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Human immunodeficiency virus type 1 (HIV) replicates only in certain primate cells. In murine cells expressing cyclin T1, a posttranscriptional block exists such that small amounts of capsid and little infectious virus are released. This block is relieved in part by fusion with human cells. Here we have tested a panel of mouse-human somatic cell hybrids for production of infectious virus. Only those containing human chromosome 2 were permissive, which correlated with capsid production. The effect was specific to HIV in that release of murine leukemia virus was minimally affected by the presence of chromosome 2. Although expression of Vpu markedly increased capsid production in the absence of chromosome 2, it did not result in a corresponding increase in infectious HIV. The presence of chromosome 2 did not have consistent effects on the amount of unspliced viral RNA, whereas the amount of cell-associated Gag p55 was increased a fewfold. These results suggest that processing of HIV Gag can be corrected by one or more genes present on human chromosome 2 to allow production of infectious HIV from murine cells.
Human immunodeficiency virus type 1 (HIV) vaccine development and pathophysiology study have been hampered by the lack of a small-animal model permissive for HIV replication. Even after the viral entry determinants (i.e., CD4 and the chemokine coreceptors) were identified, it was clear there were additional blocks to viral replication in the mouse. Expression of human cyclin T1 (cycT1) improved transcriptional elongation from the viral long terminal repeat (LTR) (14, 39) and resulted in greater amounts of viral gene products in murine cells. However, infected mouse cells that express human CD4, CCR5, and cyclin T1 release only trace amounts of capsid protein (CA) p24 and little infectious virus (2, 14, 23).
Mouse-human cell fusions have been shown to be more permissive to viral replication, suggesting that mouse cells lack one or more critical factors that permit completion of the viral life cycle (2). Because mouse cells express both unspliced and spliced viral RNAs (23), the block appears to be posttranscriptional in nature. Additionally, murine cells accumulate amorphous collections of Gag as observed by electron microscopy, suggesting that the observed replicative block is at an early, not late, assembly stage (23).
Tsg101 and other protein members of the cellular budding machinery have been shown to be directly involved in HIV assembly and budding. Tsg101 was shown to interact biochemically with the late domain of Gag (37), and inhibition of Tsg101 function resulted in extracellular tethered particles of decreased infectivity (15, 24). Reduced expression or interference with function of other proteins involved in the multivesicular body and vacuolar protein sorting pathway has resulted in a similar cellular phenotype (38). Those structures are not observed in mouse cells infected with HIV. Instead, electron-dense cytoplasmic vesicular structures collect, and extracellular or budding particles are rare (23). These observations suggest that although a late block to viral budding in mouse cells cannot be excluded, the defect appears to be earlier.
A chimeric murine leukemia virus (MLV)/HIV matrix protein (MA) p17 gene partially overcame the block to virus production in mouse cells, although viral replication was poor compared to the replication of wild-type HIV in human cells (6, 29). This suggests that in mouse cells HIV MA function may be either inefficient or inhibitory. Recently, Perez-Caballero and colleagues have demonstrated that the globular head of MA inhibits a plasma membrane targeting signal located at the amino terminus of MA (27). This inhibition was relieved by Gag multimerization, which was concentration dependent, and imparted a high degree of cooperativity to Gag-membrane association (27), suggesting that slight differences in Gag expression may have profound consequences for CA production and infectious virus release. Deletions in portions of MA increased Gag expression in both human and murine cells and also production of infectious virus (17).
In the absence of Vif, in human cells members of the APOBEC3 protein family deaminate first-strand cDNA synthesis, resulting in hypermutation (typically G-to-A changes) and catastrophe such that the level of infectious virus released is reduced by several orders of magnitude (3, 16, 20, 41). Vif promotes the degradation of APOBEC3G via interaction with cullin 5 and the proteosomal pathway (40). Murine APOBEC3 activity, however, is not neutralized by Vif (3, 22). The fact that mouse-human cell fusions partially restored infectivity might suggest that a human factor counteracts mouse APOBEC3, which has not been demonstrated. In addition, inhibition of mouse APOBEC3 would not explain the absence of CA production/release in mouse cells, since APOBEC proteins do not interfere with Gag processing.
Viral protein U (Vpu) is a phosphoprotein expressed from an intron-containing RNA late in the viral life cycle (18). Vpu interacts with CD4, targeting it for degradation via interactions with h-βTrCP (21). Vpu is also capable of enhancing particle assembly/release through an undefined mechanism, which is cell type dependent. Recently, human cells have been demonstrated to express a restrictive factor that modestly blocks particle release, which was relieved by expression of Vpu (36). It is not presently known whether mouse cells express this factor; in the absence of Vpu, however, mouse-human cell fusions allowed CA and infectious virus release (23).
Proper assembly of HIV requires nuclear export and cytoplasmic translation of fully spliced, partially spliced, and unspliced viral mRNAs. In human cells, intron-containing HIV RNAs are exported from the nucleus using the Rev/Rev response element (RRE) pathway in which Crm1 is active (8). Other retroviruses, such as Mason-Pfizer monkey virus (MPMV), rely on a separate pathway for export of unspliced mRNAs (5). In this pathway, the constitutive transport element (CTE) of MPMV binds to NFX1 (also known as Tap), and RNA export occurs via normal transport mechanisms. Swanson and colleagues recently showed that when the RRE was replaced with a multimerized CTE from MPMV, Gag trafficking to cellular membranes and CA production were both restored in mouse cells (35). This suggests that Rev/RRE-containing transcripts may be incorrectly trafficked in the cytoplasm in mouse cells, perhaps because one or more factors that interact with those "marked" RNAs are functionally deficient in some manner.
Here we have used a panel of mouse-human somatic cell hybrids (SCHs) to demonstrate that the presence of chromosome 2 allowed efficient CA and infectious virus production from murine cells, which was specific to HIV. This effect did not appear to be due to alterations in cyclin T1 function, and the increase was out of proportion to the amounts of proviral mRNA and Gag protein precursor. Although Vpu increased CA production in the absence of chromosome 2, it did not correspondingly augment infectious virus release. These results suggest that HIV Gag processing can be corrected by one or more genes present on human chromosome 2 to allow both CA and infectious virus production.
HIV-cycT1-IRES-bsd (pHIV-CIB) has been described elsewhere (7), as has pHIV-IRES-eYFP (31). pHIV-cycT1-IRES-eYFP was constructed by inserting a hemagglutinin epitope-tagged, truncated version of human cyclin T1 (residues 1 to 303, kind gift of Kathy Jones of the Salk Institute) into the unique SmaI site just upstream of the internal ribosome entry site (IRES) element of pHIV-IRES-eYFP. M-tropic HIV envelope ADA expression construct was a gift from Dan Littman, and plasmid containing full-length infectious R5 isolate JR-CSF was obtained from the NIH AIDS Research and Reference Reagent Program. pBABE-IY was derived from pBABE-MN-IRES-Blasti (7) by exchanging enhanced yellow fluorescent protein (eYFP) for bsd. pHIT60 encodes MLV Gag-Pol, driven by the cytomegalovirus (CMV) immediate-early enhancer promoter (32), and pME VSV G encodes vesicular stomatitis virus (VSV) glycoprotein, driven by the SRα promoter. CMV-driven codon-optimized gag-pol expression plasmid was obtained from Oxford Biomedica (19). The helper-dependent adenovirus encoding VSV G (HDAd-VSV G) was constructed by cleaving HDΔ28E4lacZ (26) with NheI and inserting a 2.7-kb cassette of VSV G driven by the SRα promoter with compatible cohesive ends. This plasmid has the cis-acting inverted terminal repeats and packaging sequence of adenovirus, along with a CMV-lacZ reporter, but lacks all other adenoviral genes and sequences (26).
All cells in this study were maintained in Dulbecco's modified Eagle's high-glucose medium supplemented with 10% fetal calf serum, penicillin, streptomycin, and 20 μg/ml of ciprofloxacin (complete DMEM), with other antibiotics as indicated in the text. Cells were grown in 5% CO2, 37°C water-jacketed incubators and passaged every 3 to 5 days using trypsin-EDTA. Somatic cell hybrids were obtained from the Coriell Cell Repository unless otherwise stated (exceptions given in Acknowledgments). The cells were typically maintained under antibiotic selection as indicated in Table Table1.1. 293T and HOS TK− cells were originally obtained from the American Type Culture Collection, as were A9, 3T6, and LMTK− cells. GHOST HI5 cells, which have a stably integrated HIV LTR driving enhanced green fluorescent protein (eGFP) and also express human CD4 and CCR5 so that they are highly susceptible to R5 viral isolates, were obtained from the AIDS Research and Reference Reagent Program.
Pseudotyped retroviral and lentiviral particles were produced by calcium phosphate transfection of 293T cells with the appropriate viral envelope (here typically VSV G) and vector (33). For MLV, pHIT60 was included in the transfection mix. After 72 h, vector supernatant was harvested as previously described (33). Target cells were transduced in 18- or 35-mm dish format and 48 h later passaged into selective medium (typically complete DMEM supplemented with 10 μg/ml of blasticidin S [Invitrogen]).
HDAd-VSV G was produced by initially transiently transfecting 293 cells stably expressing Cre with the HDAd-VSV G plasmid and also infecting them with a first-generation, E3-deleted helper adenovirus in which the packaging sequence is flanked by LoxP sites (AdNG163R-2) (26). Supernatant from those cells was used to coinfect the 293-Cre cells along with AdNG163R-2) to cyclically amplify the amount of HDAd-VSV G as described previously (26). Resultant HDAd-VSV G was purified on two CsCl step gradients, and the amount of contaminating helper virus was estimated by Southern blotting to be ~0.02%. The titer, based upon spectrophotometric analysis, was >1012 PFU/ml.
In order to recover and determine the titer of integrated HIV vector encoding bsd from stable cell lines, typically cells were plated 1 day prior in 35-mm or 10-cm dish format, transfected the next day at 80 to 90% confluence with 1 to 2 μg each of a viral glycoprotein envelope plasmid (e.g., pME VSV G) along with an autofluorescent control (e.g., HIV-IRES-eYFP) using Lipofectamine 2000 reagent (Invitrogen), and 48 h later, culture supernatant was harvested, centrifuged at 2,000 × g for 10 min, and inoculated onto adherent human targets, typically HOS TK− or 293T cells. Transfected cells were immediately examined by epifluorescence microscopy or subjected to flow cytometry using a FACScan (Becton-Dickinson) to determine overall transfection efficiency. Alternatively, cells were infected with HDAd-VSV G at a high multiplicity of infection (MOI) and washed extensively the following day, and after 48 h, supernatant was recovered and added to target cells. Adenoviral transduction efficiency was estimated by staining the producers with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal). After 48 h, target human cells were passaged into complete DMEM containing 10 μg/ml of blasticidin S. After 7 to 9 days, cells were fixed in methanol and acetic acid and stained with crystal violet, and colonies were enumerated.
The amount of CA produced by vector-transduced or infected cells was quantitated by using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Beckman-Coulter) or by immunoblotting. For the latter, culture supernatants or radioimmunoprecipitation assay cell lysates were size separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to nitrocellulose. The membrane was preblocked using Tris-buffered saline supplemented with 0.02% Tween 20 and 2.5% nonfat dried milk, probed using primary human anti-HIV antibody from the AIDS Research and Reference Reagent Program (catalog no. 3957) at 1:1,000 in the same buffer, washed extensively in Tris-buffered saline containing 0.02% Tween 20, probed using rabbit anti-human immunoglobulin G antibody conjugated to horseradish peroxidase (Sigma) at 1:10,000, washed, and developed using enhanced chemiluminescence.
Genomic DNA was prepared from cell lines using DNAzol (Invitrogen) and resuspended in 8 mM NaOH. The DNA PCR primers used to amplify both human and mouse β-actin were 5′-TCATTGCTCCTCCTGAGCG-3′ and 5′-CTGCGCAAGTTAGGTTTTGTCA-3′ using 100 ng genomic DNA as the template, PCR conditions 94°C for 30 s, 52°C for 30 s, and 72°C for 30 s for 25 cycles, and Taq polymerase to give rise to a ~200-bp product, which was analyzed by 1.5% agarose gel electrophoresis and ethidium bromide (EtBr) staining. The HIV DNA primers for PCR amplification were NL4-3 831L (34) and NL4-3 569U (5′-TGTTGTGTGACTCTGGTAACTAGAGATC-3′), using conditions 94°C for 30 s, 62°C for 30 s, and 68°C for 30 s for 31 cycles and Expand Long DNA polymerase (Roche) to give rise to a ~260-bp product, which was similarly analyzed. The positive-control cell line had a single HIV vector integrant. Gels were Southern blotted at high stringency using the corresponding PCR product as a 32P-labeled probe amplified from the HIV vector plasmid template.
Total RNA was prepared from cell lines using the RNeasy Protect kit from QIAGEN following the manufacturer's instructions and treated with RNase-free DNase. The first-strand reaction was performed using Superscript II reverse transcriptase (RT) (Invitrogen), 1 to 3 μg of total RNA, and oligo(dT) as the primer for 60 min at 42°C. DNA PCR for β-actin was performed as described above for 21 cycles using three different amounts of the first-strand reaction mixture. For HIV, primers spanning the tat-rev intron were 5′-CTGTGGCATTGAGCAAGCTAACAGCAC-3′ and 5′-ACAGCGACGAAGAGCTCATCAGAACAG-3′. Amplification conditions were 94°C for 30 s, 62°C for 30 s, and 68°C for 60 s for 35 cycles using Expand Long DNA polymerase and the equivalent of 15, 45, and 145 ng of total RNA from the first-strand reaction mixture. This resulted in DNA products of 1,250 and 350 bp, corresponding to unspliced and spliced viral mRNAs, respectively. The spliced 350-bp product was directly visualized by agarose gel electrophoresis and EtBr staining. For the unspliced product, Southern blotting was performed using a 0.7-kb HindIII fragment specific to the intron as a probe. For a control for contaminating DNA, Superscript II RT was omitted from the first-strand reaction mixture. Total RNA from 293T cells that had been transduced with an HIV vector carrying both luciferase and bsd genes was used as a positive control.
In order to examine why mouse cells do not produce infectious HIV, primary mouse embryo fibroblasts null for p53 (9) were stably transduced with a murine leukemia virus vector encoding a truncated form of human cyclin T1 (7). This version lacks carboxy-terminal PEST sequences and is more stable than full-length cycT1. These cells were infected with a replication-competent HIV that had its gp160 replaced with vesicular stomatitis virus G coupled to the fluorophore eYFP by an internal ribosome entry site (31). No viral spread occurred in these mouse embryo fibroblasts, and virus could not be recovered from culture supernatant, despite the fact that infected cells were easily visible by fluorescence microscopy (not shown). CA levels in the supernatant were quantitated by ELISAs and were approximately 0.5 to 1.0% of those of infected human cells. Western blotting of both supernatant and cell lysates using polyclonal anti-HIV antisera demonstrated only small amounts of CA but large amounts of Gag precursor in these mouse cells. Sucrose gradient centrifugation followed by Western blotting demonstrated that the reactive material in the cell lysates had a large S value, consistent with high-molecular-weight aggregates present in mouse cells. This work thus confirmed previously published results (2, 23).
Previously it had been shown that mouse-human cell fusions were more permissive than mouse cells to HIV replication. In order to map the responsible factor(s) or gene, a panel of monochromosomal mouse-human SCHs was obtained from the Coriell Cell Respository and other sources (Table (Table1).1). The parents of these hybrids were mouse A9, 3T6, or LMTK− cells, which are of fibroblast origin. Each of the cell lines of this panel was created by fusion with human cells containing a single marked chromosome and is essentially monochromosomal for that human chromosome. Each of these cell lines was separately transduced at a low MOI with VSV G-pseudotyped HIV vector (HIV-CIB) that encodes all cis-acting sequences, gag, pol, tat, rev, and a cassette that contains both cycT1 and blasticidin deaminase (bsd) genes. In human cells transduced with HIV-CIB, provision of a viral envelope glycoprotein is sufficient to mediate virus production. Transduced SCHs were maintained as a pool under blasticidin S selection. Each SCH pool was then transiently transfected with an HIV-eYFP reporter (to measure transfection efficiency and also confirm functionality of cyclin T1) and VSV G expression plasmid, and the titers of released virus were determined on 293T cells. Only the SCH harboring chromosome 2 (GM11712) reproducibly gave high virus titers, and the titer was occasionally greater than that obtained from similarly transfected 293T cells harboring an HIV vector carrying both luciferase and bsd genes (Table (Table1).1). GM11686, a second chromosome 2-containing cell line, gave very similar results (Table (Table11).
Since HIV does not use VSV G as its envelope, these experiments were repeated with M-tropic ADA envelope, with testing limited to a select number of hybrids. Again, infectious virus was recovered only from the chromosome 2 hybrids, as measured on GHOST HI5 cells (Fig. (Fig.1).1). As a third test, select cell lines were first transduced with an MLV-based vector encoding cyclin T1, and the functionality of cyclin T1 was confirmed by transducing those cells with HIV-eYFP(VSV G) (the vector encodes eYFP but not cyclin T1) (not shown). These lines were then transiently transfected with an R5 proviral DNA plasmid clone JR-CSF, and supernatant was used to infect GHOST HI5 cells, which were then subjected to flow cytometry. A substantial amount of R5 virus was released from GM11712 cells as judged by the activation of the integrated LTR-eGFP reporter in the target cells, almost equivalent to that from 293T cells (Fig. (Fig.22).
Several controls were performed both to demonstrate the specificity of the result obtained with the chromosome 2 SCHs and to eliminate potential sources of error. Select SCHs were transfected with an MLV vector encoding eGFP, CMV-driven MLV gag-pol expression construct, and VSV G plasmid. Differences observed in measurable infectious MLV release (based upon eGFP titer on naive targets) between the SCHs were only a fewfold and did not reflect those observed with HIV (Fig. (Fig.3A).3A). Similar results were observed after transfecting select SCHs with a replication-competent avian leukosis virus-based vector encoding eYFP (data not shown).
As a completely separate method of recovering integrated provirus from cells, select SCHs with integrated HIV-CIB were infected at a high MOI with HDAd-VSV G. This "gutted" adenoviral vector encodes only VSV G and LacZ. Adenoviral transduction efficiency, as judged by X-Gal/LacZ staining of the producers, was >95% and approximately equivalent for each of the SCHs (although 2 orders of magnitude less than that of 293T cells). At 48 h, the titers of virus from recovered supernatant were determined on naive human cells and again only the chromosome 2-containing SCH gave appreciable infectious HIV release (Fig. (Fig.3B3B).
It is conceivable that the observed results were due to differences in LTR activity secondary to variability in cyclin T1 or other Tat cofactor function. In order to test LTR transcriptional activation, select SCHs already expressing cycT1 from integrated HIV-CIB were transduced with increasing amounts of HIV-eYFP(VSV G) (the vector encodes eYFP but not cyclin T1). With the exception of the GM13259 cell line, which harbors intact chromosome 12 and the gene for cyclin T1, both transduction efficiency (as measured by eYFP) and fully spliced product expression level (as measured by mean fluorescence intensity [MFI]) were roughly equivalent for chromosome 2- and non-chromosome 2-containing SCHs (Table (Table2),2), suggesting approximately equivalent Tat cofactor function in the tested SCHs. For GM13259 cells, both transduction efficiency and MFI were greater by severalfold, consistent with the presence of chromosome 12 carrying the cyclin T1 gene.
For further confirmation of this result, we next directly transfected parental A9 cells and select SCHs with HIV-cycT1-IRES-eYFP along with VSV G, and measured by flow cytometry both transfection efficiency in the mouse cells and transduction efficiency of released virus on naïve human cells. Again, only the chromosome 2-containing cell lines gave appreciable virus release, despite similar transfection efficiencies and MFIs (Table (Table3).3). Recoverable viral titers from 293T cells were 10-fold higher, possibly due to differences in transfection efficiency or because in the mouse cells expression of cyclin T1 and all viral gene products were dependent upon expression of cyclin T1 from the transfected plasmid itself.
Despite the fact that we typically normalized infectious HIV release to transfection efficiency (especially when comparing murine cell lines), we were concerned that slight differences in transfection efficiency would yield markedly different amounts of released virus. To investigate this, the GM13259 cell line was transfected using increasing equimass amounts of pME VSV G, pHIT60, and pBABE-IY plasmid DNAs, and transfection efficiency was measured by both syncytium formation and flow cytometry. Transfection efficiency correlated with infectious MLV release from GM13259 in a linear manner, with r values ranging between 0.75 and 0.84, thereby justifying the simple normalization procedure.
We were concerned that perhaps the HIV-CIB vector copy number was much higher in the chromosome 2-containing SCHs, which would result in higher infectious virus release. We thus performed semiquantitative PCR using HIV-specific primers (β-actin serving as normalization control), and there were no discernible differences in genomic proviral copy number between the SCHs tested (not shown). To determine whether there were differences in the amounts or ratios of spliced and unspliced mRNAs, total RNA was prepared from select SCHs and subjected to RT-PCR using primers that spanned the truncated tat-rev intron. RT-PCR for β-actin again served as the normalization control. As shown in Fig. Fig.4,4, the amount of β-actin mRNA present was approximately the same for each of the SCHs tested. A9 and GM11686 cells appeared to have increased amounts of unspliced mRNA compared to the other SCHs, including GM11712 cells (Fig. (Fig.5).5). Using the same primers, however, there was little variability in the amount of fully spliced mRNA products in the SCHs tested (Fig. (Fig.4).4). 293T cells had a marked increase in unspliced viral mRNA, which had been previously noted. A similar result for unspliced viral mRNA was obtained using primers that span 2.0 kb of gag-pol, with GM11686 having at most a two- to fourfold increase in the amount of unspliced viral mRNA compared to the other SCHs tested (not shown).
Because of the known defect in Gag processing that occurs in mouse cells, we wished to see whether the defect was corrected in the chromosome 2-carrying cell line. Culture supernatants from several SCHs with integrated HIV-CIB vector were analyzed by immunoblotting for the presence of CA. Notably, GM11712 cells released substantially greater absolute amounts of CA compared to the other cell lines, similar to 293T cells (Fig. (Fig.5A,5A, top panel). In addition, the percentage of CA released in the supernatant was also much greater for GM11712 (Fig. (Fig.5A,5A, bottom panel). Similar results were obtained with GM11686 (not shown). To demonstrate that the released CA was actually part of a viral particle, the supernatant was run on a sucrose step gradient and the amount of CA was detected by an ELISA. The large amount of CA that was released from GM11712 had essentially the same buoyant density as viral particles released from 293T cells (Fig. (Fig.5B).5B). A9 and 9HIS cells released much less CA. Flow cytometric analysis of cells that had been fixed and then permeabilized demonstrated detectable amounts of intracellular CA in GM11712, whereas the parental A9 cells had very little (not shown).
There is some evidence that production of CA from Gag may not be a strictly linear phenomenon; in other words, Gag may cooperatively assemble or multimerize at the plasma membrane (27), leading to much greater CA processing and potentially infectious virus release in the presence of larger amounts of p55 precursor. To examine intracellular Gag levels, whole-cell protein lysates were prepared from select SCHs. These were denatured, electrophoresed using SDS-PAGE, transferred to nitrocellulose, and probed using HIV antisera. At most, there was a twofold increase in the amount of p55 in the chromosome 2-containing SCHs, whereas there was a marked increase in the amount of intracellular p24 in those cell lines (Fig. (Fig.5C5C).
Expression of Vpu is known to increase viral particle release up to 10-fold from certain cell types, perhaps by countering a restrictive host factor (36). A vector that is similar to HIV-CIB but that also carries the Vpu gene in its native location (within the tat-rev intron) was introduced at low MOIs into select SCHs, and culture supernatants and cell lysates were immunoblotted for CA. In the presence of Vpu, many of the SCHs released much greater amounts of CA, but the effect was more modest in the chromosome 2-containing cell line (Fig. (Fig.6A).6A). The CA yield (amount in culture supernatant divided by amount in cell lysate) also increased severalfold in the non-chromosome 2-carrying mouse cell lines, similar to the yield in HeLa cells, but increased less in the chromosome 2-carrying SCHs (Fig. (Fig.6B).6B). As a control, COS cells showed no difference in CA release in the presence of Vpu (Fig. (Fig.6A6A).
To determine whether Vpu also increased infectious virus release, select SCHs were separately transduced at low MOIs with an HIV-CIB vector with or without Vpu intact. Semiquantitative PCR confirmed that the average proviral copy numbers were approximately the same for each SCH (not shown). These were then transfected with VSV G expression plasmid and a CMV-RFP reporter (to normalize for transfection efficiency), and the titers of infectious virus were determined on HOS cells. Inclusion of Vpu in the vector did not increase the amount of infectious virus from mouse cell lines that were very nonpermissive, such as A9 and GM11715, whereas it did have a slight effect on the other SCHs tested, including GM11686 (Fig. (Fig.6C).6C). These results suggest that the presence of chromosome 2 increases both Gag processing and infectious HIV particle release, separate from any effects Vpu may have on facilitating CA release.
Previously it had been shown that transfection of mouse cells with codon-optimized, CMV-driven gag-pol (CO GP) allowed Gag to reach the plasma membrane and also increased CA release from those cells (35). Transient transfection of murine cells with a plasmid encoding codon-optimized, CMV-driven gag-eGFP fusion resulted in fluorescence localized to the plasma membrane (not shown), consistent with previous work (35). We first confirmed that CO GP had approximately equivalent activity compared to our standard packaging vector pHIV-PV (34) in producing lentiviral vector supernatant from 293T cells. Transfection of parental A9.CIB cells with CO GP, however, did not rescue infectious virus, nor did it increase the amount of infectious virus release from the chromosome 2-containing cell lines that were cotransfected with HIV-eYFP (Fig. (Fig.7).7). This result was confirmed in other murine cell lines (not shown). This suggests that neither the plasma membrane localization of Gag nor overexpression of structural gene products (independent of Rev and the RRE) is sufficient to mediate virus release from mouse cells.
Here we utilized a panel of monochromosomal mouse-human SCHs to demonstrate that the presence of human chromosome 2 is sufficient to allow infectious HIV release. This correlated with CA production and occurred whether VSV G or M-tropic envelope was used as the complementing cell surface glycoprotein. The effect appeared specific to HIV in that production of other retroviruses was minimally affected. The effect was out of proportion to the increase seen for Gag protein and unspliced mRNA. While Vpu did increase CA production, especially in the absence of chromosome 2, its expression did not correlate well with infectious virus release. A parsimonious explanation for the above results is that one or more determinants or genes that is capable of stimulating virus release from mouse cells lies on human chromosome 2.
Because production of infectious virus may not correlate linearly with the amount of Gag due to cooperative assembly effects (27), we went through some effort to demonstrate the following in select SCHs. (i) The number of integrated proviruses was similar. (ii) Cyclin T1 function was approximately thesame (with the exception of the chromosome 12-carrying SCH). (iii) The amounts of spliced mRNA were roughly equivalent. (iv) The quantities of cell-associated precursor Gag were comparable. (v) Transfection of codon-optimized, CMV promoter-driven HIV gag-pol did not rescue the mouse cell defect. We also observed inconsistent amounts of unspliced viral RNA in the SCHs in that the level in nonpermissive parental A9 cells was similar to that in permissive GM11686 cells, whereas permissive GM11712 cells had levels lower than those of other SCHs (Fig. (Fig.4).4). We cannot exclude the possibility that the differences in the amount of unspliced RNA result in variations in the amount of p55 Gag. This in turn could result in much higher levels of infectious virus release, related to highly cooperative effects of Gag at the plasma membrane (27). Consequently, it is possible that one or more factors on chromosome 2 influences the amount of unspliced mRNA and Gag expression, either through transcriptional, antisplicing, translational, or other poorly defined effects.
We also tested whether expression of Vpu is capable of alleviating the block to infectious virus release from mouse cells. Vpu is known to assist in the down-regulation of cell surface CD4 through targeting to an adapter protein (21); its role in increasing CA release from certain human cell lines is more enigmatic. Here we also demonstrated that expression of Vpu allowed increased CA production/release from mouse cells, similar to nonpermissive human cells. That effect, however, did not translate into a marked increase in infectious virus release, suggesting that Vpu functions in a separate pathway to augment CA production, independent of the action of any factors present on chromosome 2.
It is possible that Gag is mislocalized in mouse cells, which is corrected in the presence of chromosome 2. In permissive human cells, Gag may have a punctate cytosolic appearance or mainly be localized at the plasma membrane, depending upon the level of expression and tissue/cell of origin. When codon-optimized Gag was expressed as a GFP fusion protein in a CMV promoter-driven expression vector, it was localized at the plasma membrane in all cell types tested, regardless of origin (not shown). Expression of codon-optimized gag-pol was insufficient, however, to attain infectious virus release from mouse cells. When expression of codon-optimized Gag-GFP was dependent on both Rev and RRE, we observed a punctate cytosolic pattern in both permissive human cells and mouse cells with chromosome 2, whereas in other mouse cells it was more diffuse (not shown). Whether this is reflective of the trafficking of the unspliced mRNA or differences in the Gag expression level is unknown.
Cytosolic localization of proviral HIV mRNA has not been fully evaluated in mouse cells, let alone permissive human cells. Use of the ms2 phage protein binding system has suggested that MLV genomic RNA traffics along an endosomal pathway, which is dependent upon envelope and colocalizes with Gag (1). Precise determinants for mRNA entry into this pathway have not yet been fully elucidated. Because HIV has additional cis-acting nucleic acid sequences that function in RNA nuclear export, it is conceivable that these elements play a role in directing the unspliced RNA to a specific, postnuclear pathway, perhaps in concert with other viral proteins and cellular factors. Absence of one or more of these factors would abrogate infectious virus release from mouse cells. Provision of a multimerized CTE to the RNA partially corrected the defect observed in mouse cells in that Gag became localized to the plasma membrane and was processed, but it is unknown whether infectious virus was produced (35).
At least two genes lie on chromosome 2 that are involved in export of unspliced HIV RNA. Crm1, located on 2p, binds to Rev to allow export of RRE-containing mRNAs (11, 13). hRIP (4, 12) is located on 2q36. At steady state it is located in the nucleus and in the perinuclear (cytoplasmic) region. Recently by the use of dominant-negative forms and RNA interference, hRIP was shown to be specifically involved in the movement of Rev/RRE-dependent HIV RNAs from the nuclear periphery to the cytoplasm (30). Whether Crm1 or hRIP has a role in viral RNA cytoplasmic trafficking (or is involved in the "hand-off" to other as-yet uncharacterized factors) is unknown.
Both of these genes were introduced by transient transfection into murine A9.CIB cells, alone and in combination, and there was no appreciable effect on either CA production or infectious virus production (data not shown). Other cellular genes may cooperate with Rev to allow more efficient nuclear export of unspliced mRNAs, including SAM68 (25, 28). SAM68 is not present in the chromosome 2-carrying SCHs, and transient transfection of SAM68 into A9.CIB cells did not result in an increase in CA or infectious HIV release (not shown). Other genes implicated in Rev function or binding carried on chromosome 2 (10) have yet to be tested.
The results presented here do not exclude the possibility that other factors that are not encoded on chromosome 2 are critical for infectious HIV release from mouse cells. We have identified three microcell mouse-human hybrid clones extremely permissive to HIV release (not shown). Two of these clones are identical and have a complex chromosome 2-12 fusion; the third has only an intact chromosome 12. Efforts to identify a portion of chromosome 2 (or any chromosome other than 12) in this clone have thus far failed. It is therefore conceivable that non-chromosome 2-encded factors contribute to infectious virus release. For the first time, however, we have identified several murine-human hybrid cell lines that are permissive for infectious HIV release. These lines may serve as useful tools to mechanistically dissect the postintegration block to HIV production in the mouse and to identify the gene(s) responsible for this phenotype.
We thank Dan Littman (NYU School of Medicine), Larry Donehower (Baylor), and Oxford Biomedica for generous reagent gifts. We also thank Phil Ng (Baylor) for preparation of the HDAd-VSV G stocks; J. Carl Barrett (NCI) for SCHs carrying chromosome 6, 8, 9, 18, or 19; and Elton Stubblefield (MD Anderson Cancer Center) for the SCH carrying chromosome 17. The following reagents were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-immunoglobulin from NABI and NHLBI, GHOST HI5 cells from Vineet KewalRamani (NCI-Frederick) and Dan Littman, and JR-CSF proviral clone from Irvin Chen (UCLA).
This work was supported initially by the American Foundation for AIDS Research (AmfAR) and then by the National Institutes of Health. R.E.S. was an Edward Mallinckrodt, Jr., Foundation Scholar.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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A Christmas Carol: Holiday Tradition at Drury Lane
* Additional fees apply. No coupon or promo codes necessary to enjoy the displayed discount price.
The last date listed for A Christmas Carol was Saturday November 28, 2015 / 1:00pm.
Currently at Drury Lane Theatre
Deathtrap -- The Diabolically Funny Broadway Thriller
- Full Price:
- $41.40 - $57.00
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Deathtrap, the longest-running comedy-thriller on Broadway, slayed audiences for years with its… More
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Featured review from Kathy WalkerRed Velvet
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Fantastic on every level!
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A so-so production with an abbreviated plot line and time. Decent job of acting and the few sets were good as well. Good special effects. Just too much detail was cut. oaf course with the low price, it was a very good value.
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The play was very enjoyable. There were a lot of school children but they were all on their best behavior. We loved the experience.
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great show would recommend it to all kids or adults there are no bad seats at Drury Lane

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The Woes Of Me
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Baked Apricot Chicken
|Apricot preserves/Peach preserves
|10 Ounce (1 Jar)
|Lemon juice
|1⁄4 Cup (4 tbs) (From Concentrate)
|Soy sauce
|2 Teaspoon
|1⁄2 Teaspoon
|Broiler fryer chicken
|3 Pound, cut up
|Dry bread crumbs
|1 Cup (16 tbs)
|1⁄4 Cup (4 tbs), melted
Preheat oven to 350°.
In shallow dish, combine preserves, brand, soy sauce and salt.
Coat chicken with apricot mixture; roll in bread crumbs.
Set aside remaining apricot mixture.
In greased 13 x 9-inch baking dish, arrange chicken; drizzle with margarine.
Bake uncovered 1 hour or until tender.
Heat remaining apricot mixture; serve with chicken.
Refrigerate leftovers.
Recipe Summary
Rate It
Nutrition Rank
Nutrition Facts
Serving size: Complete recipe
Calories 4281 Calories from Fat 2259
% Daily Value*
Total Fat 251 g386.3%
Saturated Fat 67 g334.8%
Trans Fat 0 g
Cholesterol 1020.5 mg340.2%
Sodium 2866.9 mg119.5%
Total Carbohydrates 237 g79.1%
Dietary Fiber 2.6 g10.3%
Sugars 154.7 g
Protein 261 g521.6%
Vitamin A 107.2% Vitamin C 233.1%
Calcium 25% Iron 82.7%
*Based on a 2000 Calorie diet


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Sports Editor
The Liberty girls softball team went to the Botetourt Bash this past Friday, April 8 and the Lady Minutemen played two games and walked away 1-1.
The first game Liberty defeated Blacksburg 8-5.
Liberty scattered seven hits and scored two runs in the second, three in the third, two in the fifth and one in the sixth.
540-297-5650 / 540-874-4950
OVERVIEW- Fishing should be great this month! Water temperature will be in the 60's to mid 70's. Best times will be cloudy days. Late afternoon is usually when fish are most active and at night later in the month. Fishing the past few weeks has been good! Most of the baitfish has moved towards the upper end of the rivers, coves and the major creeks.
The Staunton River and Liberty boys soccer teams have met on the field twice and both times River walked away victorious. The Golden Eagles defeated Liberty 1-0 in the second match on Friday, April 1.
Liberty put up a tough fight against the Golden Eagles but Stuart Givens, of SR, scored at 65:00 into the match. "Aaron Plattus [the Liberty goalie] played well. He had six saves," said LHS coach Eric Seckman.
The 5th Annual Blossom to Bottle 5K run and 3K walk will take place on Saturday, April 9 at 5 p.m. at the Peaks of Otter Winery.
During Spring Break the Jefferson Forest baseball team traveled to Va Beach to play two local teams. Forest walked away 1-1 from the trip east.
The Cavaliers got off to a fast start against the Salem-Va Beach Sun Devils on Thursday, March 31. The Cavaliers defeated the Sun Devils 9-3. "We put together 15 hits against the Sun Devils and played much better offensively and defensively," said JF coach Ryan Gilleland.
The Jefferson Forest girls lacrosse team is pushing to be the best in the area and isn't letting anything stand in the way. The Lady Cavaliers defeated Rockbridge 15-9 on Tuesday, March 29.
The first half of the game went back and forth until the Lady Cavs pulled together. "Our girls played great. We were tied 6-6 at one point in the first half then we pulled ahead to an 11-7 lead by halftime," said Ava Rupert.
No doubt we've all heard the old English proverb, "The way to a man's heart is through his stomach", and the same can be said for women and children. Putting a delicious meal on the table is satisfying for both the cook and the diner regardless of gender.
By Margaret Pascale
Sports Editor
Staunton River fell to Jefferson Forest 3-2 after eight innings in a tightly contested County Clash on Friday, March 25.
Liberty struggled at the plate, losing to the Golden Eagles 8-0. Neither team was able to score a run in the first inning.
But Staunton River scored in the bottom of the second and that would be its only score until the bottom of the fifth.
After waiting an hour past the original game time, Forest, finally able to play, defeated Cave Spring 18-8 on Thursday, March 24.
This was the Cavaliers first home game on the new turf at JF.
The team struggled getting into a rhythm at the start of the game but it did not hold back the team from scoring as many goals as possible during the four quarter game.
Copyright © 2016
The Bedford Bulletin is your source for local news, sports, events, and information in Bedford County and Bedford, VA and the surrounding area.


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Capitol Hill Critics Second-Guess Agencies Using...
Co-Sourcing Improves Pharmaceutical Laboratory Productivity
Loss of Lives in Clinical Trials...
Clinical-Style Validation, Regulation, and Quality Control...
According to a just-released survey by Qiagen, sample access and data analysis are the two main choke points named by scientists using next-generation sequencing. They also called workflow complexity and regulatory constraints close seconds.
The survey, Perspectives in Advanced Genetic Analysis, Your Perspective: Results from a Survey about the Future of Sequencing, listed a number of other key findings:
Regarding this last point Qiagen reported that "Seventy percent of researchers in our survey said that current NGS workflows are not sufficiently standardized for use in a clinical diagnostic setting."
The survey also noted that most of the sequencing being carried out is either targeted sequencing or RNA sequencing. Respondents said they expect to focus more on whole-genome analysis rather than exome-level or targeted resequencing runs.
It was also revealed that most of the survey respondents (83%) use the manufacturer of their sequencing instrument for a majority of their tech support. Regarding NGS translational research, consistency of results is the main concern (55%), and 54% of respondents contract out some NGS work (1–50 samples) to a core lab or company.
The Qiagen survey was based on the responses last August of 924 researchers working in the biological sciences. They breakdown was as follows: 51% from academic/university settings, 11% from governmental laboratories, 9% each from biotechnology companies and research hospitals, and 29% from other research environments (e.g., private research institutes and pharmaceutical companies).
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MDMA (commonly known as the empathogen "ecstasy") is classified as a Schedule 1 drug, which is reserved for compounds with no accepted medical use and a high abuse potential. Two researchers from Stanford, however, call for a rigorous scientific exploration of MDMA's effects to identify precisely how the drug works, the data from which could be used to develop therapeutic compounds.Do you agree that ecstasy should be studied for its potential therapeutic benefits?
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The Trucks Debut with Abundant Attitude
Web Resources
Four women musicians from Bellingham, Wash., who call themselves "The Trucks" have released a debut album of the same name, with language and attitude that is not going to get them much airplay on mainstream radio.
The Trucks are another entry in a long line of female rock bands that know and find their audience.
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Crazy Substitute Teachers
Enjoy, NG4.06 / 5.00 1,532 Views
Vacuum Salesman
This dude and his Megazord vacuum cleaner3.98 / 5.00 2,435 Views
Markiplier Animated | Mark Of The Survey Corps
This took far too long to make...3.91 / 5.00 4,011 Views
A few tips
increase your frame rates to at least 18 or 24 to make this smoother. Not bad for a first attempt. Love the beat sounds like an instrumental off soundclick or something made by alchemist.
nto a bad concept, and sort of heartwarming, but the ending totally killed it. never write it if its not proper english. but work on it though. you need to work on animations, and start passing english.
sorry man .... but that was something even unintentionally! ^^.... e. .. is already all correct !!...
but....thanks :)
That was cute.
Im a dog person so that was perfact.
I kinda liked it.
The beginning wasn't great, but the end made up for it quite well, It wasn't bad for a first time flash
not bad at all!
The message is nice, and the music is a good selection(credit the author of the audio).
The animation was a bit choppy and needs a little bit of improvement.Anyway the flash is good for being your first post on here, so keep it up! ^_^


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#126 - sevenxninja
Reply +11(03/16/2013) [-]
#132 to #130 - eternalenki
Reply +3(03/16/2013) [-]
mimic is a recurring monster, I've seen it in a number of different games from old and newer RPGs, JRPGs mostly.


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IAS officer and Soni quizzed face-to-face in NHRM scamJaipur: It was a tough day for IAS officer Neeraj K Pawan, booked in connection with corruption in NHRM-IEC, as he was confronted with Ajit Soni, his middleman, on Sunday and asked hard-hitting questions about their connivance. Troubles for IAS ... Times of India, 2 months ago
Bribery case: IAS, RAS officers suspended by Raj govt - Press Trust of India, 1 month ago
Rajasthan IAS officer suspended in bribery case - Chandigarh Tribune, 1 month ago
NHRM scam: Kushwaha submits Rs 1.27 crore to CBI court
Former Uttar Pradesh Minister for Health and Family Planning Babu Singh Kushwaha, who is an accused in the National Rural Health Mission (NRHM) scam, today submitted Rs 1.27 crore to the CBI court as per court directions. Kushwaha, at the time of ...Business Standard, 2 months ago
MP: NRHM managers contract renewed on phone, reveals RTI
Contracts of 50 district programme managers (DPMs) with the National Rural Health Mission (NHRM) in Madhya Pradesh were renewed on phone in 2010 against the procedure, revealed a right to information query recently. The contracts should be ...Hindustan Times, 1 month ago NRHM managers' contract renewed on phone, reveals RTI HT Syndication, 1 month ago
ACB raids IAS officer, claims to have busted racket
Anti Corruption Bureau (ACB) of Rajasthan today claimed to have busted a racket in National Rural Health Mission (NHRM) as it conducted raids at the residence and office of senior IAS officer Neeraj K Pawan and held an alleged "middleman" Ajeet ...Business Standard, 2 months ago PTI - LD RAID 2 LAST - (REOPENS DES 55) Namibia Press Agency, 2 months ago ACB raids IAS officer in Jaipur, claims to have busted racket DeshGujarat, 2 months ago PTI - LD RAID - ACB raids IAS officer, claims to have busted racket Namibia Press Agency, 2 months ago
News Flash: Meeting between PM Modi and Prez Xi Jinping in Tashkent ends
New Delhi, June 23: Get the latest national and international news updates here: 5.05 pm: Decision was taken in a meet of Joint Monitoring Group (JMG) consisting of representatives of GoI, Assam Govt and NDFB(P), held in Delhi. 5.00 pm: ...Oneindia, 1 month ago
Brexit Polls: Option For Britain To Remain In European Union Gains Steam In Days Before Critical Referendum
The latest Brexit polls show a growing movement for the effort to keep Britain in the European Union, with two polls published on Saturday showing the Remain option surging back into the lead. Voters in Britain will head to the polls this week to ...Inquisitr, 1 month ago
No regularization of NRHM employees
Excelsior Correspondent Srinagar, June 16: Government today said it has no plans to regularize the employees working for the implementation of National Rural Health Mission (NHRM). In a written reply to a question of Devinder Kumal Manyal in Lower ...Daily Excelsior.com, 1 month ago
ASHA workers duped of Rs 1.5 lakh in MP
Neemuch, MP, Jun 9 (UNI) An unidentified fraud duped a number of Accredited Social Health Activist (ASHA) in the name of National Rural Health Mission in Jeeran police station limits here, police said. As many as 10 ASHA workers became victim of ...United News of India, 1 month ago
BJP Government's Utter Failure in the Health Sector
TWO years have passed since the installation of the BJP led government at the centre and there is growing frustration at the inability or unwillingness of the present government to provide leadership in key areas of public policy. The health sector ...Peoples Democracy.in, 1 month ago
News flash: War is not a solution for Indo-Pak: Abdul Basit
Bengaluru, May 30: Air passengers were stalled at IGI airport after several flights were diverted from Delhi due to bad weather, on Sunday night. Get all the latest news updates of the day: 9:32 pm: Home Minister Rajnath Singh to lead the ...Oneindia, 2 months ago
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text:
Author Affiliations: Departments of Medicine (Drs Golomb and Criqui and Ms Ritchie), Family and Preventive Medicine (Drs Golomb and Criqui), Psychiatry (Dr Dimsdale), and Economics (Dr White), University of California, San Diego, La Jolla.
Some studies have suggested reductions in blood pressure (BP)with statin treatment, particularly in persons with hypertension. Randomized trial evidence is limited.
We performed a randomized, double-blind, placebo-controlled trial with equal allocation to simvastatin, 20 mg; pravastatin sodium,40 mg; or placebo for 6 months. Nine hundred seventy-three men and women without known cardiovascular disease or diabetes mellitus, with low-density lipoprotein cholesterol screening levels of 115 to 190mg/dL, had assessment of systolic and diastolic BP (SBP and DBP, respectively). Blood pressure values were compared for placebo vs statins by intention-to-treat (ITT) analysis. Additional analyses were performed that (1) were confined to subjects with neither high baseline BP (SBP >140 mm Hg or DBP >90mm Hg) nor receiving BP medications, to exclude groups in whom BP medications or medication changes may have influenced results, and (2) separately evaluated simvastatin and pravastatin (vs placebo). The time course of BP changes after statin initiation and the effect of stopping statins on BP were examined.
Statins modestly but significantly reduced BP relative to placebo,by 2.2 mm Hg for SBP (P = .02) and 2.4mm Hg for DBP (P < .001) in ITT analysis.Blood pressure reductions ranged from 2.4 to 2.8 mm Hg for both SBP and DBP with both simvastatin and pravastatin, in those subjects with full follow-up, and without potential for influence by BP medications (ie, neither receiving nor meriting BP medications).
Reductions in SBP and DBP occurred with hydrophilic and lipophilic statins and extended to normotensive subjects. These modest effects may contribute to the reduced risk of stroke and cardiovascular events reported on statins.
Trial Registration
clinicaltrials.gov Identifier: NCT00330980
Some studies have suggested reductions in blood pressure (BP)with statin treatment, particularly in patients with hypertension. Many studies reporting BP reductions have been correlational, uncontrolled,tested against other active drugs with uncertain impact on BP, unblinded,nonrandomized, or without assessment of statistical significance.1- 9 Some double-blind, randomized studies failed to show an effect but had a small sample size.10,11 Two small, randomized, double-blind,crossover studies12,13 have shown significant (P < .05) benefit, but to our knowledge there are no published articles showing reductions in BP with statins in sizeable randomized trials.
The BP assessments performed in the double-blind, randomized,University of California, San Diego (UCSD) Statin Study were used to assess the impact of randomized assignment to statins vs placebo on systolic and diastolic BP (SBP and DBP, respectively).
The UCSD Statin Study was a randomized, double-blind, placebo-controlled trial of 6 months' duration.
Subjects were 973 men and women from Southern California. A total of 1016 screenees were eligible to participate in the larger trial by virtue of having had a low-density lipoprotein cholesterol (LDL-C) level of 115 to 190 mg/dL (inclusive) at screening; no known cardiovascular disease or diabetes mellitus; and no factors that would preclude 8 months of participation in the study (to convert LDL-C to micromoles per liter, multiply by 0.0259). Blood pressure was not an entry criterion and did not influence eligibility to participate.The 973 subjects for the present BP study are the proper randomized subset of the larger sample in whom BP was assessed at screening.A more complete description of the purpose, eligibility criteria,and study design is presented elsewhere.14,15 The study protocol was approved by the UCSD human subjects committee, and all subjects gave written informed consent to participate. The recruitment period was from April 2000 through July 2003, and all subjects were seen at UCSD.
A computer-generated, blocked, randomization sequence stratified on sex was designed by the statistician (H.L.W.) and provided to the study pharmacist who used the sequence to match assigned treatment to sequentially numbered bottles. Sequential subjects who met eligibility criteria and gave informed consent were assigned sequential study identification numbers and received the bottle with the corresponding number. Subjects received simvastatin, 20 mg; pravastatin sodium,40 mg; or placebo with equal (33%) probability. A more complete description of procedures is presented elsewhere.14,15 Simvastatin, 20 mg, and pravastatin sodium, 40 mg, were selected as the most lipophilic and hydrophilic statins, given at dosages approximately equivalent in terms of expected LDL-C reduction (the study drug was to be taken at bedtime).16 Neither subjects nor study staff were aware of subject assignment during subjects' participation.
Visits occurred at screening, baseline, and at 1 and 6 months during treatment. Subjects received an 8-month (2 months after discontinuation)follow-up visit. Primary outcomes from this study will be reported elsewhere. Brachial BP was assessed but was not a designated primary or secondary end point. Blood pressure was obtained using the auscultatory method by trained psychometrists, in the morning, with subjects seated at rest and arm at chest height using cuff sizes appropriate to the subject. Using calibrated aneroid sphygmomanometers, staff identified SBP and DBP as phase I and phase V of the Karotkoff sounds (unless heard to a BP of 0, wherein phase IV was recorded as the DBP value).17 Although BP was not a primary outcome in the UCSD Statin Study,14,15 it was measured at the screening visit (prior to randomization)and at each on-treatment and posttreatment visit. All subjects whose screening value exceeded 140 mm Hg SBP or 90 mm Hg DBP were given a letter to bring to their physicians stipulating their elevated BP (n = 210 subjects). Because of presumptive evidence suggesting a possible benefit of statins to BP and the relative ease of analysis of this end point, we received permission from the Data Safety Monitoring Board to unblind and analyze BP first, and the process of data cleaning for BP commenced prior to the last, 8-month (off-treatment) visit of the last subject. Lack of duplicate BP measurement at each time point must be considered in the context of lack of duplicate measurement of many other variables, including lipids, that show variability:this lack of duplicate measurement was nondifferential across treatment groups, and variance resulting from measurement variability, although potentially eroding power (and producing bias toward the null), can be offset by increased sample size.
Forty-three of 1016 subjects (4.2%) did not have BP measured at baseline, primarily owing to subjects' time constraints (Figure 1). The screening visit at which baseline BP was assessed comprised subjects' first trip to the study site, and some subjects were delayed in reaching the site or abridged their visit owing to other commitments. However, randomization was blinded to and independent of presence or value of screening BP. Thus,the subgroup with BP measurements (n = 973 subjects) is equivalent to a proper randomized substudy and forms the sample analyzed herein. (Baseline comparability is shown across treatment arms in this group.)
Flowchart of participants.
Change scores in BP represented the primary BP end point, subtracting baseline BP from final on-treatment BP. This may enhance power by enabling subjects to serve as their own controls.
Analyses were conducted using Stata statistical software (version 8.0; StataCorp, College Station, Texas). The primary BP measure for the present analysis was the change from baseline (screening) BP to 6-month (final on-statin) BP. All analyses were performed for both SBP and DBP.
Baseline comparability across treatment arms was assessed in subjects to address comparability by treatment assignment. We performed t tests to compare the mean change in SBP and DBP across treatment arms, provided that comparability across treatment arms was present at baseline. If baseline disparities across treatment arms were identified, then we performed regression analysis to allow adjustment for baseline disparities. Secondary analyses evaluated the effect on BP separately for pravastatin and for simvastatin vs placebo.
All subjects who received an on-treatment visit (except 3 [2who received pravastatin and 1 who received simvastatin]) had an on-treatment BP measured and were included in the intention-to-treat analysis. In those with high measured BP at baseline, an "intervention" in the form of a letter to the subjects' physicians was given—an intervention expected to contribute variance unrelated to treatment assignment, eroding power in samples in which this group is included. In addition, statin effects on BP could differentially affect implementation or modification of BP medications in the statin vs placebo groups in persons with baseline high BP or those receiving BP medications. Therefore, analyses were performed in subjects without high BP at baseline, those with neither high BP nor BP medications at baseline,and those who had never received BP medications to evaluate subjects in whom BP treatment could not have influenced results and for whom results cannot be attributed to statin interactions with BP medications. We examined subjects with higher-normal or lower-normal BP, within the subhypertensive range, by splitting SBP and DBP at the sample median among those with BP lower than 140/90 mm Hg at baseline, selecting the median to maximize power for each analysis. This suite of analyses permitted assessment of the possibility that statin effects occurred primarily at a higher BP as some have proposed12 or solely through drug interactions with hypertensive agents (eg, the "synergistic" effects others have hypothesized4). Finally, because of reports that the endothelial benefits of statins (thought to underlie BP benefits) may fail to extend to subjects with low high-density lipoprotein cholesterol (HDL-C),18 coupled with an absence of observed BP benefits in the CARE study19 (comprising a low HDL-C sample), we analyzed BP effects in those with HDL-C values above and below the median HDL-C levels. The time course of the effect of statins on BP was assessed by examining the change in BP from baseline to 1 month of treatment, to 6 months of treatment, and to 2 months after treatment was discontinued.
Subject participation occurred from April 2000 to March 2004.Analysis supported baseline comparability of analyzed treatment arms on characteristics including age, sex, ethnicity, lipid profiles, glucose, smoking status, SBP, and fraction with elevated BP at baseline (Table 1). However, the baseline difference in DBP comparing pravastatin to placebo was not significant; therefore, t tests were complemented by regression analysis, adjusting for baseline DBP values.
As Table 2 shows, in the intention-to-treat analysis, significant though modest BP reductions were present in the combined statin group relative to placebo, for SBP and for DBP. (The value P = .05is for the test viewed in isolation. Viewing the tests as an ensemble,we obtain a Bonferroni P value of P = .55; ie, 0.05 × 11 tests, for the multiple hypothesis test of no differences between placebo and statin.) This finding was stronger when subjects with high BP at baseline were excluded (this group received a letter to their physicians which may have led to treatment changes that, although nondifferential by arm, may have amplified variance and attenuated significance). Reductions in BP were seen for subjects without hypertension—those with neither high BP at baseline nor receiving BP medications. This held true, separately, for those with SBP (or DBP) above or below the sample median.
Table 2 also suggests possible effect modification for SBP based on HDL-C, splitting the sample at the median baseline HDL-C (50 mg/dL). (To convert HDL-C to micromoles per liter, multiply by 0.0259.) The reductions in SBP were indeed focused in the high HDL-C group (mean HDL-C, 65 mg/dL)relative to the low HDL-C group (mean HDL-C, 40 mg/dL). In contrast to SBP, however, DBP reductions were not focused in the high HDL-C subgroup.
Figure 2A (SBP) and Figure 2B (DBP) show the change in BP from baseline to each time point for the 2 statins relative to placebo, selecting for illustration those subjects without high BP (>140/90mm Hg) at baseline, who did not receive BP medications, and who had BP measured through the 8-month visit. At 1 month, nonsignificant (P > .05) reductions in SBP and DBP in the statin groups relative to placebo group were seen. By 6months of treatment, both SBP and DBP differences from baseline showed significant reductions (P < .05)in each of the statin groups relative to placebo. By 2 months after discontinuation of treatment, these changes had dissipated.
Difference between the statin and placebo groups. A, Systolic blood pressure (SBP), change from baseline. This figure shows values for subjects who were not hypertensive at baseline, were never prescribed BP medications, and had BP measured through the 8-month visit. This mitigates the influence of changes distinct from treatment assignment across time. The change values differed significantly from placebo (P < .05)at month 6 (only) for both pravastatin sodium and simvastatin. Standard errors for pravastatin: month 1, 1.3; month 6, 1.2; month 8, 1.2. Standard errors for simvastatin: month 1, 1.2; month 6, 1.2; month 8, 1.2. P values for pravastatin: month 1, P = .18; month 6, P = .04; month 8, P = .69. P values for simvastatin:month 1, P = .30; month 6, P = .02; month 8, P = .18. B, DBP, change from baseline. This figure shows values for subjects who were not hypertensive at baseline,were never prescribed BP medications, and had BP measured through the 8-month visit. This mitigates the influence of changes distinct from treatment assignment across time. The change values differed significantly from placebo (P ≤ .01)at month 6 (only) for both pravastatin and simvastatin. Standard errors for pravastatin: month 1, 0.98; month 6, 0.96; month 8, 1.0. Standard errors for simvastatin: month 1, 0.97; month 6, 0.98; month 8, 1.0. P values for pravastatin: month 1, P = .06; month 6, P = .004; month 8, P = .06. P values for simvastatin: month 1, P = .46; month 6, P = .01; month 8, P = .66.
Both simvastatin and pravastatin reduced SBP and DBP substantially, although the mean absolute magnitude of the change was modest in this largely nonhypertensive sample receiving relatively low statin dosages. To our knowledge, this is the first large, randomized, double-blind,placebo-controlled trial to report that statins lower both SBP and DBP relative to placebo; that the effect extends to persons with "prehypertension,"20 those with lower-normal BP, and persons not receiving BP-lowering medications; and that it occurs and seems to be comparable for similar lipid-lowering potencies of the most hydrophilic and lipophilic statins. Our data enrich information on the time course of the effect: BP reductions with statins were suggestive and not significant in this sample at 1 month of treatment but were manifest and significant at 6 months (Figure 2). At 2 months after statins were discontinued, the difference in BP between the statin and placebo groups had dissipated. These findings extend our understanding of the BP effects of statins,as underscored by a previous statement suggesting that "statins may decrease elevated but not normal blood pressure"12(p1284); the present study modifies that conclusion. Moreover, because those not receiving BP medications showed reductions in SBP and DBP with statin vs placebo that were significant (see Table 2 for P values), the effect of statins on BP could not be attributed purely to a drug interaction with existing antihypertensive medications.4
Mechanisms by which statins may reduce BP include up-regulation and/or activation of endothelial nitric oxide synthase (a potent vasodilator)21,22 and improvements in endothelial function and flow-mediated vasodilation.23- 29 Statins may reportedly induce down-regulation of angiotensin II type-I receptor expression.30 Of note,statins' benefits to endothelial function and vasodilation are thought to be founded on statins' antioxidant effects27,31 and have been absent or attenuated in some groups—such as persons with low HDL-C or diabetes mellitus.18,32- 34 In Cholesterol and Recurrent Events (CARE) study subjects—who showed no BP reduction with statins35—15% had diabetes mellitus and the mean HDL-C level was low (39 mg/dL). In contrast, our sample, evidencing a modest but significant BP reduction (see Table 2 for P values), excluded those with diabetes mellitus and had a higher mean HDL-C level (52 mg/dL). Subgroup analysis supported preferential SBP reduction in those with higher HDL-C. However, the same was not seen for DBP.
Large statin trials have seldom commented on statin effects on BP. For trials of longer duration, more BP reduction in the statin group may result over time in more BP medication use in the placebo group, undermining the ability to detect a statin effect on BP. Successful randomization produces comparability at baseline but cannot protect against differential clinical decisions arising as a result of changes induced by the treatment.
Alternatively, BP effects may genuinely fail to be sustained because physiological responses to statin effects evolve over time (eg, tachyphylaxis). Finally, through effect modification, a true absence of effect may characterize studies that differ from this study in subject selection and/or statin drug or dosage. Future studies can further evaluate the impact of statins on BP with attention to these issues.
One large, randomized, double-blind trial showed no significant effect on BP in nonhyperlipidemic subjects with cardiovascular disease.35 A recent meta-analysis of randomized controlled trials of statins reporting effects on BP, with an aggregate sample size of 828 subjects, reported significant reduction in systolic BP (only).36 The largest of the 20 included studies had a sample size of 100, raising potential concerns about publication bias among small studies. Other recent reviews and meta-analyses have also supported effects but have further emphasized the methodological shortcomings of most of the published literature in this area.37,38 A larger, parallel-design study has reported benefit to BP but has been published only as an abstract.39
We included BP assessment in our randomized controlled trial to address an important finding reported previously in observational and small crossover studies. Blood pressure was not a primary end point. Although duplicate BP measurements were not performed at each study time point, in contrast to the clinical setting (in which duplicate measurements are requisite to improve precision of the estimate for the individual), in the research setting, precision can be enhanced for group level inferences through increased sample size, averaging single values over multiple subjects. Indeed, randomized controlled trials commonly assess the impact of interventions on outcomes that have considerable test-retest variability, and this variability is typically overcome by boosting sample size rather than by duplicate assessment. Any nondifferential measurement error incurred as a result of measurement variability arising from single BP measurements produces expected bias toward the null, making the findings reported herein conservative.
The study addresses persons without diabetes mellitus, known cardiovascular disease, or extreme high or low LDL-C level; findings may not extend to excluded groups. Subjects with hypertension, those most in need of BP reduction, were not strongly represented; however,effects in persons with high-normal BP suggest clinical relevance given reported implications of prehypertension.20,40- 42
Some issues were not addressed in this study and will require different study designs. These include the impact of different statin dosages, other statin drugs, and longer duration of treatment.
This study adds to our understanding of the effects of statins,currently the best-selling prescription drugs in the world. It provides the first published confirmation in a large, parallel-design, randomized controlled trial of a finding that has been reported with other study designs: that effects of statins extend to reduction in a second primary cardiovascular risk factor, namely, BP. It suggests that this effect extends to lipophilic and hydrophilic statins. The reduction in BP seen with statins may contribute—among other identified factors43—to some of the "rapid"cardiovascular benefits of statins, arising too swiftly to be explained by effects of statins on plaque accumulation.44 Statin-induced reductions in BP, although modest, could contribute to reductions in transient ischemic attacks and stroke reported with statins,45- 50 because stroke incidence, although inconsistently related to LDL-C,is strongly related to BP.
Correspondence: Beatrice A. Golomb,MD, PhD, Department of Medicine, University of California, San Diego,9500 Gilman Dr, No. 0995, La Jolla, CA 92093-0995 (email@example.com).
Accepted for Publication: October 28,2007.
Author Contributions: Dr Golomb had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Golomb, Dimsdale, White,and Criqui. Acquisition of data: Golomb, Ritchie, and Criqui. Analysis and interpretation of data: Golomb, Dimsdale, White, and Criqui. Drafting of the manuscript: Golomb, Ritchie, and Dimsdale. Critical revision of the manuscript for important intellectual content: Golomb, Dimsdale, White, and Criqui. Statistical analysis: Golomb and White. Obtained funding: Golomb. Administrative, technical, and material support: Ritchie, Dimsdale, and Criqui. Study supervision: Golomb.
Financial Disclosure: None reported.
Funding/Support: This study was funded by the National Heart, Lung, and Blood Institute, National Institutes of Health, grant RO1 HL63055-05. The UCSD General Clinical Research Center (NIH MO1 RR00827) also helped to support this effort.
Role of the Sponsors: The funding sources did not influence the design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, and approval of the manuscript.
Previous Presentations: An abstract of this article was presented at the American Heart Association's Scientific Sessions; November 7, 2004; New Orleans, Louisiana; and published in Circulation (2004;110[17]:III-402)and Circulation (2005;112[9]:123).
Additional Contributions: We thank Julie Denenberg, MS, for data management and review of data integrity; Marcella Evans, BS, for administrative and editorial assistance; and Hanh Nguyen and Jersey Neilson for graphical assistance. We also thank the UCSD General Clinical Research Center. We are grateful to all of the Statin Study staff members—and subjects—who made this study possible.
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Foam Rolling for Swimmers
When used correctly the foam roller can provide significant benefits to swimmers and triathletes. Learn more.
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Black Eyed Pea Recipes
Looking for black-eyed pea recipes? Find delicious black-eyed pea recipes including black-eyed pea jambalaya, black-eyed pea salad, black-eyed pea chowder and more black-eyed pea recipes.
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"I've had a lot of compliments and requests for this recipe over the years. My husband loves it, and it's especially great on hot days. The salad dressing keeps the avocado from turning dark, even if you have leftovers-which doesn't happen often!" A fun alternative to pasta or potato salad. —Nancy Cariker, Bakersfield, CaliforniaGet Recipe


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Real time PV Manufacturing Diagnostic System
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Microxact Incorporated
295 Industrial Drive, Christiansburg, VA, -
Principal Investigator:
Vladimir Kochergin
(540) 394-4040
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AbstractThe main obstacle Photovoltaic (PV) industry is facing at present is the higher cost of PV energy compared to that of fossil energy. While solar cell efficiencies continue to make incremental gains these improvements are so far insufficient to drive PV costs down to match that of fossil energy. Improved in-line diagnostics however, has the potential to significantly increase the productivity and reduce cost by improving the yield of the process, as proven by industry leaders. MicroXact proposes to develop a high-throughput in-line PV manufacturing diagnostic system, which will provide fast and accurate data on the spatial uniformity of thickness, refractive indices, and the film stress of the thin films comprising the solar cell as the solar cell is processed reel-to-reel. The retrieved information will provide the opportunity to detect a wide variety of processing errors, including but not limited to thickness/composition in homogeneity in any layer comprising PV device, non-uniform scribing, thin film stress, cracking and layer separation.Commercial Applications and Other Benefits The initial target application for the proposed in-line crystalline silicon and thin film manufacturing diagnostic system is the PV cell manufacturing lines with high production capacity ( & gt;10MW/year), in which the benefits of the in-line diagnostics (such as yield improvement and cost reduction) are expected to outweigh the cost of system installation immediately. In addition, low-cost system modifications will be developed to target off-line quality assurance and quality control at smaller-scale PV cell manufacturing lines.
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Love the performing arts? Love to watch and play sports? Love great food and music? Then you're going to absolutely love Tulsa. Exceptional museums, world-class ballet and opera, professional and recreational sports, live entertainment, bargain and big-ticket shopping, gourmet and down-home cuisine—enjoy these and countless other big-city experiences in a completely different way. No long lines, no huge crowds, no big attitudes, just plenty to do.
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Located just three miles from Tulsa International Airport and other nearby attractions, such as the Tulsa Zoo.
Our hotel's downtown address places you minutes from exciting big-name music and sporting events.
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Hosted by local poets DJ Miss Joy, Misty Reign, Prinze, and A.j. Moyer
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Rock Band Blitz to release in August on PSN and XBL
Harmonix has announced Rock Band Blitz will release on PSN August 28 for $14.99 and On XBL August 29 for 1200 Microsoft Points.
Rock Band Blitz allows you to play the music game with a controller instead of an instrument peripheral, and is fully compatible with songs already available in the Rock Band Music Store, as well as tracks exported from Rock Band, Rock Band 2, and more.
It will also ship with 20 songs from artists such as Blink-182, Foster the People, Iron Maiden, and the following songs that were announced today:
- Avenged Sevenfold – "So Far Away"
- Fun. ft. Janelle Monáe – "We Are Young"
- Kool & The Gang – "Jungle Boogie"
- My Chemical Romance – "Sing"
Rock Band Blitz's songs are also immediately compatible with and playable in Rock Band 3.
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I am using Slideshow Gallery plugin. After the latest update for this SSL plugin, my slideshow does not work correctly. The only way I can get it to function is by disabling the SSL plugin. Does the SSL plugin somehow modify the javascript for the Gallery? Does it change a path or modify the code somewhere? Does it make something https that shouldn't be?
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Almarin, a Spanish manufacturer of AtoN, has completed the design and turn‐key supply of aids to navigation for Puerto Brisa (La Guajira, Colombia). The company provided 14 buoys: twelve lateral marks, one safe water and one special mark. All buoys, from its 'Guía' range, were provided with self contained lights equipped with satellite monitoring. The G2200T model was used for the channel dredged at 17m and the G2400T was selected for its additional volume in the open water marks. To help navigate the approach and channel, a sector light was installed on a 30m high tower 1,500m inland, providing precise information to the ship's lateral position with regards to the center of the channel.
The project has a 2,500m long channel that provides access to a turning basin and quay. There is no protection from the natural elements and the dredged channel is less than 250m wide, with regular strong trade winds. The information provided by the navigational aids is critical for safe access to port.
Puerto Brisa is a newly built port located in the northern coast of Colombia, in the municipality of Dibulla (La Guajira). Designed initially as a coal port, it benefits from being a free trade zone and is strategically placed to become one of the most important ports on the Colombian Caribbean coast.
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The Panama Canal has launched the Green Connection Award, a new initiative to recognize customers who demonstrate excellent environmental stewardship, and to encourage
Thyssenkrupp supplied 14 innovative elevators for expansion of the Panama Canal , seven for each new lock complex, one of which is on the Atlantic side and the other on the Pacific.
After 25 years as CEO of Antwerp Port Authority, Eddy Bruyninckx has been raised to the nobility by H.M. the king of Belgium with the rank of baron. In Belgium
With seaborne transportation accounting for the vast majority of the world's international trade, the importance of the shipping industry to the mechanics of the
US East Coast service through the Panama Canal to improve transit times significantly!
Maersk Line will transform its TP12 service in to a standalone, around-the-world
INTERSCHALT maritime systems GmbH, a provider of services for communications, navigation and bridge equipment and the maker of voyage data recorder systems, has
Authorities are building a new containment boom to fight an oil spill in a major western Canadian river, officials said on Saturday, after the spill breached a
Ecuador has paid $112 million to energy company Chevron Corp over a four-decade-old contract dispute, even though it remains in disagreement, the head of the central bank has said.
Southeast Asian nations were thrown into disarray after Cambodia on Saturday blocked them from issuing a statement referring to an international court ruling against
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The U.S. Navy conducted a series of cooperative air defense test exercises with the Spanish navy that culminated in live missile firing events using the latest Aegis Weapon System baseline July 20-21.
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For the first time, AWI scientists have found evidence of living, potentially pathogenic vibrions on microplastic particles.
With increasing water temperatures
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At high tide, the vessel "MARTIN SÆLE", the first Live Fish Carrier built by Gondan Shipyard in Figueras, was successfully launched today. Representatives of the owner of the vessel,
French supplier of systems for marine transportation of liquified natural gas (LNG) Gaztransport & Technigaz (GTT) reported strong growth in its first half results


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Hotel Staff, front desk, concierge, valet were all outstanding. The view overlooking the pool and out to the ocean was absolutely perfect. Having a refrigerator and coffee maker in the room was great for the morning , fresh papaya and pineapple from the market and coffee on the balcony watching the ocean can't be beat.
Room: the carpet was buckled up in different areas and basically needed to be replaced, baseboards in the room and bathroom were in poor shape, toilet wouldn't flush, balcony furniture appeared old and cheap. For the price; the view, staff and location were well worth it, room condition not so much. Would I stay again: absolutely yes
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