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Interstitial pneumonia is a frequent and critical manifestation of human cytomegalovirus (CMV) disease in immunocompromised patients, in particular in recipients of bone marrow transplantation. Previous work in the murine CMV infection model has identified the lungs as a major organ site of CMV latency and recurrence. It was open to question whether the viral genome is transcriptionally silent or active during latency. Transcription could be latency associated and thus be part of the latency phenotype. Alternatively, transcriptional activity could reflect episodes of reactivation. We demonstrate here that transcription of the immediate-early (IE) transcription unitie1-ie3 selectively generatesie1-specific transcripts during latency. Notably, while the latent viral DNA was found to be evenly distributed in the lungs, transcription was focal and randomly distributed. This finding indicates that IE transcription is not a feature inherent to murine CMV latency but rather reflects foci of primordial reactivation. However, this reactivation did not initiate productive infection, sinceie3 gene mRNA specifying the essential transactivator IE3 of murine CMV early gene expression was not detectable. Accordingly, transcripts encoding gB were absent during latency. By contrast, during induced virus recurrence, IE-phase transcription switched from focal to generalized and ie3-specific transcripts were generated. These data imply that latency and recurrence are regulated not only at the IE promoter-enhancer and that there exists an additional checkpoint at the level of precursor RNA splicing. We propose that focal transcription reflects random episodes of nonproductive reactivation that get terminated before IE3 is expressed and ignites the productive cycle.
Latent infection with human cytomegalovirus (CMV) entails a risk of virus recurrence and consequent multiple-organ CMV disease in immunocompromised patients, in particular in patients who have undergone hematoablative conditioning for bone marrow (BM) transplantation (BMT) (8, 51). There is a dual risk. First, latent CMV present in the donor can be transmitted with the BM cells (BMC) transplanted or with blood products and can reactivate, or become reactivated, leading to productive infection in the recipient. This implies that BMC and/or blood cells are a source of latent CMV, and there is indeed a lot of evidence for human CMV latency in hematopoietic myeloid lineage progenitors (11, 23, 27, 29, 32) and mature progeny thereof, monocytes and macrophages in particular (45, 49, 50). In major histocompatibility complex (MHC)-mismatched BMT, a graft-versus-host response may facilitate human CMV recurrence from a particular type of macrophage that expresses dendritic cell markers (45). Second, latent CMV present in the recipient can give rise to recurrent infection within organs of the recipient. That this occurs became evident from the high incidence of human CMV infection in CMV-seropositive latent CMV carriers receiving a transplant from a CMV-negative donor (reviewed in reference 4). While current research on human CMV latency is focused on latency in hematopoietic cell types, there are good arguments for the existence of a second type of latency of CMV residing in stromal and/or parenchymal cells of various tissues. First, recurrence occurs in the carriers of latent CMV after the depletion of hematopoietic cell types. Second, latent CMV is transmitted most efficiently by solid organ transplantation (6, 10), actually more efficiently than by BMT (4).
The existence of at least two independent cellular sites of CMV latency is also suggested by findings in the model of murine CMV latency. Recent work by Pollock et al. has documented murine CMV latency in peritoneal exudate macrophages (35), thereby corroborating more indirect earlier conclusions (5). Whereas spleen tissue explant cultures are a long-established method of eliciting recurrence of murine CMV (17), the latently infected cell type in the spleen has remained enigmatic. Since the spleen is rich in macrophages, they are candidates for harboring latent murine CMV. However, the latent genome as well as the potential to reactivate in explant cultures was found to copurify with class II MHC-negative stromal cells (30, 37), most likely sinusoidal endothelial cells (30). Likewise, renal peritubular endothelial cells were discussed as a cellular site of latency in the kidney (21). More recently, Koffron et al. have presented histological data supporting the view that murine CMV may become latent in macrophages and endothelial cells (22).
The problem of the cellular sites of human CMV and murine CMV latency also affects the studies on potential transcriptional activity during latency, since the molecular state and activity of the latent viral genome are likely to be influenced by the cell type. Some of the discrepant data in the literature may thus not represent controversial findings but might rather relate to a heterogeneity in the latently infected cell types. Evidence for a latency-specific transcription of human CMV was provided by Kondo et al. for the example of latency in granulocyte-macrophage progenitors. Latency-associated transcripts from the regulatory immediate-early (IE) region differed from the IE transcripts of the productive phase. A class of sense transcripts was found to use alternative start sites in the human CMV IE1/IE2 promoter-enhancer, while a second class was transcribed in the antisense direction (23, 24).
Analogous transcripts are not known for murine CMV. The occasional detection of murine CMV ie1 gene transcript sequences in the reverse transcriptase (RT) PCR analysis of organ RNA derived from operationally-defined latent mice (12, 52) was not interpreted as due to latency-associated transcription but as an indication of a persisting productive infection at a level below the detection limit of conventional infectivity assays (52).
We have shown previously that latent murine CMV DNA is harbored in a series of organs, including the salivary glands, adrenal glands, spleen, heart, kidney, and lungs (40), with a particularly high load of latent viral genome in the lungs (3, 40, 47). Notably, this "tissue latency" of murine CMV was found several months after clearance of viral DNA from BM and blood (2, 3, 25). This fact made it evident that blood leukocytes were not the source of latent CMV during that advanced stage of latency and supported the hypothesis that a stromal and/or parenchymal site of latency must exist.
Here we have employed this model to study the transcriptional activity of murine CMV in the stromal-parenchymal type of latency in the lungs.
Experimental BMT and infection.For sex-mismatched male-into-female BMT, male and female mice of the inbred strain BALB/c (MHC haplotype H-2d) were used at the age of 8 weeks as BMC donors and recipients, respectively. Hematoablative conditioning of the recipients was performed by total-body γ-irradiation with a single, sublethal dose of 6 Gy from a γ-137Cs source (OB58; Buchler, Braunschweig, Germany). Cell suspensions of donor femoral and tibial BMC were obtained by flushing medium through the bone shafts, and contaminating vascular and sinusoidal CD8 T cells were depleted as described previously (47). BMT was performed by infusion of 5 × 106 donor BMC into the tail vein of each recipient at ca. 6 h after the irradiation. Infection with 105 PFU of purified murine CMV, strain Smith ATCC VR-194/1981, was performed by subcutaneous injection at the left hind footpad at ca. 2 h after BMT (25).
High-sensitivity assay of viral infectivity.Viral latency in the lungs was verified, and virus recurrence was monitored, by the recently described RT PCR-based focus expansion assay (25). In essence, monolayers of permissive fetal mouse cells, previously often referred to as mouse "embryo fibroblasts," were inoculated with 2 ml of freeze-thawed and homogenized tissue, representing a 1/18 aliquot of the whole lungs, under the influence of a 1,000 ×g centrifugal force. Specifically, for each individual mouse tested, 2-ml homogenates were prepared from nine pieces derived from the left lung and the postcaval lobe. After a culture period of 72 h, which is equivalent to ca. three rounds of murine CMV replication in the indicator cells, poly(A)+ RNA was isolated and analyzed for the presence of viral IE1 transcripts by ie1 exon 3-exon 4 (exon 3/4)-specific RT PCR. As we have shown in great detail in a previous report (25), this assay revealed a genome-to-infectivity ratio of ca. 5:1 for purified murine CMV and consistently detected infectivity in lung homogenates supplemented with 0.05 PFU of murine CMV, which equals five infectious genomes. It is important to emphasize that all of the homogenate was plated in order to avoid a sampling error. Latency studies were performed at 1 year after primary infection, which was at least 6 months after the resolution of acute infection in organs, including the long-persisting productive infection in the salivary glands (2, 25, 40). Recurrence was monitored by the detection of infectious virus and of productive cycle IE3 transcripts on day 14 after immunoablative treatment by 6.5 Gy total-body γ-irradiation.
Isolation of DNA.Total DNA, cellular and viral, was isolated from whole blood and from BMC.
(i) Isolation of DNA from blood.Blood was taken by heart puncture (3) and was transferred immediately for DNA extraction into the extraction buffer of the NucleoSpin blood kit (Macherey & Nagel, Düren, Germany). The DNA isolation was performed according to the instructions in the supplier's method manual. In brief, for each preparation, a 200-μl sample of whole blood was incubated for 10 min at 70°C with 200 μl of extraction buffer and 0.4 mg of freshly added proteinase K. DNA was precipitated with ethanol and separated from the soluble phase by centrifugation in NucleoSpin columns, followed by extensive washing with ethanol-containing buffers. The purified DNA was eluted from the NucleoSpin column matrix by incubation with 100 μl of elution buffer at 70°C for 10 min, followed by centrifugation.
(ii) Isolation of DNA from BMC.Femoral and tibial BMC were resuspended in phosphate-buffered saline (5 × 106cells in a total volume of 200 μl) and digested for 2 h at 56°C with proteinase K. After addition of 200 μl of extraction buffer, incubation was continued for 10 min at 70°C. DNA was extracted by using the NucleoSpin method described above.
Simultaneous isolation of DNA and poly(A)+ RNA from lung tissue.Pieces of lung tissue, specifically from the superior, middle, and inferior lobes of the lungs, were dissected and were shock-frozen in liquid nitrogen. DNA as well as poly(A)+ RNA of individual pieces, each of which represented ca. 1/18 of the whole lungs, was isolated in parallel as described previously (25). Particular care was taken to reduce the time spent between sacrifice of latently infected mice and shock-freezing of tissue pieces in order to minimize the risk of postmortem transcriptional reactivation. Specifically, animals were sacrificed one-by-one, lungs were excised immediately and cut into pieces, and pieces were transferred without delay into vials containing liquid nitrogen. With some skill, this can be managed within 5 min, or within 10 min at the most. Nonetheless, it is understood that the argument that postmortem reactivation occurs can never be refuted with certainty. It is also understood that pieces cut so as to be equal in size are never absolutely equal. Therefore, after transfer of the frozen pieces into the extraction buffer, vials were weighed, and weight differences were later accounted for by appropriate adjustments of the DNA or RNA aliquots included in the assays. For the sake of clarity, we refer to a 1/18 fraction of the lungs as a "piece"; as the mouse lungs consist of ca. 60 × 106 tissue cells (25), one piece contains ca. 3.3 × 106lung cells. The average yields of cellular DNA and poly(A)+RNA per piece were found to be ca. 20 μg and ca. 2 μg, respectively.
Quantitation of viral DNA.The amount of purified organ DNA was determined from the optical density measured at 260 nm. The cellular DNA in hematopoietic cells and their mature progeny, that is, in BMC and blood cells, was quantitated in the male-into-female BMT chimeras by a PCR specific for the male sex (testes)-determining genetdy as described previously (48). The number of viral genomes present in total DNA was quantitated by diluting the DNA serially, followed by performing a PCR specific for a 363-bp sequence within exon 4 of the IE gene ie1 of murine CMV, essentially as described in previous reports (3, 47) but with further improvements. Specifically, PCR reactions were here performed in 96-well microplates in the GeneAmp PCR System 9700 thermocycler (Perkin-Elmer, Foster City, Calif.). Amplification was achieved in 30 cycles with denaturation at 95°C for 3 min in the first cycle and at 96°C for 30 s in the remaining 29 cycles, primer annealing at 58°C for 1 min, and primer extension at 72°C for 1 min with extension to 6 min in the last cycle. Cellular DNA derived from the respective specimens of uninfected mice was supplemented with defined copy numbers of plasmid pIE111 (31), which encompasses genesie1 and ie3, and was subjected to theie1 gene-specific PCR to serve as a standard in the quantification. Amplification products (20 μl thereof) were vacuum dot blotted onto nylon membrane, hybridized with a γ-32P-end-labeled internal oligonucleotide probe, and analyzed quantitatively by digital phosphorimaging as described previously (47).
Construction of recombinant plasmids for in vitro transcription.The map locations of the synthetic RNAs are illustrated in Fig. 1. Recombinant plasmids containing coding sequences of the murine CMV genes ie1, ie3, and gB were constructed as described below. For construction of pSP64 Poly(A)-ie1, a 2.1-kb EcoRI fragment containing the complete ie1 open reading frame (19) was excised from plasmid p62/3 (7), and was cloned blunt end into the SmaI site of vector pSP64 Poly(A) (GenBank/EMBL accession no. X65328 ; cat. no. P1241, Promega, Madison, Wis.). The resulting plasmid, pSP64 Poly(A)-ie1, thus includes the complete ie1 open reading frame (exons 2, 3, and 4 of the ie1-ie3 transcription unit) starting at a synthetic EcoRI site located 21 bp upstream of the initiation codon and ending at the AvaI site at the beginning of exon 5 of ie3 (31). Synthetic RNA transcribed from this plasmid has a length of 2.17 kb, including a 30-nucleotide poly(A) tail. For construction of pBlue-ie3,the vector pBluescript (pBluescript II KS(+) phagemid vector, cat. no. 212207; Stratagene, Heidelberg, Germany) was digested withEcoRV and Xba I, and a part of the ie3sequence was inserted as a 985-bp EcoRV-XbaI fragment derived from plasmid pie3 (31).EcoRV cleaves in intron 1, 58 nucleotides upstream of the ATG located in exon 2 of the ie1-ie3 transcription unit (19), and XbaI cleaves at position 534 of exon 5 (GenBank accession no. M77846 ). The resulting plasmid, pBlue-ie3, thus includes exon 2, intron 2, exon 3, and part of exon 5. Synthetic RNA transcribed from this plasmid has a length of 1.045 kb and lacks a poly(A) tail. For construction of pSP64 Poly(A)-gB, part of the open reading frame of thegB gene was amplified by PCR from plasmid pACYC-gB (38) by using oligonucleotides 5′-(CAGAATTC)AAGGACTACATCAACGATGC and 5′-(CAGAGCTC)GCGGAACGACTTGTAAAAAG as forward and reverse primers, respectively (38) (GenBank accession no. M86302 ). The amplification product was digested with SacI and inserted into the SacI site of vector pSP64 Poly(A). The resulting plasmid, pSP64 Poly(A)-gB, thus includes thegB sequence from position 1895 to position 3154 (38). Synthetic RNA transcribed from this plasmid has a length of 1.34 kb and includes a 30-nucleotide poly(A) tail.
In vitro synthesis of viral transcripts.Synthetic transcripts of the murine CMV genes ie1, ie3, andgB were prepared according to the instructions in the Promega technical manual. Plasmids pSP64 Poly(A)-ie1 and pSP64 Poly(A)-gB were linearized with EcoRI, and transcription was performed with RNA polymerase SP6 (Promega). Plasmid pBlue-ie3 was cut with XbaI, and transcription was performed with RNA polymerase T3 (Promega). Specifically, after complete linearization, plasmid DNA was purified by phenol-chloroform extraction followed by ethanol precipitation. The in vitro transcription reactions were carried out at 37°C for 2 h in a total volume of 0.1 ml, containing 2 μg of plasmid DNA, 40 U of the corresponding RNA polymerase, 100 U of RNasin, 0.5 mM of each ribonucleoside triphosphate, 10 mM dithiothreitol, 40 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 2 mM spermidine, and 10 mM NaCl. Transcripts were separated by vertical low-melting agarose (1.2%, wt/vol) gel electrophoresis. Bands were cut out, the gel was melted at 65°C, and RNA was isolated by phenol-chloroform extraction and precipitation with ethanol. The RNA was dissolved in water, and contaminating plasmid DNA was degraded by using RQ1 RNase-free DNase (M6101; Promega). The reaction was performed at 37°C for 2 h in a volume of 0.1 ml containing 4 U of the RQ1 DNase, 100 U of RNasin, 10 mM dithiothreitol, 40 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 2 mM spermidine, and 10 mM NaCl. To remove trace amounts of residual DNA, extraction was performed twice with acidic phenol. In detail, 400 μl of distilled water, 50 μl of 2.5 M potassium acetate, and 500 μl of water-saturated phenol were added to the reaction volume. After the mixture was swirled with a vortex apparatus, 100 μl of chloroform was added and the mixture was incubated for 5 min on ice. After centrifugation, this procedure was repeated, and RNA was extracted with chloroform followed by precipitation with ethanol. Finally, the purified RNA was dissolved in water, and its concentration was determined.
Analysis and quantitation of transcripts.Poly(A)+ RNA was isolated from the shock-frozen lung tissue pieces (see above) by an oligo(dT)-cellulose affinity method as described previously (1, 25). Transcripts of the viral genesie1, ie3, and gB were detected by specific RT PCRs (for a map, see Fig. 1). The reactions were carried out as described previously except that they were performed in 96-well plates in a GeneAmp 5700 thermocycler (Perkin-Elmer) and that the cDNAs were amplified in 33 cycles. In the previously describedie1-specific RT PCR (25), the probe directed against the exon 3/4 splicing junction was elongated by six nucleotides downstream in order to improve the specificity (the new probe is referred to as IE1-P). The length of the IE1-specific amplificate is 188 bp. Primers and probe for an RT PCR specific for the cellular transcript of hypoxanthine phosphoribosyltransferase (HPRT), yielding a fragment of 163 bp, have also been specified in the previous report (25). For the detection of the ie3 gene transcript, the oligonucleotide (5′) nucleotide (nt) 3445 to 3426 (located in exon 5, with the counting beginning at the 5′ transcription start site of the ie1-ie3 transcription unit) (31) was used as the RT primer as well as the reverse primer in the PCR (IE3-RT/R), the oligonucleotide (5′) nt 1361 to 1380 (exon 3, shared by ie1 and ie3) served as the forward primer (IE1/3-F), and oligonucleotide (5′) nt 1511 to 1524 (exon 3) 3379 to 3396 (exon 5) served as the probe directed against the exon 3/5 splicing junction (probe IE3-P). The fragment length of the correct amplificate is 229 bp, while an amplificate from contaminating viral DNA can readily be identified by its length of 2,085 bp. In case of a comparative quantitation of IE1 and IE3 transcripts, the exon 3 specific oligonucleotide 5′nt 1392 to 1418 was used as a common probe (probe IE1/3-P) in order to minimize the variables. The gBgene transcript was reverse transcribed by using an oligo(dT) primer. The resulting cDNA was amplified by using the oligonucleotide (5′) nt 83010 to 83029 as the forward primer (gB-F), (5′) nt 83415 to 83396 as the reverse primer (gB-R), and (5′) nt 83127 to 83146 as the probe (gB-P) (39) (GenBank accession no. MCU68299 [complete genome]). Since gB lacks introns, amplificates derived from RNA and DNA cannot be distinguished by size. Reactions performed in the absence of the enzyme RT were therefore used to verify the specific detection of RNA. Amplification products were analyzed by gel electrophoresis, Southern blotting, and hybridization with the corresponding γ-32P-end-labeled internal probe, followed by autoradiography. The radioactivity was measured by digital phosphorimaging. Defined numbers of in vitro-synthesized transcripts served as standards for quantifications.
Frequency estimation of transcriptional foci in the lungs.The frequency of transcriptional foci of murine CMV in latently infected lungs was estimated from the fraction of transcriptionally negative lung pieces, f(0), by using the Poisson distribution equation λ = −ln f(0). The fraction of pieces containing n (n = 0, 1, 2, 3, and so on) foci,F, was calculated by using the formula F(n) = λn × e−λ/n!. The estimate was made for 45 pieces derived from the superior, middle, and inferior lobes of five latently infected lungs and was then extrapolated to 18 pieces of a complete "statistical lung." For the upper estimate, neighboring positive pieces were all regarded as being individually positive. For the lower estimate, neighboring positive pieces were counted as only one positive piece assuming a single transcriptional focus located at the border between the two pieces.
Rapid exhaustion of the bone marrow pool of murine CMV DNA.After experimental BMT and concurrent murine CMV infection, prevention of lethal multiple-organ CMV disease and clearance of productive infection depend on the efficacy of lymphohematopoietic CD8 T-cell reconstitution (33, 47). Under experimental conditions that permit survival, infectious virus is cleared from organs but viral genome is retained in a nonreplicative state referred to as molecular latency (25, 36). Based on site-specific differences in the efficacy of antiviral control, molecular latency can be established in a particular organ, while productive infection persists in other organs. Typically, in the model used here and introduced previously (25), molecular latency is established after 1 to 2 months in the spleen, after 3 to 4 months in the lungs, and after 5 to 6 months in the salivary glands. Thus, by 6 months after BMT and primary infection, productive infection is resolved and viral latency is established at all potential organ sites. As we have documented previously (25) and reproduced here, viral DNA is retained in blood cells for an extended period of time after the resolution of productive infection in organs, but the viral DNA load in the blood declines progressively and viral DNA is eventually also cleared (Fig.2A). For human CMV, latently infected hematopoietic progenitor cells in the BM are currently discussed as a source of latently infected progeny in the blood (11, 45). Notably, for murine CMV, the viral genome load in the BM is much lower than is the load in the blood, and clearance from the BM precedes clearance from the blood by several months (Fig. 2B). It is pertinent to our work that all subsequent experiments and findings pertain to a parenchymal or stromal type of murine CMV latency present in the lungs many months after the clearance of viral DNA from BM and blood.
Mosaic approach to the analysis of pulmonary latency of murine CMV.Assays for viral infectivity are technically not compatible with the molecular analysis of DNA load and in vivo viral transcriptional activity. We have therefore used the "mosaic approach" illustrated in Fig. 3. The superior lobe, middle lobe, and inferior lobe of the lungs were cut into nine pieces, designated piece #1 to piece #9, which were used for the simultaneous analysis of latent viral DNA load and viral transcription in each piece. The postcaval lobe and left lung were cut into nine pieces, designated piece #10 to piece #18, which were used to verify latency by the absence of infectious virus. This mosaic approach allows one to discuss results in the context of their topographical relations in the organ. Furthermore, a statistical evaluation is greatly facilitated by the lobular anatomy of the lungs that limits the number of neighboring pieces to only one "neighbor" in the case of 10 pieces and to two "neighbors" in the case of the remaining 8 pieces.
Verification of murine CMV latency in the lungs.All assays were performed at 12 months after BMT and infection, that is, at a time when productive primary infection was resolved in all organs (25) and when, as documented in Fig. 2, viral DNA was cleared from the BM and blood. However, these general precautions did not exclude a productive infection that may result from a spontaneous recurrence, possibly occurring by chance at the time of analysis, in an individual piece of lung tissue. We therefore tested for the presence of infectious virus in lung pieces #11 to #18. An example for an individual mouse designated LM1 (latent mouse 1) is shown in Fig. 3A, and all other latently infected mice were checked in the same manner. The previously described RT PCR-based focus expansion assay detects infectivity with very high sensitivity (25). This was documented again by supplementing the homogenate of piece #10 with 0.05 PFU of purified murine CMV, which equals ca. 25 viral genomes. As shown in detail previously, as few as 5 genomes are required for initiating infection in an indicator culture of permissive cells, but 25 genomes are needed to avoid negative results for the cultures due to the Poisson statistics of the sampling (25). Infection in this positive control culture was indicated by a large amount of amplificate obtained after ie1 exon 3/4-specific RT PCR performed with poly(A)+ RNA isolated from the indicator culture after 72 h of viral replication. By contrast, no signal was obtained from the indicator cultures inoculated with the homogenates of pieces #11 to #18. Mice LM2 to LM5 included (results not shown) 40 lung pieces tested altogether, and none contained infectious virus.
Focal transcriptional activity in latently infected lungs.Based on the absence of infectious virus, the presence of productive-phase transcripts, such as the ie1 gene-derived IE1 transcript, was not expected, and transcriptional analysis was originally performed with the intention of documenting the absence of such transcripts as a further argument in favor of molecular murine CMV latency in the lungs. It was therefore a surprise to find IE1 transcripts among the poly(A)+ RNAs isolated directly from the nine pieces of the right lung lobes, as is documented for mouse LM1 in Fig. 3B. Notably, an on-or-off pattern was seen, in this particular example with five positive pieces and four virtually negative pieces. That RNA was present in all samples and in comparable amounts was verified by amplification of transcripts specified by the cellular gene coding for HPRT. The detection limit in this assay, as determined by titration of in vitro-synthesized IE1 transcripts, was ca. 10 molecules. Since an aliquot containing 1/10 of the poly(A)+RNA yield from each piece of tissue, ca. 200 ng, was included in the RT PCR, positive pieces contained >100 IE1 transcripts in total. In order to improve the sensitivity and to lower the detection limit, negative pieces #1, #4, #5, and #7 were retested with 7/10 of the poly(A)+ RNA yield (Fig. 2C). Since these four samples were again negative without exception, the corresponding tissue pieces must have contained <10 transcripts. We infer from this finding that the difference between positive pieces and negative pieces may indeed be a qualitative difference. It is understood that the numbers of transcripts represent minimum estimates and depend upon the efficacy of the isolation procedure. After supplementing of lung tissue homogenate from uninfected mice with defined numbers of either in vitro-synthesized IE1 transcripts or natural IE1 transcripts derived from productively infected lungs, the retrieval after passage through the oligo(dT) column was found to be 50% or better in either case. Because a factor of two is not meaningful in this type of assay, calculated numbers of transcripts are reported herein with no correction for isolation efficacy. Thus, a positive score is given for >100 transcripts and a negative score is given for <10 transcripts calculated per piece, with each piece representing ca. 1/18 fraction of the lungs. While the possibility that transcription occurred at a level below the limit of detection can never be excluded, significant quantitative differences between the data for the lung tissue pieces were obvious. In conclusion, the observed mosaic pattern of transcription in the latently infected lungs suggests the existence of "foci" of IE1 transcription. For clarity it should be emphasized that the physical correlate of such a focus is yet to be defined. It could represent a single cell with a detectable number of transcripts or a group of cells collectively having a detectable number of transcripts. Thus, the term focus is used here to indicate a detectable transcriptional event.
Latent viral DNA is contained in all parts of the lungs.The question arose whether the focal transcriptional activity was related to an accordingly focal distribution of the latent viral genomes. Specifically, absence of latent viral DNA in a particular piece could be a trivial explanation for the absence of IE1 transcripts. Viral DNA was therefore quantitated in lung tissue pieces #1 to #9 of mouse LM1 (Fig. 4), that is, in the very same pieces for which the transcriptional activity had been determined (Fig.3B). This was possible because DNA and RNA were isolated in parallel. All pieces contained latent viral DNA in comparable amounts. Specifically, the transcriptionally silent pieces, #1, #4, #5, and #7, did not contain less viral DNA than did the transcriptionally active pieces. In fact, the pieces with the lowest and highest DNA load, namely piece #1 and piece #7, respectively, both belonged to the group of transcriptionally silent pieces. Likewise, the high transcriptional activity found in piece #2 did not correspond to an accordingly high load of latent viral DNA. Quantitation by phosphorimaging revealed an average load of 7,500 viral genomes per 106 lung tissue cells (6 μg of cellular DNA), with a lower value of 6,000 viral genomes (for piece #1) and an upper value of 9,000 viral genomes (for piece #7). In essence, these differences in the latent viral DNA load between the pieces are minor, if at all significant, and we conclude that the total latent viral DNA load in a particular piece does not determine its transcriptional status. On the basis of the chosen resolution of the analysis, namely the mosaic of 18 pieces per lung, the latent viral DNA appeared to be evenly distributed. To avoid a misinterpretation, it should be emphasized that at higher resolution the latent viral DNA could still form hot spots critical for the initiation of transcription. The answer to this question awaits further analysis.
Foci of transcriptional activity are randomly distributed in latently infected lungs.In order to verify that mouse LM1 was not peculiar with regard to transcription during latency, transcriptional activity was tested in the same manner in mice LM2 through LM5, and the analysis for mouse LM1 was repeated with the remaining 1/10 aliquot of the poly(A)+ RNA preparations (Fig.5). In essence, the pattern was reproduced for mouse LM1, and on-or-off patterns were observed in like manner for all lungs analyzed. Topographical maps of the lung lobes illustrate the distributions of transcriptionally active and silent tissue pieces (Fig. 5, right). For mouse LM1, the particular distribution might have suggested a spread of transcriptional activity in the three lobes from the proximal portion to the distal portion relative to the bronchial tree. However, this interpretation was quickly proven wrong by the patterns observed in the other four mice. Actually, no rule can be recognized, and we hence conclude that viral transcriptional activity is randomly distributed in latently infected lungs.
Frequency of transcriptional foci in a statistically generated prototypic latent lung.By definition, silent pieces do not harbor a transcriptional focus, whereas transcriptionally active pieces must harbor at least one focus but may also contain more than one focus. The Poisson distribution statistics can be employed to estimate the frequency of transcriptional foci from the fraction of silent pieces among all pieces and to calculate the fractions of pieces containing one, two, three, or more foci. The calculations were made for the 45 pieces of the five lungs as shown in Fig. 5, and the results were then extrapolated to 18 pieces of a complete "prototypic latent lung" (Fig. 6). A problem arises from the fact that a focus may be located by chance right at the experimental cut border between two pieces, which could lead to an overestimation of the number of transcriptionally positive pieces. We thus arrived at an upper estimate by assuming that neighboring positive pieces are always both positive (Fig. 6, left). The other extreme, and actually the less likely one in our opinion, is to assume that two neighboring positive pieces always have only one focus. This gives us the lower estimate (Fig. 6, right). In conclusion, the statistical calculations show us that latently infected lungs, under the specific conditions of our experimental setting, contain 7 to 15 foci of transcription and that the number of foci per positive piece is unlikely to exceed three.
Quantitation of ie1-specific transcripts in latently infected lungs.The signal intensities were found to differ markedly between transcriptionally active pieces of lung tissue (Fig.5). This might reflect different numbers of foci per piece and/or different numbers of transcripts per focus. Since, as outlined above, the Poisson distribution statistics predicted an occupancy of positive pieces by one, two, or three but not by more foci of transcription, we classified positive pieces into those with low, intermediate, and high transcriptional activity (Fig. 5). Pools were formed for the three so defined classes, and the number of transcripts for each pool was measured by titration of poly(A)+ RNA followed byie1 exon 3/4-specific RT PCR, specific hybridization, and phosphorimaging (Fig. 7). In vitro-synthesized IE1 transcripts were titrated to serve as a standard for the quantitation. If the amounts of transcripts were determined only by the number of foci per piece, we would expect proportions of 1:2:3 for the three pools. However, the actually observed proportions were on the order of ca. 1:10:100. As a consequence, we must then conclude that the amount of transcripts is primarily determined by the number of transcripts per focus, thus suggesting a heterogeneity in the transcriptional activity per focus.
High ie1-specific transcriptional activity is not indicative of productive cycle reactivation.Even though all tissue pieces derived from the corresponding left lungs and postcaval lobes were negative in the high-sensitivity infectivity assay (Fig. 3), one might still surmise that transcriptional activity indicates a low-level persistent productive infection at a level below the detection limit of infectivity assays, an interpretation favored by other authors previously (52). The pieces containing more than 10,000 IE1 transcripts, that is, piece #5 of mouse LM4 and piece #5 of mouse LM5 (Fig. 5 and pool 3 in Fig. 7), are those which are most likely to be implicated in either persistent or recurrent productive infection. However, IE1 transcripts are not a proper indicator for initiation of the viral productive cycle. Whereas the IE1 protein contributes to the transactivation of early-phase genes, the main and essential transactivator of murine CMV is the 88-kDa protein IE3 (31), which corresponds to the 86-kDa IE2 protein of human CMV (13; for a review see reference46). Accordingly, IE3 transcripts as well as transcripts specifying a virion structural protein, such as gB (38), should be present in persistent productive infection or intermittent recurrence. RT PCRs specific for IE1, IE3, and gB transcripts detected the corresponding in vitro-synthesized transcripts with comparable sensitivities. Specifically, in each case, 10 transcripts could clearly be visualized (Fig.8, left). Whereas ca. 12,000 IE1 transcripts were again detected within the poly(A)+ RNA of the high-activity pool, pool 3, IE3 transcripts and gB transcripts were virtually absent (Fig. 8, right). To improve the sensitivity of detection, the remaining 1.4 μg of the pool 3 poly(A)+RNA was subjected to the ie3-specific RT PCR, and it was found to be negative (not shown). Based on the detection limit of eight transcripts (in a log2 titration of the standard), we conclude that the number of IE3 transcripts, if they did exist at all, was less than the number of IE1 transcripts by at least a factor of 1,000. Altogether, sensitive analyses of IE3 and gB transcripts did not give any hint of or evidence for persistent or recurrent productive infection.
Experimental induction of recurrence is associated with detectable IE3 transcripts.While the comparison between IE1 transcripts and IE3 transcripts did not provide evidence in favor of a productive cycle transcription, the observed great difference per se cannot be viewed as evidence against it. IE1 transcripts are known to be abundant and fairly stable during productive infection (18, 19). The virtual absence of IE3 transcripts during latency could thus result from an overall low level of transcriptional activity during latency in combination with a generally low abundance and/or stability of the IE3 mRNA. It is important to recall that the ie1 andie3 genes are transcribed as a transcription unit and that mRNAs are generated from a 5.1-kb IE1/3 precursor transcript by differential splicing connecting exons 1 through 3 with exon 4 for IE1 mRNA and with exon 5 for IE3 mRNA (19, 31) (illustrated in Fig. 1). It was therefore of relevance to determine the IE1 transcript: IE3 transcript ratio during productive infection. Specifically, a switch from latent nonproductive infection to recurrent productive infection should involve a significant rate of production of IE3 mRNA specifying the essential transactivator IE3. As members of our group have documented repeatedly in previous reports, rapid recurrence of infectious virus from latently infected lungs can be detected after total-body γ-irradiation (3, 25, 40, 47), and recurrence of murine CMV after other regimens of immunosuppressive treatment is a long-recognized phenomenon in this field (reviewed in reference16). We have therefore used our model of radiation-facilitated recurrence for analyzing the IE-phase transcription during reinitiation of productive infection (Fig.9). Successful induction of recurrence is documented for a representative case by the RT PCR-based focus expansion assay of infectivity, performed on day 14 after γ-irradiation with a dose of 6.5 Gy (Fig. 9A). It is worth noting that infectious virus can be found in individual pieces as early as day 4 after the irradiation, which indicates an immediate entry of latent murine CMV into the productive cycle (not shown). By day 14, infectious virus was detected in all nine tissue pieces derived from the left lung and the postcaval lobe (i.e., piece #10 through piece #18). Since none of 40 lung tissue pieces collected from five mice had been found to be positive during latency (Fig. 3), the statistical significance of the recurrence is undebatable. Tissue pieces #1 through #9 of the remaining three lobes of the lungs were used to detect IE1 and IE3 transcripts (Fig. 9B). Regarding IE1, the latency-associated on-or-off pattern of focal transcriptional activity was now replaced by the presence of a large amount of transcripts in all nine pieces. Notably, IE3 transcripts became detectable under these conditions, and, as was the case for IE1 transcripts, all nine pieces were positive.
Recurrence is characterized by an increased rate ofie3-specific splicing.Nonetheless, one could still surmise that detection of IE3 transcripts reflected merely an enhanced level of overall viral transcriptional activity during recurrence. We therefore pooled the poly(A)+ RNA of the nine pieces and quantitated IE1 and IE3 transcripts by direct comparison with the corresponding in vitro-synthesized RNAs that served as standards (Fig.10). As one would expect, the total amount of IE1 transcripts in the lungs was increased during recurrence. Notably, however, the average number of IE1 transcripts per piece, ca. 15,000 molecules, did not significantly exceed the number of IE1 transcripts determined during latency in the high-activity pool; pool 3 (compare Fig. 7 and Fig. 10, left), in which IE3 transcripts were not detectable at all (Fig. 8). By contrast, during recurrence, the average number of IE3 transcripts was calculated to be ca. 300 molecules per piece of lung tissue (Fig. 10, right). In other words, the IE1 transcript: IE3 transcript ratio was ca. 50 during recurrence and >1,000 during latency, which clearly indicates a change in the program of IE1/3 precursor transcript splicing.
In conclusion, murine CMV latency in the lungs is associated with a focal, randomly distributed generation of IE1 transcripts in the absence of productive cycle transcripts, while recurrence is associated with enhanced generation of IE3 transcripts.
While most current concepts focus on human CMV and murine CMV latency in hematopoietic cells of the myeloid differentiation lineage, our data on murine CMV latency have reconfirmed our previous conclusions regarding the significance of multiple-organ tissue latency (40), in particular of latency in the lungs (3, 25), a major organ site of CMV pathogenesis and immune control (14, 43). Specifically, at a time after acute infection of BMT recipients when the level of viral DNA in BM and blood had declined to below the detection limit (<100 copies per 106 cells), the lungs still harbored the latent viral genome, with a load of 6,000 to 9,000 copies per 106 lung cells. The maintenance of latent CMV in organs after exhaustion of the putative source in BM and blood does not formally exclude the possibility that the origin of latently infected cells in the lungs is hematopoietic lineage, but we must then postulate that the latently infected cells accumulate in organs, are extremely long-lived, and are not replaced during natural turnover by the uninfected cells delivered by the BM and circulating in the blood. While our data do not argue against the existence of a hematopoietic and leukocytic reservoir of latent CMV for a significant and most probably clinically meaningful period, we favor the interpretation that cells of the myeloid lineage are only a temporary site of CMV latency and that long-lived stromal cells are the cellular site of enduring latency in organs. The kinetics of viral DNA clearance from BM and blood indicates that the viral DNA load in the blood results primarily from the productive infection and not from exported progeny of latently infected hematopoietic progenitor cells residing in the BM. Throughout the course of the kinetics, the load in the BM was always far below the load in the blood. One may argue that a few hematopoietic progenitor cells in the BM can give rise to a huge number of progeny and that this will also lead to an amplification of the latent viral DNA. However, although true in an absolute sense, this argument ignores the fact that the uninfected hematopoietic progenitor cells also produce progeny. One might raise the interesting argument that DNA derived from the uninfected cells of the erythroid differentiation lineage may dilute the viral DNA signal in the BM, while latent viral DNA is relatively enriched in blood leukocytes due to the anuclear status of erythrocytes. However, although erythrocytes by far outnumber leukocytes in peripheral blood, the maintenance pool of erythroid-lineage cells in the BM is about the size of the myelomonocytic-lineage cell pool. We have shown this recently by a comparative histochemical quantitation of myelomonocytic and erythroid colony cells in repopulating BM after experimental BMT (48), and much earlier studies have determined a myeloid:erythroid ratio of ca. 1.5:1 for femoral BM of adult mice (26). DNA derived from erythroid-lineage cells may thus dilute the viral DNA in BM by twofold at the most, but this certainly cannot account for the observed viral load differences between BM and blood. Therefore, unless we assume that latently infected cells gain a growth advantage or that the copy number of latent viral DNA markedly increases during cell differentiation, although presently there is no evidence for this, the relative load in the blood cannot exceed the relative load in the putative BM source. In fact, our data do not exclude the possibility that the load determined for the BM resulted mainly, if not exclusively, from blood cells present in the BM capillaries and sinusoids. We have previously shown by immunohistological detection of viral IE1 protein as well as by in situ hybridization specific for viral DNA that murine CMV does indeed infect reticular stromal cells in the hematopoietic cord; however, even during acute, lethal infection of mice, the number of infected cells in the BM stroma was minute and most infected cells did not complete the productive cycle (28). The recently discussed idea that a persistent productive infection in the BM stroma may be the source from which myeloid progenitor cells or monocytes and macrophages acquire CMV (35, 45) is thus not supported by the data obtained in the murine-model system. Considering the high load of latent viral DNA in various organs, we rather propose that macrophages pick up the latent CMV genome in tissues by phagocytosis of latently infected, damaged or senescent stromal or parenchymal cells. We must admit, of course, that our arguments apply to murine CMV and that human CMV in general, or isolates with a particular cell tropism (44), may differ in this respect. For resolving this question, a quantitative and longitudinal analysis of the human CMV genome load is needed. Only a comparative study of the kinetics of the viral DNA load in the BM, blood, and organs of BMT patients will allow us to evaluate the relative contributions of myeloid-lineage cells and of tissue stromal cells to human CMV latency.
Transcriptional activity of latent human CMV has been analyzed in a model of latency established in cultures of in vitro-infected granulocyte-macrophage progenitors (23, 24). These cells do not support productive infection, but they do carry the viral DNA. Two classes of novel transcripts derived from the regulatoryie1-ie2 gene region, namely sense and antisense latency-associated transcripts, were detected in a small percentage of the cells. While infectious virus was recovered from these cultures after cocultivation with permissive cells, it remained open to question whether recurrence originated from the transcriptionally active minority or from the transcriptionally silent majority of the latently infected cells. Notably, the latency-associated transcripts could also be detected in myeloid-lineage-committed progenitor cells derived from latently infected, healthy individuals (11, 24).
In models of in vivo latency of murine CMV, ie1-specific sequences derived from spliced RNA were detected by RT PCR by some investigators (12, 52) but not by others (22). Absence of IE1 transcripts was taken as an argument in favor of molecular latency (22), whereas presence of IE1 transcripts was interpreted as being indicative of a persistent productive infection at a low level, one below the detection limit of then-available conventional infectivity assays (52). However, the assumption of low-level productive infection was not supported by any further evidence, and more recent studies employing much-improved assays for testing viral infectivity have corroborated the conclusion that murine CMV does establish molecular latency characterized by absence of infectivity (25, 36).
Data presented herein can explain these controversial previous findings. We have documented that latently infected lungs can be viewed as a mosaic, with transcriptionally active and silent pieces coexisting and randomly distributed in the very same organ at the very same time. Whereas the frequency of transcriptionally active foci was quite high, 7 to 15 foci per lung, infectious virus could not be detected with the previously described high-sensitivity focus expansion assay capable of detecting 0.01 PFU (25). Thus, apparently, IE1 transcripts are not necessarily indicative of productive infection. The IE1 protein pp89 of murine CMV (20) is not the transactivator of viral early gene expression, and its human CMV counterpart, the IE1 protein p72, is not essential for viral replication (9). Transcripts from the murine ie3 gene, which is the counterpart of the human CMV ie2 gene (13, 46) and encodes the IE3 transactivator (31), are therefore a much better marker for the initiation of the productive cycle. While IE3 transcripts were clearly detected during experimentally induced recurrent infection of the lungs, these transcripts, as well as transcripts specifying the structural glycoprotein gB, were not detectable during murine CMV latency in the lungs, not even in those individual pieces of tissue that contained an amount of IE1 transcripts comparable to the average amount present during recurrent infection. Taken together, these data demonstrated a switch in the transcriptional and splicing program from latency associated to recurrence associated and thus represent the strongest evidence presented to date against a low-level persistence of productive infection during murine CMV latency in organs. The fact that the ie1 and ie3 genes are transcribed as a transcription unit has a very important implication. While an on-position of the major IE promoter-enhancer allowing for IE1/3 precursor transcription is certainly the primary condition for recurrence to occur, a subsequent regulation at the level of splicing appears to be critical for the generation of the essential IE3 transactivator that starts the productive cycle.
So far, the small absolute amount of IE1 transcripts in the lungs has precluded their precise identification. Determination of whether the authentic, pp89-encoding 2.75-kb transcript (19) is produced at all must await further studies. An immunohistological analysis failed to detect intranuclear pp89 protein, even though hundreds of sections from latently infected lungs were screened (data not shown). We have evidence that a fraction of the ie1-specific transcripts is deficient in exon 2-encoded sequences (unpublished data). We are thus faced with a mixture of transcripts, a fact that further complicates their identification. It is worth noting that alternative start sites have not been identified upstream of theie1-ie3 transcription start site of murine CMV. Therefore, analogs of the latency-associated sense transcripts of human CMV, which use alternative start sites in the analogous IE1/IE2 promoter-enhancer region of human CMV (24), are not to be expected for murine CMV.
A comment is needed on the definition of a latency-associated transcript. We have found that IE1-phase transcriptional activity is focal and randomly distributed in the lungs, whereas the latent viral DNA is evenly distributed. Thus, apparently, the observed transcription is not a feature inherent to latency. Rather, it appears that the latent viral genome can exist in a transcriptionally active and in a transcriptionally silent state. This is not peculiar to murine CMV but appears to apply also to human CMV latency in granulocyte-macrophage progenitors, since the latency-associated transcripts of human CMV were also not found in all cells that carried latent viral DNA but actually only in a small percentage at a given time point (23). Upon experimental induction of recurrence, we have found IE3 transcripts as well as infectious virus in all pieces of the lungs. Since the absolute amount of infectious virus is quite small early in recurrence, and since antiviral antibody prevents virus spread in seropositive mice (15, 40), we consider it unlikely that the observed generalized infection of the lungs resulted from virus dissemination. If we are right, this would imply that recurrence can occur in transcriptionally active and silent pieces.
However, one has to be aware of the fact that our data can provide only a snapshot of what happens in the lungs during latency. Thus, a piece of lung tissue that was found to contain transcriptionally silent latent viral DNA at the time of assay might have been transcriptionally active the day before and a transcriptionally active piece may return to quiescence. We have no immediate approach at hand to prove this, but let us nevertheless speculate a bit as to what it might mean. Randomly distributed IE1 transcripts could indicate episodes of spontaneous reactivation of viral gene expression that is terminated before the IE3 transactivator protein is expressed at a level sufficient to initiate the productive cycle. How could reactivation be terminated? Provided that IE1 transcripts lead to protein synthesis, not necessarily of the authentic pp89, and to the presentation of the immunodominantie1 exon 4-encoded IE1 nonapeptide of murine CMV (7, 14, 42), IE1 nonapeptide-specific cytolytic T cells, which are in fact present during latency as memory cells (14, 41), could eliminate the reactivating cells and thereby prevent virus recurrence. The spontaneous recurrence observed in tissue explant cultures (reference 17 and reviewed in reference16) as well as the frequent and rapid recurrence observed in organs of latently infected mice after various protocols of immunoablative treatment (34, 40) are in accordance with this hypothesis of an immune surveillance of CMV latency.
However, we doubt that the phenomena of CMV latency and reactivation are entirely explainable by, respectively, the presence and withdrawal of immune control. The observed switch in the IE transcript splicing program, from selective IE1 splicing during latency to IE1 and IE3 splicing during reactivation, may indicate a role for splicing regulation.
This work will be part of the Ph.D thesis of S. K. Kurz at the Faculty of Biology of the Johannes Gutenberg-University. The previous scientific contributions of Angelika Ebeling-Keil, Günther Keil, and Martin Messerle have provided the essential basis of this work.
Support was provided by a grant to M.J.R. by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF), Collaborative Research Project on CMV, individual project 01Kl 9607/5. We thank all members of the collaborative project for helpful discussion.
- Received 4 August 1998.
- Accepted 15 October 1998.
- Copyright © 1999 American Society for Microbiology


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20 March 2015
Woman banned from owning
pets after sick cat left behind to die
A young woman from St. Helen's,
according to the RSPCA, on Tuseday, 17 March 2015, was
banned from keeping animals for 15 years after abandoning her sick tabby
cat in a filthy, empty home. St. Helen's Magistrates' Court heard how Lisa Marie
Tucker, from St. Helen's, is said to have:- "moved house leaving her pet cat Fluffy behind to fend
for herself." After calls reporting that a cat had been abandoned inside a property, the RSPCA
attended the house daily and were making enquiries as part of their
investigation, but despite every effort the inspector received no contact from
Miss Tucker, for several days and could not gain access to the property. After the RSPCA and the
Police had gained access to the house,
RSPCA inspector Louise Showering said:- "When I entered Miss Tucker's
property in June last year, I found Fluffy in a terrible state in the living
room. She was weak and completely emaciated but she still just about mustered
enough strength to come across to me to say hello, a testament to the fact that
she had a truly sweet nature. It was clear to me on first sight though that this
was a very sick cat and that she needed urgent attention. As I picked her up I
could literally feel her bones protruding through her fur, the poor thing must
have been starved almost to death.
I rushed Fluffy to the vet, but unfortunately she was actually suffering from a
terminal illness, made worse by the fact she had not been properly cared for,
and she sadly died not long after arriving.
Owning an animal is a big responsibility and many people just do not fully
appreciate the time and effort required to provide animals with even their basic
needs. In this case Miss Tucker not only failed to meet the very basic needs of
her pet, but she actually left this poor cat behind in disgusting conditions.
Fluffy was left completely vulnerable and dying all alone.
We just hope that this sends a message to others that not taking care of your
pets is a criminal offence and you can and will be prosecuted if caught treating
an animal in this way."
Major funding boost for
highways in Liverpool
NEW funding of �85 million is set to be
spent on major roads and transport schemes in Liverpool over the next 4 years.
A report to the City Council's Cabinet on Friday, 20 March 2015, outlines plans for:-
► A new City Centre 'connectivity' scheme including new and improved
signage and lighting, dedicated coach parking, more priority for pedestrians and
cyclists and a review of bus routes to reduce delays (to be delivered from
2017 to 2019)
► Making the Strand more pedestrian friendly to make it easier for residents and
visitors to access the waterfront by improving crossings and routes for cycles
and people on foot (to be delivered in 2018 to 2019)
► Completing the upgrade of the A565 north Liverpool corridor to improve the
flow of traffic and make Regent Road more pedestrian and cycle friendly (to be
delivered from 2016 to 2019)
► Improving the pedestrian routes in and around Lime Street (Parker
Street/Elliot Street) including links to the St John's and Clayton Square
shopping centres which are currently undergoing major refurbishments (to be
delivered from 2015 to 2017)
► Upgrading the Tunnel Road/Earle Road junction in the Baltic Triangle so there
are pedestrian crossings for each route and upgrading the pedestrian crossing on
Tunnel Road so it can accommodate cyclists (to be delivered in 2015 to 2016)
► Installing a pedestrian and cycle crossing and shared cycle/footway to connect
Everton Park to Prince Edwin Street as part of Project Jennifer as well as
east-west cycle links and crossings on Great Homer Street and Scotland Road (to
be delivered in 2015 to 2016)
► Upgrading crossings and installing bus priority measures on the A57 around the
Knowledge Quarter as well as road resurfacing and public transport
infrastructure improvements (to be delivered from 2015 to 2017)
► A bid for funding to replace the bridge on the A565 at Great Howard Street to
enable it can operate without a weight limit and new lighting and drainage on
the A59 linking the City with the M57 and M58 (to be delivered from 2015-2018)
This funding is separate and in addition to the �80 million of the Council's own
money being spent over the next 8 years, tackling the historic backlog of
highways repairs in the City. Councillor Malcolm Kennedy, Cabinet member for regeneration, said:-
"We are a growing City which is absolutely fantastic, but that means we need to
invest in our infrastructure so we are able to handle the increasing amount of
businesses, freight and people travelling to and through Liverpool. Our
proposals are aimed at improving those key routes and areas which are busiest so
that we can keep Liverpool moving and compete with other Cities. We are also to
balance the competing needs of pedestrians, cyclists and traffic, for example by
installing better pedestrian crossings, including some where both cyclists and
people on foot can cross safely."
In addition, the Commercial District (BID) and the City Council will spend a
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on Old Hall Street, Bixteth Street, Edmund Street, North John Street, Eberle
Street and Leather Lane over the next 12 months, with �500k of improvements to
Water Street taking place the following year.
Jim Gill, Chairman of Liverpool's Commercial District BID, which represents more
than 900 businesses, said:- "This multi-million investment in the public
realm of the Commercial District has been a major priority for the BID Company
and we welcome the City Council's support in this programme. We look
forward to seeing the results and building on these improvements to further
enhance the appeal of the Commercial District as the place to do business in
Money for the projects is coming from the Growth Deal (�51 million); the
Highways Challenge Fund (�16 million) and the STEP fund (�4.2 million),
supplemented by �12 million of local authority cash and �900k from the
Commercial District BID.
The Cabinet will be asked to accept the funding at its meeting.
Can you home Freya?
ON 5 March, Freshfield Animal Rescue Centre will be
marking an anniversary, which isn't a cause for celebration. Freya was brought
into the Rescue Shelter on 5 March 2010, as a young stray. Sadly, no one came
forward to claim Freya and now she has has spent the majority of her adult life
at the Centre. Freshfield are desperate to help her find the forever home she so
One of the Kennels Staff, Amy Hornsby, said:- "Freya is a
fantastic dog, full of fun with a great personality. She is the resident Kennels
Comedian, and makes us laugh here every day with her comical expressions and
unfettered ability to get into mischief. The only downside of such ebullience is
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companionship of an amazing, affectionate dog. Although Freya mixes well with
most dogs on walks, she can be strong and a little boisterous at times and
therefore is best suited to an adult-only home where she can be the only pet"
You can read more about Freya by clicking
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wonderful rescue dogs finally find happiness please call the Rescue Shelter on:-
0151 931 1604.
and Help for Heroes join forces
CRABBIE'S, the official title sponsor
of the Grand National, has unveiled a new partnership with the well known
charity, Help for Heroes.
The brand, owned by Liverpool based Halewood International, will be working with
Help for Heroes to raise a million pounds, to aid in the Charity's work
providing support to wounded, injured and sick Servicemen and women and their
The fund raising activity will officially kick off at this year's Crabbie's
Grand National Festival from the 9 April to 11 April 2015.
Peter Eaton, Deputy Chairman of Halewood International, said:-
Crabbie's Grand National Festival is one of the most iconic sporting events in
the British sporting calendar and we want to use this platform to launch this
fantastic partnership, to raise funds and awareness for the Charity. Working
with Help for Heroes is something very close to both myself and everyone at
Halewood International and we are proud to support our wounded Heroes and their
Bryn Parry, CEO and Co-Founder, Help for Heroes, added:- "We are thrilled
to be working with Crabbie's and are already looking forward to the Crabbie's
Grand National. The festival will give us a fantastic opportunity to raise
awareness of the need for lifelong support. For many of these brave men and
women their battles are just beginning and we will not let them battle alone."
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Different Security Screen Doors
In the present era of technology, the need to be safe and secure has been effectively measured through different high-tech gadgets such as CCTV cameras and alarms systems. However, some simple things are also playing a great role in providing the people with the right amount of security. One example of such things is the Screen Door that is being used in nearly all types of buildings, be it home, office or a warehouse. Screen doors provide you the protection that you need in the building, without making a negative impact on the appearance of the building. There is a variety of materials, from which, you can choose the one that suits your needs. These materials offer different qualities, while they all fulfil the basic purpose of a screen door. For example, steel screen doors are preferred for their unmatched strength while aluminium screen doors are preferred for the durability and ease in maintenance. The selection of the material depends on your application and you can get accurate guidance from an expert. However, there are some things that you should keep in mind when looking for a security screen door. Take note of these points so that you make the right purchase.
Meets Standards
This is the first consideration that you have to keep in mind and check first when you're visiting a screen door supplier. The screen door should be able to match your country's strength standards. Protection against robbery or burglary is the primary objective of a screen door so check whether the door matches the standards set for resistance against forced entry. This standard is determined through a number of tests that prove that the door is secure against any kind of intrusion.
The right frame
You can get a range of options in the frames of the screen doors, but the common choices resonate around aluminium and steel. Aluminium screen door is usually preferred for its low maintenance and high durability. It is especially preferred in coastal areas as it is highly resistant against corrosion. On the other hand, steel screen door is preferred for its high strength and sleek secure look that it adds to the building. It is nearly impossible to break through the steel screen door so it is usually preferred for warehouses. You can get a personalised advice from your supplier, based on your needs. Talk to your supplier about the purpose for which you want the screen door for. The selection of the right frame is very important.
Emergency exists
This is something most users tend to overlook or forget considering. Be it aluminium screen doors or steel screen door, it is going to add security to the house and make it difficult for the thief to intrude in the house. However, the door is going to act in the same way if you're caught in an emergency and you're trying to leave the home. The bars, grilles and the security screens do not make the door the right option in case of emergencies so you need to set another exit as emergency exit, before you install security screen door at the entrance. You can also make any window, emergency exit. The important thing is, you set an emergency exit before you install the security screen door.
As mentioned earlier, there are a number of options in screen doors and you have to choose the one that meets your needs. Aluminium screen doors are generally the ideal choice for homes where medium to high level of security is required. Aluminium screen doors are also easy to fit in the budget. If you're not satisfied by the security aluminium frame provides, you can go for steel door or an additional stainless steel mesh, which offers unmatched security. However, this is going to cost a lot so, define your budget and your requirements and then look for the best option that overlaps both.
Expert in installation
This is also something that you need to confirm before you go home with your new screen door. The person who is going to install the door, should be experienced in installing screen doors. This is usually done by a licensed professional and not just by anyone who can install a door. Make sure the person is authorised and experienced in installing the screen door.


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We are now moving into a world full of intelligent and fast-paced applications. The web development as such has taken a backseat. With that shift in the needs and focus, the expectations are that the applications should be built in a way that they turn out highly robust, scalable and stable in the long-run. So, as a developer, technologist or a project manager, how do you ensure that your application has its roots strong and future secure? If you are going in for a single page application, your prime choice shall be ASP .NET Core. Why and How? Let us look at the answers one by one.
A single page application is essentially a web application – replacing the traditional desktop applications. Interestingly enough, they are never confined to single devices for run-time. Single-page applications run within the desktop browsers and do not need multiple pages to open. This saves users from reloading and undue wait time. Gmail, Facebook and GitHub, etc are some common and prominent examples of it.
Some of the key advantages of SPAs include – streamlined development, no need to write the code for render pages, ease of debugging, faster investigation of page elements and data etc.
ASP.NET Core is a prime and youthful choice today for most developers. It offers a compact code and flexible deployment.
There are various ways you can use the two in combination. Some popular ones include:
Middleware for Angular and React, Direct build up into ASP.NET Core project and Integration of the SPA framework into the project.
Let us look at these ones by one briefly:
Using SPA Middleware
Use Visual Studio to build the approach in the new project template. See the image below to understand:
Image source: https://res.infoq.com/articles/spa-asp-dotnet-core-3/en/resources/2Single-Page-Applications-AspNet-1-1569234784593.jpg
This one involves integrating ASP.NET Core into the traditional MVC application and keeping a separate folder for integration into the two projects. Take a look here:
Image source: https://res.infoq.com/articles/spa-asp-dotnet-core-3/en/resources/1Single-Page-Applications-AspNet-3-1569234784297.jpg
This method basically involves merging the NPM configuration and then moving it into the root of the project.
Image source: https://www.infoq.com/articles/spa-asp-dotnet-core-3/
So, when you have to make a choice, you need to first get the hang of each of these approaches. It comes with practice and a skillset. The ultimate objective is to ensure the architectural stability of the application.
ASP. NET as such is one of the highest performing open-source platforms. For any SPA to perform well, its combination with ASP enhances the delivery experience for the end-user. The biggest advantage is, it never demands and changes in code.
Each time the application needs an update, you are not compelled to launch an entirely new framework. You only need to add the attributes and you are done.
The command inline application features allow support for all major platforms such as Windows, Mac, Linux, etc. These applications can readily host several other applications.
The language demands a very limited amount of coding. The developers are able to optimize the structure. The idea is to keep yourself updated.
You build an application and run it across all operating systems – that is the need of the hour. ASP.Net applications allow that luxury and relief to developers – expediting the launch of the application. You do not have to work too hard on the front-end either.
It supports building cloud-based applications. Regardless of your organization size or budget, Asp becomes your prime choice as it promises stability. All your data is stored in the cloud and you can scale up or down anytime.
It allows page-based programming. In the case of SPAs, the programming remains very concise, focussed and hence, effective.
A happy developer will always build an application effectively. Anything that simplifies their life will be welcomes and higher chances for better performance.
The only disadvantage of this combination is that it entails a few major gaps in the documentation. Hence, developers need to be a little extra mindful of covering them. Lacking support in terms of tooling can be regarded as another limitation here.
If you would like to add anything, your comments and suggestions are welcome. If you have questions, rather, we'd be glad to address them. Connect with us through the comments section below.


text: We are immensely accredited for delivering the orders within the stipulated time frame as we have the latest transportation facilities. We are supported by a well-structured warehouse that assists us wholly in keeping the lot safe and meeting urgent requirements at a time. Also, we keep it updated on regular basis. We deal in Dehydrated Pink Onion which contains various nutritional components along with a long shelf life.


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When the evening comes
we set a table
and eat.
My children laugh
then fight
then laugh again.
I look at my husband
with love
then anger
then love again.
We clean up together.
The kids watch youtube.
But there are three girls
on Sutherland tonight.
We put out bowls for breakfast.
Make their lunches.
About brushing teeth
and p.j.'s.
–we're tired.
We say our prayers.
We don't mention them.
But there are three girls
on Sutherland tonight.
And as we stay warm
the girls are there
they stand in cold air.
Cars come
and go
a steady flow
of evil
right beneath our sleeping heads.
When I wake
I pour the coffee he made
and look North.
Treaty One Territory.
Lord have mercy, Christ have mercy.
once the kids are safe at school
I choke out words for this
in living rooms
with friends.
But no one knows what to say
when morning comes.
And Sutherland is still.
This poem was written as a raw attempt at putting words to a specific tension/reality we face living at 782 Main Street.
I have lived here with my family for 2.5 years. It has been a remarkable honour to share life with the people we live with.
I will never be the same.
– Jessica Williams, Flatlanders leader


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Detectives from the Montgomery County Police Department – Major Crimes Division continue to investigate the May 19 armed robbery of a convenience store in Germantown. Today, investigators are releasing surveillance video of the suspect and are asking for the public's help in identifying him.
On Saturday, May 19 at approximately 2:49 p.m., 5th District officers responded to the 7-Eleven located at 12861 Clopper Road for the report of an armed robbery that had just occurred.
The investigation by detectives revealed that an adult male employee was behind the counter when the suspect entered the store, displayed a handgun, and demanded cash from the registers. The suspect obtained currency from the business and fled from the 7-Eleven.
Anyone with information regarding the suspect or this armed robbery is asked to call the Major Crimes Division at 240-773-5100. Those who wish to remain anonymous may call Crime Solvers of Montgomery County toll-free at 1-866-411-TIPS (8477). Crime Solvers will pay a cash reward of up to $10,000 for information provided to them that leads to an arrest in this case.


text: You know you are in the heart of summer when you spend most of your outside-time fighting mosquitos. A screened porch is the answer for these homeowners, the screened windows will allow fresh air in without the bugs. The decking use is TimberTech in brown oak, to give that warm, rustic feeling. TimberTech's low maintenance decking there is no fret about staining or power washing. This screened porch with TimberTech deck was custom designed and built by Palatine, IL screened porch and TimberTech deck designer, Archadeck of Chicagoland.


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In the mid-1970s, a young Jeff Bridges already had two decades of acting experience under his belt. Thanks to his famous father, Bridges knew the ins and outs of filming a movie, so by the time he was working on 1975's "Rancho Deluxe," he wasn't in for too many surprises on the set. Off-set, however, was a completely different story.
As Bridges tells "Oprah's Master Class," he had been filming the movie in Montana when a local woman caught his eye.
"We're sitting there doing this scene and I can't stop looking at this gorgeous [woman]. I didn't know if she was a guest at the hotel, a waitress, a maid. I couldn't figure out what she was," Bridges says.
Even though the mystery woman, named Susan, was banged up from a recent car accident, Bridges was captivated by her beauty. "She had two black eyes and a broken nose, and I just couldn't take my eyes off her," he says.
Susan had noticed Bridges staring at her, despite his best attempts to be sly and hide his gaze. After she busted him one too many times, Bridges made his move.
"Finally, I get the guts to go ask her out," he says. "I go, 'Would you like to go out tonight?'"
She flatly turned him down, adding that maybe they'd just see each other around the small town some other time. A few nights later, the two did cross paths again at a local bar.
"We danced, and that was about it, man," Bridges says. "I mean, I was head over heels. I was head over heels the first time I saw her."
They married in 1977, and about 10 years into their marriage, Bridges received one of the sweetest surprises from the man who had been the makeup artist on the set of "Rancho Deluxe." In a letter, the makeup artist had written that he was going through some old files when he found something he thought Bridges should have.
"He [writes], 'I was going through my files and I came upon this shot I took of you asking a local girl out,'" Bridges recalls. "Then, out of this envelope falls these two pictures of the first words I ever spoke to my wife."
Bridges carries these photos with him always, tucking them into his wallet and marveling at the luck to have that first conversation preserved on film.
"They're my prized possession," he says proudly. "To have pictures, what are the odds of that?"
"Oprah's Master Class" airs on OWN, the Oprah Winfrey Network.
More from Oprah.com:
Also on HuffPost:


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Contact Us
We would love to hear from you. Please fill in the information below and we will be sure to get back to you as quickly as possible. Whether you have suggestions, want the latest news, or are interested in potential offers, our team will make sure to keep you informed.
Feel free to let us know....
- Suggestions for suppliers, products, etc.
- Additional antibody info that needs to be displayed
- Additional species that need to be considered
- Additional products to list such as ELISA kits, proteins, or secondary antibodies
- ...or anything else you feel is important as feedback from our users is critical
Thank you for your interest and support,
The GenLogica Team


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Thursday 02/07/2008
Feb. 7, 2008
Clemson, SC – Cliff Hammonds of the men's basketball team has been named to the ESPN The Magazine Academic All-District team, as selected by members of CoSIDA (College Sports Information Directors of America). Hammonds is just the third All-District III selection in school history, joining Tom Wideman (1998, 1999) and Shawan Robinson (2005). Hammonds is eligible for Academic All-America recognition, which will be announced Wednesday, February 27 by CoSIDA.
Hammonds has over a 3.2 cumulative GPA as a double major in architecture and psychology. He was named to the President's List for the fall semester and is on track to earn both of his degrees in May. When he does so, he will be the first scholarship basketball player on record at Clemson to earn a degree in architecture.
On the court, Hammonds ranks in the top 10 in school history in starts, minutes played, three-point goals, steals, assists, and assist-to-turnover ratio. He is 17th in school history with 1,298 points scored. This year, he ranks first in the ACC in assist-to-turnover ratio (2.89) and third in steals per game (2.14). Hammonds is one of five players averaging in double figures for Oliver Purnell's team. He is currently at 10.5 points per game.
October 26, 2020
August 18, 2020
July 31, 2020
July 22, 2020


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GTP Technology attends BioFIT Conference in Lille in December
Published on 2018-11-29 Conferences
The GTP team will be present at the BioFIT Academia-Industry Meeting on December 4th and 5th, 2018 in Lille, France. Don't hesitate to contact us to schedule your meeting.
BioFIT, the European meeting point for biotech & academia
The BioFIT event has reached its 7th edition and is becoming the meeting point in Europe for tech transfer and early-stage innovations from public research institutions, academic spin-offs and emerging biotech companies. Together with big pharma, biotech and diagnostics companies, BioFIT serves as a platform to build partnerships for all public and private actors. The event is also the marketplace for pre-seed, seed and Series A investment opportunities in Life Sciences.
A highly attended event with a range of networking opportunities
With over 1,200 delegates from 800 organizations from over 30 countries, BioFIT is the place to be for the biotech and pharmaceutical industry. The agenda offers a range of meeting and networking opportunities:
- Pre-qualified one-to-one meetings
- Conferences and roundtable discussions
- Presentations of start-ups and licensing opportunities from academia
- Exhibition area
- Informal networking evenings
GTP's agenda is filling up quickly…
The BioFIT conference has a lot to offer and our team plans on using every opportunity… which is why our agenda is filling up quickly. Should you like to meet the GTP team to discuss your recombinant protein or antibody project, please don't hesitate to contact us and book your slot. We're already looking forward to meeting you there!


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Reunion Vacation Homes Link Exchange Programreunionvacationhomes.com - Link Exchange
We are always willing to exchange links with other websites with similar content that will benefit our users. We review all websites requesting a link and reserve the right to reject any web site that we think is not appropriate to be featured on our website.
How to Link to Us
Follow the simple steps below to exchange links with reunionvacationhomes.com
- Choose the link you would like, copy the link code and place it on your web site.
- Once you have created the link email us at [email protected] with this information:
- Web site title
- A brief description of your site
- Web site URL address
- The URL address where our web site is linked from
- Once the link is confirmed we will add your link to our listings.
Copy Link and Paste into your Website:
Reunion Vacation Homes dba Florida Vacation Homes & Villas
We provide luxury Reunion rentals that are fully furnished and exquisitely designed ideal for family reunions, corporate events or golf getaways all at affordable rates.
Code to Copy:


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Detalhe do projeto
CONTRATANTE: Ana JAckson
TÃTULO: Develop tools, planning and execution of Robot Data
ÃREA DE ATUAÃÃO: Programador & Desenvolvedor
Immediate start:
●Develop tools to improve software quality, planning and execution of Robot Data
●Provision, configure and harden robots using configuration management tools (Ansible, Puppet,etc) ●Troubleshoot a wide-variety of issues which might occur on robot systems
●Design and troubleshoot robot telemetry instrumentation
●Design and build physical and software continuous integration systems
●Collaborate with various teams to maintain robot security
●Develop automation scripts for testing
●Applied knowledge of Python 2.7+, or C++ in a Linux environment
●3+ years experience running Linux in a production environment. Ubuntu or Centos preferred ●Experience building and maintaining test environments
●Production experience with Ansible or other configuration management tools
●Working knowledge of various build and packaging systems such as CMake
●Production experience with a continuous integration system and SDLC tools
●Some experience with any containerization technology
●Strength with TLS, SSH or any VPN
●Familiarity with debugging TCP connections or REST APIâs using tools like Wireshark
Payment is monthly .
Daily Rate - 110 GBP ( around 400 reais)
VERBA:. Confidencial
TIPO DE PROFISSIONAL:Sem Preferência
LOCALIDADE DO PROFISSIONAL: Sem Preferencia
PRAZO DE DESENVOLVIMENTO: A ser acordado entre as partes
LOCAL DE DESENVOLVIMENTO: home office


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We hope you didn't just get some business cards printed, because you're going to want a new set. Square already simplified card payments with its phone-based swipe widget, and allowed businesses to take money through its Cash app. Now, it's introducing another way to fill (digital) wallets: "$Cashtags." Choose your own tag ($jondoe for example) and then anyone who wants to throw some money your way can just go to https://cash.me/$jondoe and securely wire you the green. Square has also just opened the Cash app to all US businesses (with a 1.5 percent transaction charge).
Square's 'cashtags' turn a custom web link into a tip jar
Sponsored Links
The ease of being able to quickly claim a tag and then share it makes this an obvious boost for things like generating cash in emergencies (say, after a natural disaster). It'll also help out those just looking for a memorable link to receive payment for goods and services. Of course, this isn't to be confused with the (to be fair, definitely confusing) stock tags that Twitter introduced a while back (basically hashtags with a dollar sign for stock info). Want to claim your perfect $Cashtag? You'll need the square app, and a little imagination. Just be sure to choose something easy, else $johndoe will be getting all your bunce.


text:
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ExTe's head office and main production facilities are located in Färila, Sweden, around 350 km north of the Swedish capital of Stockholm.
|Head office
|Gundbergsvägen 6
|Tel: +46 (0)651-175 00
|Box 18, SE-827 28
|Fax: +46 (0)651-175 03
|Färila, Sweden
At ExTe we have always combined the entrepreneurial spirit with innovation. This has given us a technical advantage for more than 100 years, and ExTe is the world's leading manufacturer and supplier of timber bunks and auto tensioners.
The safety of our products and our ability to make personal contact with timber hauliers, equipment installers and dealers are two important factors in our success. By listening to our customers' ideas and needs, we can create a platform for our creative product development. This has brought us to the market-leading position we occupy today.
Sales Rep.
Mobile +46 70-632 92 02
Tfn +46 651-175 07
Sales Rep.
Tfn +46 651-175 08
Sales Rep.
Mobil +46 70-244 59 07
Tfn +46 651-175 40
Manager, Railway Products
Mobile +46 70-520 17 07
Tfn +46 651-175 44
Site Manager
Tfn +46 651 175 09


text:
Professional Community for Car Dealers, Marketing, Advertising and Sales Leaders
Have you ever been in a hail storm? Many parts of our country have regularly occurring hail storms that damage cars, maybe not as bad as this one, but this situation becomes an opportunity for dealers. Especially dealers with body shops.
Dealers who have digital marketing campaigns running, know…Continue
Added by Brian Pasch on January 18, 2013 at 9:30pm — No Comments
Today I co-hosted a webinar with Patrick Workman from Facebook. The webinar was really powerful with 97% of the attendees voting that the webinar helped defined a better dealer marketing strategy for 2013.
93% of the voters said that they would start Facebook retargeting, a service that PCG Digital Marketing offers as well as…Continue
Added by Brian Pasch on January 11, 2013 at 1:21pm — 2 Comments
If you have been following Facebook stock prices in the past month, you will know that the stock has had significant activity to the upside.
Investors are starting to understand the advertising strategy that Facebook is slowly rolling out to businesses looking to leverage the world's most popular social platform.
This past week I moved into a…Continue
Added by Brian Pasch on December 17, 2012 at 4:30pm — 3 Comments
Dealers looking to expand their online brand visibility must consider retargeting.
If you are not familiar with retargeting, here is the synopsis.
Any consumer that visits a dealership website ONCE, can be tagged so that when they leave the dealership…Continue
Added by Brian Pasch on April 17, 2012 at 6:30pm — 2 Comments
If you have been around long enough, you'll remember that when the Internet first came to national attention, it was often called the Internet Highway. Business owners were told that they needed to get their business on this promising new road.
I chuckled last year when Sean Wolfington showed by a vintage automotive marketing that had…Continue
Added by Brian Pasch on November 30, 2011 at 4:19am — No Comments
The yellow banner ad on the right side of this website page is not a remarketing advertisement, but if you have visited the PCG Pit Stop website, you may have noticed that our ads are following you around the web.
If you have seen our yellow ads popping up on website, rest assured that I…Continue
Added by Brian Pasch on June 17, 2011 at 4:00am — 10 Comments
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A fractious day among the young gentleman leads to an emotional Primus storming into the house from the back yard demanding that I instantly take retribution on Secundus for threatening to throw a rock at him. When I refuse to carry out his orders -- suspecting, oh, what's the phrase, "another side to the story" -- he accuses me of lacking all human feelings for his firstborn and arranges his own revenge by hiding Secundus's favorite stuffed animals. He thus earns Daddy's threadbare lecture on the dangers of escalation and the joys of forgiveness. But Primus, in full spate, is the 11-year-old tough-on-crime candidate, requiring justice without mercy.
Later, after the annual pilgrimage to Staples with three back-to-school supply lists and four short attention spans, it's my turn to think fondly of the law-and-order policies of the late King Herod. But we're driving home at last, and I notice that P and S in the back seat are now merrily enjoying a conversation.
"You see what happens if you let a little time pass," I comment. "Now, you're best friends again. Two hours ago, you too wanted to kill each other."
"Good times," sighs Secundus nostalgically.


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Advanced Concepts and Technologies International has received a three-year, $21 million task order to provide acquisition support services to the cybersecurity and communications office within the Department of Homeland Security's national programs and protection directorate.
ACT I CEO Michael Niggel said in October the company will assist the DHS NPPD CS&C office in efforts to safeguard cyber and communication infrastructure assets in the U.S. against cyber attacks.
The order was awarded under the Pool 1 category of the General Services Administration's One Acquisition Solution for Integrated Services Small Business contract vehicle.
CS&C also works with other public and private organizations as well as international partners to address security incidents that could harm federal government systems, ACT I noted.


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Ford Pit Stop Blog
Before installing new vinyl seat covers, lay them out in the sun to loosen the material and to relieve any folds or creases in the vinyl from due to packaging. A couple of hours in direct sunlight should do the trick!
Don't forget: hog-rings and pliers, seat foam padding (optional)
I need tips on headliner installation, 47 Ford super deluxe
Does fabric windlace go on before or after headliner install on a 1962 Mercury Comet? The cleat for the headliner above the door area covers them?
The windlace should go on after the headliner.
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