR and PCR were quantitative. Immunohistochemistry or in situ hybridization techniques do not permit the quantitative assessment of c-mycERTAM levels within a given cell. In addition, the sensitivity of molecular biological techniques were defined, as a single CTX0E03 cell and ten copies of c-mycERTAM per reaction, respectively; whereas, this level of sensitivity would have been difficult, if not impossible, to achieve by immunohistochemistry or in situ hybridization techniques. Our in vivo samples were processed rapidly, immediately after harvesting, thus capturing the most accurate data. Furthermore, from a practical perspective this molecular biological approach analyzed all CTX0E03 cells present in the rodent brain, whereas, histological preparation methods would require a large number of sections to analyze all cells present. Although the average transgene expression significantly decreased in vivo, we cannot exclude the possibility that the expression observed may have arisen from a limited number of CTX0E03 cells. However, the silencing of c-mycERTAM expression observed in vivo is consistent with the absence of tumor formation derived from CTX0E03 cells when implanted in vivo [19,22].
Transgene silencing can occur at a transcriptional or post-transcriptional level. Long-term transcriptional silencing of retrovirus-delivered genes has been shown largely to result from methylation of the cytosine nucleotides within the promoter regions [27]. The factors responsible for DNA methylation is reviewed in detail elsewhere [28]. As the c-mycERTAM transgene was delivered into the CTX0E03 cell line using a retrovirus, it was reasonable to speculate that transgene silencing had occurred through methylation of CpG islands within the transgene promoter region. Bisulfite DNA sequencing was used to determine the methylation status of the CMV promoter region of the c-mycERTAM transgene within CTX0E03 cells. In culture, only 6 of the total CpG sites screened were found to be methylated. In the samples prepared from CTX0E03 grafted MCAo rodent brain 62 to 100% of the sites were methylated. These data unequivocally show that c-mycERTAM expression is silenced at a transcriptional level in vivo as a result of DNA methylation within the CMV transgene promoter region.
The c-mycERTAM technology acts at a protein rather than a transgene expression level. To mitigate the risk of the technology following intracerebral implantation of CTX0E03 cells it was important to investigate if c-mycERTAM transcription level was a predictive measure of the recombinant protein level. Western blot analysis of the in vivo samples proved impossible because the c-MycERTAM protein would be too dilute to detect in whole brain extracts. In addition, IHC analysis of the in vivo samples was impractical because of the substantial number of sections required to perform a global analysis of c-MycERTAM protein expression. To overcome these challenges we developed and in vitro model of c-mycERTAM silencing in CTX0E03 cells. The kinetics of transgene silencing in vitro was slower than that observed in vivo, which provided the ideal cell culture model to directly compare the progressive decline in c-mycERTAM transcription with the translated protein level. Although we observed a decrease in the number of c-mycERTAM positive cells by ICC, the relative percentage of positive cells did not closely follow relative transgene expression levels. These ICC findings further highlight the drawback of histological approaches because the cells were scored positive independent of the quantity of c-MycERTAM protein present within the cell. On the other hand, when total c-MycERTAM protein levels were measured by western blot they were found to decrease in parallel with transgene transcript level over the 1- to 4-week period. This data indicates that the c-mycERTAM transcript and protein levels are closely linked in CTX0E03 cells.
In conclusion, we have demonstrated that c-mycERTAM transgene expression levels in CTX0E03 cells were significantly reduced in vitro, upon growth factor and 4-OHT withdrawal, and in vivo following implantation. We demonstrated that in vivo transgene silencing occurred through DNA methylation. In addition, the in vitro results showed that c-mycERTAM transcription level was representative of the c-MycERTAM protein level. The data demonstrate that uncontrolled in vivo cell growth arising from c-mycERTAM technology is unlikely based on both the silencing of the c-mycERTAM gene expression reported here and the specific requirement for the 4-OHT ligand to activate the c-MycERTAM protein. Furthermore, these data demonstrate that the retroviral insertion of stable transgene constructs into human neural stem cells with the CMV promoter element is not an inherently unsafe procedure for the production of cellular therapies.
Ethics statement
Fetal tissue used to derive CTX0E03 cells was obtained in accordance with nationally (UK and/or USA) approved ethical and legal guidelines. All animal work was performed in accordance with the UK Animals (Scientific Procedures) Act (1986) and approved by the local animal Ethical Review Committee.
Derivation of CTX0E03
The CTX0E03 clonal cell line was derived from human fetal brain cortical tissue of 12 weeks' gestation, as described previously [19]. Briefly, a mixed culture was initiated from the dissociated cortex tissue and was infected with an amphotropic replication-incompetent retroviral vector (pLNCX-2; Clontech) encoding the c-mycERTAM transgene. The CTX0E03 clonal cell line was isolated using a ring cloning method. The CTX0E03 cells were routinely cultured as a monolayer on mouse laminin (Trevigen, AMS Biotechnology, UK) freshly coated flasks in serum free medium (RMM) containing the growth factor supplements 10 ng/ml bFGF (Peprotech), 20 ng/ml EGF (Peprotech), and 100 nM 4-OHT (Sigma). All experiments were carried out using CTX0E03 cells between passage number 33 to 37, which corresponds to a population doubling level (PDL) ranging between 65 to 75.
MCAo and cell grafting
Male Sprague-Dawley rats (Charles River) were group housed with food and water ad libitum. Briefly, animals were subjected to 60 minutes of unilateral MCAo initiated under halothane anesthesia [29]. At 50 minutes into the occlusion, animals were evaluated for behavioral dysfunction (forelimb flexion and contralateral circling behavior). Animals that did not demonstrate dysfunction were removed from the study.
CTX0E03 cells were harvested and suspended at a concentration of ~50,000 cells/μl in HBSS-NAC (HBSS without calcium, magnesium and containing 0.5 mM N-acetyl cysteine; Sigma) and implanted 4-weeks ± 3 days after the occlusion. Each grafted animal received two 4.5 μl bolus injections (approximately 225,000 cells/site with control lesioned animals receiving vehicle only) over 68 sec with 4 min rest before withdrawal. Coordinates from bregma were as follows: Site 1) AP 0.0 mm, L -3.6 mm, V -5.5 mm (from skull surface at bregma); and Site 2) AP 0.0 mm, L +3.6 mm, V -5.5 mm (from skull surface at bregma). Animal brains were collected at 1-week (6 grafted + 2 non-grafted controls) or 4-weeks (6 grafted + 3 non-grafted controls) post-implantation. All animals were administered Cyclosporin A (20 mg/kg, Sandoz Pharmaceuticals) in cremaphore L (Sigma) a day prior to grafting, the day of grafting and then three times weekly for the remaining duration of the study. In addition, Methylprednisolone (Pharmacia Upjohn) was administered at 20 mg/kg for the first 14 days, 10 mg/kg for days 15–17 and 5 mg/kg for days 18–19 post-grafting.
Alu sequence assay to detect and quantify CTX0E03 cells
The qPCR Alu assay was carried out using the LightCycler 480 and the Probes Master mix (Roche). Briefly, an average of 250 ng of genomic DNA was amplified using specific primers (Forward-TGAGGCAGGCGAATCGCTTGAA, Reverse-GACGGAGTTTCGCTCTTGTTG) and a FAM-labeled fluorogenic probe (CGCGATCTCGGCTCACTGCAACCTCCATCG) (PrimerDesign) against a conserved region of the human Alu-Sq sequence (Accession number U14573). The PCR was preformed under the following conditions: 95°C for 30 seconds, followed by 45 cycles of 95°C for 15 s, 58°C for 30 s, 72°C for 15 s. Quantification of the human CTX0E03 DNA in rat tissue was based on a standard curve using serial dilutions that ranged from 1 to 10000 CTX0E03 cells, calculated on the assumption that one human cell contains 6.6 pg of gDNA [30]. The standard curve was performed in the presence of 250 ng of rat gDNA.
CTX0E03 c-mycERTAM transgene transcription assay
Primers were designed to specifically detect the relative c-mycERTAM transgene transcription level in CTX0E03 cells using the Roche Diagnostics Lightcycler LC480 system. Using SYBR Green Master mix (Roche) and primers (Forward-AAAGGCCCCCAAGGTAGTTA, Reverse- AAGGACAAGGCAGGGCTATT), which amplified the c-myc and estrogen receptor (ER) junction, qPCR was performed under the following conditions: 95°C for 5 s, followed by 45 cycles at 95°C for 10 s, 60°C for 20 s,72°C for 20 s, 82°C for 5 s, measuring the fluorescence. A melt curve analysis was also performed after the assay to check the specificity of the reaction using the following conditions: 95°C for 5 s, 65°C for 60 s (1°C increments) followed by 97°C continuous. In order to carry out absolute quantification of c-mycERTAM transcription, a known concentration standard was required to plot a standard curve. Based on the assumption that CTX0E03 cell contains one genomic copy of the c-mycERTAM transgene [19] serial dilutions ranging from 10 to 10,000 copies of c-mycERTAM were used per reaction to produce the standard curve. The standard curve was performed in the presence of rat cDNA.
Calculation of c-mycERTAM transcription level in vivo
At 1- or 4-weeks post-implantation, the rat brains were removed, cut into 50–250 mg sections and snap frozen in liquid nitrogen. The gDNA and total RNA was then sequentially isolated from each tissue piece using the AllPrep DNA/RNA mini kit (Qiagen). Four tissue sections were processed per rat brain (i.e. two tissue sections encompassing each injection tract). In brief, the tissue samples were homogenised in the supplied lysis buffer according to sample weight. The lysed sample was processed first through an AllPrep DNA spin column, to isolate the gDNA, and subsequently through an RNeasy spin column to selectively isolate RNA. The gDNA and total RNA samples (1 μl/reaction) were then analyzed by qPCR for Alu signal and qRT-PCR for c-mycERTAM, respectively. Based on the Alu signal, the number of CTX0E03 cells present per reaction was calculated from the standard curve. Similarly, the number of c-mycERTAM transcripts present per reaction was interpolated from the c-mycERTAM copy number standard curve. A calculation was then carried out to correct for the amount analyzed and the total volume of tissue sample lysate (for example, 1 μl from a total of 650 μl). The corrected figures were then expressed as the number of c-mycERTAM transcripts present over the number of CTX0E03 cells present in each tissue section. Standard error of the mean was calculated for all tissue sections found to contain cells (Microsoft Excel version 2003).
Transgene methylation analysis by bisulfite sequencing
Bisulfite treatment of DNA followed by sequencing was used to determine the endogenous transgene promoter pattern of methylation in CTX0E03 cells. Methylation is implicated in repression of genetic activity and involves the addition of a methyl group to the carbon-5 position of cytosine residues of the dinucleotide CpG. Bisulfite sequencing is a technique used to identify specific gDNA methylation sites based on the fact that treatment of DNA with bisulfite converts cytosine residues to thymidine, but leaves 5-methylcytosine residues unaffected. Bisulfite treatment thus introduces a specific change in the DNA sequence which is dependent on the methylation status of individual cytosine residues. A bisulfite reaction was performed using Epitect Bisulfite (Qiagen). Up to 2 μg of genomic DNA was used for conversion with the bisulfite reagent. Bisulfite-converted DNA was used as a template for PCR amplification. PCR primers were designed using the web program METHPRIMER ([31]; http://www.urogene.org/methprimer/index1.html webcite). Primers used were: Forward – GGGAGTTTGTTTTGGTATTAAAATTAA and Reverse – ACTACTACTAATAAAAATTCTCCTCCTC. PCR conditions were 94°C for 5 minutes, 30 cycles of 94°C for 30 seconds, 60°C for 1 min, and 72°C for 30 seconds followed by 10 min at 72°C. PCR fragments were cloned into TA-cloning vector using TOPO TA Cloning Kit for Sequencing (Invitrogen). Ten individual clones were sequenced by GATC Biotech (Germany) using the T3 universal PCR primers. The resulting sequences were analyzed using blast software http://blast.ncbi.nlm.nih.gov/Blast.cgi webcite. Sequence analysis that identified an unchanged cytosine base (i.e. protected from the bisulfite conversion reaction) was considered to be methylated, but a cytosine conversion to thymidine in the sequence indicated no methylation.
In vitro characterisation: western blot and immunocytochemical analysis
In vitro, CTX0E03 cell samples were prepared by maintaining the cells in non-growth permitting medium alone, without the addition of EGF, bFGF, and 4-OHT, for 1- and 4-weeks. Western blot analysis for c-mycERTAM protein in CTX0E03 cells was carried out using the whole-cell lysate. To prepare cell lysate, CTX0E03 cell monolayers in a T75 flask were rinsed with cold PBS (4°C), lysed with 750 μl 1× SDS Sample Buffer and dithiothreitol (DTT) Reducing Agent (PAGEgel Inc, AMS Biotechnology, UK), and collected in a 1.5 ml centrifuge tube. Samples were triturated multiple times to lyse and shear the sample, heated at 100°C for 2 min and centrifuged. Cell lysates were loaded onto a 4%–20% Tris-Tricine gel (PAGEgel Inc, AMS Biotechnology, UK), 10 μl per well. After electrophoresis, the protein was transferred onto a nitrocellulose membrane with 0.2 μm pore size by electroelution. Immunodetection was performed with a rabbit anti-ERα polyclonal or a mouse anti-c-Myc monoclonal antibody (Santa Cruz Biotechnology Inc, both used at 1:100). Transgene product was detected using horseradish peroxidase-conjugated anti rabbit-IgG (Cell Signaling Technology, 1:2000) or anti mouse-IgG (Pierce Biotechnology Inc., 1:800) secondary antibodies, as appropriate. The nitrocellulose membrane was then processed using chemiluminescence detection reagents (Thermo scientific). The blots were then stripped and reprobed using anti-α-tubulin (Sigma, 1:1000) to act as an internal loading level standard. Western blot images were captured using BioRad FluorS Imaging Unit and densitometry carried out using Scion Image software (Scion Corporation, version Alpha 220.127.116.11, USA).
Immunocytochemistry was carried out on CTX0E03 cells fixed in a 96-well plate (BD) using 4% PFA/4% sucrose in PBS for 15 min after 3 days growth (control) and following 1- and 4-weeks in medium containing no growth factors (EGF, bFGF) or 4-OHT. Anti-c-myc (1:100) and anti-ERα (1:500) were incubated in 1xPBS containing 1% normal goat serum (Vector laboratories) overnight at room temperature. After rinsing, the CTX0E03 cells were incubated for 1.5 h at room temperature using anti-mouse conjugated Alexa Fluor 488 (Invitrogen, 1:2000) for c-myc and anti-rabbit conjugated Alexa Fluor 568 (Invitrogen, 1:2500) for ERα. CTX0E03 cells were then counterstained by incubating with Hoechst 33342 (Sigma, 1 μM) for 2 min. Quantification of c-myc and ERα was carried out by manual counting of three representative fields using an Olympus IX70 fluorescent microscope.
Authors' contributions
EM, LS, AH and JS conceived the experiments. LS, EM, RC, JH and PS designed and performed the experiments and analyzed the data. All authors contributed to the preparation of the written manuscript.
We thank Prof. Jack Price (King's College London, United Kingdom) for his general support and helpful discussions. In addition, our gratitude extends to Dr. Aaron Jeffries (King's College London, United Kingdom) for advice and guidance on DNA methylation analysis; and to PrimerDesign for their help in developing the Alu primers. We also extend our thanks to Susan Hines (Imperial College London, United Kingdom) and Dr. Marc Oliver Baradez (Laboratory of the Government Chemist, LGC, United Kingdom) for their kind assistance in the preparation of the cells used in this study. This study was supported and designed by ReNeuron (RENE.L) who where involved in study design, data collection, analysis, and interpretation, as well as manuscript preparation and decision to submit for publication.
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Modo M, Stroemer RP, Tang E, Patel S, Hodges H: Effects of implantation site of stem cell grafts on behavioral recovery from stroke damage.
Stroke 2002, 33(9):2270-2278. PubMed Abstract | Publisher Full Text
Veizovic T, Beech JS, Stroemer RP, Watson WP, Hodges H: Resolution of stroke deficits following contralateral grafts of conditionally immortal neuroepithelial stem cells.
Stroke 2001, 32(4):1012-1019. PubMed Abstract | Publisher Full Text
De Filippis L, Ferrari D, Rota Nodari L, Amati B, Snyder E, Vescovi AL: Immortalization of human neural stem cells with the c-myc mutant T58A.
PLoS ONE 2008, 3(10):e3310. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Bai Y, Hu Q, Li X, Wang Y, Lin C, Shen L, Li L: Telomerase immortalization of human neural progenitor cells.
Neuroreport 2004, 15(2):245-249. PubMed Abstract | Publisher Full Text
Villa A, Navarro-Galve B, Bueno C, Franco S, Blasco MA, Martinez-Serrano A: Long-term molecular and cellular stability of human neural stem cell lines.
Exp Cell Res 2004, 294(2):559-570. PubMed Abstract | Publisher Full Text
Donato R, Miljan EA, Hines SJ, Aouabdi S, Pollock K, Patel S, Edwards FA, Sinden JD: Differential development of neuronal physiological responsiveness in two human neural stem cell lines.
BMC Neurosci 2007, 8:36. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
De Filippis L, Lamorte G, Snyder EY, Malgaroli A, Vescovi AL: A novel, immortal, and multipotent human neural stem cell line generating functional neurons and oligodendrocytes.
Stem Cells 2007, 25(9):2312-2321. PubMed Abstract | Publisher Full Text
Cho T, Bae JH, Choi HB, Kim SS, McLarnon JG, Suh-Kim H, Kim SU, Min CK: Human neural stem cells: electrophysiological properties of voltage-gated ion channels.
Neuroreport 2002, 13(11):1447-1452. PubMed Abstract | Publisher Full Text
Ryder EF, Snyder EY, Cepko CL: Establishment and characterization of multipotent neural cell lines using retrovirus vector-mediated oncogene transfer.
J Neurobiol 1990, 21(2):356-375. PubMed Abstract | Publisher Full Text
Snyder EY, Deitcher DL, Walsh C, Arnold-Aldea S, Hartwieg EA, Cepko CL: Multipotent neural cell lines can engraft and participate in development of mouse cerebellum.
Cell 1992, 68(1):33-51. PubMed Abstract | Publisher Full Text
Rao MS, Anderson DJ: Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells.
J Neurobiol 1997, 32(7):722-746. PubMed Abstract | Publisher Full Text
Jung M, Kramer EM, Muller T, Antonicek H, Trotter J: Novel pluripotential neural progenitor lines exhibiting rapid controlled differentiation to neurotransmitter receptor-expressing neurons and glia.
Eur J Neurosci 1998, 10(10):3246-3256. PubMed Abstract | Publisher Full Text
Villa A, Snyder EY, Vescovi A, Martinez-Serrano A: Establishment and properties of a growth factor-dependent, perpetual neural stem cell line from the human CNS.
Exp Neurol 2000, 161(1):67-84. PubMed Abstract | Publisher Full Text
Nakafuku M, Nakamura S: Establishment and characterization of a multipotential neural cell line that can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro.
J Neurosci Res 1995, 41(2):153-168. PubMed Abstract | Publisher Full Text
Miljan EA, Hines SJ, Pande P, Corteling RL, Hicks C, Zbarsky V, Umachandran M, Sowinski P, Richardson S, Tang E, et al.: Implantation of c-mycERTAM immortalized human mesencephalic derived clonal cell lines ameliorates behaviour dysfunction in a rat model of Parkinson disease.
Stem Cells Dev 2008, 18(2):307-319. Publisher Full Text
Pollock K, Stroemer P, Patel S, Stevanato L, Hope A, Miljan E, Dong Z, Hodges H, Price J, Sinden JD: A conditionally immortal clonal stem cell line from human cortical neuroepithelium for the treatment of ischemic stroke.
Exp Neurol 2006, 199(1):143-155. PubMed Abstract | Publisher Full Text
Maurer J, Fuchs S, Jager R, Kurz B, Sommer L, Schorle H: Establishment and controlled differentiation of neural crest stem cell lines using conditional transgenesis.
Differentiation 2007, 75(7):580-591. PubMed Abstract | Publisher Full Text
Littlewood TD, Hancock DC, Danielian PS, Parker MG, Evan GI: A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins.
Nucleic Acids Res 1995, 23(10):1686-1690. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Stroemer P, Patel S, Hope A, Oliveira C, Pollock K, Sinden J: The Neural Stem Cell Line CTX0E03 Promotes Behavioral Recovery and Endogenous Neurogenesis after Experimental Stroke in a Dose-Dependent Fashion.
Zhang F, Thornhill SI, Howe SJ, Ulaganathan M, Schambach A, Sinclair J, Kinnon C, Gaspar HB, Antoniou M, Thrasher AJ: Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells.
Blood 2007, 110(5):1448-1457. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lee HJ, Kim KS, Kim EJ, Choi HB, Lee KH, Park IH, Ko Y, Jeong SW, Kim SU: Brain transplantation of immortalized human neural stem cells promotes functional recovery in mouse intracerebral hemorrhage stroke model.
Stem Cells 2007, 25(5):1204-1212. PubMed Abstract | Publisher Full Text
Nicklas JA, Buel E: Development of an Alu-based, real-time PCR method for quantitation of human DNA in forensic samples.
J Forensic Sci 2003, 48(5):936-944. PubMed Abstract
Schneider T, Osl F, Friess T, Stockinger H, Scheuer WV: Quantification of human Alu sequences by real-time PCR – an improved method to measure therapeutic efficacy of anti-metastatic drugs in human xenotransplants.
Clin Exp Metastasis 2002, 19(7):571-582. PubMed Abstract | Publisher Full Text
Lavie L, Kitova M, Maldener E, Meese E, Mayer J: CpG methylation directly regulates transcriptional activity of the human endogenous retrovirus family HERV-K(HML-2).
J Virol 2005, 79(2):876-883. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Strathdee G, Brown R: Aberrant DNA methylation in cancer: potential clinical interventions.
Expert Rev Mol Med 2002, 4(4):1-17. PubMed Abstract | Publisher Full Text
Longa EZ, Weinstein PR, Carlson S, Cummins R: Reversible middle cerebral artery occlusion without craniectomy in rats.
Stroke 1989, 20(1):84-91. PubMed Abstract | Publisher Full Text
Applied_Biosystems: Creating standard curves with genomic DNA or plasmid DNA templates for use in quantitative PCR. [http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf] webcite
Li LC, Dahiya R: MethPrimer: designing primers for methylation PCRs.
Bioinformatics 2002, 18(11):1427-1431. PubMed Abstract | Publisher Full Text
Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Granzow M, Ragg T: The RIN: an RNA integrity number for assigning integrity values to RNA measurements.
BMC Mol Biol 2006, 7:3. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text


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How to collect VMware info in ProSphere
Posted on 10. Jan, 2013 by vTexan.
Hopefully you had a chance to review my "How to install ProSphere" blog post and are now ready to continue adding more discovery objects into ProSphere to get a larger overview of your environment. The next object we will be adding is our VMware vSphere infrastructure. This will get us one step closer to […]
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How To Install EMC ProSphere
Posted on 08. Jan, 2013 by vTexan.
<Update May 21st ProSphere was updated to 2.0, the install is still the same) <Updated Jan 14th to include links to setting up VMware with ProSphere, and Cisco Switches – links at the bottom> In December, the Storage Resource Management Suite was updated and with that came ProSphere 1.7. If you are not familiar with […]
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How to setup Solutions Enabler with SMI-S for Windows
Posted on 07. Dec, 2012 by vTexan.
From time to time I've needed to get Solutions Enabler setup for various different reasons, on various different lab environments, as well as helping customers get this setup for things like VASA and ProSphere. I figured I might as well document it and publish it so that in another 3 months, when I need […]
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Storage Resource Management Suite
Posted on 27. Nov, 2012 by vTexan.
Hopefully you read my blog post around IT Transformation. If you recall, the whole movement around IT Transformation is the understanding that various companies' differentiated value is coming more and more from their customer facing Applications. Because of this, more and more attention needs to be focused on these applications. This is driving the […]
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Introducing EMC Elect
Posted on 15. Nov, 2012 by vTexan.
One of the things that drove me to social media (Twitter+Blogging+Facebook+Linkedin etc) was a desire to share with the community, good, bad or indifferent, but sharing none-the less. It's probably a lot of the same reasons others gravitated towards this medium. It's not for everyone, but for those that embrace it, they can certainly […]
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IT Transformation
Posted on 23. Oct, 2012 by vTexan.
Lead, follow or get the hell out of the way. This is pretty true when describing trends in IT over the last few years. Every week, IT groups battle for relevancy within the organization they support. Ever heard the running joke that IT puts the "NO" in innovation? Being able to respond quickly to […]
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VSI Install Guide – Path Management Plugin
Posted on 25. Sep, 2012 by vTexan.
The final installment of my VSI Install Guide Series is our Path Management Plugin. This is another free plugin that allows you to set, or change global multiplathing settings in your VMware vSphere environment as it relates to connecting to EMC storage solutions. If you've kept up with my other guides I took you […]
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Sizing EMC VNX Series for VDI Workloads
Posted on 20. Sep, 2012 by vTexan.
HOT off the presses !! EMC just released today a new whitepaper called "Sizing EMC VNX Series for VDI Workloads" <Click here to download if you don't have a Powerlink Account> that goes into some GREAT detail on how to properly understand some of the metrics you need to pay attention to when designing Virtual […]
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The Legendary VMworld 2012 Hands On Labs list
Posted on 21. Aug, 2012 by vTexan.
<Updated Aug 22> Hot off the presses – in the immortal words of Barney Stinson ! This years VMword HoL is indeed going to be legendary !! For those of you that have been to VMworlds in the past, you understand the value of the VMware HoL. In most cases, you schedule your sessions […]
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VMworld 2012 Tips Tricks and General FYIs
Posted on 20. Aug, 2012 by vTexan.
I recently published "EMC Guide to VMworld 2012" with lots of information focused on EMC customers, partners and employees that might be at VMworld I'll probably publish one more later this week with a consolidate list of "Tweetups" that you might find interesting. In the mean time, I thought I would follow that blog […]
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ACE Fit Share
Add some diversity to your fitness program with these butt and hip exercises from the American Council on Exercise. We'll help you tone up those glutes and get that butt into shape. Choose from a wide variety of hip flexibility and strengthening exercises including glute bridges, BOSU lateral jumps and more. These exercises come with detailed instructions, step-by-step guidance and tutorials on proper equipment use. For full workout routines featuring butt and hip exercises, be sure to visit our Workouts section.
Fit Life Articles
Stretching is arguably the most frequently overlooked component of a fitness program, but that doesn't mean it isn't important. Even a few minutes of stretching can offer big benefits. Try the following short routine at the end of your next workout for a whole-body stretch...
If you're new to exercise, or if it's been some time since you performed resistance-training exercise, here is a quick review of some basic—but very effective—upper-body exercises. And if you've already mastered the basics, options are given to increase the challenge.
If you want to make exercise a regular habit, joining a health club could be just what you need to achieve your goal. Here are seven things to consider if you find yourself in the market for joining a health club this year.
ACE Pro Homepage
Copyright © 2015 The American Council on Exercise. All Rights Reserved.4851 Paramount Drive, San Diego, California 92123 1 (888)-825-3636


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Contact EPA Pacific Southwest Water Division
Pacific Southwest, Region 9
Serving: Arizona, California, Hawaii, Nevada, Pacific Islands, Tribal Nations
NPDES Wastewater & Stormwater Permits
|NPDES Quick Finder
|NPDES Home Compliance & Enforcement State Programs
|NEPA Projects Guidance
|Permits and Public Notices Pretreatment
|Sewage Sludge (biosolids) Stormwater
Region 9 Draft NPDES Permits and Public Notices
National Information
You will need Adobe Reader to view some of the files on this page. See EPA's PDF page to learn more about PDF, and for a link to the free Adobe Reader.
This page lists Public Notices for Draft Permits for all areas where EPA Region 9 is the permitting authority. EPA Region 9 is the permitting authority for Tribal lands in Arizona, California, Nevada, and also all Navajo lands; the Pacific Island territories of Guam, American Samoa, Commonwealth of the Northern Mariana Islands; and any discharges into federal ocean waters beyond state boundaries. This page includes permits currently open for public comment as well as permits for which the public comment period has ended.
For permits that EPA has issued, visit Region 9 NPDES permits page.
Arizona, California, Hawaii and Nevada issue NPDES permits for discharges on lands (other than Tribal lands) within those states. State-issued permits and related information are accessible through state program websites.
Public Notices and Draft Permits Open for Comment
|Permit #
|Permit Name / Location
Tristar Guam F-1 Pier Terminal
|Public Notice (PDF) (1 pp 117K)
Comment Period Closes: April 5, 2015
Draft Fact Sheet (PDF) (10 pp, 173K)
Draft Permit (PDF) (38 pp, 903K)
Tristar Guam Agat Terminal
Public Notice (PDF) (1 pp 117K)
RGI Inc. dba Goulding's Lodge Wastewater Treatment Facility
|Public Notice (PDF) (1 pp, 59K)
Comment Period Closes: April 18, 2015
Draft Fact Sheet (PDF) (11 pp, 252K)
Draft Permit (PDF) (14 pp, 207K)
Baza Gardens Sewage Treatment Plant
|Public Notice (PDF) (1 pp 11)
Comment Period Closes: April 19, 2015
Draft Fact Sheet (PDF) (31 pp, 545k)
Draft Permit (PDF) (43 pp, 1.4MB)
Umatac-Merizo Wastewater Treatment Plant
|Public Notice (PDF) (1 pp 11K)
Comment Period Closes: April 19, 2015
Draft Fact Sheet (PDF) (13 pp, 468K)
Draft Pyermit (PDF) (41 pp, 1MB)
Public Notices for Which Comment Period has Ended
|Pacific Southwest NewsroomPacific Southwest Programs
|Grants & FundingUS-Mexico Border
|Media CenterCareers
|About EPA Pacific SouthwestA-Z Index


text:
Play The Game Here! -->>> http://www.roblox.com/Slender-Mans-Revenge-Port-Early-Access-place?id=201055875
[Updates: Added a new GUI in which you can control the sounds playing during the game. You can turn off and on Crickets, etc)
[/e Commands: split, russian, flap, zombie, relax, leap, noob]
{SCARY WARNING} -Blood, Gore, Jump Scares, Scary Scenes!
{SEIZURE WARNING} -Flashy Lights, Quick Movements!
Play At Own Risk!
{CREEPYPASTAS} -Slender, Maxwell!
(All CreepyPastas that have nothing to do with Slender, aren't going to be in this game.)
{MODERATORS}-anthonygerman30, grunt295, maddog08, Goku543210
{INTRO MUSIC: http://www.roblox.com/Work-It-Tavi-Boosted-Bass-item?id=187873176
==The Official Slender Man's Revenge ROBLOX Game==
==Created By Puzzled Productions==
Share with your friends
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ROBLOX, "Online Building Toy", characters, logos, names, and all related indicia are trademarks of ROBLOX Corporation, ©2015. Patents pending.
ROBLOX is not sponsored, authorized or endorsed by any producer of plastic building bricks, including The LEGO Group, MEGA Brands, and K'Nex, and no resemblance to the products of these companies is intended. Use of this site signifies your acceptance of the Terms and Conditions.
ROBLOX is now loading. Get ready to play!
You're moments away from getting into the game!
Check Remember my choice and click in the dialog box above to join games faster in the future!


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Change Location
ACE Fit Share
Fit Life Articles
Are you struggling to stick with your New Year's resolutions? If so, it might be time to recruit the help of your significant other. After all, the key to behavior change is making your goals as attainable as possible, so having the support of your partner is an important...
Injuries occur at any time and for any reason. If you get injured, you may not know how to proceed and prepare for exercise post-injury. This mind-and-body guide can help prepare you for a brighter, injury-free future in fitness and performance.
If you're just getting started with a fitness routine, it can be a challenge to know how to properly perform exercises for the various muscles of the body. Here are four introductory lower-body movements for beginners—once you've mastered these exercises you can continue...
ACE Pro Homepage
Copyright © 2015 The American Council on Exercise. All Rights Reserved.4851 Paramount Drive, San Diego, California 92123 1 (888)-825-3636


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Home > Accessories > Office Supplies > Labels > Office Labels
Avery Laser White Address and Shipping Labels
Usually ships in 5-7 business days. Limited availability. May not be in stock at time of order. No back orders.
Avery Dennison is the leading supplier of office products. It makes a wide variety of products for the office and home, including labels, binders, dividers, cards, pens and pencils, highlighters and markers, and more. Avery Dennison makes things easier for you in other ways, too.
Product Information
Mfr Part#
Label Specifications
Paper Size
3 1/3" x 4"
6-labels per sheet, 100-sheets per pack, 600 total labels
Printer Type
For Laser Printers
Paper Color
Manufacturer Warranty
1 Year
1 Year
Important Add-Ons
Questions and Answers
More Info
You may be business savvy, but what about your mailings? These White Shipping Labels with TrueBlock® Technology save time by allowing you to include both sender and recipient information on one label. The process is quite clever: Fill in a free template from avery.com. No need to adjust margins or spaces—just enter names, addresses, company logos and barcodes and print with your laser printer. And only Avery offers bright white labels with TrueBlock® Technology to cover any markings underneath. Soon you'll have pages of professional-looking labels that completely cover everything underneath, and print without a jam or smudge in sight. Now you have a mailing solution that's as smart as you are.
Product Views
Warranty & Support
Vendor Warranty:
- Parts: 1 Year
- Labor: 1 Year
Vendor Support:
- Main Website: www.avery.com/avery/en_us/
- Support Website: www.avery.com/avery/en_us/Help-Center
- Support Phone #: 888-835-8379
Micro Center Return Policy:
We guarantee your satisfaction on every product we sell with a full refund — and you won't even need a receipt.* We want you to be satisfied with your Micro Center purchase. However, if you need help or need to return an item, we're here for you!
If an item you have purchased from us is not working as expected, please visit one of our in-store Knowledge Experts for free help, where they can solve your problem or even exchange the item for a product that better suits your needs.
If you need to return an item, simply bring it back to any Micro Center store for a full refund or exchange.
*If you are a Micro Center Insider or if you have provided us with validated contact information (name, address, email address), you won't even need your receipt.
Desktop / notebook computers, tablets, processors, motherboards, digital cameras, camcorders and projectors, and CD/DVD duplicators may be returned within 15 days of purchase. All other products may be returned within 30 days of purchase. Merchandise must be in new condition, with original carton/UPC, and all packaging/accessories/materials. If you need to return software, a game or a movie, remember that you may not retain any copies — it's not legal, and it's not nice.Refunds will be credited to the credit card or debit card account used for the original purchase.
Micro Center Support:
|Free Walk-in Tech Support
|As a service to our customers, Micro Center offers free in-store technical support. Stop in any of our store locations during regular business hours, and our expert staff will answer your computer questions or help you with issues regarding:
|Support from Home or Office
If you need help with hardware or software products purchased from Micro Center, please contact one of our knowledgeable tech support reps by live chat or email.
|Free assistance is available for the first 30 days on new purchases, excluding internal hardware installations or networking support. Your invoice reference number will be required for free assistance.
We accept the following credit cards: Micro Center VISA® credit card with WorldPoints® Rewards; VISA; MasterCard; American Express; Novus/Discover.
|Learn more about our exclusive Priority Care Program. You'll have access to our in-house Technical Support team of certified technology professionals trained to assist you on a variety of PC and Mac related support issues.


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Member Only Content
By Date
AICPA News Update Vol. 14 No. 40
Free Webinar: XBRL for Practitioners
Published on September 30, 2011
AICPA News Update - April 27, 2012
In this issue: AICPA Praises House Passage of Bill Requiring XBRL Use, International Panel Meets at AICPA to Discuss the Future of Accounting Standards, Regulatory Alert: Upcoming PCAOB Forums on Auditing Smaller Broker-Dealers, Comment Period on AICPA Accounting and...
Published on April 27, 2012
AICPA News Update Vol. 14 No. 46
Free Webinar Offering CPE: Understand FAF's Proposal on Private Company Financial Reporting and the AICPA's Response ASEC Extends Comment Period on XBRL Exposure Draft to November 30 AICPA Releases White Paper on Fair Value Measurement for NFPs AICPA Accounting Competition Semi-finalist Teams Announced Catch up
Published on October 28, 2011
AICPA News Update Vol.12 No.31
IRS Extends FBAR Filing Deadline for Certain Persons, Biggest Changes to Compilation and Review Services Since 1978 May Be Ahead; Hear What's Proposed, Follow XBRL from Birth to Present; AICPA Releases Chronology, AICPA Named Green Plus Mover by Institute for Sustainable Developmen, Ethics Rules Revisions to Be Proposed Later this
Published on June 24, 2011
AICPA News Update Vol.12 No.20
Non-SEC Issuer Entities Subject to SEC Independence Rules, Supreme Court Agrees to Hear Appeal Challenging Constitutionality of PCAOB, SOP 09-1 Issued on Performing AUP Engagements Addressing XBRL-Tagged Data, CPA Financial Executives, CFOs Cautiously Optimistic on Economy's Future, AICPA Survey: CPAs Gaining IFRS Knowledge but Want SEC to Allow More Time
Published on June 24, 2011
AICPA News Update - January 22, 2010
January 22, 2010: Filing Essentials for this Year's Tax Season; Provide Help to Haiti through AICPA's CPAs in Support of America Fund; Read AICPA Confirmation Letter to SEC Staff on Day 2 Loan Accounting; CPAs in Government Alerted to Ways to Combat Fraud, Waste and Abuse; FASB Seeking Nominations for
Published on March 08, 2010
AICPA News Update Vol. 13 No. 46
IRS Announces Return Preparer Office; AICPA Annual Report Available
Published on October 29, 2010
AICPA News Update Vol.12 No.40
Enterprise Risk Management Extends to Board of Directors, AICPA Issues Working Draft of Updated Audit and Accounting Guide, Entities With Oil and Gas Producing Activities, CPA Firm Identity Theft Prevention Program Guidance Released, Resources Available for CPAs to Prepare for H1N1 Outbreaks, Presentation Prepared to Recruit Accounting Students for Work in Government,
Published on June 24, 2011
AICPA News Update Vol.12 No.28
Vote on Consumer Financial Protection Agency Delayed Until September; AICPA Joins Chamber of Commerce and Others Urging More Time, AICPA, GAA Host Roundtable for Accounting Leaders to Discuss Simplifying Financial Reporting, What Government Entities Need to Know about American Reinvestment and Recovery Act of 2009; New Member Resource Online for
Published on June 24, 2011
AICPA News Update Vol. 13 No. 42
Member Ballots Due October 8; Mail Today
Published on October 01, 2010


text:
- Description
DetailsAndis UltraEdge Hair Clipper Blade Size 50 64185
Whether you are looking to replace clippers blades or add to your current collection, you've come to the right place. Big Daddy Beauty carries a full line of dog clipper blades, including many chrome-plated, carbon-edged Andis UltraEdge Hair Clipper Blade, such as this Andis UltraEdge Hair Clipper Blade Size 50 64185.
This size 50 blade cuts hair to 1/125" (0.2mm) and is used for extremely close cutting for show poodles.
Shop Big Daddy Beauty today for a whole host of Andis products, including hair clippers, accessories and even blades for dog clippers.
Manufacturer: AndisSKU: ANDIS-64185Weight: 0.25 lbs100% Guaranteed Authentic
- Additional Information
Additional Information
Ingredients N/A Ingredients
- Reviews


text:
Put cod into a skillet or saucepan.
Cover fish with cold water.
Cover skillet.
Bring water to simmering over low heat and simmer 1 minute.
Repeat process once or twice depending on saltiness of fish.
Simmer in last water until fish is cooked.
Allow about 10 minutes cooking time per inch thickness of fish.
Put cooked cod into a greased baking dish and pour milk over cod.
Bake at 350°F 10 minutes, or until milk is hot.


text:
Baby Gear
Baby Carriers, Car Seat Accessories, Car Seats & Booster Seats, more...
Sign up & be the first to hear about exclusive offers,
new arrivals & more.
We're sorry, this product is no longer available!
Shop for other products in Premium Dog Treats
Ingredients: Turkey, Rice Flour, Maple Syrup, Potato Protein, Glycerin, Barley Flour, Potato Starch, Guar Gum, Cane Molasses, Carrots, Peas, Salt, Natural Smoke Flavor, Phosphoric Acid, Sorbic Acid (natural preservative), Mixed Tocopherols.
Crude Protein 15.0% minimum
Crude Fat 9.0% minimum
Crude Fiber 2.0% maximum
Moisture 30.0% maximum


text:
Staying Informed--Getting Weather Warnings
In today's digital world, weather warnings can be received by NOAA Weather Radio, email, cell phones, internet, commercial radio and television. It is best to use multiple means of getting weather warnings.
NOAA Weather Radio is broadcast on frequencies ranging from 162.40 MHz to 162.55 MHz. This frequency range is just above the normal FM radio band. Special NOAA weather radio receivers can be purchased. Many multi band radios and scanners also include the NOAA weather radio frequencies. You can check for your nearest transmitter at weather.gov/nwr.
Email alerts: You can subscribe to email or text message weather alerts through a variety of providers listed on the internet.
Cell phones: The commercial mobile alert system is new and allows cell phone users to get warnings for severe thunderstorms, tornadoes and flash floods on their phones. Contact your cell phone company for specific details.
Internet: Warnings are available using graphics and text at weather.gov.and also from many commercial internet weather providers.
Commercial Radio and Television use the Emergency Alert System (EAS): to receive and relay weather warnings from the National Weather Service and all hazards information from government officials. The EAS system is tested on a weekly and monthly basis. The National Weather Service also conducts tests on the NOAA weather radio usually on Wednesday morning. Listen for the tests so that you recognize what an EAS activation sounds like. When a real activation takes place, you will be able to immediately recognize the EAS sound.
This week is severe weather awareness week in the Pacific Northwest. Now is the time to get prepared for thunderstorms, tornadoes and flash floods.
Additional information can be found on the web at:
US Dept of Commerce
National Oceanic and Atmospheric Administration
National Weather Service
Portland Weather Forecast Office
5241 NE 122nd Avenue
Portland, OR 97230-1089
Tel: (503) 261-9246


text:
When I use phpMyAdmin to see my online tables, it uses collation method "latin_swedish_ce". Why? What is the default? And, for what collation method is used?
A string in MySQL has a character set and a collation. Utf8 is a character set, and utf8_bin is one of its collations. The collation determines how MySQL compares strings.
For example, here's two ways to compare a UTF8 string:
The first uses the utf8_bin collation, which is case sensitive. The second uses utf8_roman_ci, which is case insensitive.
A list of all character sets and collations can be found with:
The collation is used when you compare strings, e.g. for sorting them. For example, in Swedish the letter "Ä" comes after "Z", but in German it is treated like "AE". So, depending on the collation, strings are sorted differently.
Since MySQL comes from Sweden, Swedish is the default.
You can find more information in MySQL Reference Manual.
In general this collation is considered a reasonable default for any not-too-fussy application using the ISO-8859-1 (Western European) character set. It makes most diacriticals insignificant in a comparison:
however having said that it does have a few quirks specific to Swedish:
See the gory details for more.
Of course these days one would hope you'd be using one of the
Do you mean
It is used when performing sorting/comparison.
To change it, here's the reference:


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Lovitz, Miller replacing Ritter and Winkler in legiter
Jon Lovitz and Larry Miller are skedded to make their Broadway debuts in Neil Simon's "The Dinner Party" at the Music Box Theatre on June 12.Lovitz, who starred on "Saturday Night Live" for five seasons, replaces Henry Winkler in the comedy, with Miller taking over for John Ritter. Miller had recurring roles on "Mad About You" and "Law & Order." Both Ritter and Winkler have been with the show since its world premiere in Los Angeles at the Mark Taper Forum in December 1999. The production then played the Kennedy Center in summer 2000 before opening in Gotham on Oct. 19. Lovitz and Miller will join original Broadway cast members Len Cariou, Penny Fuller, Veanne Cox and Jan Maxwell.
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As the Head of Global Benefits Strategy for Airbnb, you will: Develop and lead an RFP for a Global Benefits Broker Lead the design, implementation, and hands on management of global benefits for all countries Airbnb employees work, helping Airbnb maintain its competitive1/27/2015 San Francisco, CA Save This Job
Location : Woodlawn MD US 21207 Job Type : Full Time Shift/Hours : First Shift (Day) Career Level : Experienced (Non-Manager) Education : Bachelor's Degree Skill : Marketing -> Graphic Design Specialist, Graphics & Desktop Publishing Category : IT/Software Development Job3/17/2015 Woodlawn, MD Save This Job
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Providence, RI Full Time Client Services Experienced SHARE ShapeUp is looking for an experienced account management professional to foster its continued growth. This is an exciting opportunity to manage and support a subset of ShapeUp?s clients. This is a dynamic role3/17/2015 Providence, RI Save This Job
Location:Mid-Atlantic Region (Eastern PA, MD, DC of VA) Position Description The Regional Sales Engineer is an indoor and outdoor network design and product specialist that serves in a customer facing role. The RSE?s primary responsibility is to help ensure that ExteNet is2/7/2015 PA Save This Job
Location:CA-Pleasanton Job Code:799 Description Ellie Mae (NYSE:ELLI) is a leading provider of enterprise on-demand solutions, including an online network, software and services for the residential mortgage industry. Ellie Mae is leading the mortgage industry into the future,3/25/2015 Pleasanton, CA Save This Job
Manager, Business Services At LegalZoom, we?re passionate about our products and services because we know they help customers achieve their dreams. The Core and Special Filings Manager will oversee the daily operation of LegalZoom?s Business Formation Services Filing and Core3/10/2015 Austin, TX Save This Job
Location: (Multiple states)Type: Full TimeMin. Experience: Experienced InTouch Health is the leader in robotic telehealth solutions for healthcare. We are looking for an eager, self-starting Senior Region Account Manager to join our team! This role is responsible for supporting3/4/2015 Santa Barbara, CA Save This Job
Location: (Multiple states)Type: Full TimeMin. Experience: Experienced InTouch Health is the leader in robotic telehealth solutions for healthcare. We are looking for an eager, self-starting Senior Region Account Manager to join our team! This role is responsible for supporting3/4/2015 Santa Barbara, CA Save This Job
This role is responsible for supporting and managing all client needs in pre-sale activities and after contract closure. This includes but is not limited to; customer needs analysis, program development, leading the implementation process and ongoing project management. The2/2/2015 Santa Barbara, CA Save This Job
Information Technology San Jose, CA Send Jobvite Network and application security specialist providing direction to the help desk group. This technical team lead will interface with the Cyber Security and Information Technology groups to ensure business solutions are secure and2/21/2015 San Jose, CA Save This Job
Location: Minneapolis Department: Clinical Services Reports to: Manager, Coaching Services What is RedBrick Health? RedBrick Health is a health technology and services company founded in 2006 and based in Minneapolis. With half of health care costs driven by behavior, the best2/11/2015 Minneapolis, MN Save This Job
Job Description Senior Business System Analyst - (00679) Description Location: Belcamp, MD Summary: Under general direction responsible for the design, analysis, documentation, development, implementation and maintenance of the Oracle eBusiness Suite environment version3/27/2015 Belcamp, MD Save This Job
Job ID 2015-2306 Location US-NC-Morrisville Category Professional Services Type Regular Full-Time More information about this job: Overview: SciQuest is the largest publicly-held provider of Software-as-a-Service (SaaS) procure-to-pay technology. We?re on a Quest to develop3/19/2015 Morrisville, NC Save This Job
The Position Wellcentive is looking for experienced, client facing Implementation Managers in Atlanta, GA, Dallas, TX or New York, NY. As health systems are leveraging more and more disparate technologies to improve their clinical and financial outcomes; they are turning to1/31/2015 Roswell, GA Save This Job


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Interview: Alex Mustard at the Natural History Museum
Wetpixel Associate Editor Alex Mustard was interviewed by Charlotte Coales in January about his underwater photography and his winning images in the 2013 Wildlife Photographer Of The Year competition. The presentation was a question and answer session held in the Attenborough Studio at the Natural History Museum, London.
Reminder: DEEP Indonesia 2014
If you haven't done so yet, just a reminder that you have 4 days left to enter the 2014 DEEP Indonesia Contest. Hosted by Wetpixel and DivePhotoGuide, DEEP is, together with Our World Underwater, the pinnacle of underwater competitions.
Coral reefs come to life in underwater time lapse video
Underwater time lapse photography can tell scientists a lot about what happens on a coral reef. Over the past five years Dr. Pim Bongaerts of the University of Queensland's Global Change Institute has used time lapse photography to document coral reefs.
Bluefin Tuna underwater by Brian Skerry
National Geographic photo of the day for March 3, 2014 is a stunning capture of a wide mouthed bluefin tuna by NG photographer Brian Skerry. The photo is released in tandem with an article on bluefin tuna in the print magazine this month.
CM Products releases polecam
CM Products has started shipping their new pole cam set up. The pole can be disassembled to fit into most carrying cases. The pole's head angle can be adjusted and will fit most housings. The polecam is normally supplied with a triggering system, which can also be used a remote trigger off the pole.
Call for entries: Nudibranch Photo Competition 2014
The Nudibranch Photo Competition 2014 (NPC) is calling for entries. The contest has been founded to celebrate nudibranchs and their allies and there are just two simple categories in this inaugural contest: British Nudibranchs and Nudibranchs of the Rest of the World.
Video: California Dreaming by Howard Hall
Filmmakers Howard and Michelle Hall have re-released a short film filmed entirely within 100 miles of Los Angeles. It was originally made: "To present at a Reef Check fund raiser a few years ago. The event was in Los Angeles and Reef Check wanted to feature California marine life."
Tutorial: Erin Quigley on the Adjustment brush in LR4
Lightroom guru and underwater photographer Erin Quigley has reposted one of her Lightroom tutorials about using the Adjustment brush in Lightroom 4. Using the brush is fundamentally the same in Lightroom 5, so the advice is current.
Recent News
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- Issue 27 of African Diver is now available to download (via African Diver)
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- Think Tank launches the "About a Photography" video… (via )


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Christmas Cookie Gift Tags
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Making the Dough
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Three days before the Day of Absolute Silence, the most sacred relics of each temple for miles around are carried to the beach for purification and cleansing accompanied by thunderous, almost cacophonic gamelan music with many devotees wailing in a trance state and ceremonially pressing the points of their sharp long swords into their chests. Colorful banners and parasols fill the sky and one has the impression one is in some war movie of Kurosawa
Asia is the biggest continent on Earth, a darling little gem floating around in space.Hm, what is Asia? Who's in on it?China and India are safe bets for Asian nations. Korea, Japan, Thailand, you're fine.Europe? No. Europe would sort of be on the "Asian continent" if not for those pesky Ural mountains dividing things up in the middle, and then also the whole lineage of kings and wealth and nations and the EU and all that "give me my respect" stuff.Russia would probably be happiest as its own continent, so for now we'll leave it in "Eurasia" and just hope the natural gas supplies keep flowing.Pakistan and Afghanistan are dang close to Asia, but politically they show up in "Middle East" news stories an awful lot. Verdict: Eurasia.I guess we'll actually have to consider everything from Turkey on eastward to be "Eurasian", although the moniker seems overlappitory of the territory.The Asian economy is now officially raging like a wild furnace of lava that consumes everything in its path. Japan has had the largest individual economy in Asia for decades, but it is forecast that both India and China will outstrip Japan within twenty years.China is the largest holder of United States debt and is positioned to become the world's next superpower, provided that Godzilla doesn't return and decide to stomp everybody back into the Shang Dynasty.Text by Steve Smith.


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About Calypto
Calypto® Design Systems family of products enables ASIC, SOC and FPGA designers to quickly create fully-verified, power-optimized RTL for downstream synthesis and physical design. With Calypto's CatapultTM High-Level Synthesis products, designers have the option of using SystemC or C++ to dramatically shorten the design cycle by producing correct-by-construction, error-free, PPA optimized RTL. Calypto's PowerProTM product line enables users to analyze both static and dynamic power usage at RTL and either automatically or manually create a power optimized RTL that includes memory and leakage power optimization. The SLECTM family of products formally verifies the complete RTL without the need for time consuming simulation and complex testbenches. The end result is dramatic reduction in time to market with up to 60% reduction in power usage.
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|*High Level Synthesis Report 2014
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This is Calypto's sixth annual High Level Synthesis survey report. 750 SoC, IC, and FPGA design professionals responded to the survey. This year's emphasis is on verification.
|*Closing Functional and Structural Coverage on RTL Generated by High-Level Synthesis
|White Paper
Most hardware design teams have a verification methodology that requires a deep understanding of the RTL to reach their verification goals, but this type of methodology is difficult to apply to the machine generated RTL from High-level Synthesis (HLS). This paper describes innovative techniques to use with existing methodologies, for example the Universal Verification Methodology (UVM), to close functional and structural coverage on HLS generated code.
|*Clock Domain Crossing Aware Sequential Clock Gating
|White Paper
In this paper, we present an algorithm to handle CDC violations as part of the objective function for sequential clock gating optimizations. With the proposed algorithm, we have obtained an average of 22% sequential power savings — this is within 3% of the power savings obtained by the CDC unaware sequential clock gating. In comparison, the state-of-the-art twopass solution is leading to an almost complete loss of power savings.
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Enjoy natural, farm-to-table foods in their purest forms. Vitamix helps you create delicious meals while enjoying the benefits of whole, uncooked ingredients.
The TurboBlend VS includes a filtration bag and raw recipes.
Citrus can be as delicious as it is healthy when paired with certain dishes.
read more
You will be pleasantly surprised at the selection of raw food recipes that will satisfy the hibernator in you.


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Does anyone know an online store where I can get some love temperature controllers models tcs, tss2, etc. and if you bought from them or more important if they are reliable
I want to do son DYI projects like a fermentation chamber and try to automate the burners of the HLT and MLT, and if you know were I can find some schematics, diagrams, examples, let me now the URL.
Not sure if they are available from any other retailer but you can get them straight from the manufacturer here...http://www.dwyer-inst.com/Products/P...eName=Ordering
A few retailers carry some models, but Dwyer obviously has the full line up. I had some issues with the shipping charges/methods but overall transaction was ok.
Most of what you seek is on HBT, you need to search & read.
If you order from Dwyer, beware the fine print regarding "wait to ship consolidated items" etc... I got charged 3 times for shipping and had to raise issue (which they corrected, thanks dwyer). Order everything you need at once and don't think "I'll get that later". Also, I wish I'd gotten the 10' probes instead of the 5' for an extra $. Had to extend them.
S/H on an extra $6 probe if you need one that weighs .5 oz will be $14.50. That's right.
Just know that the price you "confirm" does not have Shipping charges added. You get a "bill" in the mail with that added after the fact. Certainly an industrial supply house sort of customer service, not as much a retail one.
Alternatively, you can buy probes from someone like brewershardware.com.
I'm going to give a thumbs up with Sanke. They treated me right.
Cole Parmer is who I got mine from. Just beware, you will receive 700lb catalogs several times throughout the year. They make great fire starters though.
Regarding sensors/probes for a Love -
Digi-Key has PTC Thermistors for 90 cents - and they charge about 2 bucks to put them into a padded envelope and mail them to you. (1000 ohms at 25C)
Solder one to a little speaker wire the length you want, heat-shrink each leg for electrical isolation, drop it into a piece of brass hobby tubing, epoxy both ends, voila!
here's the LINK to the DigiKey page - part # 568-1003-ND
hi there just recive my love tss2 controller, i ordered from grainger.com, super fast purchase, highly recomended.
Although I will say this re: pricing. (my experience, anyways)
It was interesting- the difference in pricing between Dwyer direct and Cole Parmer. The prices for the TSS2 itself was higher from Cole and lower on the TS2. The probes were much higher. But when calculating in the S/H from Dwyer vs Cole, they were within one or two dollars. Just shed's a light on the business model. So, obviously if I'm gonna order 2500 units, it's better to go direct from Dwyer, but one or two... it's either I guess. Guess Dwyer plumps up the wholesale price to distributors.
The other oddity was lead time from being out of stock (meaning when Dwyer is gonna send them more units) is lower priority than Dwyer having some to sell internally. I was told OOS from Cole Parmer w/ 1 mo lead time a few mo ago, and in stock a week earlier at Dwyer. Could be reading too much into that, but I thought it was interesting.


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Preguntas Para El Examen De Manejo En Espanol in Massachusetts
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Massachusetts Suspended Drivers License Information and Procedures | DMV.orgSuspended Massachusetts drivers license - find all of the information you need for reinstating your suspended MA drivers license.
Massachusetts DMV Locations, MA DMV Hours, Phone Numbers, & RMV Appointments | DMV.org: The DMV Made SimpleMassachusetts RMV Locations & Hours - Find Massachusetts RMV Office Phone Numbers, Locations, Hours, & Appointments.
MA Online Driver License Prep Course - Guide to Written License and Permit TestsPrepare For Your Massachusetts Driver's License Written Exam With An Online Prep Course. This One-Hour Class Covers Everything You Need To Know To Pass Your CDL, Motorcycle, Driver License Or Permit Test.
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Motor Vehicle Reports (MVR) and Car Insurance | DMV.orgMotor vehicle reports, or driving records, are used to determine your car insurance rates. Learn more about what's in your record.
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County Government Websites in TexasFind Texas County Websites .
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Student Housing Forms
All forms can be filled out and submitted entirely online.
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Housing assignments for the Spring, Summer, and Fall 2015 quarters will be sent via email on the following dates:
Summer 2015: May 1st
Fall 2015: July 15th
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Summer Tour Ticketing Onsale Dates And Times
Tickets go sale to the general public for Phish's 2015 Summer Tour beginning tomorrow, Thursday, April 2nd at 10am ET and continuing into the weekend. For complete tour and ticketing information click here. A list of public on sale dates and times are listed below. Phish returns to Watkins Glen International in Watkins Glen, NY


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Is Painful Urination A Sign Of HIV
Here's a taste of what TheBody.com has to offer on this topic:
Get the Facts
Caring for Your Gut: Dealing With Diarrhea, Nausea and Other Stomach Problems if You Have HIV/AIDS... Table of Contents Gut Basics The Gut and HIV Diarrhea and HIV Coping With Diarrhea Nausea, Vomiting and Anorexia Gas, Bloating and Heartburn Taking Care of Yourself Gut Basics The gut includes the stomach, small intestine, large intestine, rectum,
Ask the Experts
The kidneysI was just reading a question to you about kidney issues. I was wondering what the signs of problems are. I've read one sign is if your urine gets really foamy. I've only noticed my urine a little bit foamy when I first pee in the morning or when...
Response from Keith Henry, M.D.
University of Minnesota, School of Medicine...Key indicators of kidney problems in the setting of HIV Infection include abnormal blood tests (increases in Creatinine and BUN levels and decrease in calculated GFR). Protein or glucose Read more »
HIV and Herpes III have been tested positive for Herpes I and II but negative for HIV 5 months after a high risk encounter. However I have never shown any signs of Herpes II, no sores on penis and no pain during urination. On the other hand I am showing signs of HIV...
Response from Ryan M. Kull, C.S.W
Columbia University's Health & Related Services...Testing for antibodies to herpes virus and HIV are two distinct processes. Testing positive for herpes in no way suggests that you are HIV infected.It is important that you Read more »
Sometimes being careful is not enoughDear Dr.Bob,I think it's time for me to go and get tested. Being only 20, and having a wonderful educational life with a bright future I can't believe that I've been so stupid in the last 8 months. I've slept with my ex-girlfriend several times and...
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation..."being careful" is defined as having safer sex by properly using latex condoms, then it may very well be "enough" to prevent HIV and a host of other STDs!To specifically address your questions:1.Burning during urination can indeed be a sign of an... Read more »
Negative HIV Test Trumps Symptoms Everytime, But Why Do I Still Have The Symptoms?Hi Dr.How are you? I want to tell you that you are the best!My question is, when you say that a negative hiv test trumps symptoms everytime, why do I still have symptoms?I took a 6 month test after a possible exposure (received unprotected oral sex...
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation...six-month HIV test, I can with great certainty tell you what's not causing them: It's not HIV. No way. No how.If your symptoms are painful urination, dribbling, pain in the bladder and low back, and you know your prostate feels "boggy," why are... Read more »
Caring for Your Liver if You Have HIV/AIDS... Table of Contents Liver Basics Symptoms of Liver Damage Tests for Liver Damage The Liver and HIV Liver Disease and Women Caring for Your Liver Taking Care of Yourself Liver Basics Your liver is one of the Read more »
Crixivan...diarrhea, stomach pains, loss of appetite, yellowing of skin/eyes, changed skin color, dry mouth/sore throat, taste changes, painful urination, indigestion, joint pain, hives, and liver toxicity. Itchy/dry skin, ingrown toe nails, and hair loss... Read more »
How to Spot HIV/AIDS Fraud... What Is HIV/AIDS Fraud? Warning Signs of an HIV Fraud Common Sense About Unproven Treatments Questions You Should Ask Take Your Read more »
Peripheral Neuropathy and HIV/AIDS...Have PN? PN Treatments Finding Support References What Is Peripheral Neuropathy? Approximately 60 percent of HIV+ people will develop problems that involvethe nervous system. The nervous system controls thinking, movement,... Read more »
Connect With Others
why am I still STRESSED, your opinions please
Posted by Anonymous...masterbation, which I did not finish as I realised I was not gay) I became convinced that he had precum on his fingers which contained hiv , I convinced myself that I had contracted hiv via exposure through my uretha which he accidently touched I... Read more »
some symptoms, time frame question
Posted by Anonymous, 2 Replies...exposure about a week and a half ago. Shortly after I experienced a discharge and pain during urination. I main concern is HIV. About 7-8 days later I started to feel very slugish with a bit of a sore throat. I have had mono and feel very... Read more »
some symptoms, time frame question
Posted by Anonymous, 1 Reply...exposure about a week and a half ago. Shortly after I experienced a discharge and pain during urination. I main concern is HIV. About 7-8 days later I started to feel very slugish with a bit of a sore throat. I have had mono and feel very... Read more »
8 days...
Posted by Anonymous, 1 Reply...recently had unprotected sex with a prostitute in Argentina. (I know Im an idiot) About 3 days later I experienced drainage and painful urination. The day I had the sympotoms I started taking Penicillin. I just got home, and have been feeling... Read more »
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